Vortioxetine impurity as well as preparation method and application thereof

Abstract

The invention discloses a vortioxetine (English name is Vortioxetine) impurity as well as a preparation method and application thereof, the impurity adopts vortioxetine or a pharmaceutically acceptable salt form thereof as a raw material, the raw material is easy to obtain, the operation is simple and convenient, and the reaction condition is mild. The synthesized impurity is characterized by methods of nuclear magnetic resonance, high-resolution mass spectrometry, X-ray single crystal diffraction and the like, and meanwhile, hepatotoxicity research shows that the compound disclosed by the invention has important significance for researching adverse reaction of vortioxetine. The reference substance obtained according to the method provided by the invention can be used for qualitative and quantitative analysis of vortioxetine impurities, so that the medication safety of vortioxetine is improved.

Description

Technical Field

[0001] The invention belongs to the field of pharmacy, and specifically relates to a vortioxetine impurity and a preparation method and application thereof. Background Art

[0002] Drug impurities refer to other chemical substances other than the active ingredients that are introduced or generated during the production and storage of drugs. According to the 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 the introduction of starting materials or reagents; intermediates, by-products, degradation impurities and residues of materials and reagents in the production process; degradation impurities in the storage process and other channels. Impurity research and control is one of the key links in drug registration and marketing in drug research and development; the impurity limit requirements of drugs are closely related to the safety of drug users. Impurity research needs to consider whether the production process is reliable and stable, and whether the quality standards are reasonable; ultimately, it is also to ensure that the quality of drugs meets the requirements of safety, reliability and controllable quality.

[0003] Vortioxetine (also known as vortioxetine) has the Chinese chemical name 1-[2-(2,4-methylphenylthio)phenyl]piperazine. It is a new type of antidepressant. Its hydrobromide form is used clinically, mainly for the treatment of major depression in adults. The drug has a unique mechanism of action and is classified as a multi-action antidepressant. Its main mechanism is to selectively inhibit 5-hydroxytryptamine transporters, thereby increasing the concentration of 5-hydroxytryptamine in the synaptic cleft. In addition, vortioxetine also exhibits the characteristics of partial 5-HT1A receptor agonists and 5-HT3 receptor antagonists. This dual effect helps improve the mood and cognitive function of patients with depression. Its structure is shown below:

[0004]

[0005] Currently, there are many reported synthesis processes of vortioxetine, such as the synthesis route reported in CN 112759562 A as follows: 1-fluoro-2-nitrobenzene reacts with piperazine to obtain 1-(2-nitrophenyl)piperazine, which is then protected with a sulfonyl chloride group, reduced, iodinated, and reacted with 2,4-dimethylthiophenol to remove the sulfonyl protection to obtain the vortioxetine hydrobromide raw material.

[0006]

[0007] LG is methanesulfonyl, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl or o-nitrobenzenesulfonyl. The inventors found in the quality study of the vortioxetine API and its preparations obtained based on the synthetic route that unknown impurities were produced at retention times of 33.72min and 45.08min in the liquid chromatogram, with a content exceeding 0.1%. The results of nuclear magnetic resonance, high-resolution mass spectrometry, and X-ray single crystal diffraction analysis determined that the impurities were vortioxetine bromide impurities that had not been reported. Since impurity research is an important part of drug research and development, it runs through the entire research and development process and is directly related to the safety, effectiveness, and quality control of the drug. Therefore, it is of great significance to conduct impurity research on vortioxetine. Summary of the invention

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

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

[0010]

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

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

[0013]

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

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

[0016]

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

[0018] Preferably, the pharmaceutically acceptable salt of the compound represented by formula II is the hydrobromide of formula II-I:

[0019]

[0020] The preparation method of vortioxetine impurities of the present invention comprises reacting vortioxetine with a brominating agent in an organic solvent under an acidic catalyst condition to obtain a compound of formula I; the brominating agent is N-bromosuccinimide or bromine; the acidic catalyst is at least one of aluminum chloride, concentrated sulfuric acid, and acetic acid:

[0021]

[0022] Preferably, the bromination agent is bromine.

[0023] Preferably, the acidic catalyst in the acidic catalytic conditions is aluminum chloride and acetic acid.

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

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

[0026] Further preferably, vortioxetine is reacted with a bromination agent in an organic solvent in the presence of an acidic catalyst and at a certain temperature to obtain the compound of formula I; the temperature condition is 0 to 50°C, preferably 0 to 20°C.

[0027] The preparation method of the vortioxetine impurity of the present invention comprises dissolving vortioxetine in an organic solvent, reacting with sulfonyl chloride under alkaline conditions at a certain temperature to obtain an intermediate III, dissolving the intermediate III in an acetic acid solution of hydrobromic acid and heating to react to remove the protecting group, and simultaneously undergoing a bromination reaction and salt formation to obtain a compound of formula I-I, wherein LG represents a sulfonyl protecting group; the certain temperature condition is 0 to 40° C.; LG in the compound III is a sulfonyl group that can be removed under acidic conditions, including methanesulfonyl, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl or o-nitrobenzenesulfonyl; the base under the alkaline conditions is at least one of pyridine, triethylamine and dimethylamine:

[0028]

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

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

[0031] Preferably, the base of the alkaline condition is triethylamine.

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

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

[0034] More preferably, the temperature condition is 0-10°C.

[0035] More preferably, the reaction of vortioxetine with sulfonyl chloride further comprises controlling the temperature conditions for heat preservation reaction, and the temperature conditions are selected from 0 to 40°C, preferably 20 to 30°C.

[0036] Preferably, the hydrobromic acid 33% acetic acid solution is 10 to 50 times that of the compound of formula III.

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

[0038] In order to understand its molecular structure more clearly, the present invention further cultivated single crystals of the obtained vortioxetine bromide impurity, and performed single crystal analysis by X-ray single crystal diffraction method to determine the substitution position of bromine and unit cell related parameters of the vortioxetine bromide impurity. The technical scheme adopted is as follows:

[0039] The preparation method of the vortioxetine impurity of the present invention comprises the steps of heating and dissolving the compound of formula I-I in an organic solvent, filtering, standing and culturing, and crystallizing to obtain a single crystal of the vortioxetine impurity (formula 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°C-60°C.

[0042] Preferably, the static culture temperature is -10°C to 30°C.

[0043] Preferably, the static culture time is 2-5 days.

[0044] The preparation method of vortioxetine impurities of the present invention comprises reacting vortioxetine with a brominating agent in an organic solvent under acidic catalytic conditions to obtain a compound of formula II; the brominating agent 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 agent is bromine.

[0047] Preferably, the acidic catalyst in 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, vortioxetine is reacted with a bromination agent in an organic solvent under acidic catalytic conditions and at a certain temperature to obtain the compound of formula II; the temperature condition is 30-100°C, preferably 60-80°C.

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

[0052]

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

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

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

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

[0057] The method for preparing the vortioxetine impurity of the present invention comprises reacting the compound represented by formula I and formula II as claimed in claim 1 or 4 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 of the present invention is to control the quality of vortioxetine raw materials or vortioxetine preparations or to use it as a standard or reference substance.

[0060] Preferably, the application is as a pharmaceutical standard or reference substance, more preferably as a quality research standard or reference substance for vortioxetine bulk drug or vortioxetine preparation.

[0061] The vortioxetine impurity of the present invention is used as an impurity reference substance in the content analysis and quality control of vortioxetine tablets.

[0062] The method for detecting the compounds of Formula I and Formula II or their pharmaceutically acceptable salts comprises using liquid chromatography, such as high performance liquid chromatography, to detect the compounds of Formula I and Formula II or their pharmaceutically acceptable salts in the test sample solution; 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 anti-octadecylsilane bonded silica gel; preferably, the mobile phase of the high performance liquid chromatography is a mixture of water and an 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, the preferred specification of the chromatographic column is 4.6×250 mm, and the particle size is 5 μm; mobile phase A: 0.05% formic acid (adjusting pH to 3.5 with ammonia water)-acetonitrile (90:10); mobile phase B: acetonitrile-water (80:20); gradient elution; preferably, the gradient elution is eluted 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 HPLC also include

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

[0066] The present invention finally obtains vortioxetine brominated impurities and a preparation method thereof through structural confirmation and directional synthesis, and through toxicological studies, it is found that the brominated impurities have obvious hepatotoxicity. Compared with the benzene ring fragment, the brominated benzene fragment in the drug structure usually has greater toxicity and side effects. On the one hand, the introduction of bromine atoms on the benzene ring will increase the lipophilicity of the drug, so that it may accumulate in the body and increase toxicity, and its metabolites in the body are also more toxic. On the other hand, the brominated benzene fragment may also cause allergic reactions or cell damage, so compared with the benzene ring fragment, the brominated benzene ring fragment may bring greater safety risks.

[0067] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention provides two new impurities of vortioxetine (compounds of formula I and formula II) or their pharmaceutically acceptable salts and a preparation method, which are of great significance to the quality research and quality control of vortioxetine tablets. The preparation method is simple to operate, and the obtained new impurities of vortioxetine can be used as a reference substance for qualitative and quantitative analysis, providing a guarantee for the quality control of vortioxetine tablets. Through the study of the adverse reactions of the compounds of the present invention, it is shown that the compounds of the present invention have a large hepatotoxicity, which is particularly serious under high-dose conditions, so it is necessary to strictly control the relative content of the compounds in the vortioxetine raw material. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 The H NMR spectrum of the compound of formula Ⅰ-Ⅰ of the present invention;

[0069] Figure 2 The carbon-NMR spectrum of the compound of formula Ⅰ-Ⅰ of the present invention;

[0070] Figure 3 The mass spectrum of the compound of formula Ⅰ-Ⅰ of the present invention;

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

[0072] Figure 5 The mass spectrum of the compound of formula II of the present invention;

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

[0074] Figure 7 The results of the hepatotoxicity experiment of Formula Ⅰ-Ⅰ of the present invention are statistically analyzed, wherein the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of rats were compared with those in the control group under the conditions of oral administration of different doses of Compound Ⅰ-Ⅰ; wherein, Control is a normal saline blank control group, and the rest are experimental groups taking different doses (28, 54, 80 mg / kg) of Compound Ⅰ-Ⅰ. DETAILED DESCRIPTION

[0075] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0076] Example 11 Preparation of 1-[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] Vortioxetine (4 g), 30 mL of dichloromethane and triethylamine (1.5 g) were added to a 100 mL round-bottom flask and stirred at 0°C. p-Toluenesulfonyl chloride (2.84 g) was added and stirred for 10 minutes, and then the temperature was raised to 10°C and stirred for 2 hours. After the reaction was completed, 20 mL of 30% sodium hydroxide aqueous solution was added and continued to stir for 10 minutes. 30 mL of dichloromethane was added to extract the aqueous phase three times. The organic phases were combined and washed with 1 mol / L hydrochloric acid and saturated brine successively. After column purification, the organic phase was dried and concentrated to obtain 6.12 g of compound III, with a yield of 98%, MS (m / z): 453.6394 [M+H] + .

[0080] (2) Synthesis of 1-[4-bromo-2-(2,4-methylphenylthio)phenyl]piperazine hydrobromide (compound of formula Ⅰ-Ⅰ) The above-mentioned compound of formula Ⅲ (6 g) and 20 mL of 33% hydrogen bromide acetic acid solution were added to a 100 mL round-bottom flask. The mixture was heated and stirred at 70°C for 1 hour. The reaction solution was cooled to room temperature, 25 mL of water was added and stirred for 10 minutes to precipitate a white solid. 30 mL of petroleum ether and 10 mL of methyl tert-butyl ether were added to the flask to make a slurry. After 10 minutes, the mixture was filtered, and the filter cake was rinsed with petroleum ether and methyl tert-butyl ether. After drying, 4.36 g of a white solid (compound of formula Ⅰ-Ⅰ) was obtained. The yield was 72%. The hydrogen nuclear magnetic resonance spectrum was as follows: Figure 1 As shown, 1 H NMR (400 MHz, Chloroform-d) δ7.33 (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); carbon nuclear magnetic resonance spectrum as shown Figure 2 As shown, 13 C NMR (100MHz, CDCl3) δ146.07 (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 related fragment signal: δ C 146.07(C) and δ H 6.53 (s, 1H) and δ H 7.16 (t, J = 2.6 Hz, 2H) hydrogen resonance peak HMBC correlation; δ H7.16 (t, J = 2.6 Hz, 2H) and δ H 6.94 (d, J = 8.3 Hz, 1H) proton 1 H- 1 H COSY related; δ H 6.53(s,1H) and δ C 146.07(C) and δ C The carbon resonance peak of 128.57 (CH) is HMBC related; δ H 7.16 (t, J = 2.6 Hz, 2H) and δ C The carbon resonance peak at 146.07 (C) is related to HSQC. Figure 3 As 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 when the sulfonyl protection is removed, the generated sulfonyl bromide undergoes a reduction reaction under the condition of hydrobromic acid to release bromine, and further undergoes an 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] Vortioxetine (320 mg) and 10 mL of pyridine were added to a 100 mL round-bottom flask and stirred at 0°C. p-Toluenesulfonyl chloride (230 mg) was added and stirred for 10 minutes, and then the temperature was raised to 10°C and stirred for 3.5 hours. After the reaction was completed, 10 mL of 30% sodium hydroxide aqueous solution was added and stirred, and then dichloromethane was added to extract 3 times, and then saturated brine was used for extraction. After drying over anhydrous sodium sulfate, it was purified by column 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] Compound III (120 mg) and 8 mL of 33% acetic acid solution of hydrogen bromide were added to a 100 mL round-bottom flask. After heating and stirring at 80°C for 1 hour, the reaction solution was cooled to room temperature, 10 mL of water was added and stirred until a white solid precipitated. 10 mL of methyl tert-butyl ether and 20 mL of petroleum ether were added to the mixture for pulping, and then filtered and the filter cake was washed. 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] Example 3 Preparation of 1-[4-bromo-2-(2,4-methylphenylthio)phenyl]piperazine hydrobromide (compound of formula Ⅰ-Ⅰ)

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

[0090] Vortioxetine (169 mg), dimethylamine (38 mg), and 10 mL of dichloromethane were added to a 100 mL round-bottom flask and stirred at 0°C. p-Toluenesulfonyl chloride (138 mg) was added and stirred for 10 minutes, and then the temperature was raised to 10°C and 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 3 times. The organic phases were combined, washed with dilute hydrochloric acid and saturated brine, and purified by column, and dried to obtain 39 mg of the intermediate of formula III, with a yield of 15%.

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

[0092] Compound III (75 mg) and 6 mL of 33% acetic acid solution of hydrogen bromide were added to a 100 mL round-bottom flask. After heating and stirring at 60°C for 1 hour, the reaction solution was cooled to room temperature, water was added and stirred until a white solid precipitated. 10 mL of methyl tert-butyl ether and 15 mL of petroleum ether were added to make a pulp, and then filtered. The filter cake was rinsed with methyl tert-butyl ether and petroleum ether successively. 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] Example 4 Preparation of 1-[4-bromo-2-(2,4-methylphenylthio)phenyl]piperazine (compound of formula I)

[0094]

[0095] Vortioxetine (50 mg), aluminum chloride (18 mg), and 3 mL of acetic acid were added to a 100 mL round-bottom flask, and bromine (21 mg) was added dropwise at 10°C. After reacting for 2 hours, a large amount of water was added and stirring was continued for 10 minutes. After filtration, a small amount of water was rinsed to obtain a light yellow solid mixture. After purification by column chromatography, 12 mg of the compound of formula I was obtained, with a yield of 19%.

[0096] Example 5 Preparation of 1-[4-bromo-2-(2,4-methylphenylthio)phenyl]piperazine (compound of formula I)

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

[0098] Example 6 Synthesis of 1-[4-bromo-2-(2,4-methylphenylthio)phenyl]piperazine hydrobromide (compound of formula Ⅰ-Ⅰ)

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

[0100]

[0101] Example 7 Single Crystal Cultivation of Vortioxetine Brominated Impurity (Formula Ⅰ-Ⅰ)

[0102] 20 mg of the crude vortioxetine bromide impurity (Formula Ⅰ-Ⅰ) obtained in 1-6 is placed in a vial, dichloromethane is added to dissolve it, and then n-hexane is slowly added dropwise to make the liquid slightly turbid, and then heated to 40 degrees Celsius to completely dissolve it, filtered, and the obtained clarified filtrate is placed in a vial, sealed with a sealing film, and holes are pierced in the sealing film with a capillary tube. The vial is allowed to stand and cultured for 2-3 days, and crystals are precipitated to obtain needle-shaped single crystals of vortioxetine bromide impurity (Formula Ⅰ-Ⅰ).

[0103] The vortioxetine bromide impurity single crystal belongs to the monoclinic system, and the unit cell parameters are: α / °90, β / °98.855(4), γ / °90.

[0104] X-ray single crystal diffraction test results are attached Figure 4

[0105] The single crystal structure data is shown below:

[0106]

[0107]

[0108] Example 8 Preparation of 1-[4,5-dibromo-2-(2,4-methylphenylthio)phenyl]piperazine hydrobromide (compound of formula II-I)

[0109]

[0110] In a 100 mL round-bottom flask, 1-[4-bromo-2-(2,4-methylphenylthio)phenyl]piperazine (100 mg) and 5 mL of hydrobromic acid 33% acetic acid solution were added, heated to 80°C and stirred for reaction, 2 mL of tert-butyl hydroperoxide was slowly added dropwise, and the reaction was continued for 2 hours. After that, 10 mL of water and 10 mL of ethyl acetate were added and stirred for 10 minutes. After separation, the mixture was dried over anhydrous sodium sulfate and concentrated, and then purified by column chromatography to obtain 23 mg of an off-white solid (compound of formula II-I), with a yield of 16%. 1 H NMR (400 MHz, Chloroform-d) δ7.37 (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 Figure 5 As shown, Q-TOF LC-MS (m / z): 454.9779 [M+H] + .

[0111] The formation of 1-[4,5-dibromo-2-(2,4-methylphenylthio)phenyl]piperazine is produced by the further disubstitution reaction of the bromine released by the reduction of p-toluenesulfonyl bromide and the compound of formula Ⅰ-Ⅰ:

[0112]

[0113] Example 9 Preparation of 1-[4,5-dibromo-2-(2,4-methylphenylthio)phenyl]piperazine hydrobromide (compound of formula II-I)

[0114] 1-[4-bromo-2-(2,4-methylphenylthio)phenyl]piperazine (100 mg) and 5 mL of hydrobromic acid 33% acetic acid solution were added to a 100 mL round-bottom flask, heated to 80°C and stirred for reaction, 0.5 mL of sodium hypochlorite was slowly added dropwise, and the reaction was continued for 1.5 hours. 10 mL of water and 10 mL of ethyl acetate were added and stirred for 10 minutes. After extraction, drying and concentration, 12 mg of off-white solid (compound of formula II-I) was obtained by column chromatography purification, with a yield of 8%, MS (m / z): 454.9779 [M+H] + .

[0115] Example 10 Preparation of 1-[4,5-dibromo-2-(2,4-methylphenylthio)phenyl]piperazine (Compound of Formula II)

[0116]

[0117] Vortioxetine (45 mg), aluminum chloride (32 mg) and 3 mL of acetic acid were added to a 50 mL round-bottom flask. Bromine (40 mg) was added dropwise at room temperature and the temperature was raised to 65°C. After stirring for 2 hours, 10 mL of water was added and stirring was continued for 10 minutes. 10 mL of ethyl acetate was added for extraction and concentration, and then column chromatography was performed to obtain 14 mg of a light yellow solid (compound of Formula II), with a yield of 20%.

[0118] Example 11 Preparation of 1-[4,5-dibromo-2-(2,4-methylphenylthio)phenyl]piperazine (Compound of Formula II)

[0119] In a 50 mL round-bottom flask, vortioxetine (38 mg), 3 mL of acetonitrile and 3 mL of concentrated sulfuric acid were added. After stirring and heating to 80°C, NBS (23 mg) was added. After reacting for 4 hours, 10 mL of water and 10 mL of dichloromethane were added. After extraction, drying and concentration, the product was purified by column chromatography to obtain 6 mg of solid (compound of Formula II), with a yield of 10%.

[0120] Example 12 Preparation of 1-[4,5-dibromo-2-(2,4-methylphenylthio)phenyl]piperazine hydrobromide (compound of formula II-I)

[0121] 20 mg of compound II was added to 5 mL of ethyl acetate, and 3 mL of hydrobromic acid solution was added dropwise. After the addition was completed, the reaction was continued to stir for 1 h. The reaction solution was concentrated to dryness, filtered, and the filter cake was collected and dried in vacuo to obtain 18 mg of product, namely compound II-I, which is the hydrobromide salt of compound II, with a yield of 78%, MS (m / z): 454.9779 [M+H] + .

[0122]

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

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

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

[0126] Mobile phase A: 0.05% formic acid (pH adjusted to 3.5 with aqueous ammonia)-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°C, detection wavelength: 226 nm, 254 nm

[0131] The results show that if Figure 6 As shown, chromatographic peaks with retention times of 33.72 and 45.08 can be detected in vortioxetine, which are consistent with the retention times in the HPLC spectra of compounds Ⅰ-Ⅰ and Ⅱ-Ⅰ, proving the presence of compounds Ⅰ-Ⅰ and Ⅱ-Ⅰ in vortioxetine.

[0132] Example 14 Evaluation of hepatotoxicity of compound Ⅰ-Ⅰ and vortioxetine in rats

[0133] Experimental methods: 30 healthy SPF SD male rats, 6-10 weeks old, weighing 220-280g (provided by Pizhou Dongfang Breeding Co., Ltd.), were divided into five groups. The content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined using the alanine aminotransferase (ALT) activity fluorescence test kit (sensitivity 0.01U / L) and aspartate aminotransferase (AST) activity fluorescence test kit (sensitivity 0.01U / L), respectively. The PerkinElmer Envision multifunctional microplate reader was used for detection, with an excitation wavelength of 535nm and an emission wavelength of 587nm;

[0134] 1. Control group (G1): Normal saline was given by gavage for three weeks.

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

[0136] 3. Medium-dose compound Ⅰ-Ⅰ (G3) test group: Compound Ⅰ-Ⅰ was administered by gavage for three consecutive weeks (dose of 54 mg / kg / day);

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

[0138] 5. Vortioxetine test group (G5): Vortioxetine (at a dose of 80 mg / kg / day) was administered by oral gavage for three consecutive weeks.

[0139] After three weeks of continuous administration, blood samples were collected from the retroorbital vein of rats and centrifuged at 4°C, 3500 rpm for 10 minutes. The supernatant was then tested. The differences between the experimental groups were compared by one-way analysis of variance (ANOVA). Figure 7As shown, the difference between the control group and the experimental group was highly statistically significant (P < 0.0001, ****). The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of rats in each experimental group increased significantly, indicating that the rats had liver damage after oral administration.

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

[0141] At a dose of 80 mg / kg of vortioxetine (group G5), the AST and ALT values ​​of rats were 38.20±0.94 U / mL and 174.55±1.85 U / mL, which were similar to the low-dose compound Ⅰ-Ⅰ group 28 mg / kg (group G2) (ns, no significant difference). The above results show that compared with the vortioxetine API, the hepatotoxicity of compound Ⅰ-Ⅰ is more obvious. Therefore, by reducing the content of brominated impurities or their pharmaceutically acceptable salts in vortioxetine through quality control, the side effects caused by brominated impurities or their pharmaceutically acceptable salts can be effectively avoided or reduced.

Claims

1. A vortioxetine impurity, characterized in that: The vortioxetine impurity 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 hydrobromide single crystal of formula Ⅰ-Ⅰ belongs to the monoclinic system, and the unit cell parameters are: α / °90, β / °98.855(4), γ / °90.

4. A vortioxetine impurity, characterized in that The vortioxetine impurity 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 method comprises reacting vortioxetine with a bromination reagent in an organic solvent under acidic catalyst conditions to obtain a compound of formula I; the bromination reagent 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 according to claim 2, characterized in that: The method comprises dissolving vortioxetine in an organic solvent, reacting it with sulfonyl chloride under alkaline conditions at a certain temperature to obtain an intermediate III, dissolving the intermediate III in an acetic acid solution of hydrobromic acid and heating it to remove the protecting group, and simultaneously undergoing a bromination reaction and forming a salt to obtain a compound of formula Ⅰ-Ⅰ, wherein LG represents a sulfonyl protecting group: The certain temperature condition is 0-40°C; LG in the compound III is a sulfonyl group that can be removed under acidic conditions, including methanesulfonyl, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl or o-nitrobenzenesulfonyl; the base in the alkaline condition is at least one of pyridine, triethylamine and dimethylamine.

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

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

10. A use of the vortioxetine impurity according to any one of claims 1 to 5; the use is for controlling the quality of vortioxetine API or vortioxetine preparation or as a standard or reference substance.

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