CN115707687A

CN115707687A - Related impurity compound of varenicline tartrate and preparation, application and detection methods thereof

Info

Publication number: CN115707687A
Application number: CN202110961260.5A
Authority: CN
Inventors: 魏彦君; 裴慧; 孙芳慧; 杜文峰; 张争艳; 王建; 孔猛; 刘希望; 邢艳平; 徐青景; 胡青燕
Original assignee: Shandong Weizhi Zhongke Pharmaceutical Co ltd; Weizhi Pharmaceutical Co ltd
Current assignee: Shandong Weizhi Zhongke Pharmaceutical Co ltd; Weizhi Pharmaceutical Co ltd
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2023-02-21

Abstract

The invention provides related impurity compounds of varenicline tartrate, and preparation, application and detection methods thereof. The impurity compound is shown as formula II, wherein R ₁₁ 、R ₁₂ Independently selected from nitro (-NO) ₂ ) Or amino (-NH) ₂ ). The impurity compound is converted to have a gene with the progress of the reactionThe toxic nitrosamine impurity P08 is used as a standard substance, a reference substance or a detection item for impurity research, quality control and the like of the valnemulin tartrate or raw materials and intermediates thereof, so that qualitative and/or quantitative detection of the impurity is realized, and further an active and effective technical means is adopted to control the content of the nitrosamine genotoxic impurity in the valnemulin tartrate bulk drug or preparation to a lower level, thereby meeting the safety requirement and the drug product supervision regulation and better ensuring the medication safety of patients.

Description

Related impurity compound of varenicline tartrate and preparation, application and detection methods thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to related impurity compounds of varenicline tartrate, and preparation, application and detection methods thereof.

Background

Vanninkel, an aryl fused azapolycyclic compound capable of modulating cholinergic energy, is sold as the tartrate salt under the trade name Vanninkel

Or

Can bind to the specific receptor site of neuronal nicotinic acetylcholine and can be used for the following diseases:

treatment of inflammatory bowel diseases (including but not limited to: ulcerative colitis, pyoderma gangrenosum and Crohn's disease, irritable bowel syndrome, spastic dystonia, chronic pain, acute pain, diarrhea (celiac disease), cystitis (pouchitis), vasoconstriction, anxiety, panic disorder (panic disorder), depression, bipolar disorder (bipolar disorder), autism, sleep disorders, jet lag (jet lag), amyotrophic Lateral Sclerosis (ALS), cognitive dysfunction, drug/toxin-induced cognitive impairment (e.g., due to alcohol, barbiturates, vitamin deficiencies, recreational drugs (cognitive drug), lead, arsenic, mercury), disease-induced cognitive impairment (e.g., caused by Alzheimer's disease (Alzheimer's disease), vascular dementia (vascular dementia), parkinson's disease, multiple sclerosis, AIDS, (macro) encephalitis, trauma, renal and hepatic encephalopathy, hypothyroidism, pick's disease, korsakoff's syndrome and prefrontal and subcortical dementia (subcortical dementia)), hypertension, bulimia, anorexia, obesity, arrhythmia, hyperacidity, ulcers, pheochromocytoma, progressive supranuclear palsy (progressive subaccular palsy), chemical dependence and addiction (e.g. to nicotine (and/or tobacco products), alcohol, benzodiazepines

Steroids, barbiturates, opiate or cocaine dependence and addiction), headache, migraine, stroke, traumatic Brain Injury (TBI), obsessive-compulsive disorder (OCD)Psychosis, huntington's chorea, tardive dyskinesia, hyperkinesia, dyslexia, schizophrenia, multiple sclerosis dementia, age-related cognitive decline, epilepsy, including seizure deficiency epilepsy (attention deficit epilepsy), attention Deficit Hyperactivity Disorder (ADHD), tourette's Syndrome (Tourette's Syndrome); in particular, nicotine dependence, addiction and withdrawal, including use in smoking cessation therapy (see: CN 1509174A).

Processes for the preparation of Vanillan, and the resolution of racemates thereof, are disclosed in U.S. Pat. No. 6,410,550 and International patent application WO 01/62736, the contents of which are also incorporated herein by reference.

Nitrosamine compounds, including nitroso (N (R1) (R2) -N = O) structures bound to amines, are genotoxic substances in certain animals, some of which have been classified as possible or potential human carcinogens by the international agency for research on cancer (IARC).

These nitrosamine compounds, referred to as "focus group" compounds in the International Conference on harmony, ICH for short) instruction document M7 (R1) for human drug registration technology, evaluate and control DNA reactive (mutagenic) impurities in pharmaceutical products to limit potential carcinogenic risk (3 months 2018). ICH guidelines suggest that any known mutagenic/carcinogenic agent, including nitrosamine compounds, be controlled at levels of uptake where the risk of cancer in humans is negligible.

Currently, seven nitrosamine impurities have been identified by the U.S. Food and Drug Administration (FDA), which may theoretically be present in pharmaceutical products because of the use of manufacturing processes and materials that may lead to the formation of nitrosamines: n, N-dimethylnitrosamine (NDMA), N-Nitrosodiethylamine (NDEA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-Nitrosoisopropylamine (NIPEA), N-Nitrosodiisopropylamine (NDIPA), N-Nitrosodibutylamine (NDBA) and N-Nitrosomethylaniline (NMPA). Of these, five impurities (NDMA, NDEA, NMBA, NIPEA and NMPA) have been actually detected in bulk drugs or medicines.

For example, some preliminary results of FDA testing indicate that the NDMA content in certain ranitidine products has exceeded acceptable levels. In recent years, the FDA has also found that metformin products contain NDMA impurities, and in certain batches NDMA levels above the FDA recommended acceptable intake limits have been detected.

In view of the above, the invention is especially provided to control the nitrosamine impurity content in the valnemulin tartrate bulk drug or preparation to a lower level capable of meeting the safety requirement.

Disclosure of Invention

In view of the problems and/or disadvantages of the prior art, it is an object of the present invention to provide a novel compound related to varenicline tartrate: a compound of formula II. The new compound can be converted into nitrosamine impurity P08 with genotoxicity along with the reaction process, and needs to be detected and controlled in the whole process of the synthesis of the varenicline tartrate, so that the content of the nitrosamine genotoxicity impurity in the pharmaceutical raw material or preparation of the varenicline tartrate can be controlled at a lower level by adopting an active and effective technical means, and the safety requirement is met.

The invention provides a compound shown as a formula II:

wherein R is ₁₁ 、R ₁₂ Independently selected from nitro (-NO) ₂ ) Or amino (-NH) ₂ )。

The invention also provides a preparation method of the compound shown in the formula II, which comprises the following steps: carrying out nitrosamine reaction on the compound M01 and nitrite in the presence of acid and a mixed solvent to generate a compound P06; optionally including thereafter: the compound P06 and a reducing agent are subjected to reduction reaction in the presence of a catalyst and a halogenated hydrocarbon solvent to generate a compound P07;

the compound shown in the formula II is a compound P06 or a compound P07;

preferably, the first and second electrodes are formed of a metal,

the nitrite is potassium nitrite or sodium nitrite;

the acid is acetic acid;

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

the reducing agent is hydrogen;

the catalyst is a palladium-carbon catalyst;

the halogenated hydrocarbon solvent is liquid halogen substituted C ₁ ～C ₄ Alkanes, more preferably dichloromethane;

more preferably still, the first and second liquid crystal compositions are,

the molar ratio of the compound M01 to the nitrite is 1.5-5, and preferably 1:2-4;

the volume-mass ratio of the mixed solvent to the compound M01 is 5-20 mL/g, and preferably 10-15 mL/g;

the mass ratio of the acid to the compound M01 is 0.1 to 1, and preferably 0.4 to 0.8;

the reaction temperature of the nitrosamine reaction is 30-60 ℃;

the mass ratio of the compound P06 to the catalyst is 1;

the volume-mass ratio of the halogenated hydrocarbon solvent to the compound P06 is 5-20 mL/g, and preferably 10-15 mL/g;

the reaction temperature of the reduction reaction is 10-40 ℃.

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

wherein，R ₁₁ 、R ₁₂ Independently selected from nitro or amino.

The invention also provides a detection method of the compound shown in the formula II in the Vanilla tartrate, which takes the compound shown in the formula II as a standard substance or a reference substance and adopts liquid chromatography-tandem mass spectrometry to carry out detection;

wherein R is ₁₁ 、R ₁₂ Independently selected from nitro or amino.

In one embodiment of the present invention, the chromatographic conditions of the liquid chromatography-tandem mass spectrometry include:

a chromatographic column: a C18 chromatography column, preferably Waters Xbridge C18;

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

preferably, the first and second electrodes are formed of a metal,

the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method further comprise:

the specifications of the chromatographic column are as follows: 4.6 mm. Times.150mm, 3.5 μm;

column temperature: 15-35 ℃, preferably 20 ℃, 25 ℃, 30 ℃ or 32 ℃;

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

sample introduction volume: 1 to 35. Mu.1, more preferably 2. Mu.1, 3. Mu.1, 4. Mu.1, 5. Mu.1, 6. Mu.1, 7. Mu.1, 8. Mu.1, 9. Mu.1, 10. Mu.1, 15. Mu.1, 20. Mu.1, 25. Mu.1 or 30. Mu.1.

In one aspect of the present invention, the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry include:

an ion source: ESI (electrospray ionization source) or APCI (atmospheric pressure chemical ionization source), preferably ESI;

a positive ion mode;

scanning mode: SIM (single ion detection scanning), SRM (selective interaction detection) or MRM (multiple interaction detection scanning), preferably SIM or MRM;

preferably, the first and second electrodes are formed of a metal,

the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry method further comprise: ion source temperature: 350 ℃; capillary voltage: 6000V: spraying air flow rate: 13L/min.

In one embodiment of the present invention, the compound represented by formula II is

(P06) wherein the volume ratio of mobile phase a to mobile phase B is 55%:45% to 75%:25%, said mobile phase a is formic acid-purified water, and the volume ratio of formic acid to purified water is 0.05 to 0.2 (for example, 0.06; preferably, the volume ratio of the mobile phase A to the mobile phase B is 63% to 37%, and the volume ratio of formic acid to purified water is 0.1.

(P07) a volume ratio of mobile phase a to mobile phase B is 90%: 10%: 98%:2%, said mobile phase a is an aqueous ammonium acetate solution having a concentration of 0.005 to 0.02mol/L (e.g., 0.006mol/L, 0.007mol/L, 0.008mol/L, 0.009mol/L, 0.01mol/L, 0.011mol/L, 0.012mol/L, 0.015mol/L, etc.), and pH is adjusted with formic acid =2 to 3; preferably, the volume ratio of the mobile phase A to the mobile phase B is 95% to 5%, the concentration of the ammonium acetate aqueous solution is 0.01mol/L, and the pH is adjusted to be 2.5 by formic acid.

In one scheme of the invention, the detection method is qualitative detection or quantitative detection, the solvents of the standard substance, the reference substance and the varenicline tartrate are water or water-acetonitrile, and the volume ratio of the water to the acetonitrile is 90 percent to 10 percent to 98 percent to 2 percent, preferably 95 percent to 5 percent.

In one scheme of the invention, an internal standard method or an external standard method is adopted for quantitative detection;

preferably, the first and second liquid crystal display panels are,

the external standard method comprises the following steps: taking the compound shown in the formula II as a standard substance or a reference substance, preparing standard substance solutions or reference substance solutions with different concentrations, carrying out sample injection detection to obtain corresponding peak area data, and carrying out linear regression on the concentration and the peak area to obtain a linear equation; substituting peak area detection data of the compound shown in the formula II in the detection sample solution of the varenicline tartrate into a linear equation to obtain concentration data of the compound shown in the formula II in the detection sample solution; and dividing the data by the concentration data of the sample solution to obtain a quantitative detection result.

The invention has the following beneficial effects: the compound shown in the formula II can be converted into nitrosamine impurity P08 with genotoxicity along with the reaction process, and is used as a standard substance, a reference substance or a detection item for impurity research, quality control and the like of varenicline tartrate or raw materials and intermediates thereof, so that qualitative and/or quantitative detection of the impurity is realized, and further, an active and effective technical means is adopted to control the content of the nitrosamine genotoxicity impurity in the valenicline tartrate bulk drug or preparation to a lower level, thereby meeting the safety requirement and the drug product supervision regulation and better ensuring the drug safety of patients.

Drawings

FIG. 1 is a chromatogram diagram of detecting nitrosamine impurity compound P06 in compound A by high performance liquid chromatography.

Detailed Description

While the present invention will be described more fully hereinafter with reference to the accompanying specific embodiments, it is to be understood by those skilled in the art that the following descriptions are provided for purposes of illustration only and are not intended to limit the scope of the present invention.

In the present invention, those who do not specify specific conditions are performed according to conventional conditions or conditions recommended by the manufacturer, and those who do not specify the reagents or instruments used are conventional products commercially available.

With respect to definitions of terms used herein, the initial definitions provided for the terms herein apply to the terms throughout, unless otherwise specified; terms not specifically defined herein should be given their meanings to those skilled in the art in light of the disclosure and/or the context.

Example 1

Preparation of nitrosamine Compound P06

To a100 mL three-necked flask, 5g of Compound M01 and 52mL of Tetrahydrofuran (THF) were added and the mixture was stirred to dissolve; 10mL of NaNO was added ₂ Heating an aqueous solution (with the concentration of 0.4 g/mL) and 3g of acetic acid to 50 ℃ for reaction, monitoring the reaction by TLC or HPLC, concentrating, adding 50mL of Dichloromethane (DCM) and 50mL of saturated saline solution, stirring to dissolve, standing, separating, taking an organic phase, washing with saturated saline solution once, drying with anhydrous sodium sulfate, filtering, and concentrating to dryness to obtain the nitrosamine compound P06 with the purity of 93.69% (area normalization method).

Of nitrosamine compounds P06 ¹ H-NMR、 ¹³ C-NMR and IR spectrum data, shown in tables 1-3, respectively; m + H ⁺ =279.10; UV: the maximum absorption wavelength in acetonitrile solution was 223.80nm.

TABLE 1 preparation of nitrosamine Compound P06 ¹ H-NMR spectrum

TABLE 2 preparation of nitrosamine Compound P06 ¹³ C-NMR spectrum

TABLE 3 IR spectrum of nitrosamine Compound P06

Absorption wave number (cm) ^-1 )	Attribution
		3034.5	Ar-H
2920.2,2879.0	Saturated C-H stretching vibration
		1667.2	Benzene ring C = C stretching vibration
1546.9,1428.2,1344.9	N = O, N-O stretching vibration
		845.2	C-H bending vibration of benzene ring

Example 2

Preparation of nitrosamine compound P07

To a100 mL three-necked flask, 4.0g of compound P06 and 50mL of Dichloromethane (DCM) were added and stirred to dissolve; adding 1.0g of palladium-carbon catalyst (Pd/C), replacing with nitrogen, then continuously introducing hydrogen, heating to 25-30 ℃, reacting overnight, monitoring by TLC, filtering, and concentrating to dryness to obtain nitrosamine compound P07 with purity of 96.59% (area normalization method).

Of nitrosamine compounds P07 ¹ H-NMR、 ¹³ C-NMR and IR spectrum data, see tables 4-6, respectively; m + H ⁺ =219.20; UV: the maximum absorption wavelength in acetonitrile water =1:1 solution was 212.00nm.

TABLE 4 preparation of nitrosamine Compound P07 ¹ H-NMR spectrum

TABLE 5 preparation of nitrosamine Compound P07 ¹³ C-NMR spectrum

TABLE 6 IR spectra of nitrosamine Compound P07

Example 3

Preparation of nitrosamine compound P08

To a100 mL three-necked flask, 4.0g of Compound M02 (Vanillan) and 45mL of Tetrahydrofuran (THF) were added and the mixture was stirred to dissolve; add 8.2mL of NaNO ₂ Heating the aqueous solution (with the concentration of 0.4 g/mL) and 2.9g of acetic acid to 52 ℃, monitoring the reaction by TLC, separating out solids, filtering, and drying in vacuum to obtain the nitrosamine compound P08 with the purity of 98.87% (area normalization method).

Of nitrosamine compounds P08 ¹ H-NMR、 ¹³ C-NMR and IR spectrum data, see tables 7-9, respectively; m + H ⁺ =241.20; UV: the maximum absorption wavelength in the acetonitrile solution was 204.40nm.

TABLE 7 preparation of nitrosamine Compound P08 ¹ H-NMR spectrum

TABLE 8 preparation of nitrosamine Compound P08 ¹³ C-NMR spectrum

TABLE 9 IR spectra of nitrosamine Compound P08

Absorption wave number(cm ^-1 )	Attribution
		3048.8	Ar-H
2961.4,2931.8	Saturated C-H stretching vibration
		1925.2	Benzene ring C = C stretching vibration
1575.2	N = O telescopic vibration
		885.2	C-H bending vibration of benzene ring

Example 4

Chinese patent application 202010698570.8 filed on 20/07/2020 (title: a process for the preparation of varenicline intermediate, varenicline and salts thereof), the entire contents of which are incorporated herein by reference in their entirety.

1. Preparation of varenicline intermediates

(1) Adding 3kg (about 8.69 mol) of compound A and 0.35kg of palladium-carbon (Pd/C, pd 5%) catalyst into a reaction kettle containing 36kg of isopropanol and 15kg of purified water, controlling the reaction temperature to be 25-35 ℃, introducing hydrogen and stirring for reacting for 6 hours, stopping the reaction (detecting the residual amount of the compound A in the reaction solution to be less than or equal to 0.5 percent by HPLC), and filtering by using kieselguhr to remove the palladium-carbon catalyst to obtain a reaction solution containing a compound B;

(2) adding 75g of sodium bicarbonate (about 0.89mol, showing alkalinity) into the reaction liquid obtained in the step (1) under the protection of inert gas (nitrogen in the embodiment), then slowly adding an aqueous solution of glyoxal (0.556 kg (about 9.58 mol) of glyoxal and 6.5kg of water, showing acidity), controlling the reaction temperature at 20-30 ℃, stirring for 8h (including the adding time of the aqueous solution of glyoxal), then stopping the reaction (the residual amount of the compound B in the reaction liquid is less than or equal to 0.5% by HPLC detection), carrying out reduced pressure distillation (the vacuum degree is less than or equal to-0.09MPa, 40-50 ℃) until no fraction is generated basically, adding 90kg of purified water, controlling the temperature in a kettle at 20-30 ℃, carrying out heat preservation for 2h, then centrifuging, drying (45-50 ℃) to obtain 2.34kg of a Vannite intermediate (compound C) which is a white-like solid with the HPLC purity of 99.1%, and related substance detection items: less than or equal to 0.1 percent of compound A, less than or equal to 0.1 percent of compound B and less than or equal to 0.1 percent of other maximum single unknown impurities.

2. Preparation of valnemulin tartrate

a. Under the protection of inert gas (nitrogen in the embodiment), adding 20kg of purified water, 0.81kg (about 20.25 mol) of sodium hydroxide, 8kg of dichloromethane and 2kg (about 6.51 mol) of compound C (prepared in the previous step) into a reaction kettle, controlling the reaction temperature at 20-35 ℃, stirring for reaction for 8h, stopping the reaction (HPLC detects that the residual amount of the compound C in the reaction solution is less than or equal to 0.5%), then adding 18.5kg of dichloromethane into the reaction kettle, stirring, standing, separating liquid, taking an organic phase, extracting a water phase by using dichloromethane (15 kg multiplied by 2), combining the organic phase, adding 5kg of anhydrous sodium sulfate into the organic phase for drying, filtering, distilling the filtrate under reduced pressure (the vacuum degree is less than or equal to-0.09MPa and 40-50 ℃) until no fraction is generated basically, adding 10kg of anhydrous ethanol, continuing distilling under reduced pressure (the vacuum degree is less than or equal to-0.09MPa and 40-50 ℃) until no fraction is generated basically, and obtaining mother liquor containing Vannickel;

b. and c, slowly adding 30kg of absolute ethyl alcohol and 1.16kg (about 7.73 mol) of L- (+) -tartaric acid into the mother liquor containing the Vanilla obtained in the step a, controlling the reaction temperature to be between 20 and 30 ℃, stirring and reacting for 16h (the adding time of the absolute ethyl alcohol and the L- (+) -tartaric acid is included), stopping the reaction, centrifuging, and drying (70 to 75 ℃) to obtain 2.08kg of Vanilla tartrate which is a white-like solid.

Example 5

According to the FDA (Food and Drug Administration), nitrosamine impurities in the medicine need to be detected, and are generally required to be controlled to be less than or equal to 7.5ppm.

Therefore, an ultra performance liquid chromatography triple quadrupole mass spectrometry (UPLC-MS/MS) is creatively developed for qualitative and/or quantitative detection of the nitrosamine impurity (compound P08) in the valnemulin tartrate bulk drug. The chromatographic conditions and mass spectrum conditions of UPLC-MS/MS are shown in Table 10.

TABLE 10 chromatographic and mass spectral conditions for UPLC-MS/MS

The content of nitrosamine impurity compound P08 in the valnemulin tartrate prepared in example 4 was found to be 114ppm (test results show that the retention time of the impurity is about 3.47 min); this indicates that if varenicline tartrate is prepared according to the conventional method without control and treatment, the content of nitrosamine impurity compound P08 in the obtained product is generally in the order of hundreds of ppm, which cannot meet the limited requirement of FDA on nitrosamine impurity.

Example 6

1. Preparation of varenicline intermediates

(1) Adding 3kg of compound A (the content of a nitrosamine impurity compound P06 in the compound A is 722ppm by detection) and 0.35kg of palladium-carbon (Pd/C, pd 5%) catalyst into a reaction kettle containing 36kg of isopropanol and 15kg of purified water, controlling the reaction temperature at 25-35 ℃, introducing hydrogen and stirring for reaction, stopping the reaction when the residual amount of the compound A in the reaction solution is less than or equal to 0.5% by HPLC detection, and filtering by using kieselguhr to remove the palladium-carbon catalyst to obtain a reaction solution containing a compound B;

(2) adding 75g of sodium bicarbonate into the reaction solution obtained in the step (1) under the protection of inert gas (nitrogen), then slowly adding aqueous solution of glyoxal (0.56 kg of glyoxal and 6.5kg of water), controlling the reaction temperature at 20-30 ℃, stirring for reaction, stopping the reaction when the residual quantity of the compound B in the reaction solution is less than or equal to 0.5% by HPLC (high performance liquid chromatography), carrying out reduced pressure distillation (the vacuum degree is less than or equal to-0.09MPa and 40-50 ℃) until no fraction is generated basically, adding 90kg of purified water, controlling the temperature in a kettle at 20-30 ℃, preserving the temperature for 2h, then centrifuging and drying (45-50 ℃) to obtain a compound C.

2. Preparation of varenicline tartrate

a. Under the protection of inert gas (nitrogen), adding 20kg of purified water, 0.81kg of sodium hydroxide, 8kg of dichloromethane and 2kg of compound C (prepared in the previous step) into a reaction bottle, controlling the reaction temperature to be 20-35 ℃, detecting the residual amount of the compound C in the reaction solution to be less than or equal to 0.5% by HPLC (high performance liquid chromatography), stopping the reaction, then adding 18.5kg of dichloromethane into a reaction kettle, stirring, standing, separating, taking an organic phase, extracting an aqueous phase by using dichloromethane (15 kg multiplied by 2), combining the organic phases, adding 5kg of anhydrous sodium sulfate into the organic phase for drying, filtering, carrying out reduced pressure distillation (the vacuum degree is less than or equal to-0.09MPa and 40-50 ℃) on a filtrate until no fraction is generated basically, adding 10kg of anhydrous ethanol, and continuing the reduced pressure distillation (the vacuum degree is less than or equal to-0.09MPa and 40-50 ℃) until no fraction is generated basically, thus obtaining a mother solution containing Vannite;

b. b, slowly adding 30kg of anhydrous ethanol and 1.16kg of L- (+) -tartaric acid into the mother liquor containing the valnemadectin obtained in the step a, controlling the reaction temperature to be between 20 and 30 ℃, stirring for reacting for 16 hours, stopping the reaction, centrifuging, drying (70 to 75 ℃) to obtain the valnemadectin tartrate;

adding the prepared varenicline tartrate into a pulping solvent according to the dosage of 8ml of the solvent per gram of the varenicline tartrate, pulping for 1.5 hours at the temperature of between 30 and 40 ℃, centrifuging and drying to obtain the valenicline tartrate; through detection, in the tartaric acid Vannickel after the dichloromethane pulping treatment, the content of a nitrosamine impurity compound P08 is 5.94ppm, and the content of nitrosamine impurity compounds P06 and P07 is not detected (is smaller than the detection limit), so that the limit requirement of FDA on nitrosamine impurities is met.

Example 7

1. Preparation of varenicline intermediates

(1) Adding 3kg of compound A (generally, in order to better ensure the product quality, the requirement is that the content of a nitrosamine impurity compound P06 in the compound A is less than or equal to 500ppm, and the detection value is 485 ppm) and 0.35kg of palladium-carbon (Pd/C, pd 5%) catalyst into a reaction kettle containing 36kg of isopropanol and 15kg of purified water, controlling the reaction temperature to be 25-35 ℃, introducing hydrogen and stirring for reaction, stopping the reaction when HPLC detects that the residual amount of the compound A in the reaction solution is less than or equal to 0.5%, and filtering by using kieselguhr to remove the palladium-carbon catalyst to obtain a reaction solution containing a compound B;

(2) adding 75g of sodium bicarbonate into the reaction solution obtained in the step (1) under the protection of inert gas (nitrogen), then slowly adding aqueous solution of glyoxal (0.56 kg of glyoxal and 6.5kg of water), controlling the reaction temperature to be 20-30 ℃, stirring for reaction, stopping the reaction when the residual quantity of the compound B in the reaction solution is less than or equal to 0.5% by HPLC (high performance liquid chromatography), carrying out reduced pressure distillation (the vacuum degree is less than or equal to-0.09MPa and the temperature is 40-50 ℃) until no fraction is generated basically, adding 90kg of purified water, controlling the temperature in a kettle to be 20-30 ℃, preserving heat for 2 hours, then centrifuging, and drying (45-50 ℃) to obtain a compound C.

2. Preparation of valnemulin tartrate

b. b, slowly adding 30kg of anhydrous ethanol and 1.16kg of L- (+) -tartaric acid into the mother liquor containing the Vanilla obtained in the step a, controlling the reaction temperature to be between 20 and 30 ℃, stirring for reaction for 16 hours, stopping the reaction, centrifuging, drying (between 70 and 75 ℃) to obtain Vanilla tartrate;

adding the prepared varenicline tartrate into a pulping solvent according to the dosage of 8ml of the solvent per gram of the varenicline tartrate, pulping for 1.5 hours at the temperature of between 30 and 40 ℃, centrifuging and drying to obtain the valenicline tartrate; through detection, in the valnemulin tartrate after the ethyl acetate pulping treatment, the content of a nitrosamine impurity compound P08 is 3.04ppm, and the content of nitrosamine impurity compounds P06 and P07 is not detected (is smaller than the detection limit).

Example 8

Preparation of valnemulin tartrate

a. Under the protection of inert gas (nitrogen), adding 20kg of purified water, 0.81kg of sodium hydroxide, 8kg of dichloromethane and 2kg of compound C (prepared in example 7) into a reaction bottle, controlling the reaction temperature to be between 20 and 35 ℃, detecting that the residual amount of the compound C in the reaction solution is less than or equal to 0.5 percent by HPLC (high performance liquid chromatography), stopping the reaction, then adding 18.5kg of dichloromethane into a reaction kettle, stirring, standing, separating, taking an organic phase, extracting an aqueous phase by using dichloromethane (15 kg multiplied by 2), combining the organic phases, adding 5kg of anhydrous sodium sulfate into the organic phase for drying, filtering, distilling the filtrate under reduced pressure (the vacuum degree is less than or equal to-0.09MPa and between 40 and 50 ℃) until no fraction is generated basically, adding 10kg of anhydrous ethanol, and continuing distilling under reduced pressure (the vacuum degree is less than or equal to-0.09MPa and between 40 and 50 ℃) until no fraction is generated basically, thus obtaining mother liquor containing Vannite;

adding the prepared varenicline tartrate into a pulping solvent according to the dosage of 8ml of the solvent per gram of the varenicline tartrate, pulping for 1.5 hours at the temperature of between 30 and 40 ℃, centrifuging and drying to obtain the valenicline tartrate; through detection, in the varenicline tartrate subjected to the pulping treatment of the dichloromethane, the content of a nitrosamine impurity compound P08 is 4.47ppm, and the content of nitrosamine impurity compounds P06 and P07 is not detected (is smaller than the detection limit).

Example 9

Preparation of valnemulin tartrate

adding the prepared varenicline tartrate into a pulping solvent according to the dosage of 8ml of the solvent per gram of the varenicline tartrate, pulping for 1.5 hours at the temperature of between 30 and 40 ℃, centrifuging and drying to obtain the valenicline tartrate; through detection, in the valnemulin tartrate after the isopropanol beating treatment, the content of a nitrosamine impurity compound P08 is 2.89ppm, and the content of nitrosamine impurity compounds P06 and P07 is not detected (is smaller than the detection limit).

Example 10

Preparation of valnemulin tartrate

adding the prepared varenicline tartrate into a pulping solvent according to the dosage of 8ml of the solvent per gram of the varenicline tartrate, pulping for 1.5 hours at the temperature of between 30 and 40 ℃, centrifuging and drying to obtain the valenicline tartrate; through detection, the nitrosamine impurity compound P08 content in the valnemulin tartrate after n-heptane pulping treatment is 24.29ppm.

Example 11

Preparation of varenicline tartrate

a. Under the protection of inert gas (nitrogen), adding 20kg of purified water, 0.81kg of sodium hydroxide, 8kg of dichloromethane and 2kg of compound C (prepared in example 7) into a reaction bottle, controlling the reaction temperature to be between 20 and 35 ℃, detecting the residual amount of the compound C in the reaction solution by HPLC (high performance liquid chromatography) to be less than or equal to 0.5 percent, stopping the reaction, then adding 18.5kg of dichloromethane into the reaction kettle, stirring, standing, separating, taking an organic phase, extracting an aqueous phase by using dichloromethane (15 kg multiplied by 2), combining the organic phases, adding 5kg of anhydrous sodium sulfate into the organic phase, drying, filtering, distilling a filtrate under reduced pressure (the vacuum degree is less than or equal to-0.09MPa and between 40 and 50 ℃) until no fraction is generated basically, adding 10kg of anhydrous ethanol, continuing distilling under reduced pressure (the vacuum degree is less than or equal to-0.09MPa and between 40 and 50 ℃) until no fraction is generated basically, and obtaining mother liquor containing Vannickel blue;

adding the prepared valnemulin tartrate into a pulping solvent according to the dosage of the solvent of 8ml per gram of valnemulin tartrate, pulping for 1.5 hours at the temperature of 30-40 ℃, centrifuging and drying to obtain the valnemulin tartrate; through detection, in the tartaric acid varenicline subjected to the ethanol pulping treatment, the content of the nitrosamine impurity compound P08 is 2.60ppm, and the content of the nitrosamine impurity compounds P06 and P07 is not detected (is smaller than the detection limit).

Examples 12 to 14

By further controlling the content of the nitrosamine impurity compound in the starting material of the present invention (for example, controlling the content of the nitrosamine impurity compound P06 in the compound A in the range of 250ppm or less, 200ppm or less, 150ppm or less, 100ppm or less), and increasing the number of beating treatments, etc., we also produced Vanillan tartrate having a content of the nitrosamine impurity compound P08 of 0.511ppm, 0.205ppm or 0.118ppm, respectively.

If the content of the nitrosamine impurity compound P06 in the compound a is too high, the content of P06 can be reduced by the following beating treatment method, for example: adding 3.3kg of compound A (the content of nitrosamine impurity compound P06 is 722 ppm) into a pulping solvent (8.00 kg (11.7L) of n-heptane and 2.70kg (3L) of ethyl acetate), pulping and stirring for 4-5 h at 15-35 ℃, centrifuging, washing a filter cake with water, centrifuging again, and drying to obtain the nitrosamine compound A. Through detection, the content of the nitrosamine impurity compound P06 in the compound A after pulping treatment is 0.03% (315 ppm). This indicates that the content of the nitrosamine impurity compound P06 in the compound a can be reduced by at least half by one beating treatment as described above.

The working principle of the pulping treatment is as follows: the pulping solvent can dissolve trace impurities in the target compound to enable the trace impurities to remain in liquid, and the insoluble target compound is still in a solid form, and then solid-liquid separation is carried out to further reduce the content of the impurities in the target compound.

Example 15

And (3) detecting a nitrosamine impurity compound P08 in the bulk drug of the varenicline tartrate by UPLC-MS/MS (ultra performance liquid chromatography-Mass Spectrometry/Mass Spectrometry) and carrying out methodology verification.

1. Solution preparation

Diluent agent: purified water

Control stock solutions: 19.970mg nitrosamine compound P08 (purity is more than or equal to 98%) is weighed and placed in a10 ml measuring flask, 8ml acetonitrile is added, then diluent is added to dissolve and dilute to scale, and the mixture is mixed evenly.

Mother liquor of a reference product: measuring 37.5 μ l of the control stock solution in a10 ml measuring flask, diluting with diluent to scale, and mixing.

2. System applicability

System applicability solution (100% limit level control solution): measuring 100.0 μ l of the control mother liquor in a10 ml measuring flask, diluting with diluent to scale, and mixing.

The 6-pin system suitability solution was continuously fed, and RSD of the peak area of the target in the 6-pin system suitability solution was calculated, and the result was 4%, and the system suitability was good.

3. Linearity

Standard curve solution: respectively weighing control mother liquor 50.0 μ L, 100.0 μ L, 150.0 μ L and 200.0 μ L, placing in 410 ml measuring bottles, diluting with diluent to scale, mixing, and sequentially making into linear solutions L-3, L-4, L-5 and L-6. Measuring the L-3 solution in a measuring flask of 800 mu 1 to 10ml, diluting the diluent to scale, and uniformly mixing to obtain L-2. Measuring the L-2 solution into a measuring flask with the volume of 1000 mu 1 to 10ml, diluting the solution to the scale with a diluent, and uniformly mixing to obtain the L-1.

Respectively sampling and analyzing the standard curve solution, performing linear regression by taking the concentration (X) of the target object as a horizontal coordinate (unit: ng/ml) and the peak area (Y) as a vertical coordinate, and calculating to obtain a linear equation and a linear correlation coefficient r; the results show that the linear equation: y =7370X-12900, linear correlation coefficient r =0.999, good linearity, in the concentration range of 0.2951ng/ml to 147.6ng/ml (i.e., 0.02951ppm to 14.76 ppm).

4. Detection Limit (LOD)

LOD solution: measuring the L-1 solution into a measuring flask with 5000 mu 1 to 10ml, diluting the solution to the scale with a diluent, and uniformly mixing the solution to obtain an LOD solution.

2 needles of LOD solution are continuously injected, S/N (signal to noise ratio) of a target peak is respectively 11 and 8, the requirement of not less than 3 is met, and the method is sensitive; LOD =0.01476ppm.

5. Quantitative Limit (LOQ)

Taking a quantitative limiting solution (L-1 solution) for continuous sampling 6 needles, wherein S/N of a target peak is 23, 24, 17, 12, 24 and 14 respectively, the requirement of not less than 10 is met, and the method is sensitive; LOQ =0.02951ppm.

6. Accuracy of

Sample solution: weighing about 100mg of sample (such as purchased or synthesized valnemulin tartrate bulk drug) and placing in a10 ml measuring flask, adding diluent to dilute to the scale, and mixing well. 2 parts are prepared in parallel.

Accuracy solution: weighing about 100mg of sample, placing in a10 ml measuring flask, adding control mother liquor 50.0 μ l (50% limit level), 100.0 μ l (100% limit level), and 150.0 μ l (150% limit level), diluting with diluent to scale, and mixing. Each level of accuracy was formulated in 3 parts in parallel.

The results show that the recovery of the solution at the 50% limit level accuracy is between 95% and 102%, the RSD is 4%; the recovery rate of the solution at the 100% limit level accuracy is between 90% and 103%, and the RSD is 7%; the recovery rate of the solution at the accuracy of 150% limit level is between 98% and 100%, the RSD is 1%, the solution meets the acceptance standard, and the accuracy is good.

7. Repeatability of

Weighing about 100mg of sample, placing in a10 ml measuring flask, adding 100.0 μ l of control mother liquor, diluting with diluent to scale, and mixing. 6 parts are prepared in parallel.

The result shows that the RSD of the content of the nitrosamine impurity compound P08 in 6 parts of the repetitive solution is 6 percent, the acceptable standard is met, and the repeatability is good.

8. Specificity

Blank solution (diluent), 100% limit level control solution, sample solution (same as "repetitive" sample solution), and 100% limit level accuracy solution were injected and analyzed separately.

The results show that the target retention time of the sample solution, the 100% limit level control solution and the 100% limit level accuracy solution is 3.47min, the peak position of the target has no obvious interference, the requirement of the separation degree is met, and the specificity is good.

9. Intermediate precision

Intermediate precision solution: weighing about 100mg of sample, placing in a10 ml measuring flask, adding 100.0 μ l of control mother liquor, diluting with diluent to scale, and mixing. 6 parts are prepared in parallel. Plus 6 portions of "7, duplicate" solution.

The concentration RSD of 12 parts of solution in total of 6 parts of intermediate precision solution and 6 parts of repetitive solution is calculated to be 9 percent, the requirement of not more than 15 percent is met, the receiving standard is met, and the intermediate precision is good.

10. Stability of solution

And (3) placing the solution with the 100% limit horizontal accuracy at 15 ℃, carrying out sample injection analysis at intervals, and calculating the concentration ratio of each time point to a zero point, wherein the ratio is 96-113% in 21 h.

11. Durability

The method has the advantages that when the temperature change of a chromatographic column is +/-2 ℃, the initial proportion of an organic phase is +/-1%, and the flow rate is +/-0.01 ml/min, the concentration value of a target object in a solution with 100% limit horizontal accuracy is respectively inspected, 2 needles are respectively injected under each condition, and in the calculation of the same parameter fluctuation, the RSD of the concentration value of the target object in 6 parts of solution is 4% -6%, and the method has good durability.

Calculating the content of a nitrosamine impurity compound P08 in a sample (a bulk drug of varenicline tartrate) according to the following formula, detecting for 2 times, and taking an average value:

p08 content: c (ppm) = P08 concentration in sample solution (ng/ml)/[ m (mg)/V (ml) ]

m: sample mass, mg;

v: sample dilution volume, ml.

Example 16

The liquid chromatography-tandem mass spectrometry method with the conditions as shown in the table 11 is adopted for the qualitative and/or quantitative detection of the nitrosamine impurity compound P07 in the bulk drug of the varenicline tartrate, and the methodological verification is carried out and passed.

TABLE 11 chromatographic and mass spectrometric conditions for the detection of nitrosamine impurity compound P07

1. Solution preparation

Blank (diluent): water-acetonitrile (95

Control stock solutions: weighing 25mg of nitrosamine compound P07 (the purity is more than or equal to 95 percent), placing the nitrosamine compound P07 into a 50ml volumetric flask, adding acetonitrile to dissolve the nitrosamine compound P07, fixing the volume to the scale, and uniformly mixing (fixing solution I). And (3) putting 1.0ml of the fixed solution I into a100 ml volumetric flask, adding a diluent to a constant volume to a scale, and uniformly mixing to obtain a reference substance stock solution.

Control solution: taking 0.45ml of reference substance stock solution, placing in a100 ml volumetric flask, adding a diluent to a constant volume to reach a scale, and mixing uniformly to obtain the finished product.

Sample solution: taking 100mg of the bulk drug of the valnemulin tartrate, putting the bulk drug of the valnemulin tartrate in a10 ml volumetric flask, adding a diluent to dissolve the bulk drug, fixing the volume to a scale, and uniformly mixing the bulk drug and the diluent.

2. System applicability

The RSD of the peak area of the nitrosamine impurity compound P07 is less than or equal to 15 percent and the RSD of the retention time is less than or equal to 2.0 percent in 5 continuous reference solutions, and the reference solutions can be well separated from adjacent peaks, so that the system applicability is good.

3. Linearity

Preparing control solutions with the concentrations of 0.002688 mug/ml, 0.020157 mug/ml, 0.033594 mug/ml, 0.053751 mug/ml, 0.067189 mug/ml and 0.100783 mug/ml (based on the nitrosamine compound P07);

respectively sampling and analyzing the reference substance solutions with different concentrations, performing linear regression by taking the concentration (X) of the target substance as a horizontal coordinate (unit: mu g/ml) and the peak area (Y) as a vertical coordinate, and calculating to obtain a linear equation and a linear correlation coefficient r; the results show that the linear equation: y =3000000X +2671.7, linear correlation coefficient r =0.998, good linearity, in the concentration range of 0.002688 μ g/ml to 0.100783 μ g/ml (i.e., 0.2688ppm to 10.0783 ppm).

4. Detection Limit (LOD)

LOD =0.1344ppm, S/N (signal-to-noise ratio) of a target peak is 14.8, 13.1 and the like, the requirement of being more than 3 is met, and the method is sensitive.

5. Quantitative Limit (LOQ)

LOQ =0.2688ppm, the S/N of the target peak is 20.5, 24.9 and the like, the requirement of being more than 10 is met, and the method is sensitive.

6. Specificity property

The result shows that the retention time of the nitrosamine impurity compound P07 is about 14.487min, no interference exists between adjacent peaks, the requirement of the separation degree is met, and the specificity is good.

In addition to this, the present invention is,

through verification, the accuracy, precision, repeatability, durability and the like of the detection method also meet the rules and requirements of methodology verification, and qualitative and/or quantitative detection of the nitrosamine impurity compound P07 in the valnemulin tartrate bulk drug can be well realized.

Example 17

The liquid chromatography-tandem mass spectrometry method with the conditions as shown in the table 12 is adopted for the qualitative and/or quantitative detection of the nitrosamine impurity compound P06 in the bulk drug of the varenicline tartrate, and the methodological verification is carried out and passed.

TABLE 12 chromatographic and mass spectrometric conditions for the detection of nitrosamine impurity compound P06

1. Solution preparation

Blank (diluent): purified water

Control stock solutions: weighing 25mg of nitrosamine compound P06 (the purity is more than or equal to 92%), placing the nitrosamine compound P06 in a 50ml volumetric flask, adding acetonitrile to dissolve the nitrosamine compound P06, fixing the volume to the scale, and uniformly mixing (fixing solution II). And (3) putting 1.0ml of the fixed solution II into a100 ml volumetric flask, adding a diluent to a constant volume to a scale, and uniformly mixing to obtain a reference substance stock solution.

Control solution: putting 4.5ml of the reference substance stock solution into a 50ml volumetric flask, adding a diluent to a constant volume to scale, and uniformly mixing.

Sample solution: taking about 1000mg of the bulk drug of the varenicline tartrate, putting the bulk drug into a 20ml headspace bottle, accurately measuring 5.0ml of diluent for dissolution, and uniformly mixing.

2. System applicability

The RSD of the peak area of the nitrosamine impurity compound P06 is less than or equal to 15 percent and the RSD of the retention time is less than or equal to 2.0 percent in the continuous 5-needle reference solution, and the RSD can be well separated from the adjacent peak, so that the system applicability is good.

3. Linearity

Respectively sampling and analyzing reference substance solutions with different concentrations, performing linear regression by taking the concentration (X) of a target object as a horizontal coordinate (unit: mu g/ml) and a peak area (Y) as a vertical coordinate, and calculating to obtain a linear equation and a linear correlation coefficient r; the result shows that the linear correlation coefficient r is more than 0.99, and the linear relation is good.

4. Detection Limit (LOD)

LOD =0.0145ppm, S/N (signal-to-noise ratio) of the target peak is 5.8, the requirement of more than 3 is met, and the method is sensitive.

5. Quantitative Limit (LOQ)

LOQ =0.0289ppm, S/N of the target peak is 12.0, the requirement of more than 10 is met, and the method is sensitive.

6. Specificity property

The result shows that the retention time of the nitrosamine impurity compound P06 is about 14.648min, no interference exists between adjacent peaks, the requirement of the separation degree is met, and the specificity is good.

In addition to this, the present invention is,

the verification proves that the accuracy, precision, repeatability, durability and the like of the detection method also meet the rules and requirements of methodology verification, and the qualitative and/or quantitative detection of the nitrosamine impurity compound P06 in the bulk drug of the valnemulin tartrate can be well realized.

Example 18

In order to better ensure the limit requirements of nitrosamine impurity compounds (including P06, P07, P08; wherein P08 is converted from P06 through intermediate product P07; therefore, P08 is the main existing form of nitrosamine impurity in valnemulin tartrate bulk drug or preparation, P06 is the main existing form of nitrosamine impurity in compound A and other raw materials, and P07 is intermediate product in preparation process), the nitrosamine impurity compound P06 in the starting raw material compound A needs to be qualitatively and/or quantitatively detected, for example: the high performance liquid chromatography was used, and the chromatographic conditions are shown in Table 13.

TABLE 13 chromatographic conditions for detection of nitrosamine impurity Compound P06 in Compound A

1. Making a standard curve

Blank (diluent): acetonitrile-water (60

Control solution: weighing 25mg of nitrosamine compound P06 (the purity is more than or equal to 92%), placing the nitrosamine compound P06 in a 25ml volumetric flask, adding a diluent to dissolve the nitrosamine compound P06, fixing the volume to a scale, and uniformly mixing to obtain a reference solution.

Preparing control solutions with the concentrations of 0.0001394mg/ml, 0.0004645mg/ml, 0.0007432mg/ml, 0.000929mg/ml, 0.001115mg/ml, 0.001394mg/ml and 0.001858mg/ml (calculated as nitrosamine compound P06);

respectively sampling and analyzing the reference substance solutions with different concentrations, performing linear regression by taking the concentration (X) of the target substance as a horizontal coordinate (unit: mg/ml) and the peak area (Y) as a vertical coordinate, and calculating to obtain a linear equation and a linear correlation coefficient r; the results show that the linear equation: y =228.94X +0.0025, and the linear correlation coefficient r =0.999, the linearity is good, and the linearity is good in the concentration range.

Detection limit: LOD =0.004% (i.e. 40 ppm), S/N (signal-to-noise ratio) of the target peak is 6.4, the requirement of more than 3 is met, and the method is sensitive.

And (4) quantitative limit: LOQ =0.014% (i.e. 140 ppm), S/N of the target peak is 18.9, satisfying the requirement of more than 10, the method is sensitive.

The specificity is as follows: the detection result of the mixed solution of the sample and the reference shows that the retention time of the nitrosamine impurity compound P06 is about 4.592min (separation degree R = 13.18), the retention time of the compound A is about 7.587min (separation degree R = 6.82), the retention time of the impurity M01 is about 9.373min (separation degree R = 7.24), the retention time of the impurity M02 is about 12.45min (separation degree R = 5.25), no interference exists among peaks, the requirement of the separation degree is met, and the specificity is good.

In addition, in the case of the present invention,

the detection method is verified to be in accordance with the rules and requirements of methodology verification on accuracy, precision, repeatability, durability and the like, and can be used for qualitative and/or quantitative detection of the nitrosamine impurity compound P06 in the compound A.

2. Detecting the sample

Sample solution: taking about 25mg of the compound A, placing the compound A in a 25ml volumetric flask, adding a diluent to dissolve the compound A, fixing the volume to a scale, and uniformly mixing.

And (3) carrying out sample injection detection on the sample solution to obtain peak area data and a chromatogram (see figure 1), substituting the peak area data into the linear equation obtained from the reference solution, and calculating to obtain concentration data of the nitrosamine impurity compound P06 in the detected sample solution.

And calculating the content of the nitrosamine impurity compound P06 in the sample (compound A) according to the following formula:

p06 content: c (in% or ppm) = concentration of P06 in sample solution (mg/ml)/[ m (mg)/V (ml) ]

m: sample mass, mg;

v: sample dilution volume, ml.

Example 19

And (3) detecting whether the nitrosamine impurity compound P08 has genotoxicity (can cause mutation or carcinogenesis).

1 materials and methods

1.1 test strains

Salmonella typhimurium TA97a, TA98, TA100, TA102, TA1535, origin: MOLT OX, USA, available from Shanghai Bao Bingshi Co. The identified Salmonella typhimurium strain meeting the requirements is adopted for the test.

1.2 Metabolic activation System

Rat liver S ₉ And (3) source: shanghai Bao Bian Biotech Co., ltd.

1.3 test substances

The name is as follows: nitrosamine impurity compound P08, property: white powder.

1.4 Main instruments and reagents

X SR-204 electronic balance (Mettler, switzerland); an autoclave (SANYO, japan); a water-insulated constant temperature incubator (Shanghai-constant technology Co., ltd.); stuart temperature controlled shaker incubators (Stuart, uk); BHC-1300 II A2 biosafety cabinet (Sujing group Suzhou Antai air technologies, inc.).

Glucose-6-phosphate, source: beijing Bailingwei science and technology Co., ltd; coenzyme II, source: national chemical group chemical agents, ltd; nutrient broth, source: beijing Luqiao technology GmbH; technical agar powder, source: kyoto Tokay microbial science and technology Co., ltd; sterilized water for injection, source: chenxin pharmaceutical industry, gmbh; dexon, source: CHEMSERVICE; methyl methanesulfonate, source: beijing Bailingwei science and technology Co., ltd; 2-aminofluorene, source: shanghai pure reagents, inc.; 1,8-dihydroxyanthraquinone, source: SIGMA-ALDRICH, USA; cyclophosphamide, source: SIGMA-ALDRICH, USA; sodium azide, source: RIEDEL-seeleie.

1.5 test methods (Ames test plate incorporation method)

The experiment was performed using a plate incorporation method.

1.5.l dose selection and test formulation

Dose design: the tests were carried out at 5mg, 2.5mg, 1.25mg, 0.625mg, 0.3125mg, 0.15625mg per dish.

Preparing a test substance: taking the product, preparing the product into 50mg/ml solution by using dimethyl sulfoxide, and sequentially diluting the solution into 25mg/ml, 12.5mg/ml, 6.25mg/ml, 3.125mg/ml and 1.5625mg/ml by using the dimethyl sulfoxide.

1.5.2 test methods.

The frozen and preserved bacteria solutions TA97a, TA98, TA100, TA102 and TA 1535.1 ml were inoculated into 10ml nutrient broth medium, and cultured at 37 ℃ for 10 hours with shaking (100 times/min). 2.0ml of top layer culture medium containing 0.5mmol/L histidine to 0.5mmol/L biotin is subpackaged in test tubes, sterilized at 0.103MPa for 20min, and kept warm in 50 ℃ water bath. Then, 0.1ml of the test solution and S are added into each tube in turn ₉ Mixing the mixed solution 0.5ml (when needing metabolism activation) and the bacterial solution 0.1ml, mixing well, pouring onto the bottom agar plate quickly, rotating the plate to make it distribute uniformly. Horizontally placing for condensation and solidification, inversely placing in an incubator at 37 ℃ for incubation for 48h, and counting the number of bacterial colonies changed per dish. Three parallel plates were made for each dose, with a self-priming regression group, a solvent control group, and a positive control group.

1.6 statistics of test data

Data are averaged. + -. Standard deviation

Data analysis calculations are shown using SPSS 19.0.

1.7 determination of results

If the number of the test object retrogradation colonies is two or more times of the number of the solvent control retrogradation colonies and shows a dose-response relationship, the test object is judged to be mutagenic positive; and (3) judging the test object as positive mutagenic if the test object has positive reaction and repeatability under any dosage condition. If the test substance is tested by the test strain, no matter whether S is added ₉ And without addition of S ₉ If all conditions are negative, the test substance is mutagenic negative.

2 results of the test

The results are shown in Table 14.

TABLE 14 Ames test results

Table of Ames test results

As can be seen from the Ames test results, the S is not added ₉ At a nitrosamine impurity compound P08 dose of 625 μ g/dish, the number of TA1535 colonies exceeded twice that of the solvent control. Adding S ₉ Under the condition, the TA98 colony count exceeds twice of the solvent control group when the dosage is 2500 mu g/dish, and the TA1535 colony count exceeds twice of the solvent control group when the dosage is 5000, 2500, 1250 and 625 mu g/dish, and the dosage-response relationship is formed. This indicates that the nitrosamine impurity compound P08 is mutagenically positive for salmonella typhimurium with and without the addition of a metabolic activation system.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore intended that all such changes and/or modifications be considered within the scope of the appended claims.

Claims

1. A compound of formula II:

2. A method for preparing a compound shown as a formula II is characterized by comprising the following steps: carrying out nitrosamine reaction on the compound M01 and nitrite in the presence of acid and a mixed solvent to generate a compound P06; optionally including thereafter: the compound P06 and a reducing agent are subjected to reduction reaction in the presence of a catalyst and a halogenated hydrocarbon solvent to generate a compound P07;

the compound shown in the formula II is a compound P06 or a compound P07;

preferably, the first and second liquid crystal display panels are,

the nitrite is potassium nitrite or sodium nitrite;

the acid is acetic acid;

the reducing agent is hydrogen;

the catalyst is a palladium-carbon catalyst;

more preferably still, the first and second liquid crystal compositions are,

the reaction temperature of the nitrosation reaction is 30-60 ℃;

the mass ratio of the compound P06 to the catalyst is 1;

the reaction temperature of the reduction reaction is 10-40 ℃.

3. The compound shown in the formula II is used as a standard substance, a reference substance or a detection item in the impurity research, quality control or detection method of the varenicline tartrate; preferably, the detection method is liquid chromatography-tandem mass spectrometry;

wherein R is ₁₁ 、R ₁₂ Independently selected from nitro or amino.

4. The detection method of the compound shown in the formula II in the Vanilla tartrate is characterized in that the compound shown in the formula II is used as a standard substance or a reference substance, and liquid chromatography-tandem mass spectrometry is adopted for detection;

wherein R is ₁₁ 、R ₁₂ Independently selected from nitro or amino.

5. The method of claim 4, wherein the chromatographic conditions of the liquid chromatography-tandem mass spectrometry method comprise:

mobile phase: the mobile phase A-mobile phase B, the volume ratio of the mobile phase A to the mobile phase B is 55 percent, 45 percent to 98 percent and 2 percent; the mobile phase A is formic acid-purified water or ammonium acetate water solution, and the mobile phase B is acetonitrile;

preferably, the first and second electrodes are formed of a metal,

column temperature: 15-35 ℃, and preferably 20 ℃, 25 ℃, 30 ℃ or 32 ℃;

6. The method for detecting the compound represented by the formula II in the varenicline tartrate according to claim 4 or 5, wherein the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry comprise:

an ion source: ESI or APCI, preferably ESI;

a positive ion mode;

scanning mode: SIM, SRM or MRM, preferably SIM or MRM;

preferably, the first and second electrodes are formed of a metal,

7. The method of detecting a compound of formula II in Vanilla tartrate according to claim 4 or claim 5, wherein the compound of formula II is

When the volume ratio of the mobile phase A to the mobile phase B is 55 percent to 45 percent to 75 percent to 25 percent, the mobile phase A is formic acid-purified water, and the volume ratio of the formic acid to the purified water is 0.05-0.2; preferably, the volume ratio of the mobile phase A to the mobile phase B is 63% to 37%, and the volume ratio of formic acid to purified water is 0.1.

8. The method of detecting a compound of formula II in Vanilla tartrate according to claim 4 or claim 5, wherein the compound of formula II is

When the volume ratio of the mobile phase A to the mobile phase B is 90 percent to 10 percent to 98 percent to 2 percent, the mobile phase A is ammonium acetate aqueous solution, and the concentration of the ammonium acetate aqueous solution is0.005-0.02 mol/L, and adjusting the pH value to be 2-3 by formic acid; preferably, the volume ratio of the mobile phase A to the mobile phase B is 95% to 5%, the concentration of the ammonium acetate aqueous solution is 0.01mol/L, and the pH is adjusted by formic acid =2.5.

9. The method for detecting the compound shown in the formula II in the varenicline tartrate according to claim 4 or 5, wherein the detection method is qualitative detection or quantitative detection, the solvents of the standard product, the reference product and the varenicline tartrate are water or water-acetonitrile, and the volume ratio of the water to the acetonitrile is 90% -98% to 2%, preferably 95% -5%.

10. The method for detecting the compound represented by the formula II in Vanilla tartrate according to claim 9, wherein the quantitative detection is performed by an internal standard method or an external standard method;

preferably, the first and second electrodes are formed of a metal,

the external standard method comprises the following steps: taking the compound shown in the formula II as a standard substance or a reference substance, preparing standard substance solutions or reference substance solutions with different concentrations, carrying out sample injection detection to obtain corresponding peak area data, and carrying out linear regression on the concentration and the peak area to obtain a linear equation; substituting peak area detection data of the compound shown in the formula II in the detection sample solution of the varenicline tartrate into a linear equation to obtain concentration data of the compound shown in the formula II in the detection sample solution; then dividing the data by the concentration data of the sample solution to obtain the quantitative detection result.