Vortioxetine impurities, processes for their preparation and uses thereof

Abstract

The application discloses a vortioxetine impurity, a preparation method and application thereof, and adopts vortioxetine or a pharmaceutically acceptable salt form thereof as raw material, the raw material is easy to obtain and simple to operate, and the reaction condition is mild. The synthesized impurity is characterized by nuclear magnetic resonance, high-resolution mass spectrometry and X-ray single crystal diffraction, and liver toxicity research shows that the compound disclosed by the application has important significance for researching adverse reactions of vortioxetine. The control sample obtained by the method provided by the application can be used for qualitative and quantitative analysis of the vortioxetine impurity, so that the drug safety of vortioxetine is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pharmacy, and particularly relates to a vortioxetine impurity and a preparation method and application thereof. BACKGROUND

[0002] A drug impurity refers to a chemical substance other than an effective component introduced or generated in the production and storage of a drug. According to physical and chemical properties and sources, drug impurities can be divided into three categories: organic impurities, inorganic impurities and residual solvents. The sources of impurities include starting materials or reagents, intermediates, by-products, degradation impurities and residual materials and reagents in the production process, and degradation impurities in the storage process. Impurity research and control are one of the key links in drug research and development for drug registration; the impurity limit requirement of a drug is closely related to the safety of a drug user. Impurity research needs to consider whether the production process is reliable and stable, and whether the quality standard is reasonable; ultimately, it is to ensure that the drug quality meets the requirements of safety and reliability, and quality control.

[0003] The Chinese chemical name of vortioxetine (also known as vortioxetine) is 1-[2-(2,4-methylphenylthio)phenyl]piperazine, which is a new type of antidepressant. The hydrogen bromide salt form thereof is clinically used, mainly for the treatment of major depressive disorder in adults. The mechanism of action of the drug is unique, and it is classified as a multiple action antidepressant. Its main mechanism is to selectively inhibit the 5-hydroxytryptamine transporter, thereby increasing the concentration of 5-hydroxytryptamine in the synaptic cleft. In addition, vortioxetine also exhibits partial 5-HT1A receptor agonist and 5-HT3 receptor antagonist properties. This dual action helps to improve the mood and cognitive function of patients with depression. Its structure is shown as follows:

[0004]

[0005] Currently, there are several reported synthesis processes of vortioxetine. For example, the synthesis route reported in CN 112759562 A is as follows: 1-fluoro-2-nitrobenzene is reacted with piperazine to obtain 1-(2-nitrophenyl)piperazine, then a sulfonyl chloride group is protected, and then reduced, iodized, reacted with 2,4-dimethylphenylthiol, and then the sulfonyl protection is removed to obtain vortioxetine hydrobromide raw material.

[0006]

[0007] LG is methanesulfonyl, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl or o-nitrobenzenesulfonyl. The inventors found unknown impurities at the retention time of 33.72 min and 45.08 min in the liquid chromatogram of the vortioxetine bulk drug and its formulations prepared based on the synthetic route, and the content of the unknown impurities was more than 0.1%. The unknown impurities were determined to be a bromide impurity of vortioxetine which has not been reported by nuclear magnetic resonance, high resolution mass spectrometry and X-ray single crystal diffraction analysis. Since impurity research is an important part of drug research and development, and runs through the entire research and development process, it is directly related to the safety, effectiveness and quality control of the drug. Therefore, it is of great significance to carry out impurity research on vortioxetine. SUMMARY

[0008] The purpose of the present application is to provide a new impurity of vortioxetine, a preparation method and application thereof, and to provide strong support for the quality research and quality control of vortioxetine.

[0009] Technical solution: The vortioxetine impurity of the present application is a compound (1-[4-bromo-2-(2,4-methylphenylthio)phenyl]piperazine) or a pharmaceutically acceptable salt thereof as shown in formula I:

[0010]

[0011] Preferably, the pharmaceutically acceptable salt of the compound of formula I is a pharmaceutically acceptable salt of the compound of formula I with an acid known in the art, including hydrochloride, hydrobromide, hydroiodide, p-toluenesulfonate, methanesulfonate, maleate or citrate of the compound of formula I.

[0012] Preferably, the pharmaceutically acceptable salt of the compound of formula I is a hydrobromide of formula I-I:

[0013]

[0014] Preferably, the single crystal of the hydrobromide of formula I-I belongs to a monoclinic system, and the cell parameters are: 16.3374(6), 6.2652(2), 19.7807(8), α / ° 90, β / ° 98.855(4), γ / ° 90.

[0015] The vortioxetine impurity of the present application is a compound (1-[4,5-dibromo-2-(2,4-methylphenylthio)phenyl]piperazine) or a pharmaceutically acceptable salt thereof as shown in formula II:

[0016]

[0017] Preferably, the pharmaceutically acceptable salt of the compound of Formula II is a pharmaceutically acceptable salt of the compound of Formula II with an acid known in the art, including hydrochloride, hydrobromide, hydroiodide, p-toluenesulfonate, methanesulfonate, maleate or citrate of the compound of Formula II.

[0018] Preferably, the pharmaceutically acceptable salt of the compound of Formula II is a hydrobromide of Formula II-I:

[0019]

[0020] The method for preparing the vortioxetine impurity of the present application comprises reacting vortioxetine with a brominating agent in an organic solvent under acidic catalysis to obtain a compound of Formula I; the brominating agent is N-bromosuccinimide or bromine; the acidic catalyst is at least one of aluminum trichloride, concentrated sulfuric acid or acetic acid:

[0021]

[0022] Preferably, the brominating agent is bromine.

[0023] Preferably, the acidic catalyst of the acidic catalysis is aluminum trichloride and acetic acid.

[0024] Preferably, the organic solvent is at least one of acetonitrile, concentrated sulfuric acid or acetic acid.

[0025] Preferably, the molar ratio of vortioxetine to the brominating agent is 1:0.8-1.5, preferably 1:1.

[0026] Further preferably, the method for preparing the vortioxetine impurity of the present application comprises reacting vortioxetine with a brominating agent in an organic solvent under acidic catalysis and under a certain temperature condition to obtain a compound of Formula I; the temperature condition is 0-50°C, preferably 0-20°C.

[0027] The method for preparing the vortioxetine impurity of the present application comprises dissolving vortioxetine in an organic solvent, and reacting with sulfuryl chloride under alkaline condition at a certain temperature to obtain an intermediate III, and then dissolving the intermediate III in a hydrobromic acid-acetic acid solution and heating to remove the protecting group, at the same time, bromination reaction and salt formation occur to obtain a compound of Formula I-I, wherein LG represents a sulfuryl protecting group; the certain temperature condition is 0-40°C; the LG in the compound III is a sulfuryl group capable of being removed under acidic condition, including methylsulfonyl, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl or o-nitrobenzenesulfonyl; the base of the alkaline condition is at least one of pyridine, triethylamine or dimethylamine:

[0028]

[0029] Preferably, the hydrobromic acid-acetic acid solution is a 33% hydrobromic acid-acetic acid solution.

[0030] Preferably, the LG in the compound III is p-toluenesulfonyl.

[0031] Preferably, the base in the basic condition is triethylamine.

[0032] Preferably, the organic solvent is at least one of dichloromethane, tetrahydrofuran, pyridine, toluene, preferably dichloromethane.

[0033] Preferably, the molar ratio of the vortioxetine, the sulfonyl chloride, the base is 1:1.0-2.0:1.0-3.0, preferably 1:1.1-1.2:1.2-1.5.

[0034] Further preferably, the temperature condition is 0-10℃.

[0035] Further preferably, the temperature condition for the incubation reaction in the process of the reaction of vortioxetine and the sulfonyl chloride is selected from 0-40℃, preferably 20-30℃.

[0036] Preferably, the 33% hydrobromic acid solution is 10-50 times of the compound III.

[0037] Preferably, the heating reaction temperature is 50-100℃, preferably 60-80℃.

[0038] For a clearer understanding of the molecular structure, the obtained vortioxetine bromide impurity is further cultured into a single crystal, and X-ray single crystal diffraction method is used for single crystal analysis to determine the substitution position of bromine in the vortioxetine bromide impurity and the cell related parameters, and the technical scheme is as follows:

[0039] The preparation method of the vortioxetine impurity in the application comprises the following steps: heating and dissolving the compound I-I in an organic solvent, filtering, standing and culturing, and crystallizing to obtain a single crystal of the vortioxetine impurity (compound I-I); the organic solvent is at least one of dichloromethane, chloroform, methanol, ethanol, acetonitrile, tetrahydrofuran, ethyl acetate, n-hexane, n-heptane, cyclohexane, petroleum ether, toluene and DMF.

[0040] Preferably, the organic solvent is dichloromethane and n-hexane.

[0041] Preferably, the heating reflux temperature is 30℃-60℃.

[0042] Preferably, the standing and culturing temperature is-10℃-30℃.

[0043] Preferably, the standing and culturing time is 2-5 days.

[0044] The preparation method of the vortioxetine impurity comprises reacting vortioxetine with a bromination reagent in an organic solvent under acidic catalytic conditions to obtain a compound of formula II; the bromination reagent is at least one of N-bromosuccinimide and bromine; the acidic catalyst of the acidic catalytic conditions is at least one of aluminum chloride, concentrated sulfuric acid and acetic acid:

[0045]

[0046] Preferably, the bromination reagent is bromine.

[0047] Preferably, the acidic catalyst of the acidic catalytic conditions is aluminum chloride and acetic acid.

[0048] Preferably, the organic solvent is at least one of acetonitrile, concentrated sulfuric acid and acetic acid.

[0049] Preferably, the molar ratio of vortioxetine to the bromination reagent is 1:2-3, preferably 1:2.1 or 1:2.3.

[0050] Preferably, the vortioxetine is reacted with the bromination reagent in the organic solvent under the acidic catalytic conditions and at a temperature to obtain the compound of formula II; the temperature is 30-100 DEG C, preferably 60-80 DEG C.

[0051] The preparation method of the vortioxetine impurity comprises dissolving a compound of formula I in an acetic acid solution of hydrobromic acid and then adding an oxidizing agent to obtain a compound of formula II-I; the oxidizing agent is at least one of tert-butyl hydroperoxide, hydrogen peroxide and sodium hypochlorite:

[0052]

[0053] Preferably, the acetic acid solution of hydrobromic acid is 33% hydrobromic acid in acetic acid.

[0054] Preferably, the oxidizing agent is tert-butyl hydroperoxide.

[0055] Preferably, the molar ratio of the compound of formula I to the oxidizing agent is 1:1-3, preferably 1:1.3.

[0056] Preferably, the compound of formula I is dissolved in the acetic acid solution of hydrobromic acid and then the oxidizing agent is added to react at a temperature to obtain the compound of formula II-I; the temperature is 60 DEG C-120 DEG C, preferably 80 DEG C-100 DEG C.

[0057] The preparation method of the vortioxetine impurity comprises reacting the compounds of formula I and formula II with an acid to obtain a pharmaceutically acceptable salt thereof.

[0058] Preferably, the acid is hydrobromic acid, hydrochloric acid, hydroiodic acid, p-toluenesulfonic acid, methanesulfonic acid, maleic acid or citric acid.

[0059] The application of the vortioxetine impurity described in the present application; the application is the application in controlling the quality of vortioxetine bulk drug or vortioxetine preparation or the use as a standard or control.

[0060] Preferably, the application is the use as a pharmaceutical standard or control, more preferably the use as a quality research standard or control of vortioxetine bulk drug or vortioxetine preparation.

[0061] The application of the vortioxetine impurity described in the present application as an impurity control in the content analysis and quality control of vortioxetine tablets.

[0062] The detection method of the compound represented by formula I and formula II or its pharmaceutically acceptable salt, comprising detecting the compound represented by formula I and formula II or its pharmaceutically acceptable salt in the test sample solution by using liquid chromatography, such as high performance liquid chromatography; preferably, the stationary phase of the chromatographic column in the liquid chromatography, such as high performance liquid chromatography, is silica gel, such as octadecylsilane bonded silica gel, preferably reversed octadecylsilane bonded silica gel; preferably, the mobile phase of the high performance liquid chromatography is a mixture of water and organic solvent; preferably, the organic solvent can be selected from methanol, acetonitrile or a mixture thereof, preferably acetonitrile. The test conditions of the high performance liquid chromatography are as follows: chromatographic column: YMC-Pack ODS-A chromatographic column, preferably the specification of the chromatographic column is 4.6x250mm, and the particle size is 5μm; mobile phase A: 0.05% formic acid (pH adjusted to 3.5 with ammonia water)-acetonitrile (90:10); mobile phase B: acetonitrile-water (80:20); gradient elution; preferably, the gradient elution is carried out according to the following gradient:

[0063] T(min) 0 50 50.1 60 A(％) 100 0 100 100 B(％) 0 100 0 0

[0064] Preferably, the test conditions of the high performance liquid chromatography further comprise

[0065] Flow rate: 1.0ml / min, column temperature: 30℃, detection wavelength: 226nm, 254nm.

[0066] The vortioxetine bromo impurity and its preparation method are finally obtained through structure confirmation and directional synthesis, and through toxicology research, it is found that the bromo impurity has obvious hepatotoxicity. Compared with the benzene ring fragment, the bromo benzene fragment in the drug structure usually has greater toxicity and side effects. On the one hand, the introduction of bromine atom on the benzene ring will increase the lipophilicity of the drug, which may accumulate in the body and increase the toxicity, and its metabolites in the body are also more toxic. On the other hand, the bromo benzene fragment may also cause allergic reactions or cell damage, so compared with the benzene ring fragment, the bromo benzene ring fragment may bring greater safety risk.

[0067] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: the present application provides two new impurities (formula I and formula II compounds) or pharmaceutically acceptable salts of vortioxetine and a preparation method, which has important significance for the quality research and quality control of vortioxetine tablets. The preparation method is simple to operate, and the prepared new impurities of vortioxetine can be used as a control for qualitative and quantitative analysis, thereby providing a guarantee for the quality control of vortioxetine tablets. Through the study of the adverse reactions of the compounds of the present application, it is shown that the compounds of the present application have greater hepatotoxicity, especially under high-dose conditions, so it is necessary to strictly control the relative content of the compounds in vortioxetine raw materials. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 NMR of the compound of formula I-I of the present application;

[0069] Figure 2 NMR of the compound of formula I-I of the present application;

[0070] Figure 3 Mass spectrum of the compound of formula I-I of the present application;

[0071] Figure 4 X-ray single crystal diffraction of the compound of formula I-I of the present application;

[0072] Figure 5 Mass spectrum of the compound of formula II of the present application;

[0073] Figure 6 High performance liquid chromatography of the compound of formula I-I and formula II-I of the present application;

[0074] Figure 7 Statistical results of hepatotoxicity experiment of the compound of formula I-I of the present application, wherein the contents of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of rats under the condition of oral administration of different doses of compound I-I are compared with those of the control group; wherein, Control is the blank control group of physiological saline, and the rest are experimental groups of different doses (28, 54, 80 mg / kg) of compound I-I. DETAILED DESCRIPTION

[0075] The technical solutions of the present application will be further described below in combination with the drawings and examples.

[0076] Preparation of 11-[4-bromo-2-(2,4-methylphenylthio)phenyl]piperazine hydrobromide (compound of formula I-I)

[0077]

[0078] (1) Synthesis of intermediate compound of formula III

[0079] To a 100 mL round bottom flask was added vortioxetine (4 g), dichloromethane 30 mL, triethylamine (1.5 g) stirred at 0 °C, added p-toluenesulfonyl chloride (2.84 g) stirred for 10 minutes, then raised the temperature to 10 °C and stirred the reaction for 2 hours. After the reaction was completed, 20 mL of 30% sodium hydroxide aqueous solution was added and stirred for 10 minutes, extracted the aqueous phase with dichloromethane 30 mL for 3 times, combined the organic phase, washed with 1 mol / L hydrochloric acid and saturated brine in turn, dried and concentrated to obtain 6.12 g of compound III, yield 98%, MS (m / z): 453.6394 [M+H] + .

[0080] (2) Synthesis of 1-[4-bromo-2-(2,4-dimethylphenylthio)phenyl]piperazine hydrobromide (compound of formula I-I) To a 100 mL round bottom flask was added the above compound of formula III (6 g), hydrogen bromide 33% acetic acid solution 20 mL, heated and stirred at 70 °C for 1 hour, then cooled the reaction solution to room temperature, added water 25 mL and stirred for 10 minutes to precipitate white solid, added petroleum ether 30 mL and methyl tert-butyl ether 10 mL to the flask to make slurry, filtered after 10 minutes, washed the filter cake with petroleum ether and methyl tert-butyl ether, dried to obtain 4.36 g of white solid (compound of formula I-I), yield 72%, the proton nuclear magnetic resonance spectrum is shown in Figure 1 , 1 H NMR (400 MHz, Chloroform-d) δ 7.33 (d, J = 7.8 Hz, 1H), 7.16 (t, J = 2.6 Hz, 2H), 7.04 (d, J = 8.1 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.53 (s, 1H), 3.53 (s, 4H), 3.42 (s, 4H), 2.37 (s, 3H), 2.28 (s, 3H); the carbon nuclear magnetic resonance spectrum is shown in Figure 2 , 13 CNMR (100 MHz, CDCl3) δ 146.07 (C), 142.52 (C), 140.41 (C), 137.80 (C), 136.51 (CH), 132.26 (CH), 128.57 (CH), 128.37 (CH), 128.28 (CH), 125.80 (C), 122.02 (CH), 119.20 (C), 48.44 (CH2), 44.47 (CH2), 21.38 (CH3), 20.70 (CH3). 2D-NMR correlation fragment signals: δ C 146.07 (C) with δ H 6.53 (s, 1H) and δ H 7.16 (t, J = 2.6 Hz, 2H) hydrogen resonance peaks HMBC correlation; δ H7.16 (t, J = 2.6 Hz, 2H) and δ H 6.94 (d, J = 8.3 Hz, 1H) of proton 1 H- 1 H COSY correlation; δ H 6.53 (s, 1H) and δ C 146.07 (C) and δ C 128.57 (CH) of carbon resonance HMBC correlation; δ H 7.16 (t, J = 2.6 Hz, 2H) and δ C 146.07 (C) of carbon resonance HSQC correlation. Mass spectrum as Figure 3 shown, Q-TOF LC-MS (m / z): 377.0671 [M+H] + .

[0081] According to the analysis of the reaction mechanism, it is speculated that the generation of 1-[4-bromo-2-(2,4-methylphenylthio) phenyl] piperazine hydrobromide (compound of formula I-I) is that the sulfuryl bromide generated in the removal of the sulfuryl protection group is reduced to release bromine under the condition of hydrobromic acid, and further undergoes electrophilic substitution reaction with the benzene ring of vortioxetine under acidic conditions:

[0082]

[0083] Example 2 Preparation of 1-[4-bromo-2-(2,4-methylphenylthio) phenyl] piperazine hydrobromide (compound of formula I-I)

[0084] (1) Synthesis of intermediate compound of formula III

[0085] Into a 100 mL round bottom flask, vortioxetine (320 mg) was added, 10 mL of pyridine was stirred at 0 °C, p-toluenesulfonyl chloride (230 mg) was added, and after stirring for 10 minutes, the temperature was raised to 10 °C, and the reaction was stirred for 3.5 hours. After the reaction was completed, 10 mL of 30% sodium hydroxide aqueous solution was added, stirred, and then extracted with dichloromethane three times, and then extracted with saturated brine. After drying over anhydrous sodium sulfate, column purification was performed to obtain 243 mg of compound III, with a yield of 50%, MS (m / z): 453.6394 [M+H] + .

[0086] (2) Synthesis of 1-[4-bromo-2-(2,4-methylphenylthio) phenyl] piperazine hydrobromide (compound of formula I-I)

[0087] Into a 100 mL round bottom flask was added compound III (120 mg), hydrogen bromide 33% acetic acid solution 8 mL, heated to 80 °C and stirred for 1 hour. The reaction solution was cooled to room temperature, 10 mL of water was added and stirred until white solids were precipitated. 10 mL of methyl tert-butyl ether and 20 mL of petroleum ether were added, and the mixture was slurried, filtered, and the filter cake was rinsed with methyl tert-butyl ether and petroleum ether. After drying, 102 mg of compound of formula I-I was obtained, with a yield of 84%, MS (m / z): 377.0671 [M+H] + .

[0088] Preparation of 1-[4-bromo-2-(2,4-dimethylphenylthio)phenyl]piperazine hydrobromide (compound of formula I-I)

[0089] (1) Synthesis of intermediate compound of formula III

[0090] Into a 100 mL round bottom flask was added vortioxetine (169 mg), dimethylamine (38 mg), dichloromethane 10 mL, stirred at 0 °C, and then p-toluenesulfonyl chloride (138 mg) was added and stirred for 10 minutes. The temperature was then raised to 10 °C and the reaction was stirred for 2 hours. After the reaction was completed, 30% sodium hydroxide aqueous solution was added and stirred, and then dichloromethane was added for extraction three times. The organic phase was combined, washed with dilute hydrochloric acid and saturated brine, and then purified by column chromatography to obtain 39 mg of the intermediate compound of formula III, with a yield of 15%.

[0091] (2) Synthesis of 1-[4-bromo-2-(2,4-dimethylphenylthio)phenyl]piperazine hydrobromide (compound of formula I-I)

[0092] Into a 100 mL round bottom flask was added compound III (75 mg), hydrogen bromide 33% acetic acid solution 6 mL, heated to 60 °C and stirred for 1 hour. The reaction solution was cooled to room temperature, and water was added and stirred until white solids were precipitated. Methyl tert-butyl ether 10 mL and 15 mL of petroleum ether were added, and the mixture was slurried, filtered, and the filter cake was rinsed with methyl tert-butyl ether and petroleum ether. After drying, 23 mg of compound of formula I-I was obtained, with a yield of 30%, MS (m / z): 377.0671 [M+H] + .

[0093] Preparation of 1-[4-bromo-2-(2,4-dimethylphenylthio)phenyl]piperazine (compound of formula I)

[0094]

[0095] Into a 100 mL round bottom flask was added vortioxetine (50 mg), aluminum trichloride (18 mg), acetic acid 3 mL, and bromine (21 mg) was added dropwise at 10 °C. The reaction was stirred for 2 hours, and then a large amount of water was added and stirred for 10 minutes. After filtration, the product was rinsed with a small amount of water to obtain a yellowish solid mixture. After purification by column chromatography, 12 mg of compound of formula I was obtained, with a yield of 19%.

[0096] Preparation of 1-[4-bromo-2-(2,4-dimethylphenylthio)phenyl]piperazine (compound of formula I)

[0097] In a 100 mL round bottom flask, vortioxetine (50 mg), acetonitrile 3 mL, concentrated sulfuric acid 2 mL, after stirring and heating to 50 °C, NBS (32 mg) was added, after 1 hour of reaction, 10 mL of water and 10 mL of dichloromethane were added, after extraction, drying and concentration, column chromatography purification, 5 mg of compound of formula I was obtained, with a yield of 8%.

[0098] Synthesis of 1-[4-bromo-2-(2,4-dimethylphenylthio)phenyl]piperazine hydrobromide (compound of formula I-I)

[0099] 50 mg of compound I was added to 5 mL of ethyl acetate, 3 mL of hydrobromic acid solution was added dropwise, after the dropwise addition was completed, the stirring was continued for 1 h, the reaction liquid was concentrated to dryness, the filter cake was collected and vacuum dried to obtain 46 mg of product, which was the hydrobromide salt of compound I, with a yield of 76%, MS (m / z): 377.0671 [M+H] + .

[0100]

[0101] Example 7 Single crystal culture of vortioxetine bromo impurity (compound of formula I-I)

[0102] The crude vortioxetine bromo impurity (compound of formula I-I) obtained in Example 1-6, 20 mg, was placed in a Schlenk flask, dichloromethane was added to make it clear, then n-hexane was slowly added dropwise to make the liquid slightly turbid, and then heated to 40 °C to make it completely clear. Filtration was performed, and the obtained clear filtrate was placed in a Schlenk flask, sealed with a sealing film, and a capillary tube was used to pierce the sealing film. After standing for 2-3 days, crystals were precipitated, and needle-shaped single crystals of vortioxetine bromo impurity (compound of formula I-I) were obtained.

[0103] The single crystal of vortioxetine bromo impurity belongs to monoclinic system, and the cell parameters are as follows: 16.3374(6), 6.2652(2), 19.7807(8), α / ° 90, β / ° 98.855(4), γ / ° 90.

[0104] The X-ray single crystal diffraction test results are shown in the following table: Figure 4

[0105] The single crystal structure data is shown as follows:

[0106]

[0107]

[0108] Preparation of 1-[4,5-dibromo-2-(2,4-dimethylphenylthio)phenyl]piperazine hydrobromide (compound of formula II-I)

[0109]

[0110] In a 100 mL round bottom flask was added 1-[4-bromo-2-(2,4-dimethylphenylthio)phenyl]piperazine (100 mg), 5 mL of 33% hydrobromic acid in acetic acid, heated to 80 °C and stirred for 2 hours. Then 2 mL of t-butyl hydroperoxide was added dropwise slowly and the reaction was continued for 2 hours. Then 10 mL of water and 10 mL of ethyl acetate were added and stirred for 10 minutes. After separation, it was dried over anhydrous sodium sulfate and concentrated. Then it was purified by column chromatography to obtain 23 mg of white solid (compound of formula II-I) with a yield of 16%, MS (m / z): 454.9779 [M+H] 1 H NMR (400 MHz, Chloroform-d) δ 7.37 (d, 1H), 7.23 (d, 1H), 7.12 (d, 1H), 6.99 (s, 1H), 6.92 (s, 1H), 3.49 (s, 4H), 3.40 (t, J = 5.0 Hz, 4H), 2.36 (s, 3H), 2.29 (s, 3H). Mass spectrum was shown as Figure 5 Q-TOF LC-MS (m / z): 454.9779 [M+H] + .

[0111] 1-[4,5-dibromo-2-(2,4-dimethylphenylthio)phenyl]piperazine was generated by the further double substitution reaction of the released bromine from the reduction of p-toluenesulfonyl bromide and formula I-I:

[0112]

[0113] Preparation of 1-[4,5-dibromo-2-(2,4-dimethylphenylthio)phenyl]piperazine hydrobromide (compound of formula II-I)

[0114] In a 100 mL round bottom flask was added 1-[4-bromo-2-(2,4-dimethylphenylthio)phenyl]piperazine (100 mg), 5 mL of 33% hydrobromic acid in acetic acid, heated to 80 °C and stirred for 2 hours. Then 2 mL of t-butyl hydroperoxide was added dropwise slowly and the reaction was continued for 2 hours. Then 10 mL of water and 10 mL of ethyl acetate were added and stirred for 10 minutes. After separation, it was dried over anhydrous sodium sulfate and concentrated. Then it was purified by column chromatography to obtain 23 mg of white solid (compound of formula II-I) with a yield of 16%, MS (m / z): 454.9779 [M+H] + .

[0115] Example 10 Preparation of l-[4,5-dibromo-2-(2,4-dimethylphenylthio)phenyl]piperazine (Compound of Formula II)

[0116]

[0117] In a 50 mL round bottom flask, vortioxetine (45 mg), aluminium chloride (32 mg), acetic acid 3 mL, bromine (40 mg) was added drop wise at room temperature and the temperature was raised to 65 °C and stirred for 2 h. 10 mL water was added and stirred for 10 min. 10 mL ethyl acetate was added and concentrated and column chromatography was performed to get 14 mg of light yellow solid (Compound of Formula II) with 20% yield.

[0118] Example 11 Preparation of l-[4,5-dibromo-2-(2,4-dimethylphenylthio)phenyl]piperazine (Compound of Formula II)

[0119] In a 50 mL round bottom flask, vortioxetine (38 mg), acetonitrile 3 mL, concentrated sulphuric acid 3 mL was added and stirred and heated to 80 °C. NBS (23 mg) was added and stirred for 4 h. 10 mL water and 10 mL dichloromethane was added and extracted, dried, concentrated and column chromatography was performed to get 6 mg of solid (Compound of Formula II) with 10% yield.

[0120] Example 12 Preparation of l-[4,5-dibromo-2-(2,4-dimethylphenylthio)phenyl]piperazine hydrobromide (Compound of Formula II-I)

[0121] To a solution of 20 mg of compound II in 5 mL of ethyl acetate, 3 mL of hydrobromic acid solution was added drop wise. The reaction mixture was stirred for 1 h after the addition was complete. The reaction mixture was concentrated to dryness. The filter cake was collected and dried under vacuum to get 18 mg of product, which was compound II-I, the hydrobromide salt of compound II, with 78% yield, MS (m / z): 454.9779 [M+H] + .

[0122]

[0123] Example 13 Detection of related impurities in vortioxetine raw material

[0124] This example is the HPLC determination of impurities II-I and II-I in vortioxetine

[0125] YMC-Pack ODS-A column (4.6 x 250 mm, 5 μm);

[0126] Mobile phase A: 0.05% formic acid (pH adjusted to 3.5 with ammonia water) - acetonitrile (90:10);

[0127] Mobile phase B: acetonitrile - water (80:20);

[0128] Gradient elution

[0129] T(min) 0 50 50.1 60 A(％) 100 0 100 100 B(％) 0 100 0 0

[0130] Flow rate: 1.0 ml / min, column temperature: 30℃, detection wavelength: 226 nm, 254 nm

[0131] The results show that, as shown in Figure 6 the retention time of 33.72 and 45.08 can be detected in vortioxetine, which is consistent with the retention time in the HPLC spectrum of compounds I-I and II-I, proving that compounds I-I and II-I exist in vortioxetine.

[0132] Example 14 Rat hepatotoxicity evaluation of compound I-I and vortioxetine

[0133] Experimental method: 30 healthy SPF level SD male rats, 6-10 weeks, body weight 220-280g (provided by Pizhou Dongfang Breeding Co., Ltd.), were divided into five groups. The contents of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using alanine aminotransferase (ALT) activity fluorescence test box (sensitivity 0.01 U / L) and aspartate aminotransferase (AST) activity fluorescence test box (sensitivity 0.01 U / L), respectively. PerkinElmer Envision multifunctional enzyme label instrument was used for detection, excitation wavelength 535 nm, emission wavelength 587 nm;

[0134] 1. Control group Control (G1): intragastrically administered with normal saline for three weeks.

[0135] 2. Low-dose compound I-I (G2) test group: intragastrically administered with compound I-I (dose 28 mg / kg / day) for three consecutive weeks;

[0136] 3. Medium-dose compound I-I (G3) test group: intragastrically administered with compound I-I (dose 54 mg / kg / day) for three consecutive weeks;

[0137] 4. High-dose compound I-I (G4) test group: intragastrically administered with compound I-I (dose 80 mg / kg / day) for three consecutive weeks;

[0138] 5. Vortioxetine test group (G5): intragastrically administered with vortioxetine (dose 80 mg / kg / day) for three consecutive weeks.

[0139] After three weeks of continuous administration, the retro-orbital venous blood samples of rats were taken, centrifuged at 4℃, 3500 rpm for 10 minutes, and the supernatant was detected. The differences between each experimental group were compared by one-way analysis of variance (ANOVA). The results are as follows Figure 7As shown, the difference between the control group and the experimental groups was statistically significant (P<0.0001, ****). The serum AST and ALT levels of the rats in each experimental group were significantly increased, indicating that the rats developed liver damage after intragastric administration.

[0140] The average AST and ALT levels of the normal control group were 22.55 ± 0.98 U / mL and 71.47 ± 0.89 U / mL, respectively. For compound I-I, the average AST and ALT levels were 36.78 ± 1.17 U / mL and 42.73 ± 0.94 U / mL at low dose (G2 group), 178.65 ± 1.35 U / mL and 186.61 ± 1.15 U / mL at medium dose (G3 group), and 55.85 ± 1.18 U / mL and 202.0 ± 1.17 U / mL at high dose, respectively, indicating that the liver function of the rats was gradually damaged as the dose increased.

[0141] At a dose of 80 mg / kg (G5 group), the AST and ALT levels of the rats were 38.20 ± 0.94 U / mL and 174.55 ± 1.85 U / mL, which were similar to those of the low-dose compound I-I group (28 mg / kg, G2 group) (ns, no significant difference). The above results indicated that the liver toxicity of compound I-I was more obvious than that of vortioxetine. Therefore, by quality control, reducing the content of brominated impurities or pharmaceutically acceptable salts of vortioxetine can effectively avoid or reduce the side effects caused by brominated impurities or pharmaceutically acceptable salts thereof.

Claims

1. A vortioxetine impurity, characterized in that, The impurity of vortioxetine is a compound represented by Formula I or a pharmaceutically acceptable salt thereof:

2. The vortioxetine impurity according to claim 1, characterized in that, The pharmaceutically acceptable salt of the compound represented by Formula I is the hydrobromide of Formula I-I:

3. The vortioxetine impurity according to claim 2, characterized in that, The hydrobromate single crystal of Formula I-I belongs to the monoclinic crystal system, and its unit cell parameters are: α / °90, β / °98.855(4), γ / °90.

4. A vortioxetine impurity, characterized in that, The impurity of vortioxetine is a compound represented by Formula II or a pharmaceutically acceptable salt thereof:

5. The vortioxetine impurity according to claim 4, characterized in that, The pharmaceutically acceptable salt of the compound represented by Formula II is the hydrobromide of Formula II-I:

6. A method for preparing the vortioxetine impurity according to claim 1, characterized in that, The reaction involves reacting vortioxetine with a brominizing agent in an organic solvent under acidic catalytic conditions to obtain a compound of formula I; wherein the brominizing agent is N-bromosuccinimide or bromine; and the acidic catalyst is at least one of aluminum trichloride, concentrated sulfuric acid, and acetic acid.

7. A method for preparing the vortioxetine impurity as described in claim 2, characterized in that, The process involves dissolving vortioxetine in an organic solvent, reacting it with sulfonyl chloride under alkaline conditions at a specific temperature to obtain intermediate III, then dissolving intermediate III in an acetic acid solution of hydrobromic acid and heating it to remove the protecting group, simultaneously undergoing bromination and salt formation to obtain compound I-I. The specified temperature conditions are 0–40°C; LG in compound III is a sulfonyl group that can be removed under acidic conditions, selected from methanesulfonyl, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, or o-nitrobenzenesulfonyl; the base in the specified alkaline conditions is at least one of pyridine, triethylamine, and dimethylamine.

8. A method for preparing the vortioxetine impurity as described in claim 4, characterized in that, The reaction involves reacting vortioxetine with a bromide in an organic solvent under acidic catalytic conditions to obtain a compound of formula II; the bromide is at least one of N-bromosuccinimide and bromine; and the acidic catalyst under the acidic catalytic conditions is at least one of aluminum trichloride, concentrated sulfuric acid, and acetic acid.

9. A method for preparing the vortioxetine impurity as described in claim 5, characterized in that, This includes dissolving compound I in an acetic acid solution of hydrobromic acid and then adding an oxidizing agent to obtain compound II-I: The oxidant is at least one of tert-butyl hydroperoxide, hydrogen peroxide, and sodium hypochlorite.

10. An application of any one of the impurities of vortioxetine according to claims 1 to 5; said application being its use in controlling the quality of vortioxetine raw material or vortioxetine preparation, or its use as a standard or reference.

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