Nitrosamine compounds, their preparation methods, their use in detecting varenicline tartrate raw materials or preparations, and their detection methods

Abstract

This invention discloses nitrosamine compounds, their preparation methods, their application in the detection of varenicline tartrate raw materials or preparations, and detection methods. Specifically, this invention discloses providing the compound shown in formula P08, its preparation method, detection method, and its application in the preparation of bactericidal drugs, as an insecticide, and further, its application as a standard, reference standard, or detection item in the impurity study, quality control, or detection methods of varenicline tartrate raw materials or preparations.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to nitrosamine compounds, their preparation methods, their application in the detection of varenicline tartrate raw materials or preparations, and detection methods. Background Technology

[0002] Vareniclan is an aryl fused nitrogen heterocyclic compound that modulates cholinergic activity. It is sold as a tartrate salt under the trade name [trade name missing]. and It can bind to the specific receptor site for nicotinic acetylcholine on neurons and can be used to treat the following diseases:

[0003] Treatment of inflammatory bowel disease (including but not limited to: ulcerative colitis, pyoderma gangrenosa, 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 impairment, drug / toxin-induced cognitive impairment (e.g., caused by alcohol, barbiturates, vitamin deficiency, recreational drugs, lead, arsenic, mercury), and disease-induced cognitive impairment (e.g., caused by Alzheimer's disease, vascular dementia). Dementia, Parkinson's disease, multiple sclerosis, AIDS, (major) encephalitis, trauma, renal encephalopathy and hepatic encephalopathy, hypothyroidism, Pick's disease, Korsakoff syndrome and frontal and subcortical dementia, hypertension, hyperphagia, anorexia, obesity, arrhythmia, hyperacidity, ulcers, pheochromocytoma, progressive supramuscular palsy, chemical dependence and addiction (e.g., to nicotine (and / or tobacco products), alcohol, benzodiazepines). Dependence and addiction to nicotine, barbiturates, opioids or cocaine, headache, migraine, stroke, traumatic brain injury (TBI), obsessive-compulsive disorder (OCD), psychosis, Huntington's disease, tardive dyskinesia, hyperkinesis, 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, addiction and withdrawal from nicotine, including its use in smoking cessation treatment (see: CN 1509174A).

[0004] The preparation method of vareniclan and the method for resolving its racemic compounds have been disclosed in U.S. 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 nitroso groups (N(R1)(R2)-N=O) bound to amines, are genotoxic to some animals, and some have been classified as possible or potential human carcinogens by the International Agency for Research on Cancer (IARC).

[0006] These nitrosamine compounds are designated as "group of concern" compounds in International Conference on Harmonization (ICH) guidance document M7(R1), which assesses and controls DNA-reactive (mutagenic) impurities in pharmaceutical products to limit potential carcinogenic risks (March 2018). The ICH guidance recommends that any known mutagenic / carcinogenic substances, including nitrosamine compounds, be controlled at intake levels where the risk of cancer in humans is negligible.

[0007] Currently, the U.S. Food and Drug Administration (FDA) has identified seven nitrosamine impurities that could theoretically be present in pharmaceutical products due to the use of manufacturing processes and materials that may lead to nitrosamine formation: N,N-dimethylnitrosamine (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). Of these, five impurities (NDMA, NDEA, NMBA, NIPEA, and NMPA) have actually been detected in active pharmaceutical ingredients (APIs) or finished products.

[0008] For example, some preliminary results from FDA trials have indicated that NDMA levels in certain ranitidine products have exceeded acceptable levels. In recent years, the FDA has also found NDMA impurities in some metformin products, and in some batches, NDMA levels have been detected above the FDA's recommended acceptable intake limits.

[0009] In view of this, the present invention is proposed to control the nitrosamine impurity content in varenicline tartrate raw materials or preparations to a low level that meets safety requirements. Summary of the Invention

[0010] In view of the problems and / or deficiencies of the existing technology, the purpose of the present invention is to provide a nitrosamine compound, its preparation method, its application in the detection of varenicline tartrate raw material or preparation, and a detection method thereof, and further to use the nitrosamine compound of the present invention as a standard, reference standard or detection item in the impurity research, quality control or detection method of varenicline tartrate raw material or preparation.

[0011] This invention provides a compound of formula P08, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof:

[0012]

[0013] The present invention also provides a method for preparing the compound shown in formula P08, which includes the following steps: vareniclan reacts with nitrite under acid and solvent conditions to undergo a nitrosamine reaction to generate the compound shown in formula P08;

[0014]

[0015] In the preparation method, the nitrite is preferably sodium nitrite and / or potassium nitrite.

[0016] In the preparation method, the nitrite is preferably an aqueous solution of nitrite, and more preferably, the concentration of the aqueous solution of nitrite is 0.2 to 0.6 g / mL, for example, 0.4 g / mL.

[0017] In the preparation method, the acid is preferably acetic acid.

[0018] In the preparation method, the solvent is preferably tetrahydrofuran and water, and the volume ratio of tetrahydrofuran to water is 1:4 to 6.

[0019] In the preparation method, the volume-to-mass ratio of the solvent to the vareniclan is preferably 8-16 g / mL, for example, 12.5 g / mL.

[0020] In the preparation method, the molar ratio of vareniclan to nitrite is preferably 1:(1-4), for example 1:2.5.

[0021] In the preparation method, the reaction temperature is preferably 40-60°C, for example, 52°C.

[0022] This invention also provides the application of the compound shown in formula P08 as a standard, reference, or detection item in the impurity study, quality control, or detection method of a nitrogen-containing polycyclic aromatic hydrocarbon (NAPA) active pharmaceutical ingredient or preparation; wherein the NPA is varenicline or a pharmaceutically acceptable salt thereof; preferably, the detection method is liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0023]

[0024] This invention also provides a method for detecting the compound represented by formula P08. First, qualitative detection is achieved, and then quantitative detection is further achieved based on the qualitative detection. This method monitors the residue of the compound represented by formula P08 (genotoxic impurity) in the active pharmaceutical ingredient or formulation of varenicline or its pharmaceutically acceptable salt, thereby ensuring the product quality and medication safety of the drug. The detection method includes the following steps: using the compound represented by formula P08 as a standard or reference, liquid chromatography-tandem mass spectrometry is used to detect the compound represented by formula P08 in the sample to be tested.

[0025]

[0026] Furthermore, the sample to be tested is a raw material or preparation of a nitrogen-containing polycyclic compound, wherein the nitrogen-containing polycyclic compound is varenicline or a pharmaceutically acceptable salt thereof, preferably, the sample to be tested is: varenicline tartrate raw material or varenicline tartrate preparation.

[0027] Furthermore, when the sample to be tested is varenicline tartrate raw material, the chromatographic conditions for the liquid chromatography-tandem mass spectrometry method include:

[0028] Column: ACQUITY UPLC BEH C18 or equivalent column;

[0029] Mobile phase A: formic acid - purified water, with a volume ratio of 0.01 to 0.2:100 (including but not limited to: 0.02:100, 0.03:100, 0.04:100, 0.05:100, 0.06:100, 0.07:100, 0.08:100, 0.09:100, 0.1:100, 0.11:100, 0.12:100, 0.15:100, 0.18:100, etc.), preferably 0.1:100;

[0030] Mobile phase B: Acetonitrile;

[0031] Preferably, gradient elution is performed according to the table below:

[0032]

[0033] Furthermore, when the sample to be tested is varenicline tartrate raw material, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: the column specifications are: 2.1 mm × 100 mm, 1.7 μm.

[0034] Furthermore, when the sample to be tested is varenicline tartrate raw material, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: column temperature: 15-35℃ (including but not limited to: 20℃, 25℃, 30℃ or 32℃, etc.).

[0035] Furthermore, when the sample to be tested is varenicline tartrate raw material, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: injection volume: 1-25 μL (including but not limited to: 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL or 22 μL, etc.).

[0036] Furthermore, when the sample to be tested is varenicline tartrate raw material, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: flow rate: 0.1-1 ml / min (including but not limited to: 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 or 0.9 ml / min, etc.).

[0037] Furthermore, when the sample to be tested is varenicline tartrate raw material, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: column temperature: 30℃; injection volume: 2μl; flow rate: 0.4ml / min.

[0038] Furthermore, when the sample to be tested is varenicline tartrate raw material, the mass spectrometer is switched in before the compound shown in formula P08 elutes; for example, the mass spectrometer is switched in at 2 to 3 minutes of the gradient elution program, including but not limited to: 2 min, 2.1 min, 2.2 min, 2.3 min, 2.4 min, 2.5 min, 2.6 min, 2.7 min, 2.8 min, 2.9 min or 3 min.

[0039] Furthermore, when the sample to be tested is varenicline tartrate raw material, the detection instrument is UPLC-MS / MS (ACQuity I CLASS & TRIPLE QUAD 5500+).

[0040] Furthermore, when the sample to be tested is the varenicline tartrate raw material, the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry method also include: ion source: ESI (electrospray ionization source) or APCI (atmospheric pressure chemical ionization source); jet gas: 9 psi; curtain gas: 30 psi; ionization voltage: 5500 V; temperature: 500 °C; spray gas: 50 psi; auxiliary heating gas: 50 psi.

[0041] Furthermore, when the sample to be tested is varenicline tartrate raw material, the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry method also include: positive ion mode; scanning mode: SIM (single ion monitoring), SRM (selective reaction monitoring), or MRM (multi-reaction monitoring); preferably SIM or MRM.

[0042] Furthermore, when the sample to be tested is a varenicline tartrate preparation, the chromatographic conditions for the liquid chromatography-tandem mass spectrometry method include:

[0043] Chromatographic column: ChromCore Biphenyl or equivalent column;

[0044] Mobile phase A: formic acid - purified water, with a volume ratio of 0.01–0.2:100 (including but not limited to: 0.02:100, 0.03:100, 0.04:100, 0.05:100, 0.06:100, 0.07:100, 0.08:100, 0.09:100, 0.1:100, 0.11:100, 0.12:100, 0.15:100, 0.18:100, etc.), preferably 0.05:100; Mobile phase B: acetonitrile;

[0045] Preferably, gradient elution is performed according to the table below:

[0046]

[0047] Furthermore, when the sample to be tested is a varenicline tartrate preparation, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: the column specifications are: 2.1 mm × 100 mm, 3 μm.

[0048] Furthermore, when the sample to be tested is a varenicline tartrate preparation, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: column temperature: 15-35℃ (including but not limited to: 20℃, 25℃, 30℃ or 32℃, etc.).

[0049] Furthermore, when the sample to be tested is a varenicline tartrate preparation, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: injection volume: 1 to 25 μL (including but not limited to: 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL or 22 μL, etc.).

[0050] Furthermore, when the sample to be tested is a varenicline tartrate preparation, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: flow rate: 0.1 to 1 ml / min (including but not limited to: 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 or 0.9 ml / min, etc.).

[0051] Furthermore, when the sample to be tested is a varenicline tartrate preparation, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method also include: column temperature: 25℃; injection volume: 20μL; flow rate: 0.4ml / min.

[0052] Furthermore, when the sample to be tested is a varenicline tartrate formulation, the mass spectrometer is switched in before the compound represented by formula P08 elutes; for example, the mass spectrometer is switched in at 6-7 min of the gradient elution program.

[0053] Furthermore, the mass spectrometry conditions for the liquid chromatography-tandem mass spectrometry method include:

[0054] Positive ion mode;

[0055] Scanning modes: SIM (single ion monitoring), SRM (selective reaction monitoring), or MRM (multi-reaction monitoring); SIM or MRM is preferred.

[0056] Furthermore, when the sample to be tested is a varenicline tartrate preparation, the detection instrument is a Vanquish HPLCQ-Exactive-MS / MS.

[0057] Furthermore, when the sample to be tested is a varenicline tartrate preparation, the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry method also include: spray voltage: 3.9 kV; sheath gas flow rate: 35 arb; auxiliary gas flow rate: 10 arb; capillary temperature: 320 °C; auxiliary gas temperature: 320 °C.

[0058] Furthermore, when the sample to be tested is a varenicline tartrate preparation, the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry method also include: scanning mode: SIM, single ion detection scan; scanning range: 240.3-241.9 m / z; polarity: positive ion mode.

[0059] Furthermore, the varenicline tartrate formulation comprises varenicline tartrate and pharmaceutically acceptable excipients; wherein the excipients comprise one or more of diluents, disintegrants, glidants, and lubricants.

[0060] Furthermore, the diluent is selected from one or more of microcrystalline cellulose, silicified microcrystalline cellulose, dicalcium phosphate (including anhydrous form and hydrate), mannitol, copovidone, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, starch, maltodextrin, agar, and guar gum; preferably, the diluent comprises microcrystalline cellulose and / or anhydrous dicalcium phosphate.

[0061] Furthermore, the weight ratio of varenicline tartrate to the diluent is 1:(100-300); preferably 1:(150-250); more preferably 1:(185-195).

[0062] Furthermore, the disintegrant is selected from one or more of croscarmellose sodium, glycolic acid starch sodium, croscarmellose, corn or pregelatinized starch, sodium carboxymethyl cellulose and calcium carboxymethyl cellulose; preferably, the disintegrant is croscarmellose sodium.

[0063] Furthermore, the weight ratio of varenicline tartrate to the disintegrant is 1:(1-10); preferably 1:(2-6); more preferably 1:(3.5-4.5); for example, 1:4.

[0064] Furthermore, the flow aid is selected from one or more of colloidal silica, fumed silica, silica, colloidal silica, corn starch, talc, calcium silicate, magnesium silicate, tricalcium phosphate, and silica hydrogel; preferably, the flow aid is colloidal silica.

[0065] Furthermore, the weight ratio of varenicline tartrate to the glidant is 1:(0.1-8); preferably 1:(0.2-5); more preferably 1:(0.5-2.5); and most preferably 1:(0.8-2), for example 1:0.8, 1:1 or 1:2.

[0066] Furthermore, the lubricant is selected from one or more of stearate, stearic acid, talc, mineral oil, malt, glyceryl benzoate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, and sodium lauryl sulfate; preferably, the lubricant is stearate; more preferably, the stearate is magnesium stearate.

[0067] Furthermore, the weight ratio of varenicline tartrate to the lubricant is 1:(0.1-8); preferably 1:(0.2-5); more preferably 1:(0.5-2.5); and most preferably 1:(1-2), for example 1:1, 1:1.4 or 1:2.

[0068] Furthermore, the varenicline tartrate formulation is a solid dosage form; preferably tablets or granules.

[0069] Furthermore, the tablets are plain tablets or coated tablets.

[0070] Furthermore, the coating agent of the coated tablet is an Opadry coating agent (including but not limited to: white, blue, yellow, red, green, orange, etc. Opadry coating agents); preferably. White and / or Blue.

[0071] Furthermore, the detection method is either qualitative or quantitative.

[0072] Furthermore, when the detection method is quantitative detection, the compound shown in formula P08 is used as a standard or reference standard to prepare standard or reference standard solutions of different concentrations. These solutions are then injected for detection to obtain the corresponding peak area data. A linear regression is performed between the concentration and the peak area to obtain a linear equation. The sample solution to be tested is then injected for detection to obtain the corresponding peak area data. Substituting the peak area detection data of the compound shown in formula P08 in the sample solution to be tested into the linear equation, the concentration data of the compound shown in formula P08 in the sample solution to be tested can be obtained.

[0073] Furthermore, when the sample to be tested is the varenicline tartrate raw material, the concentration data of the compound shown in formula P08 in the sample solution to be tested is divided by the concentration data of varenicline tartrate in the sample solution to obtain the content data of the compound shown in formula P08 in the varenicline tartrate raw material.

[0074] Furthermore, when the sample to be tested is the varenicline tartrate preparation, the concentration data of the compound shown in formula P08 in the sample solution to be tested is divided by the concentration data of varenicline tartrate in the sample solution to obtain the content data of the compound shown in formula P08 in the varenicline tartrate preparation.

[0075] Furthermore, the solvent for the standard or reference solution is acetonitrile and / or purified water; the solvent for the test sample solution is acetonitrile and / or purified water.

[0076] Furthermore, the solvent of the standard or reference solution is the same as the solvent of the sample solution to be tested.

[0077] The present invention also provides the use of the compound of formula P08 or its pharmaceutically acceptable salt, crystal form or solvate as a bactericide and / or insecticide;

[0078]

[0079] The positive and progressive effects of this invention are as follows: the compound shown in formula P08 of this invention can have bactericidal and insecticidal activities, and the compound of this invention can be used as a standard, reference standard or detection item in the impurity research, quality control or detection method of varenicline tartrate raw material or preparation.

[0080] There are no particular restrictions on the administration method of the drug of the present invention. Representative administration methods include, but are not limited to, oral, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0081] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), or with the following components: (a) filler or compatibilizer; (b) thickener, such as carboxymethyl cellulose and its salts; (c) humectant; (d) disintegrant; (e) slowing agent; (f) absorption accelerator; (g) wetting agent; (h) adsorbent; and (i) lubricant. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0082] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shell materials, such as casings and other materials known in the art. They may contain opaque agents, and the release of the active compound or compound in such dosage forms can be delayed in a portion of the digestive tract. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0083] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, as well as solubilizers and emulsifiers.

[0084] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention. Attached Figure Description

[0085] Figure 1 The chromatogram of compound PO6, a nitramine impurity compound, is shown for the detection of compound A by high performance liquid chromatography. Detailed Implementation

[0086] The present invention will now be clearly and completely described in conjunction with specific embodiments. Those skilled in the art will understand that the embodiments described below are some, but not all, embodiments of the present invention, and are only used to illustrate the present invention, and should not be regarded as a limitation on the scope of protection of the present invention.

[0087] In this invention, unless otherwise specified, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0088] Regarding the definitions of terms used in this invention, unless otherwise stated, the initial definitions provided herein apply to the term throughout the text; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and / or context.

[0089] Example 1

[0090] Varenicline tartrate tablets (Formulation 1):

[0091] According to the formulation in Table 1, the colloidal silicon dioxide (part I) and anhydrous dibasic calcium phosphate (part I) were mixed together. Varenicline tartrate was then added and mixed thoroughly. Next, the colloidal silicon dioxide (part II) and anhydrous dibasic calcium phosphate (part II) were added and mixed thoroughly. Then, the intragranular portions of microcrystalline cellulose, croscarboxymethyl cellulose sodium, and magnesium stearate were added and mixed thoroughly. The mixture was then dry-granulated, and the granules were mixed with the extragranular portions of magnesium stearate. The mixture was compressed into tablets and then coated with Opadry coating powder: milky white and / or blue (…). White and / or Blue, which can also be abbreviated as: Oppa Daibai, Oppa Dailan, etc.

[0092] In the above formulation, microcrystalline cellulose and anhydrous dicalcium phosphate are used as diluents, croscarmellose sodium cellulose is used as a disintegrant, colloidal silica is used as a flow aid, magnesium stearate is used as a lubricant, and ethanol and / or purified water are used as coating solvents.

[0093] Table 1. Formulation 1 (Uncoated tablets)

[0094]

[0095] Among them, varenicline tartrate 1.71mg / tablet can be converted into varenicline 1mg / tablet, that is: each tablet contains 1mg of active ingredient (varenicline), which can also be expressed as: 1mg plain tablet (or 1mg coated tablet, etc.); while 0.5mg plain tablet is based on 1mg plain tablet, in which the amount of each component is reduced by half.

[0096] Example 2

[0097] Varenicline tartrate tablets (formulation 2):

[0098] According to the formulation in Table 2, the colloidal silicon dioxide (part I) and anhydrous dibasic calcium phosphate (part I) were mixed together. Varenicline tartrate was then added and mixed thoroughly. Next, the colloidal silicon dioxide (part II) and anhydrous dibasic calcium phosphate (part II) were added and mixed thoroughly. Then, the intragranular portions of microcrystalline cellulose, croscarboxymethyl cellulose sodium, and magnesium stearate were added and mixed thoroughly. The mixture was then dry-granulated, and the granules were mixed with the extragranular portions of magnesium stearate. The mixture was compressed into tablets and then coated with Opadry coating powder: milky white and / or blue (…). White and / or Blue).

[0099] In the above formulation, microcrystalline cellulose and anhydrous dicalcium phosphate are used as diluents, croscarmellose sodium cellulose is used as a disintegrant, colloidal silica is used as a flow aid, magnesium stearate is used as a lubricant, and purified water is used as a coating solvent.

[0100] Table 2, Formulation 2 (Uncoated tablets)

[0101]

[0102] Example 3

[0103] Varenicline tartrate tablets (formulation 3):

[0104] According to the formulation in Table 3, the colloidal silicon dioxide (part I) and microcrystalline cellulose (part I) were mixed together. Varenicline tartrate was then added and mixed thoroughly. Next, the colloidal silicon dioxide (part II) and microcrystalline cellulose (part II) were added and mixed thoroughly. Then, the intragranular portions of anhydrous calcium phosphate, croscarboxymethyl cellulose sodium, and magnesium stearate were added and mixed thoroughly. This mixture was then combined with the exogranular portion of magnesium stearate, compressed into tablets, and coated with Opadry coating powder: milky white and / or blue (…). White and / or Blue).

[0105] In the above formulation, microcrystalline cellulose and anhydrous dicalcium phosphate are used as diluents, croscarmellose sodium cellulose is used as a disintegrant, colloidal silica is used as a flow aid, magnesium stearate is used as a lubricant, and purified water is used as a coating solvent.

[0106] Table 3, Formulation 3 (Uncoated tablets)

[0107]

[0108] Example 4

[0109] Varenicline tartrate tablets (formulation 4): Prepared according to the formulation in Table 4, using a process similar to that in Example 3.

[0110] Table 4, Formulation 4 (Uncoated tablets)

[0111]

[0112] Example 5

[0113] Varenicline tartrate tablets (formulation 5): Prepared according to the formulation in Table 5, using a process similar to that in Example 3.

[0114] Table 5, Formulation 5 (Uncoated tablets)

[0115]

[0116] Example 6

[0117] Varenicline tartrate tablets (formulation 6): Prepared according to the formulation in Table 6, using a process similar to that in Example 3 (dry granulation).

[0118] Table 6, Formulation 6 (Uncoated tablets)

[0119]

[0120] Example 7

[0121] The nitrosamine impurity P08 in varenicline tartrate raw material was detected using UPLC-MS / MS (equipment type: ACQuity I CLASS & TRIPLE QUAD 5500+), and its structural formula is shown below. The nitrosamine impurity P08 in varenicline tartrate tablets (plain tablets or coated tablets) was detected using HPLC Q-Exactive-MS / MS (Vanquish HPLC Q-Exactive-MS / MS).

[0122]

[0123] The test results are shown in Table 7.

[0124] Table 7. Detection results of nitrosamine impurity P08 in unprocessed tablets

[0125]

[0126]

[0127] ppm: parts per million, defined as one part per million, 1 ppm is one part per million.

[0128] Example 8

[0129] Coating process optimization: According to Table 8, the uncoated tablets obtained in Examples 2 to 6 were coated to obtain varenicline tartrate coated tablets.

[0130] Table 8. Varenicline Tartrate Coated Tablets

[0131]

[0132] Among them, #1W indicates that 1mg of the raw tablet (#1) is used with 2% White coating is used; #2W indicates that 1mg of unprocessed tablets (#2) are coated with 2% [a specific coating]. White is used for coating, and so on for other types.

[0133] Example 9

[0134] According to FDA (Food and Drug Administration) requirements, nitrosamine impurities in drugs need to be tested, and the level is generally required to be controlled at ≤7.5ppm.

[0135] Nitrosamine impurity P08 in coated tablets was detected using HPLC Q-Exactive-MS / MS (Vanquish HPLC Q-Exactive-MS / MS). The method employed was Chanpike (USA). RLD) and China Changpe ( RLD was used as a control; RLD is an abbreviation for Reference Listed Drug. The test results are shown in Table 9.

[0136] Table 9. Detection results of nitrosamine impurity P08 in coated tablets

[0137]

[0138]

[0139] Example 10

[0140] Preparation of nitrosamine compound PO8

[0141]

[0142] To a 100 mL three-necked flask, add 4.0 g of compound MO2 (vareniclan) and 45 mL of tetrahydrofuran (THF), and stir until dissolved. Add 8.2 mL of NaNO2 aqueous solution (concentration 0.4 g / mL), add 2.9 g of acetic acid, heat to 52 °C, and monitor the reaction by TLC until complete. A solid precipitates out. Filter and dry under vacuum to obtain nitrosamine compound PO8 with a purity of 98.87% (area normalization method).

[0143]

[0144] Nitrosamine compound P08 1 H-NMR, 13 C-NMR and IR spectra are shown in Tables 10-12, respectively; M+H + =241.20; UV: The maximum absorption wavelength in acetonitrile solution is 204.40 nm.

[0145] Table 10. Nitrosamine compound P08 1 H-NMR spectrum

[0146]

[0147] Table 11. Nitrosamine compound P08 13 C-NMR spectrum

[0148]

[0149] Table 12. IR spectrum of nitrosamine compound P08

[0150] <![CDATA[Absorption wave number (cm -1 )]]> Belonging 3048.8 Ar-H 2961.4,2931.8 Saturated CH stretching vibration 1925.2 C=C stretching vibration of benzene ring 1575.2 N=O stretching vibration 885.2 Benzene ring CH bending vibration

[0151] Example 11

[0152] We have creatively developed an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS) method for the qualitative and / or quantitative detection of nitrosamine impurities (compound P08) in varenicline tartrate raw material, and the method has been validated.

[0153] The chromatographic and mass spectrometric conditions for UPLC-MS / MS are detailed in Table 13.

[0154] Table 13. Chromatographic and mass spectrometric conditions for UPLC-MS / MS

[0155]

[0156] 1. Solution preparation

[0157] Diluent: Purified water

[0158] 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, then add diluent to dissolve and dilute to the mark, and mix well.

[0159] Reference stock solution: Measure 37.5 μl of the reference stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and mix well.

[0160] 2. System Applicability

[0161] System suitability solution (100% limit level reference solution): Measure about 100.0 μl of the reference stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and mix well.

[0162] Six consecutive injections of system suitability solution were administered, and the RSD of the target peak area in the six injection system suitability solutions was calculated. The result was 4%, indicating good system suitability.

[0163] 3. Linear

[0164] Standard curve solutions: Measure 50.0 μl, 100.0 μl, 150.0 μl, and 200.0 μl of the reference stock solution into four 10 mL volumetric flasks, respectively. Dilute to the mark with diluent and mix well. These are the linear solutions L-3, L-4, L-5, and L-6, respectively. Measure 800 μl of solution L-3 into a 10 mL volumetric flask, dilute to the mark with diluent, and mix well. This is L-2. Measure 1000 μl of solution L-2 into a 10 mL volumetric flask, dilute to the mark with diluent, and mix well. This is L-1.

[0165] The standard curve solutions were injected and analyzed separately. Linear regression was performed with the target analyte concentration (X) as the abscissa (unit: ng / ml) and peak area (Y) as the ordinate 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 within the concentration range of 0.2951 ng / ml to 147.6 ng / ml (i.e., 0.02951 ppm to 14.76 ppm).

[0166] 4. Limit of Detection (LOD)

[0167] LOD solution: Measure 5000 μL of L-1 solution into a 10 ml volumetric flask, dilute to the mark with diluent, mix well, and use as the LOD solution.

[0168] Two consecutive injections of the LOD solution were performed, and the S / N (signal-to-noise ratio) of the target peak were 11 and 8, respectively, which meet the requirement of not less than 3, indicating that the method is sensitive; LOD = 0.01476 ppm.

[0169] 5. Limit of Quantification (LOQ)

[0170] Six consecutive injections of the limit of quantitation solution (L⁻¹ solution) yielded target peak S / N values ​​of 23, 24, 17, 12, 24, and 14, respectively, meeting the requirement of not less than 10, indicating method sensitivity; LOQ = 0.02951 ppm.

[0171] 6. Accuracy

[0172] Sample solution: Weigh approximately 100 mg of the sample (e.g., purchased or synthesized varenicline tartrate raw material) into a 10 ml volumetric flask, dilute to the mark with diluent, and mix well. Prepare two parallel solutions.

[0173] Accuracy solution: Weigh approximately 100 mg of sample into a 10 ml volumetric flask, add 50.0 μl (50% limit level), 100.0 μl (100% limit level), and 150.0 μl (150% limit level) of the reference standard stock solution, respectively, and dilute to the mark with diluent. Mix well to obtain the solution. Prepare three replicates for each accuracy level.

[0174] The results showed that the recoveries of the 50% limit level accuracy solution were between 95% and 102%, with an RSD of 4%; the recoveries of the 100% limit level accuracy solution were between 90% and 103%, with an RSD of 7%; and the recoveries of the 150% limit level accuracy solution were between 98% and 100%, with an RSD of 1%, which met the acceptance criteria and indicated good accuracy.

[0175] 7. Repeatability

[0176] Weigh approximately 100 mg of sample into a 10 ml volumetric flask, add 100.0 μl of the reference standard stock solution, dilute to the mark with diluent, and mix well. Prepare 6 parallel aliquots.

[0177] The results showed that the RSD of the nitrosamine compound PO8 content in the six reproducible solutions was 6%, which met the acceptance criteria and indicated good repeatability.

[0178] 8. Exclusivity

[0179] Inject blank solution (diluent), 100% limit level reference solution, sample solution (same as the "reproducibility" sample solution), and 100% limit level accuracy solution into the sample solution for analysis.

[0180] The results showed that the retention time of the target analyte in the sample solution, the 100% limit level reference solution, and the 100% limit level accuracy solution was 3.47 min. There was no obvious interference at the peak position of the target analyte, which met the separation requirements and showed good specificity.

[0181] 9. Intermediate precision

[0182] Intermediate precision solution: Weigh approximately 100 mg of sample into a 10 ml volumetric flask, add 100.0 μl of the reference standard stock solution, dilute to the mark with diluent, and mix well. Prepare 6 parallel aliquots. Add the 6 aliquots from section "7. Repeatability".

[0183] The concentration RSD of 12 solutions (6 intermediate precision solutions and 6 repeatability solutions) was calculated to be 9%, which meets the requirement of not exceeding 15% and is in line with the acceptance criteria, indicating good intermediate precision.

[0184] 10. Solution stability

[0185] Take a 100% limit level accuracy solution and place it at 15°C. Inject the sample for analysis at regular intervals and calculate the concentration ratio of each time point to the zero point. Within 21 hours, this ratio should be between 96% and 113%.

[0186] 11. Durability

[0187] The concentration of the target analyte in the 100% limit level accuracy solution was investigated under different conditions: column temperature change ±2℃, initial organic phase ratio ±1%, and flow rate change ±0.01 ml / min. Two injections were performed under each condition. The RSD of the target analyte concentration in the six solutions was calculated to be between 4% and 6% under the same parameter fluctuation, indicating good robustness of the method.

[0188] Calculate the content of nitrosamine compound P08 in the sample using the following formula, perform the test twice, and take the average value:

[0189] PO8 content: C (ppm) = PO8 concentration in sample solution (ng / ml) / [m (mg) / V (ml)]

[0190] m: Sample mass, mg;

[0191] V: Sample dilution volume, ml.

[0192] Example 12

[0193] Meanwhile, we have also developed an HPLC Q-Exactive-MS / MS (Vanquish HPLC Q-Exactive-MS / MS) method for the qualitative and / or quantitative detection of nitrosamine impurity P08 in varenicline tartrate tablets, and have conducted and passed method validation.

[0194] The chromatographic and mass spectrometric conditions for HPLC Q-Exactive-MS / MS are detailed in Table 14.

[0195] Table 14. Chromatographic and mass spectrometric conditions for HPLC Q-Exactive-MS / MS

[0196]

[0197]

[0198] 1. Solution preparation

[0199] Reference stock solution: Weigh 7.5 mg of nitrosamine compound P08 into a 100 ml volumetric flask, dissolve in acetonitrile, and dilute to volume (solution 1A). Take 1.0 ml of solution 1A into a 100 ml volumetric flask, and dilute to volume with acetonitrile (solution 1B). Take 1.0 ml of solution 1B into a 20 ml volumetric flask, and dilute to volume with acetonitrile to obtain the reference stock solution.

[0200] Reference solution: Take 1.0 ml of the reference stock solution and place it in a 10 ml volumetric flask. Add acetonitrile to make up to the volume to obtain the reference solution.

[0201] Sample solution: Place 10 varenicline tartrate tablets in a 10ml volumetric flask, add 3ml purified water and 3ml acetonitrile, sonicate for 30s, dilute to the mark with acetonitrile, and mix well.

[0202] Sample spiking solution: Place 10 varenicline tartrate tablets in a 10ml volumetric flask, add 3ml purified water and 3ml acetonitrile, sonicate for 30s, then add 1.0ml of reference stock solution, add acetonitrile to the mark, and mix well.

[0203] Placebo solution: Weigh 1g of placebo (equivalent to a tablet with the active ingredient removed) into a 10ml volumetric flask, add 3ml of purified water and 3ml of acetonitrile, sonicate for 30s, dilute to the mark with acetonitrile, and mix well.

[0204] Placebo spiked solution: Weigh 1g of placebo into a 10ml volumetric flask, add 3ml of purified water and 3ml of acetonitrile, sonicate for 30s, then add 1.0ml of standard stock solution, dilute to the mark with acetonitrile, and mix well.

[0205] Blank: Acetonitrile.

[0206] 2. Exclusivity

[0207] Inject blank, control solution, sample solution, placebo solution, spiked sample solution, and spiked placebo solution into the sample for analysis.

[0208] The results showed that the retention time of the target analyte in the reference solution, sample solution, and spiked solution was approximately 7.80 min. There was no significant interference at the peak position of the target analyte, which met the separation requirements and demonstrated good specificity.

[0209] 3. Solution stability

[0210] Take the reference solution and sample solution and place them at room temperature. Inject and analyze them at regular intervals: 0, 2, 4, 6, 8, 10, and 12 hours. Calculate the peak area ratio (i.e., peak area change rate) at each time point compared to the zero point.

[0211] The results showed that the peak area change rate of nitrosamine compound PO8 in the reference solution was <12% within 12 hours, and the peak area change rate of nitrosamine compound PO8 in the sample solution was <3.5% within 12 hours.

[0212] 4. System Applicability

[0213] The reference solution was injected and analyzed 5 times. The RSD of the peak area of ​​the target analyte was calculated, and the result was 1.41%, indicating good system suitability.

[0214] 5. Repeatability

[0215] Six sample solutions were prepared in parallel and analyzed separately. The results showed that the RSD of the impurity compound PO8 in the six sample solutions was 3.49%, which met the acceptance criteria and demonstrated good repeatability.

[0216] 6. Intermediate precision

[0217] Two analysts weighed six sample solutions at different times, injected them for analysis, and calculated the average value and RSD. The results showed that the RSD was 5.93%.

[0218] 7. Linear

[0219] Reference solutions with concentrations of 0.015 ng / ml, 0.38 ng / ml, 1.88 ng / ml, 3.77 ng / ml, 5.65 ng / ml, 18.83 ng / ml, 37.66 ng / ml, and 753.2 ng / ml were prepared and injected for analysis. Linear regression was performed with the concentration of the target analyte (X) on the x-axis and the peak area (Y) on the y-axis to calculate the linear equation and the linear correlation coefficient r.

[0220] The results showed that the linear equation was Y = 462785X + 1000000, with a linear correlation coefficient r = 0.9999, indicating good linearity within the concentration range of 0.015 ng / ml to 753.2 ng / ml.

[0221] 8. Accuracy

[0222] The results showed that the recovery rate of the spiked solution was between 85% and 95%, with an RSD of 2.03%, which met the acceptance criteria and demonstrated good accuracy.

[0223] 9. Limit of Detection (LOD)

[0224] LOD = 13.2 ppb, and the average S / N (signal-to-noise ratio) of the target peak is 75.94, which meets the requirement of being greater than 3, indicating that the method is sensitive.

[0225] 10. Limit of Quantification (LOQ)

[0226] The LOQ is 17.6 ppb, and the average S / N of the target peak is 103.35, which meets the requirement of being greater than 10, indicating that the method is sensitive.

[0227] 11. Durability

[0228] When the column temperature was varied by ±2℃, the results showed that the method had good robustness.

[0229] Calculate the content of nitrosamine impurity compound P08 in the sample using the following formula:

[0230] C (ppm) = P08 concentration in sample solution (ng / ml) / Varenicline tartrate concentration in sample solution (mg / ml)

[0231] Example 13

[0232] Preparation of nitrosamine compound PO6

[0233]

[0234] Add 5g of compound MO1 and 52mL of tetrahydrofuran (THF) to a 100mL three-necked flask and stir until dissolved. Add 10mL of NaNO2 aqueous solution (concentration 0.4g / mL) and 3g of acetic acid. Heat to 50℃ and react. Monitor the reaction by TLC or HPLC until complete. Concentrate the solution and add 50mL of dichloromethane (DCM) and 50mL of saturated saline solution. Stir until dissolved and allow to stand. Separate the liquid and take the organic phase. Wash once with saturated saline solution, dry with anhydrous sodium sulfate, filter, and concentrate to dryness to obtain nitrosamine compound PO6 with a purity of 93.69% (area normalization method).

[0235]

[0236] Nitrosamine compound PO6 1 H-NMR, 13 C-NMR and IR spectra are shown in Tables 15–17, respectively; M+H + =279.10; UV: The maximum absorption wavelength in acetonitrile solution is 223.80 nm.

[0237] Table 15. Nitrosamine compound P06 1 H-NMR spectrum

[0238]

[0239] Table 16. Nitrosamine compound P06 13 C-NMR spectrum

[0240]

[0241] Table 17. IR spectra of nitrosamine compound PO6

[0242] <![CDATA[Absorption wave number (cm -1 )]]> Belonging 3034.5 Ar-H 2920.2,2879.0 Saturated CH stretching vibration 1667.2 C=C stretching vibration of benzene ring 1546.9,1428.2,1344.9 N=O,NO stretching vibration 845.2 Benzene ring CH bending vibration

[0243] Example 14

[0244] Preparation of nitrosamine compound PO7

[0245]

[0246] Add 4.0 g of compound PO6 and 50 mL of dichloromethane (DCM) to a 100 mL three-necked flask and stir until dissolved. Add 1.0 g of palladium on carbon catalyst (Pd / C), purge with nitrogen first, then continuously purge with hydrogen, heat to 25–30 °C, and react overnight. Monitor the reaction completion by TLC, filter, and concentrate to dryness to obtain nitrosamine compound PO7 with a purity of 96.59% (area normalization method).

[0247]

[0248] Nitrosamine compound PO7 1 H-NMR, 13 C-NMR and IR spectra are shown in Tables 18-20, respectively; M+H + =219.20; UV: The maximum absorption wavelength in an acetonitrile:water = 1:1 solution is 212.00 nm.

[0249] Table 18. Nitrosamine compound P07 1 H-NMR spectrum

[0250]

[0251] Table 19. Nitrosamine compound P07 13 C-NMR spectrum

[0252]

[0253]

[0254] Table 20. IR spectra of nitrosamine compound P07

[0255] <![CDATA[Absorption wave number (cm -1 )]]> Belonging 3432.6 NH stretching vibration 3008.2 Ar-H 2942.2,2870.9 Saturated CH stretching vibration 1655.5 C=C stretching vibration of benzene ring 1495.2 N=O stretching vibration 866.5 Benzene ring CH bending vibration

[0256] Example 15

[0257] The entire contents of Chinese patent application 202010698570.8 (invention title: a method for preparing varenicline intermediate, varenicline and its salt), filed on July 20, 2020, are incorporated herein by reference.

[0258] 1. Preparation of Varenicline intermediates

[0259]

[0260] ① Add 3 kg (approximately 8.69 mol) of compound A and 0.35 kg of palladium on carbon (Pd / C, Pd 5%) catalyst to a reactor containing 36 kg of isopropanol and 15 kg of purified water. Control the reaction temperature at 25-35℃, pass hydrogen gas and stir for 6 hours, then stop the reaction (HPLC detection shows that the remaining amount of compound A in the reaction solution is ≤0.5%), filter with diatomaceous earth to remove the palladium on carbon catalyst, and obtain a reaction solution containing compound B.

[0261]

[0262] ② Under the protection of an inert gas (nitrogen in this example), add 75g of sodium bicarbonate (approximately 0.89mol, alkaline) to the reaction solution obtained in step ①, then slowly add an aqueous solution of glyoxal (0.556kg (approximately 9.58mol) glyoxal and 6.5kg water, acidic). Control the reaction temperature at 20-30℃ and stir the reaction for 8 hours (including the time for adding the glyoxal aqueous solution). Then stop the reaction (HPLC detection shows that the remaining amount of compound B in the reaction solution is ≤0.5g). The solution was distilled under reduced pressure (vacuum degree ≤ -0.09MPa, 40-50℃) until almost no fraction was produced. 90kg of purified water was added, and the temperature inside the vessel was controlled at 20-30℃ for 2 hours. After that, it was centrifuged and dried (45-50℃) to obtain 2.34kg of varenicline intermediate (compound C), which was an off-white solid with an HPLC purity of 99.1%. Related substances test items: compound A ≤ 0.1%, compound B ≤ 0.1%, and the content of other maximum single unknown impurities ≤ 0.1%.

[0263] 2. Preparation of Vareniclan tartrate

[0264]

[0265] a. Under the protection of an inert gas (nitrogen in this example), add 20 kg of purified water, 0.81 kg (approximately 20.25 mol) of sodium hydroxide, 8 kg of dichloromethane, and 2 kg (approximately 6.51 mol) of compound C (obtained in the previous step) to the reaction vessel. Control the reaction temperature at 20-35°C and stir the reaction for 8 hours. Then stop the reaction (HPLC detection shows that the remaining amount of compound C in the reaction solution is ≤0.5%). Afterward, add 18.5 kg of purified water to the reaction vessel. Add g of dichloromethane, stir, let stand, separate the liquids, take the organic phase, extract the aqueous phase with dichloromethane (15kg×2), combine the organic phases, add 5kg of anhydrous sodium sulfate to the organic phase to dry, filter, distill the filtrate under reduced pressure (vacuum degree ≤ -0.09MPa, 40-50℃) until basically no distillate is produced, add 10kg of anhydrous ethanol, continue to distill under reduced pressure (vacuum degree ≤ -0.09MPa, 40-50℃) until basically no distillate is produced, to obtain the mother liquor containing vareniclan;

[0266]

[0267] b. Slowly add 30 kg of anhydrous ethanol and 1.16 kg (approximately 7.73 mol) of L-(+)-tartaric acid to the mother liquor containing varenicline obtained in step a. Control the reaction temperature at 20-30℃ and stir the reaction for 16 h (including the time for adding anhydrous ethanol and L-(+)-tartaric acid). Then stop the reaction, centrifuge, and dry (70-75℃) to obtain 2.08 kg of varenicline tartaric acid, which is an off-white solid.

[0268] Example 16

[0269] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS, see Table 13) was used for the qualitative and / or quantitative detection of nitrosamine impurities (compound P08) in varenicline tartrate raw material.

[0270] The content of nitrosamine impurity compound PO8 in the varenicline tartrate prepared in Example 15 was found to be 114 ppm (the test results showed that the retention time of this impurity was about 3.47 min). This indicates that if varenicline tartrate is prepared using conventional methods without control and treatment, the content of nitrosamine impurity compound PO8 in the product is usually in the hundreds of ppm range, which cannot meet the FDA's limit requirements for nitrosamine impurities.

[0271] Example 17

[0272] 1. Preparation of Varenicline intermediates

[0273] ① Add 3 kg of compound A (the content of nitrosamine impurity compound PO6 in compound A was found to be 722 ppm) and 0.35 kg of palladium on carbon (Pd / C, Pd 5%) catalyst to a reactor containing 36 kg of isopropanol and 15 kg of purified water. Control the reaction temperature at 25-35℃, pass hydrogen gas and stir to carry out the reaction. Stop the reaction when the remaining amount of compound A in the reaction solution is ≤0.5% as determined by HPLC. Filter with diatomaceous earth to remove the palladium on carbon catalyst to obtain a reaction solution containing compound B.

[0274] ② Under inert gas (nitrogen) protection, add 75g of sodium bicarbonate to the reaction solution obtained in step ①, then slowly add an aqueous solution of glyoxal (0.56kg glyoxal and 6.5kg water), control the reaction temperature at 20-30℃, stir the reaction, and stop the reaction when the remaining amount of compound B in the reaction solution is ≤0.5% as detected by HPLC. Distill under reduced pressure (vacuum degree ≤-0.09MPa, 40-50℃) until almost no distillate is produced, add 90kg of purified water, control the temperature inside the reactor at 20-30℃, keep warm for 2h, then centrifuge and dry (45-50℃) to obtain compound C.

[0275] 2. Preparation of Vareniclan tartrate

[0276] a. Under inert gas (nitrogen) protection, 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) to the reaction flask. Control the reaction temperature at 20-35℃. Stop the reaction when the remaining amount of compound C in the reaction solution is ≤0.5% as detected by HPLC. Then add 18.5 kg of dichloromethane to the reaction vessel, stir, let stand, separate the liquids, 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 to dry, filter, and distill the filtrate under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50℃) until basically no distillate is produced. Add 10 kg of anhydrous ethanol, continue to distill under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50℃) until basically no distillate is produced, and obtain the mother liquor containing varenicline.

[0277] 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 20-30℃, stir the reaction for 16 h, then stop the reaction, centrifuge and dry (70-75℃) to obtain varenicline tartaric acid.

[0278] The prepared varenicline tartrate was then added to the pulping solvent at a solvent volume of 8 ml per gram of varenicline tartrate. The mixture was pulped at 30–40°C for 1.5 h, then centrifuged and dried. Testing showed that the varenicline tartrate treated with dichloromethane contained 5.94 ppm of nitrosamine impurity compound PO8, and the contents of nitrosamine impurities PO6 and PO7 were undetectable (below the detection limit), meeting the FDA's limit requirements for nitrosamine impurities.

[0279] Example 18

[0280] 1. Preparation of Varenicline intermediates

[0281] ① Add 3 kg of compound A (to better ensure product quality, it is generally required that the content of nitrosamine impurity compound PO6 in compound A is ≤500 ppm, and the detection value is 485 ppm) and 0.35 kg of palladium on carbon (Pd / C, Pd 5%) catalyst to a reaction vessel containing 36 kg of isopropanol and 15 kg of purified water. Control the reaction temperature at 25-35℃, pass hydrogen gas and stir to carry out the reaction. Stop the reaction when the remaining amount of compound A in the reaction solution is ≤0.5% by HPLC. Filter with diatomaceous earth to remove the palladium on carbon catalyst to obtain a reaction solution containing compound B.

[0282] ② Under inert gas (nitrogen) protection, add 75g of sodium bicarbonate to the reaction solution obtained in step ①, then slowly add an aqueous solution of glyoxal (0.56kg glyoxal and 6.5kg water), control the reaction temperature at 20-30℃, stir the reaction, and stop the reaction when the remaining amount of compound B in the reaction solution is ≤0.5% as detected by HPLC. Distill under reduced pressure (vacuum degree ≤-0.09MPa, 40-50℃) until almost no distillate is produced, add 90kg of purified water, control the temperature inside the reactor at 20-30℃, keep warm for 2h, then centrifuge and dry (45-50℃) to obtain compound C.

[0283] 2. Preparation of Vareniclan tartrate

[0284] a. Under inert gas (nitrogen) protection, 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) to the reaction flask. Control the reaction temperature at 20-35℃. Stop the reaction when the remaining amount of compound C in the reaction solution is ≤0.5% as detected by HPLC. Then add 18.5 kg of dichloromethane to the reaction vessel, stir, let stand, separate the liquids, 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 to dry, filter, and distill the filtrate under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50℃) until basically no distillate is produced. Add 10 kg of anhydrous ethanol, continue to distill under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50℃) until basically no distillate is produced, and obtain the mother liquor containing varenicline.

[0285] 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 20-30℃, stir the reaction for 16 h, then stop the reaction, centrifuge and dry (70-75℃) to obtain varenicline tartaric acid.

[0286] The prepared varenicline tartrate was then added to the pulping solvent at a solvent volume of 8 ml per gram of varenicline tartrate, and pulped at 30–40°C for 1.5 h. After centrifugation and drying, the product was ready. The results showed that the nitrosamine impurity compound PO8 in the ethyl acetate pulped varenicline tartrate was 3.04 ppm, while the contents of nitrosamine impurities PO6 and PO7 were not detected (below the detection limit).

[0287] Example 19

[0288] Preparation of Vareniclan tartrate

[0289] a. Under inert gas (nitrogen) protection, 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 18) 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 is ≤0.5% as detected by HPLC. Then add 18.5 kg of dichloromethane to the reaction vessel, stir, let stand, separate the liquids, 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 to dry, 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 anhydrous ethanol, continue to distill under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50°C) until basically no distillate is produced, and obtain the mother liquor containing vareniclan.

[0290] 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 20-30℃, stir the reaction for 16 h, then stop the reaction, centrifuge and dry (70-75℃) to obtain varenicline tartaric acid.

[0291] The prepared varenicline tartrate was then added to the pulping solvent at a solvent volume of 8 ml per gram of varenicline tartrate. The mixture was pulped at 30–40 °C for 1.5 h, then centrifuged and dried. The results showed that the nitrosamine impurity compound PO8 in the varenicline tartrate treated with dichloromethane was 4.47 ppm, while the contents of nitrosamine impurities PO6 and PO7 were not detected (below the detection limit).

[0292] Example 20

[0293] Preparation of Vareniclan tartrate

[0294] a. Under inert gas (nitrogen) protection, 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 18) 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 is ≤0.5% as detected by HPLC. Then add 18.5 kg of dichloromethane to the reaction vessel, stir, let stand, separate the liquids, 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 to dry, 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 anhydrous ethanol, continue to distill under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50°C) until basically no distillate is produced, and obtain the mother liquor containing vareniclan.

[0295] 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 20-30℃, stir the reaction for 16 h, then stop the reaction, centrifuge and dry (70-75℃) to obtain varenicline tartaric acid.

[0296] The prepared varenicline tartrate was then added to the pulping solvent at a solvent volume of 8 ml per gram of varenicline tartrate. The mixture was pulped at 30–40 °C for 1.5 h, then centrifuged and dried. The results showed that the nitrosamine impurity compound PO8 in the isopropanol pulped varenicline tartrate was 2.89 ppm, while the contents of nitrosamine impurities PO6 and PO7 were not detected (below the detection limit).

[0297] Example 21

[0298] Preparation of Vareniclan tartrate

[0299] a. Under inert gas (nitrogen) protection, 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 18) 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 is ≤0.5% as detected by HPLC. Then add 18.5 kg of dichloromethane to the reaction vessel, stir, let stand, separate the liquids, 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 to dry, 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 anhydrous ethanol, continue to distill under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50°C) until basically no distillate is produced, and obtain the mother liquor containing vareniclan.

[0300] 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 20-30℃, stir the reaction for 16 h, then stop the reaction, centrifuge and dry (70-75℃) to obtain varenicline tartaric acid.

[0301] Add the prepared varenicline tartrate to the pulping solvent according to the solvent volume of 8 ml per gram of varenicline tartrate, pulp at 30-40℃ for 1.5 h, then centrifuge and dry. The content of nitrosamine impurity compound PO8 in the varenicline tartrate after n-heptane pulping treatment was found to be 24.29 ppm.

[0302] Example 22

[0303] Preparation of Vareniclan tartrate

[0304] a. Under inert gas (nitrogen) protection, 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 18) 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 is ≤0.5% as detected by HPLC. Then add 18.5 kg of dichloromethane to the reaction vessel, stir, let stand, separate the liquids, 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 to dry, 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 anhydrous ethanol, continue to distill under reduced pressure (vacuum degree ≤ -0.09 MPa, 40-50°C) until basically no distillate is produced, and obtain the mother liquor containing vareniclan.

[0305] 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 20-30℃, stir the reaction for 16 h, then stop the reaction, centrifuge and dry (70-75℃) to obtain varenicline tartaric acid.

[0306] The prepared varenicline tartrate was then added to the pulping solvent at a solvent volume of 8 ml per gram of varenicline tartrate. The mixture was pulped at 30–40 °C for 1.5 h, then centrifuged and dried. The results showed that the nitrosamine impurity compound PO8 in the ethanol-pulped varenicline tartrate was 2.60 ppm, while the contents of nitrosamine impurities PO6 and PO7 were not detected (below the detection limit).

[0307] Examples 23-25

[0308] By further controlling the content of nitrosamine impurity compounds in the starting materials of this invention (for example, controlling the content of nitrosamine impurity compound PO6 in compound A to ≤250ppm, ≤200ppm, ≤150ppm, ≤100ppm or lower), and increasing the number of pulping treatments, we also prepared vareniclan tartrate with nitrosamine impurity compound PO8 contents of 0.511ppm, 0.205ppm and 0.118ppm, respectively.

[0309] If the content of nitrosamine impurity compound PO6 in compound A is too high, the content of PO6 can be reduced by the following pulping treatment method. For example, add 3.3 kg of compound A (containing 722 ppm of nitrosamine impurity compound PO6) to a pulping solvent (8.00 kg (11.7 L) of n-heptane and 2.70 kg (3 L) of ethyl acetate), and pulp and stir at 15-35°C for 4-5 hours. After centrifugation, wash the filter cake with water, centrifuge again, and dry. The result shows that the content of nitrosamine impurity compound PO6 in compound A after pulping is 0.03% (315 ppm). This indicates that the content of nitrosamine impurity compound PO6 in compound A can be reduced by at least half through a single pulping treatment.

[0310] The working principle of pulping is: the pulping solvent can dissolve trace impurities in the target compound, leaving them in the liquid, while the undissolved target compound remains in solid form. Then, through solid-liquid separation, the impurity content in the target compound is reduced.

[0311] Example 26

[0312] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) under the conditions described in Table 21 was used for the qualitative and / or quantitative detection of nitrosamine impurity compound P07 in varenicline tartrate raw material of the present invention, and the methodology was validated.

[0313] Table 21. Chromatographic and mass spectrometric conditions for detecting nitrosamine impurity compound P07

[0314]

[0315] 1. Solution preparation

[0316] Blank (diluent): Water-acetonitrile (95:5)

[0317] Reference stock solution: Weigh 25 mg of nitrosamine compound P07 (purity ≥95%), place it in a 50 ml volumetric flask, add acetonitrile to dissolve, and dilute to the mark. Mix well (Fixed solution I). Take 1.0 ml of Fixed solution I, place it in a 100 ml volumetric flask, add diluent to the mark, and mix well to obtain the reference stock solution.

[0318] Reference solution: Take 0.45 ml of reference stock solution, place it in a 100 ml volumetric flask, add diluent to the mark, mix well, and the solution is ready.

[0319] 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.

[0320] 2. System Applicability

[0321] For five consecutive reference solutions, the RSD of the peak area of ​​the nitrosamine impurity compound PO7 was ≤15%, and the RSD of the retention time was ≤2.0%. It could be well separated from adjacent peaks, indicating good system suitability.

[0322] 3. Linear

[0323] 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 PO7);

[0324] The above-mentioned reference solutions of different concentrations were injected and analyzed. Linear regression was performed with the concentration (X) of the target analyte as the abscissa (unit: μg / ml) and the peak area (Y) as the ordinate 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 r = 0.998, indicating good linearity within the concentration range of 0.002688 μg / ml to 0.100783 μg / ml (i.e., 0.2688 ppm to 10.0783 ppm).

[0325] 4. Limit of Detection (LOD)

[0326] LOD = 0.1344ppm, and 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, indicating that the method is sensitive.

[0327] 5. Limit of Quantification (LOQ)

[0328] The LOQ is 0.2688 ppm, and the S / N of the target peaks are 20.5, 24.9, etc., which meet the requirement of being greater than 10, indicating that the method is sensitive.

[0329] 6. Exclusivity

[0330] The results showed that the retention time of the nitrosamine impurity compound P07 was about 14.487 min, with no interference between adjacent peaks, meeting the separation requirements and exhibiting good specificity.

[0331] also,

[0332] The accuracy, precision, repeatability, and robustness of this detection method have been verified to meet the requirements of method validation, and it can effectively achieve the qualitative and / or quantitative detection of nitrosamine impurity compound P07 in varenicline tartrate raw material.

[0333] Example 27

[0334] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) under the conditions described in Table 22 was used for the qualitative and / or quantitative detection of nitrosamine impurity compound P06 in varenicline tartrate raw material of the present invention, and the methodology was validated.

[0335] Table 22. Chromatographic and mass spectrometric conditions for detecting nitrosamine impurity compound P06

[0336]

[0337] 1. Solution preparation

[0338] Blank (diluent): Purified water

[0339] Reference stock solution: Weigh 25 mg of nitrosamine compound PO6 (purity ≥92%), place it in a 50 ml volumetric flask, add acetonitrile to dissolve, dilute 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 the mark, and mix well to obtain the reference stock solution.

[0340] Reference solution: Take 4.5 ml of reference stock solution and place it in a 50 ml volumetric flask. Add diluent to the mark and mix well.

[0341] Sample solution: Take about 1000 mg of varenicline tartrate raw material, place it in a 20 ml headspace vial, accurately measure 5.0 ml of diluent to dissolve it, and mix well.

[0342] 2. System Applicability

[0343] For five consecutive reference solutions, the RSD of the peak area of ​​the nitrosamine impurity compound PO6 was ≤15%, and the RSD of the retention time was ≤2.0%. It could be well separated from adjacent peaks, and the system had good suitability.

[0344] 3. Linear

[0345] Different concentrations of reference solutions were injected for analysis. Linear regression was performed with the concentration (X) of the target analyte as the abscissa (unit: μg / ml) and the peak area (Y) as the ordinate. The linear equation and linear correlation coefficient r were calculated. The results showed that the linear correlation coefficient r > 0.99, indicating a good linear relationship.

[0346] 4. Limit of Detection (LOD)

[0347] LOD = 0.0145ppm, and the S / N (signal-to-noise ratio) of the target peak is 5.8, which meets the requirement of being greater than 3, indicating that the method is sensitive.

[0348] 5. Limit of Quantification (LOQ)

[0349] LOQ = 0.0289 ppm, the S / N of the target peak is 12.0, which meets the requirement of being greater than 10, indicating that the method is sensitive.

[0350] 6. Exclusivity

[0351] The results showed that the retention time of the nitrosamine impurity compound PO6 was about 14.648 min, with no interference between adjacent peaks, meeting the separation requirements and exhibiting good specificity.

[0352] also,

[0353] The accuracy, precision, repeatability, and robustness of this detection method have been verified to meet the requirements of method validation, and it can effectively achieve the qualitative and / or quantitative detection of nitrosamine impurity compound P06 in varenicline tartrate raw material.

[0354] Example 28

[0355] To better ensure the limits of nitrosamine impurities (including PO6, PO7, and PO8) in varenicline tartrate raw materials or preparations, where PO8 is derived from PO6 via intermediate product PO7, and therefore PO8 is the main form of nitrosamine impurities in varenicline tartrate raw materials or preparations, PO6 is the main form of nitrosamine impurities in raw materials such as compound A, and PO7 is an intermediate product in the preparation process), it is necessary to perform qualitative and / or quantitative detection of nitrosamine impurity compound PO6 in starting material compound A. For example, high performance liquid chromatography (HPLC) can be used, and the chromatographic conditions are shown in Table 23.

[0356] Table 23. Chromatographic conditions for detecting nitrosamine impurity compound P06 in compound A

[0357]

[0358]

[0359] 1. Create a standard curve

[0360] Blank (diluent): Acetonitrile-water (60:40)

[0361] Reference solution: Weigh 25 mg of nitrosamine compound PO6 (purity ≥ 92%), place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, mix well to obtain the reference solution.

[0362] Reference solutions were prepared 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 as nitrosamine compound PO6), respectively.

[0363] The above-mentioned reference solutions of different concentrations were injected and analyzed. The concentration of the target analyte (X) was used as the abscissa (unit: mg / ml) and the peak area (Y) was used 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 = 228.94X + 0.0025 and the linear correlation coefficient r = 0.999, indicating good linearity within the concentration range.

[0364] The detection limit is LOD = 0.004% (i.e., 40 ppm), and the S / N (signal-to-noise ratio) of the target peak is 6.4, which meets the requirement of being greater than 3, indicating that the method is sensitive.

[0365] Limit of quantitation: LOQ = 0.014% (i.e. 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.

[0366] Specificity: The results of the mixed solution of sample and reference showed that the retention time of nitrosamine impurity compound PO6 was about 4.592 min (resolution R = 13.18), the retention time of compound A was about 7.587 min (resolution R = 6.82), the retention time of impurity M01 was about 9.373 min (resolution R = 7.24), and the retention time of impurity M02 was about 12.45 min (resolution R = 5.25). There was no interference between the peaks, which met the resolution requirements and showed good specificity.

[0367] also,

[0368] The accuracy, precision, repeatability, and robustness of this detection method have been verified to meet the requirements and specifications for method validation, and it can be used for the qualitative and / or quantitative detection of nitrosamine impurity compound PO6 in compound A.

[0369] 2. Test the samples.

[0370] 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.

[0371] Sample solution was injected and analyzed to obtain peak area data and chromatograms (see: Figure 1 Substituting the peak area data into the linear equation obtained from the reference solution, the concentration data of nitrosamine impurity compound P06 in the test sample solution was calculated.

[0372] Then, calculate the content of nitrosamine impurity compound PO6 in the sample (compound A) using the following formula:

[0373] PO6 content: C (expressed as % or ppm) = PO6 concentration in sample solution (mg / ml) / [m(mg) / V(ml)]

[0374] m: Sample mass, mg;

[0375] V: Sample dilution volume, ml.

[0376] Example 29:

[0377] To determine whether the nitrosamine impurity compound P08 is genotoxic (mutagenic or carcinogenic).

[0378] 1. Materials and Methods

[0379] 1.1 Experimental strains

[0380] Salmonella Typhimurium TA97a, TA98, TA100, TA102, and TA1535, sourced from MOLT OX, USA, and purchased from Shanghai Baolu Biotechnology Co., Ltd. Experiments were conducted using Salmonella Typhimurium strains that had been identified as meeting the requirements.

[0381] 1.2 Metabolic Activation System

[0382] Rat liver S9, source: Shanghai Baolu Biotechnology Co., Ltd.

[0383] 1.3 Test substance

[0384] Name: Nitrosamine impurity compound P08; Appearance: White powder.

[0385] 1.4 Main Instruments and Reagents

[0386] X SR-204 electronic balance (Mettler AG, Switzerland); autoclave (SANYO Corporation, Japan); water-jacketed constant temperature incubator (Shanghai Yiheng Technology Co., Ltd.); Stuart temperature-controlled shaker incubator (Stuart Ltd., UK); BHC-1300II A2 biosafety cabinet (Suzhou Antai Air Technology Co., Ltd., Suzhou Jingjing Group).

[0387] 6-Phosphogluconate, 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 Microbial Technology Co., Ltd.; Sterile water for injection, source: Chenxin Pharmaceutical Co., Ltd.; Dichlorophenate, source: CHEMSERVICE; Methyl mesylate, 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.

[0388] 1.5 Test Method (Ames Plate Incorporation Method)

[0389] The experiment used the plate doping method.

[0390] 1.5.1 Dosage selection and test substance preparation

[0391] Dosage design: Experiments were conducted at doses of 5 mg, 2.5 mg, 1.25 mg, 0.625 mg, 0.3125 mg, and 0.15625 mg per dish.

[0392] Preparation of the test substance: Dissolve the product in dimethyl sulfoxide to prepare a 50 mg / ml solution, and then dilute it with dimethyl sulfoxide to prepare 25 mg / ml, 12.5 mg / ml, 6.25 mg / ml, 3.125 mg / ml and 1.5625 mg / ml respectively.

[0393] 1.5.2 Test methods.

[0394] Cryopreserved bacterial suspensions TA97a, TA98, TA100, TA102, and TA1535 (0.1 ml each) were inoculated into 10 ml of nutrient broth and incubated at 37°C with shaking (100 times / min) for 10 h. 2.0 ml of top-layer medium containing 0.5 mmol / L histidine and 0.5 mmol / L biotin was dispensed into test tubes, sterilized at 0.103 MPa for 20 min, and incubated in a 50°C water bath. Then, 0.1 ml of the test solution, 0.5 ml of S9 mixture (if metabolic activation is required), and 0.1 ml of bacterial suspension were added to each tube sequentially, mixed thoroughly, and quickly poured onto the bottom agar plate. The plate was rotated to ensure even distribution. After solidification, the plates were inverted and incubated at 37°C for 48 h, and the number of revertant colonies per plate was counted. Three parallel plates were prepared for each dose, including a spontaneous reversion group, a solvent control group, and a positive control group.

[0395] 1.6 Statistical Analysis of Experimental Data

[0396] Data expressed as mean ± standard deviation This indicates that SPSS 19.0 was used for data analysis and calculation.

[0397] 1.7 Result Determination

[0398] If the number of revertant colonies of the test substance is twice or more than twice the number of revertant colonies of the solvent control, and a dose-response relationship is observed, the test substance is considered mutagenic positive. If the test substance shows a positive reaction at any dose condition and the reaction is reproducible, the test substance is considered mutagenic positive. If the test substance is negative after testing with the above-mentioned test strains, regardless of whether S9 is added or not, the test substance is mutagenic negative.

[0399] 2. Experimental Results

[0400] The results are shown in Table 24.

[0401] Table 24. Results of the Ames test

[0402]

[0403] Continued from the table of Ames test results

[0404]

[0405] The Ames test results showed that, without the addition of S9, the TA1535 colony count at a dose of 625 μg / plate of the nitrosamine impurity compound P08 was more than twice that of the solvent control group. With the addition of S9, the TA98 colony count was more than twice that of the solvent control group at a dose of 2500 μg / plate, and the TA1535 colony count was more than twice that of the solvent control group at doses of 5000, 2500, 1250, and 625 μg / plate, showing a dose-response relationship. This indicates that, with and without the addition of the metabolic activation system, the nitrosamine impurity compound P08 is mutagenic to *Salmonella typhimurium*.

[0406] Example 30

[0407] The fungicidal or insecticidal activity of the nitrosamine impurity compound P08 was determined in accordance with existing technical standards or specifications (e.g., Chinese agricultural industry standard NY / T 1156.4-2006 Pesticide Indoor Bioassay Test Guidelines Fungicides Part 4: Pot Method for Controlling Wheat Powdery Mildew; Chinese agricultural industry standard NY / T 1156.5-2006 Pesticide Indoor Bioassay Test Guidelines Fungicides Part 5: Broad Bean Leaf Method for Inhibition of Rice Sheath Blight; Chinese agricultural industry standard NY / T 1154.6-2006 Pesticide Indoor Bioassay Test Guidelines Insecticides Part 6: Insecticide Activity Test Immersion Method; NY / T 1154.9-2008 Pesticide Indoor Bioassay Test Guidelines Insecticides Part 9: Spraying Method, etc.).

[0408] Of course, the present invention can also have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and / or modifications according to the present invention, and these corresponding changes and / or modifications should all fall within the protection scope of the appended claims.

Claims

1. A method for detecting the compound shown in formula P08, characterized in that, Using the compound shown in formula P08 as a standard or reference, the compound shown in formula P08 in the test sample was detected by liquid chromatography-tandem mass spectrometry. Formula P08 The sample to be tested was: varenicline tartrate raw material; The chromatographic conditions for the liquid chromatography-tandem mass spectrometry method include: Column: ACQUITY UPLC BEH C18; The chromatographic column specifications are: 2.1 mm × 100 mm, 1.7 µm; Mobile phase A: formic acid - purified water, volume ratio 0.01-0.2:100; Mobile phase B: Acetonitrile; Perform gradient elution according to the table below: The chromatographic conditions for the liquid chromatography-tandem mass spectrometry method meet the following conditions A1 to A3: A1: Column temperature is 15~35℃; A2: Injection volume is 1–25 µL; A3: Flow rate is 0.1–1 ml / min; The mass spectrometry conditions for the liquid chromatography-tandem mass spectrometry method meet the following conditions B1 to B4: B1: Switch to mass spectrometry before the compound shown in formula P08 elutes its peak; B2: The testing instrument is UPLC-MS / MS, ACQuity I CLASS & TRIPLE QUAD 5500+; B3: Ion source: ESI or APCI; jet gas: 9psi; curtain gas: 30psi; ionization voltage: 5500V; temperature: 500°C; spray gas: 50psi; auxiliary heating gas: 50psi; B4: Positive ion mode; Scanning mode: SIM, SRM, or MRM.

2. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, Mobile phase A: formic acid - purified water, with a volume ratio of 0.02:100, 0.03:100, 0.04:100, 0.05:100, 0.06:100, 0.07:100, 0.08:100, 0.09:100, 0.1:100, 0.11:100, 0.12:100, 0.15:100, or 0.18:

100.

3. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, Mobile phase A: formic acid - purified water, volume ratio 0.1:

100.

4. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, The column temperature is 20℃, 25℃, 30℃ or 32℃.

5. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, The injection volume is 2µl, 3µl, 4µl, 5µl, 6µl, 7µl, 8µl, 9µl, 10µl, 15µl, 20µl or 22µl.

6. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, The flow rate is 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 or 0.9 ml / min.

7. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, The chromatographic conditions for the liquid chromatography-tandem mass spectrometry method are as follows: column temperature 30℃; injection volume 2µL; flow rate 0.4ml / min.

8. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, Enter the mass spectrometer 2–3 min into the gradient elution program.

9. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, Cut into the mass spectrometer at 2 min, 2.1 min, 2.2 min, 2.3 min, 2.4 min, 2.5 min, 2.6 min, 2.7 min, 2.8 min, 2.9 min, or 3 min in the gradient elution program.

10. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, The scanning mode is SIM or MRM.

11. The method for detecting the compound represented by formula P08 as described in claim 1, characterized in that, The detection method described is either qualitative or quantitative.

12. The method for detecting the compound represented by formula P08 as described in claim 11, characterized in that, When the detection method is quantitative detection, the compound shown in formula P08 is used as a standard or reference to prepare standard or reference solutions of different concentrations, which are then injected for detection to obtain the corresponding peak area data. The concentration and peak area are then linearly regressed to obtain a linear equation. The sample solution to be tested is then injected for detection to obtain the corresponding peak area data. Substituting the peak area detection data of the compound shown in formula P08 in the sample solution to be tested into the linear equation, the concentration data of the compound shown in formula P08 in the sample solution to be tested can be obtained.

13. The method for detecting the compound represented by formula P08 as described in claim 12, characterized in that, The detection method also satisfies one or more of the following conditions F1 to F3: F1: Divide the concentration data of the compound shown in formula P08 in the sample solution by the concentration data of varenicline tartrate in the sample solution to obtain the content data of the compound shown in formula P08 in the varenicline tartrate raw material. F2: The solvent for the standard or reference solution is acetonitrile and / or purified water; the solvent for the test sample solution is acetonitrile and / or purified water; F3: The solvent of the standard or reference solution is the same as the solvent of the sample solution to be tested.

14. A method for detecting the compound shown in formula P08, characterized in that, Using the compound shown in formula P08 as a standard or reference, the compound shown in formula P08 in the test sample was detected by liquid chromatography-tandem mass spectrometry. Formula P08 The sample to be tested was: varenicline tartrate preparation; The chromatographic conditions for the liquid chromatography-tandem mass spectrometry method include: Chromatographic column: ChromCore Biphenyl; The chromatographic column specifications are: 2.1 mm × 100 mm, 3 µm; Mobile phase A: formic acid - purified water, volume ratio 0.01-0.2:100; Mobile phase B: Acetonitrile; Perform gradient elution according to the table below: The chromatographic conditions for the liquid chromatography-tandem mass spectrometry method meet the following conditions C1 to C3: C1: Column temperature is 15-35℃; C2: Injection volume is 1–25 µL; C3: Flow rate is 0.1–1 ml / min; The mass spectrometry conditions for the liquid chromatography-tandem mass spectrometry method satisfy the following conditions D1 to D4: D1: Switch to mass spectrometry before the compound shown in formula P08 elutes; D2: Spray voltage: 3.9kV; Sheath gas velocity: 35arb; Auxiliary gas velocity: 10arb; Capillary temperature: 320℃; Auxiliary gas temperature: 320℃; D3: The detection instrument was Vanquish HPLC Q-Exactive-MS / MS; D4: Scan mode: SIM, SRM, or MRM; Polarity: Positive ion mode.

15. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, Mobile phase A: formic acid - purified water, with a volume ratio of 0.02:100, 0.03:100, 0.04:100, 0.05:100, 0.06:100, 0.07:100, 0.08:100, 0.09:100, 0.1:100, 0.11:100, 0.12:100, 0.15:100, or 0.18:

100.

16. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, Mobile phase A: formic acid - purified water, volume ratio 0.05:

100.

17. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, The column temperature is 20℃, 25℃, 30℃ or 32℃.

18. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, The injection volume is 2µl, 3µl, 4µl, 5µl, 6µl, 7µl, 8µl, 9µl, 10µl, 15µl, 20µl or 22µl.

19. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, The flow rate is 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 or 0.9 ml / min.

20. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, The chromatographic conditions for the liquid chromatography-tandem mass spectrometry method are as follows: column temperature is 25℃; injection volume is 20µL; flow rate is 0.4ml / min.

21. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, The mass spectrometer was switched in at 6–7 min during the gradient elution program.

22. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, The scanning mode is SIM.

23. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, The varenicline tartrate formulation comprises varenicline tartrate and pharmaceutically acceptable excipients; wherein the excipients comprise one or more of diluents, disintegrants, glidants, and lubricants.

24. The method for detecting the compound represented by formula P08 as described in claim 23, characterized in that, The varenicline tartrate formulation meets one or more of the following conditions E1 to E5: E1: The diluent is selected from one or more of the following: microcrystalline cellulose, silicified microcrystalline cellulose, dicalcium phosphate, mannitol, copovidone, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, starch, maltodextrin, agar, and guar gum. E2: The disintegrant is selected from one or more of the following: sodium croscarmellose, sodium glycolate starch, crosvidone, corn or pregelatinized starch, sodium carboxymethyl cellulose and calcium carboxymethyl cellulose; E3: The flow aid is selected from one or more of the following: silica, colloidal silica, corn starch, talc, calcium silicate, magnesium silicate, tricalcium phosphate, and silica hydrogel. E4: The lubricant is selected from one or more of the following: stearate, stearic acid, talc, mineral oil, malt, glyceryl benzoate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, and sodium lauryl sulfate. E5: The varenicline tartrate formulation is a solid dosage form.

25. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The diluent comprises microcrystalline cellulose and / or anhydrous calcium hydrogen phosphate.

26. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the diluent is 1:(100-300).

27. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the diluent is 1:(150-250).

28. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the diluent is 1:(185-195).

29. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The disintegrant is croscarmellose sodium.

30. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the disintegrant is 1:(1-10).

31. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the disintegrant is 1:(2-6).

32. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the disintegrant is 1:(3.5-4.5).

33. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the disintegrant is 1:

4.

34. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The flow aid is selected from one or more of colloidal silica, fumed silica, colloidal silica, corn starch, talc, calcium silicate, magnesium silicate, tricalcium phosphate, and silica hydrogel.

35. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The flow aid is colloidal silica.

36. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the glidin is 1:(0.1-8).

37. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the glidin is 1:(0.2-5).

38. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the glidin is 1:(0.5-2.5).

39. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the glidin is 1:(0.8-2).

40. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the glidant is 1:0.8, 1:1, or 1:

2.

41. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The lubricant is stearate.

42. The method for detecting the compound represented by formula P08 as described in claim 41, characterized in that, The stearate mentioned is magnesium stearate.

43. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the lubricant is 1:(0.1-8).

44. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the lubricant is 1:(0.2-5).

45. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the lubricant is 1:(0.5-2.5).

46. ​​The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the lubricant is 1:(1-2).

47. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The weight ratio of varenicline tartrate to the lubricant is 1:1, 1:1.4, or 1:

2.

48. The method for detecting the compound represented by formula P08 as described in claim 24, characterized in that, The varenicline tartrate preparation is in tablet or granule form.

49. The method for detecting the compound represented by formula P08 as described in claim 48, characterized in that, The tablets are plain tablets or coated tablets.

50. The method for detecting the compound represented by formula P08 as described in claim 49, characterized in that, The coating agent for the coated tablets is Opadry coating agent.

51. The method for detecting the compound represented by formula P08 as described in claim 50, characterized in that, The coating agent of the coated tablet is a white, blue, yellow, red, green or orange Opadry coating agent.

52. The method for detecting the compound represented by formula P08 as described in claim 50, characterized in that, The coating agent for the coated tablets is Opadry. ® White and / or Opalry ® Blue.

53. The method for detecting the compound represented by formula P08 as described in claim 14, characterized in that, The detection method described is either qualitative or quantitative.

54. The method for detecting the compound represented by formula P08 as described in claim 53, characterized in that, When the detection method is quantitative detection, the compound shown in formula P08 is used as a standard or reference to prepare standard or reference solutions of different concentrations, which are then injected for detection to obtain the corresponding peak area data. The concentration and peak area are then linearly regressed to obtain a linear equation. The sample solution to be tested is then injected for detection to obtain the corresponding peak area data. Substituting the peak area detection data of the compound shown in formula P08 in the sample solution to be tested into the linear equation, the concentration data of the compound shown in formula P08 in the sample solution to be tested can be obtained.

55. The method for detecting the compound represented by formula P08 as described in claim 54, characterized in that, The detection method also satisfies one or more of the following conditions F1 to F3: F1: Divide the concentration data of the compound shown in formula P08 in the sample solution by the concentration data of varenicline tartrate in the sample solution to obtain the content data of the compound shown in formula P08 in the varenicline tartrate preparation. F2: The solvent for the standard or reference solution is acetonitrile and / or purified water; the solvent for the test sample solution is acetonitrile and / or purified water; F3: The solvent of the standard or reference solution is the same as the solvent of the sample solution to be tested.

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