Clofazimine impurity C, salt and preparation method thereof, and clofazimine detection method

Abstract

The invention belongs to the technical field of material synthesis, analysis and detection, and provides a clofazimine impurity C, a salt and a preparation method thereof, and a clofazimine detection method. The invention provides a clofazimine impurity C and a salt thereof. The clofazimine impurity C has a structure as shown in a formula I. The applicant predicts the toxicity of the clofazimine impurity C through a Cramerrules model, and the result shows that the grade of the toxic harmful substance of the clofazimine impurity C is class III, and the clofazimine impurity C has significant toxicity or reactive functional groups. Meanwhile, according to the prediction of a Carcinogene model (IRFMN-ISSCAN-CGX) 1.2. 2 model, the impurity C of the clofazimine is a cancerogen, and three cancerogenic alarms exist. The clofazimine impurity C has potential genetic toxicity and carcinogenicity. The clofazimine impurity C and the salt thereof can provide convenience for impurity analysis and research of clofazimine bulk drugs and preparations thereof.

Description

Technical Field

[0001] The present invention relates to the technical field of substance synthesis and analysis and detection, and in particular to a clofazimine impurity C and a salt thereof, a preparation method thereof, and a clofazimine detection method. Background Art

[0002] The chemical name of clofazimine is 10-(p-chlorophenyl)-2,10-dihydro-3-(p-chlorophenylamino)-2-isopropyliminophenazine, and its structural formula is as shown in Formula 1:

[0003]

[0004] The common preparation formula of clofazimine is shown in Formula 2:

[0005]

[0006] The European Pharmacopoeia includes clofazimine impurities A and B. However, in actual production, it is found that there are far more than two types of impurities in clofazimine, and no matter how it is purified, it cannot be completely removed.

[0007]

[0008] According to the provisions of the Chinese Pharmacopoeia <9102 Guidelines for Analysis of Pharmaceutical Impurities>, in the study of pharmaceutical impurities, impurities should be separated, purified, prepared or synthesized for safety and quality research. Summary of the Invention

[0009] In light of this, the present invention aims to provide a clofazimine impurity C, its salt, preparation methods, and a clofazimine detection method. This invention identifies a new impurity, clofazimine impurity C, in crude clofazimine, facilitating impurity analysis and research in clofazimine APIs and their preparations.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a clofazimine impurity C and a salt thereof. The clofazimine impurity C has a structure shown in Formula I:

[0012]

[0013] Preferably, the salt of clofazimine impurity C is clofazimine impurity C trifluoroacetate, and the clofazimine impurity C trifluoroacetate has the structure shown in Formula II:

[0014]

[0015] The present invention also provides a method for preparing the clofazimine impurity C and its salt described in the above technical solution, comprising the following steps:

[0016] The crude clofazimine solution is separated and extracted by semi-preparative liquid chromatography to obtain the clofazimine impurity C trifluoroacetate;

[0017] The parameters of the semi-preparative liquid chromatography include: a C18 chromatographic column; mobile phases include mobile phase A and mobile phase B; mobile phase A is a trifluoroacetic acid aqueous solution with a volume concentration of 0.03%, and mobile phase B is acetonitrile; the elution method is gradient elution, and the gradient elution program is: from 0 to 120 minutes: the volume fraction of mobile phase A changes at a constant rate from 55% to 45%.

[0018] Preferably, the flow rate of the mobile phase is 15 mL / min, the injection volume is 5 mL; the column temperature of the chromatographic column is 25-35°C;

[0019] The detector of the semi-preparative liquid chromatography is an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 280 nm.

[0020] Preferably, the crude clofazimine solution is an acetonitrile solution of crude clofazimine.

[0021] Preferably, the method for obtaining the crude clofazimine comprises the following steps:

[0022] The intermediate 3 and isopropylamine are subjected to a condensation reaction in a solution system, and the solvent in the obtained reaction solution is removed and then dried to obtain the crude clofazimine;

[0023]

[0024] Preferably, the molar ratio of the intermediate 3 to isopropylamine is 1:5-25.

[0025] Preferably, the condensation reaction temperature is 120-140° C., and the time is 6-12 hours.

[0026] The present invention also provides a method for detecting clofazimine, comprising the following steps:

[0027] Dissolve the clofazimine to be tested to obtain a test solution;

[0028] Performing analytical liquid chromatography on the test liquid to obtain an analytical liquid chromatogram;

[0029] Based on the analytical liquid chromatogram and peak area normalization method, the content of each substance in the crude clofazimine product is obtained; the each substance includes clofazimine, clofazimine impurity A, clofazimine impurity B, clofazimine impurity C, unknown impurity I and unknown impurity II;

[0030] The parameters of the analytical liquid chromatography detection include: the chromatographic column is a C8 chromatographic column, the filler of the C8 chromatographic column is octylsilane bonded silica gel; the mobile phase includes mobile phase A and mobile phase B; the mobile phase A is a phosphate buffer, and the pH value of the phosphate buffer is 3; the mobile phase B is acetonitrile; the elution mode is isocratic elution, and during the isocratic elution process, the volume ratio of mobile phase A and mobile phase B in the mobile phase is 35:65.

[0031] Preferably, the raw materials for preparing the phosphate buffer include: sodium lauryl sulfate, tetrabutylammonium hydrogen sulfate, disodium hydrogen phosphate dodecahydrate and water, and the usage ratio of the sodium lauryl sulfate, tetrabutylammonium hydrogen sulfate, disodium hydrogen phosphate dodecahydrate and water is 0.475g:0.935g:0.885g:500mL;

[0032] The flow rate of the mobile phase was 1.0 mL / min, the injection volume was 20 μL, and the column temperature of the chromatographic column was 25-35°C;

[0033] The detector for the analytical liquid chromatography detection is an ultraviolet detector, and the wavelength of the ultraviolet detector is 280 nm.

[0034] The present invention provides a clofazimine impurity C and a salt thereof, which have the structure shown in Formula I.

[0035] The applicant used the Cramerrules model to predict the toxicity of clofazimine impurity C, and the results showed that clofazimine impurity C was classified as a Class III toxic hazard, indicating significant toxicity or reactive functional groups. Furthermore, the Carcinogenicity model (IRFMN-ISSCAN-CGX) 1.0.2 model predicted that clofazimine impurity C was a carcinogen with three carcinogenicity alerts. This indicates that clofazimine impurity C has potential genotoxicity and carcinogenicity. The clofazimine impurity C and its salts of the present invention can facilitate the analysis and research of impurities in clofazimine APIs and their preparations, provide a basis for determining the production and medication safety of clofazimine APIs and their preparations, and provide a theoretical basis for the synthesis and preparation of impurity reference substances, thus possessing excellent practical application value.

[0036] The present invention also provides a method for preparing the clofazimine impurity C and its salt described in the above technical solution. The present invention uses semi-preparative liquid chromatography to derivatize a crude clofazimine product with trifluoroacetate, separate the clofazimine impurity C trifluoroacetate from other substances, and prepare the clofazimine impurity C trifluoroacetate.

[0037] The present invention also provides a method for detecting clofazimine, and the detection method of the present invention can accurately detect impurities in clofazimine. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the analytical liquid chromatogram of crude clofazimine;

[0039] Figure 2 This is a semi-preparative liquid chromatogram of crude clofazimine;

[0040] Figure 3 This is the analytical liquid chromatogram of the new impurity C at 30.252 min of semi-preparative liquid chromatography;

[0041] Figure 4 This is the analytical liquid chromatogram of unknown impurity 1 at 44.279 min of semi-preparative liquid chromatography;

[0042] Figure 5 This is the analytical liquid chromatogram of unknown impurity 2 at 67.496 min of semi-preparative liquid chromatography;

[0043] Figure 6 This is the analytical liquid chromatogram of impurity A at 73.288 min of semi-preparative liquid chromatography;

[0044] Figure 7 This is the analytical liquid chromatogram of impurity B at 78.601 min of semi-preparative liquid chromatography;

[0045] Figure 8 This is the analytical liquid chromatogram of clofazimine at 108.180 min of semi-preparative liquid chromatography;

[0046] Figure 9 This is the H NMR spectrum of clofazimine impurity C trifluoroacetate;

[0047] Figure 10 This is the carbon NMR spectrum of clofazimine impurity C trifluoroacetate;

[0048] Figure 11 is the mass spectrum of clofazimine impurity C trifluoroacetate;

[0049] Figure 12 This is a semi-preparative liquid chromatography (without trifluoroacetic acid) diagram of crude clofazimine;

[0050] Figure 13 This is the Toxtree prediction graph;

[0051] Figure 14 It is the VEGA prediction graph;

[0052] Figure 15 This is the analytical liquid chromatogram of commercially available clofazimine soft capsules. DETAILED DESCRIPTION

[0053] The present invention provides a clofazimine impurity C and a salt thereof. The clofazimine C has a structure shown in Formula I:

[0054]

[0055] In the present invention, the salt of clofazimine impurity C is preferably clofazimine impurity C trifluoroacetate, and the clofazimine impurity C trifluoroacetate has the structure shown in Formula II:

[0056]

[0057] The present invention also provides a method for preparing the clofazimine impurity C and its salt described in the above technical solution, comprising the following steps:

[0058] The crude clofazimine solution is separated and extracted by semi-preparative liquid chromatography to obtain the clofazimine impurity C trifluoroacetate;

[0059] The parameters of the semi-preparative liquid chromatography include: a C18 chromatographic column; mobile phases include mobile phase A and mobile phase B; mobile phase A is a trifluoroacetic acid aqueous solution with a volume concentration of 0.03%, and mobile phase B is acetonitrile; the elution method is gradient elution, and the process of the gradient elution is: from 0 to 120 minutes: the volume fraction of mobile phase A changes at a constant speed from 55% to 45%.

[0060] In the present invention, the method for obtaining the crude clofazimine preferably comprises the following steps:

[0061] The intermediate 3 and isopropylamine are subjected to a condensation reaction in a solution system, and the solvent in the obtained reaction solution is removed and then dried to obtain the crude clofazimine;

[0062]

[0063] In the present invention, the solution system preferably includes intermediate 3, isopropylamine and a reaction medium; the reaction medium is preferably toluene, dioxane and ethanol, more preferably dioxane; the ethanol is preferably anhydrous ethanol. In the present invention, the molar ratio of intermediate 3 to isopropylamine is preferably 1:5 to 25, specifically preferably 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24 or 1:25. In the present invention, the amount ratio of intermediate 3 to the reaction medium is preferably 10g:70 to 90mL, specifically preferably 10g:70mL, 10g:80mL or 10g:90mL. In the present invention, the solution system is preferably prepared under stirring conditions, and the stirring speed is preferably 200-500 r / min, specifically preferably 200 r / min, 300 r / min, 400 r / min or 500 r / min. In the present invention, the temperature of the condensation reaction is preferably 120-140°C, specifically preferably 120°C, 130°C or 140°C, and the time is preferably 6-12 hours, specifically preferably 6 hours, 8 hours, 10 hours or 12 hours. In the present invention, the condensation reaction is preferably carried out under stirring and oil bath conditions. In the present invention, the condensation reaction is preferably carried out under closed conditions.

[0064] In the present invention, the method for removing the solvent from the obtained reaction liquid is preferably rotary evaporation, and the temperature of the rotary evaporation is preferably 50°C; the present invention does not specifically limit the time of the rotary evaporation, as long as there is substantially no solvent. In the present invention, the drying is preferably vacuum drying, and the vacuum degree of the vacuum drying is preferably -0.07 to -0.09 MPa, specifically preferably -0.07 MPa, -0.08 MPa or -0.09 MPa, the temperature is preferably 60 to 80°C, specifically preferably 60°C, 70°C or 80°C, the time is preferably 5 to 7 hours, specifically preferably 5 hours, 6 hours or 7 hours; the vacuum drying is preferably carried out in a vacuum drying oven.

[0065] In the present invention, the crude clofazimine solution is preferably an acetonitrile solution of crude clofazimine. In the present invention, the concentration of the crude clofazimine solution is preferably 1 to 5 mg / mL, specifically preferably 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL. In the present invention, the method for preparing the crude clofazimine solution preferably comprises the steps of dissolving crude clofazimine in acetonitrile and filtering to obtain the crude clofazimine solution. In the present invention, the filtration is preferably membrane filtration, and the membrane pore size of the membrane filtration is preferably 0.45 μm.

[0066] In the present invention, the parameters of the semi-preparative liquid chromatography include: the chromatographic column is a C18 chromatographic column, and the C18 chromatographic column is preferably GS-120-8-C18-AP; the size of the C18 chromatographic column is preferably 250mm×30mm; the mobile phase includes mobile phase A and mobile phase B; the mobile phase A is a trifluoroacetic acid aqueous solution with a volume concentration of 0.03%, and the mobile phase B is acetonitrile; the elution mode is gradient elution, and the program of the gradient elution is: 0-120min: the volume fraction of mobile phase A changes from 55% to 45% at a uniform rate; the flow rate of the mobile phase is preferably 15mL / min, and the injection volume is preferably 5mL; the column temperature of the chromatographic column is preferably 25-35°C, and more preferably 30°C; the detector of the semi-preparative liquid chromatography is preferably an ultraviolet detector, and the detection wavelength of the ultraviolet detector is preferably 280nm.

[0067] In the present invention, semi-preparative liquid chromatography is used to separate and extract the clofazimine crude product solution, and trifluoroacetic acid in the mobile phase can derivatize the clofazimine impurity C to form clofazimine impurity C trifluoroacetate; due to the different adsorption capacities of the substances (clofazimine, clofazimine impurity A, clofazimine impurity B, clofazimine impurity C, unknown impurity I, unknown impurity II) and the chromatographic column in the semi-preparative liquid chromatography, the substances in the clofazimine crude product solution elute different substances at different times, and the eluates at different time periods are collected to obtain different substances; the clofazimine impurity C trifluoroacetate is the first eluate to flow out.

[0068] The present invention also provides a method for detecting clofazimine, comprising the following steps:

[0069] Dissolve the clofazimine to be tested to obtain a test solution;

[0070] Performing analytical liquid chromatography on the test liquid to obtain an analytical liquid chromatogram;

[0071] Based on the analytical liquid chromatogram and peak area normalization method, the content of each substance in the crude clofazimine product is obtained; the each substance includes clofazimine, clofazimine impurity A, clofazimine impurity B, clofazimine impurity C, unknown impurity I and unknown impurity II;

[0072] The parameters of the analytical liquid chromatography detection include: the chromatographic column is a C8 chromatographic column, the filler of the C8 chromatographic column is octylsilane bonded silica gel; the mobile phase includes mobile phase A and mobile phase B; the mobile phase A is a phosphate buffer, and the pH value of the phosphate buffer is 3; the mobile phase B is acetonitrile; the elution mode is isocratic elution, and during the isocratic elution process, the volume ratio of mobile phase A to mobile phase B is 35:65.

[0073] The present invention dissolves the clofazimine to be tested to obtain a test liquid. In the present invention, the reagent for dissolving the clofazimine to be tested is preferably a mobile phase, and the composition of the mobile phase is preferably described later and will not be repeated here. After the dissolution, the present invention preferably further includes filtration, and the parameters of the filtration are preferably consistent with the above technical solution and will not be repeated here. In the present invention, the concentration of the test liquid is preferably 1 to 5 mg / mL, specifically preferably 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL or 5 mg / mL.

[0074] After obtaining the liquid to be tested, the present invention performs analytical liquid chromatography on the liquid to be tested to obtain an analytical liquid chromatogram.

[0075] In the present invention, the parameters of the analytical liquid chromatography detection include: the chromatographic column is a C8 chromatographic column, the filler of the C8 chromatographic column is octylsilane bonded silica gel, the C8 chromatographic column is further preferably Caprisil C8-P, the size of the chromatographic column is preferably 4.6mm×250mm, 5μm; the mobile phase includes mobile phase A and mobile phase B; the mobile phase A is phosphate buffer, the pH value of the phosphate buffer is 3, and the mobile phase B is acetonitrile; the elution mode is isocratic elution, and during the isocratic elution, the volume ratio of mobile phase A and mobile phase B is 35:65; the flow rate of the mobile phase is preferably 1.0mL / min, and the injection volume is preferably 20μL; the column temperature of the chromatographic column is preferably 25-35°C, and more preferably 30°C; the detector of the analytical liquid chromatography detection is a UV detector, and the wavelength of the UV detector is preferably 280nm,

[0076] In the present invention, the raw materials for preparing the phosphate buffer preferably include sodium lauryl sulfate, tetrabutylammonium hydrogen sulfate, disodium hydrogen phosphate dodecahydrate, and water. The ratio of sodium lauryl sulfate, tetrabutylammonium hydrogen sulfate, disodium hydrogen phosphate dodecahydrate, and water is preferably 0.475 g:0.935 g:0.885 g:500 mL. In the present invention, the reagent for adjusting the pH value of the phosphate buffer is preferably phosphoric acid.

[0077] After obtaining the analytical liquid chromatogram, the present invention obtains the content of each substance in the crude clofazimine product based on the analytical liquid chromatogram and the peak area normalization method; the each substance includes clofazimine, clofazimine impurity A, clofazimine impurity B, clofazimine impurity C, unknown impurity I and unknown impurity II.

[0078] The present invention does not specifically limit the specific method of the peak area normalization method, and operations familiar to those skilled in the art can be used.

[0079] The following examples describe in detail the clofazimine impurity C and its salt, the preparation method, and the detection method of clofazimine provided by the present invention. However, they should not be construed as limiting the scope of protection of the present invention.

[0080] Experimental materials and instrument conditions Experimental materials: acetonitrile, Tianjin Kangkede Technology Co., Ltd.; purified water, homemade; trifluoroacetic acid, Tianjin Zhiyuan Chemical Reagent Co., Ltd.; tetrabutylammonium hydrogen sulfate, Shanghai MacLean Biochemical Technology Co., Ltd.; disodium hydrogen phosphate dodecahydrate, Shanghai Aladdin Biochemical Technology Co., Ltd.; sodium dodecyl sulfate, Tianjin Zhiyuan Chemical Reagent Co., Ltd.; phosphoric acid, Tianjin Komiou Chemical Reagent Co., Ltd.; microporous filter membrane (0.45μm), LuerSyringe Filter; instrument: semi-preparative liquid chromatography, Dalian Yilite Analytical Instrument Co., Ltd.; electronic balance, Sartorius SQP; semi-preparative liquid chromatography column: GS-120-8-C18-AP, Unimicro Technologies Ins; high performance liquid chromatography, Agilent; analytical liquid chromatography column: Caprisil C8-P, Morhchemtechnologies inc.

[0081] Commercially available clofazimine soft capsules were purchased from NOVARTIS, batch number: MG7165.

[0082] Preparation of crude clofazimine: 80 mL of dioxane, 10 g of intermediate 3, and 30 g of isopropylamine were weighed and added to a 200 mL glass pressure tube. A stirrer was added and stirring was started (300 rpm). The oil bath temperature was set to 130°C and maintained at this temperature for 10 hours. After the reaction, the mixture was evaporated on a rotary evaporator at 50°C until the dioxane was essentially eliminated. The mixture was then dried in a vacuum oven at -0.08 MPa and 70°C for 6 hours to yield 18.3 g of crude clofazimine.

[0083] Example 1

[0084] Semi-preparative liquid chromatography conditions: GS-120-8-C18-AP was used as the chromatographic column (250 mm × 30 mm), mobile phase A was 0.03% trifluoroacetic acid in water, mobile phase B was acetonitrile, the elution mode was gradient elution, and the gradient elution conditions were as shown in Table 1; the flow rate of the mobile phase was 15 mL / min, the detection wavelength was 280 nm, the injection volume was 5 mL, and the column temperature was 30°C.

[0085] Table 1 Gradient elution conditions

[0086] Time (min) 0.03% trifluoroacetic acid-water (%) Acetonitrile (%) 0 55 45 120 45 55

[0087] Analytical liquid chromatography conditions: The filler of the Caprisil C8-P column is octylsilane bonded silica gel, and the size of the Caprisil C8-P column is 4.6 mm × 250 mm, 5 μm; phosphate buffer is used as mobile phase A, and acetonitrile is used as mobile phase B; isocratic elution is performed, and the volume ratio of mobile phase A to mobile phase B is 35:65; the flow rate of the mobile phase is 1.0 mL / min; the detection wavelength is 280 nm; the injection volume is 20 μL, and the column temperature is 30°C; wherein, the preparation method of phosphate buffer is as follows: take 2.475 g of sodium dodecyl sulfate, 0.935 g of tetrabutylammonium hydrogen sulfate, and 0.885 g of disodium hydrogen phosphate dodecahydrate, add 500 mL of water to dissolve, and adjust the pH value to 3.0 with phosphoric acid.

[0088] Semi-preparative liquid phase sample preparation method: Accurately weigh 0.3 g of the above crude clofazimine product, place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well to obtain a crude clofazimine solution with a concentration of 3 mg / mL.

[0089] The crude clofazimine solution was analyzed by analytical liquid chromatography. The analytical liquid chromatogram of the crude clofazimine obtained was as follows: Figure 1 Table 2 shows the analytical liquid chromatography information of the crude clofazimine.

[0090] Table 2 Analytical liquid chromatography information of crude clofazimine

[0091]

[0092] Next, the crude clofazimine solution was separated and extracted by semi-preparative liquid chromatography to obtain various eluates. Figure 2 is a semi-preparative liquid chromatogram of the crude product of clofazimine, and Table 3 is the semi-preparative liquid chromatogram information of the crude product of clofazimine.

[0093] Table 3 Semi-preparative liquid chromatography information of crude clofazimine

[0094] # Retention time [min] Peak height [mAU] Peak height [%] Peak area [mAU.s] Peak area [%] Peak width at 0.05 peak height [min] 1 21.381 9.749 1.6 504.783 0.3 1.778 2 30.252 152.682 25.6 19670.951 11.4 4.426 3 44.279 163.752 27.5 45227.099 26.3 9.343 4 67.496 49.435 8.3 8956.329 5.2 5.698 5 73.288 23.166 3.9 2549.114 1.5 3.203 6 78.601 110.571 18.6 56544.078 32.8 15.625 7 108.180 86.486 14.5 38759.924 22.5 12.799 total 595.841 100.0 172212.278 100.0

[0095] After the eluents were concentrated, they were analyzed using analytical liquid chromatography conditions to obtain Figures 3 to 8 shown.

[0096] Figure 3 This is the analytical liquid chromatogram of the new impurity C at 30.252 min of semi-preparative liquid chromatography. Figure 4 This is the analytical liquid chromatogram of unknown impurity 1 at 44.279 min of semi-preparative liquid chromatography. Figure 5 This is the analytical liquid chromatogram of unknown impurity 2 at 67.496 min of semi-preparative liquid chromatography. Figure 6This is the analytical liquid chromatogram of impurity A at 73.288 min of semi-preparative liquid chromatography; Figure 7 This is the analytical liquid chromatogram of impurity B at 78.601 min of semi-preparative liquid chromatography; Figure 8 This is the analytical liquid chromatogram of clofazimine at 108.180 min of semi-preparative liquid chromatography.

[0097] Figure 9 is the H NMR spectrum of clofazimine impurity C trifluoroacetate, Figure 10 This is the C-NMR spectrum of clofazimine impurity C trifluoroacetate. Figure 11 The mass spectrum of clofazimine impurity C trifluoroacetate. Figures 9 to 11 It can be determined that the structural formula of the obtained clofazimine impurity C is:

[0098]

[0099] Comparative Example 1

[0100] The difference from Example 1 is that the mobile phase A in the semi-preparative liquid chromatography condition is replaced by water, and trifluoroacetic acid is not added; the rest is the same as Example 1.

[0101] Figure 12 This is a semi-preparative liquid chromatography (without trifluoroacetic acid) diagram of clofazimine crude product. Figure 2 and Figure 12 It can be seen that without adding trifluoroacetic acid to mobile phase A, the impurities in the crude clofazimine cannot be separated, and all impurities are in one peak. After adding trifluoroacetic acid to mobile phase A, the clofazimine impurity C can be effectively separated.

[0102] Example 2

[0103] The expert rule-based software Toxtree 2.6.13 was used to predict the toxicity and harmfulness level of clofazimine impurity C using the Cramerrules model. The results are as follows: Figure 13 As shown, from Figure 13 It can be seen that the toxicity and harmfulness level of clofazimine impurity C is class III, which has significant toxicity or reactive functional groups.

[0104] The carcinogenicity of clofazimine impurity C was evaluated using the Carcinogenicity model (IRFMN-ISSCAN-CGX) 1.0.2 model based on the statistical software VEGA 1.2.4. The results are as follows: Figure 14 As shown, from Figure 14 It can be seen that the obtained clofazimine impurity C is a carcinogen and has 3 carcinogenic alerts.

[0105] Example 3

[0106] Based on 0.1 g of clofazimine, accurately weigh the contents of the clofazimine soft capsules, place them in a 20 mL volumetric flask, add the mobile phase for analytical liquid chromatography to dissolve and dilute to the scale, and shake the clofazimine solution; accurately measure an appropriate amount of the clofazimine solution, dilute it with the mobile phase for analytical liquid chromatography to obtain a dilution solution with a concentration of 0.5 mg / mL based on clofazimine, and filter to obtain a sample for the machine.

[0107] The above sample was analyzed by analytical liquid chromatography, and the analytical liquid chromatogram of the commercially available clofazimine soft capsules was as follows: Figure 15 Table 4 shows the analytical liquid chromatography information of commercially available clofazimine soft capsules.

[0108] Table 4 Analytical liquid chromatography information of commercially available clofazimine soft capsules

[0109]

[0110] from Figure 15 As can be seen from Table 4, the commercially available clofazimine soft capsules contain clofazimine impurity C.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A clofazimine impurity C and a salt thereof, wherein the clofazimine impurity C has the structure shown in Formula I:

2. The clofazimine impurity C and its salt according to claim 1, characterized in that The salt of the clofazimine impurity C is the clofazimine impurity C trifluoroacetate, and the clofazimine impurity C trifluoroacetate has a structure shown in Formula II:

3. The method for preparing the clofazimine impurity C and its salt according to claim 1 or 2, characterized in that: The following steps are involved: The crude clofazimine solution is separated and extracted by semi-preparative liquid chromatography to obtain the clofazimine impurity C trifluoroacetate; The parameters of the semi-preparative liquid chromatography include: a C18 chromatographic column; mobile phases include mobile phase A and mobile phase B; mobile phase A is a trifluoroacetic acid aqueous solution with a volume concentration of 0.03%, and mobile phase B is acetonitrile; The elution mode is gradient elution, and the program of the gradient elution is: 0-120 min: the volume fraction of the mobile phase A changes at a constant speed from 55% to 45%.

4. The preparation method according to claim 3, characterized in that The flow rate of the mobile phase was 15 mL / min, the injection volume was 5 mL, and the column temperature of the chromatographic column was 25-35°C; The detector of the semi-preparative liquid chromatography is an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 280 nm.

5. The preparation method according to claim 3, characterized in that The crude clofazimine solution is an acetonitrile solution of crude clofazimine.

6. The preparation method according to claim 3, characterized in that The method for obtaining the crude clofazimine product comprises the following steps: The intermediate 3 and isopropylamine are subjected to a condensation reaction in a solution system, and the solvent in the obtained reaction solution is removed and then dried to obtain the crude clofazimine; 7. The preparation method according to claim 6, characterized in that The molar ratio of the intermediate 3 to isopropylamine is 1:5-25.

8. The preparation method according to claim 6 or 7, characterized in that The condensation reaction temperature is 120-140° C., and the reaction time is 6-12 hours.

9. A method for detecting clofazimine, characterized in that: The following steps are involved: Dissolve the clofazimine to be tested to obtain a test solution; Performing analytical liquid chromatography on the test liquid to obtain an analytical liquid chromatogram; Based on the analytical liquid chromatogram and peak area normalization method, the content of each substance in the crude clofazimine product is obtained; the each substance includes clofazimine, clofazimine impurity A, clofazimine impurity B, clofazimine impurity C, unknown impurity I and unknown impurity II; The parameters of the analytical liquid chromatography detection include: the chromatographic column is a C8 chromatographic column, the filler of the C8 chromatographic column is octylsilane bonded silica gel; the mobile phase includes mobile phase A and mobile phase B; the mobile phase A is a phosphate buffer, and the pH value of the phosphate buffer is 3; the mobile phase B is acetonitrile; the elution mode is isocratic elution, and during the isocratic elution process, the volume ratio of mobile phase A to mobile phase B is 35:

65.

10. The detection method according to claim 9, characterized in that: The raw materials for preparing the phosphate buffer solution include: sodium lauryl sulfate, tetrabutylammonium hydrogen sulfate, disodium hydrogen phosphate dodecahydrate and water, and the usage ratio of the sodium lauryl sulfate, tetrabutylammonium hydrogen sulfate, disodium hydrogen phosphate dodecahydrate and water is 0.475g:0.935g:0.885g:500mL; The flow rate of the mobile phase was 1.0 mL / min, and the injection volume was 20 μL; the column temperature of the chromatographic column was 25-35°C; The detector for the analytical liquid chromatography detection is an ultraviolet detector, and the wavelength of the ultraviolet detector is 280 nm.

Images