CN107305202B

CN107305202B - HPLC method for analyzing impurities of levovatinib mesylate and preparation thereof and application of impurities as reference standard

Info

Publication number: CN107305202B
Application number: CN201610258464.1A
Authority: CN
Inventors: 贾慧娟; 何学敏
Original assignee: Beijing Creatron Institute Of Pharmaceutical Research Co ltd
Current assignee: Beijing Creatron Institute Of Pharmaceutical Research Co ltd
Priority date: 2016-04-22
Filing date: 2016-04-22
Publication date: 2020-04-17
Anticipated expiration: 2036-04-22
Also published as: CN107305202A

Abstract

A method for analyzing lenvatinib (4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide) or a lenvatinib-containing preparation, comprising chromatographically determining impurities in a sample, said impurities being selected from one or more of compounds A-I and LVTN-1, said method comprising the steps of: (1) dissolving lenvatinib mesylate or a drug containing lenvatinib mesylate in a solvent to prepare a sample solution; (2) dissolving a sample of any one or more of compounds a to I and intermediate-1 (LVTN-1) in a solvent to prepare a reference standard solution or a control solution; (3) subjecting the sample solution and the reference standard solution to a chromatographic technique; and (4) determining the presence of any one or more of compound a-I and intermediate-1 (LVTN-1) in lenvatinib mesylate or a medicament containing lenvatinib mesylate by reference to one or more of compound a-I and intermediate-1 (LVTN-1) in the reference standard solution.

Description

HPLC method for analyzing impurities of levovatinib mesylate and preparation thereof and application of impurities as reference standard

Technical Field

The invention belongs to the field of drug synthesis. In particular, the invention relates to impurities occurring during the preparation of lenvatinib (4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide) and a method for analyzing a preparation containing the lenvatinib.

Background

Lenvatinib was developed and developed by defense corporation and currently has no standard Chinese translation name, so the applicant has translated it here as "Levatinib". The drug was approved by the FDA in the united states 2 months in 2015 for treatment of patients with locally recurrent or metastatic, progressive, radioiodine refractory differentiated thyroid cancer, under the trade name LENVIMA. In 2015, 3 months, lenvatinib was approved by the japan ministry of Kyoho and became the first molecular targeted therapeutic drug for the treatment of unresectable thyroid cancer (including differentiated thyroid cancer, medullary thyroid cancer, and undifferentiated thyroid cancer); 5 months 2015, approved by the European food and drug administration for marketing. Lenima is a molecularly targeted formulation for oral administration. There is no standard Chinese translation so the applicant has translated it here as "Levatinib mesylate".

The chemical name of lenvatinib is: 4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide, the mesylate salt of which has the formula:

an HPLC method for analyzing the chemical purity of lenvatinib or various salts of lenvatinib is disclosed in patent CN 200480036184.1. Crude lenvatinib (prepared in example 2 of this patent without ethanol recrystallization) was analyzed according to the method of experimental example 3 of this patent using an efficient liquid chromatograph (HPLC) Agilent1260, PDA detector (available from Agilent) and the results are shown in fig. 1, in which the highest peak in fig. 1 is the peak of lenvatinib. As shown in fig. 1, the analysis method of this patent has the following disadvantages:

(1) the impurities contained in the crude lenvatinib product (the retention time under the method is about: 5.063min, 5.180min and 5.273min) can not realize baseline separation and accurate quantification can not be carried out; the separation degree between the impurities does not meet the requirement of the related substance methodology validation item in the ICHQ3A on specificity, namely the separation degree between the impurities is less than 1.5;

(2) the method definitely uses water-methanol (v/v, 3: 1) as a solvent, and the concentration of a test solution is 0.1mg/ml, so that the low concentration of the test solution inevitably causes that impurities with weak ultraviolet absorption at a certain wavelength cannot be detected, and the underestimation of the impurities is caused. When the concentration of the sample solution under the condition is increased to 0.5mg/ml, the sample solution can not be completely dissolved by using the solvent in the patent, and after the dimethyl sulfoxide (DMSO) is added for assisting the dissolution, the sample injection analysis is carried out under the method, and a plurality of spectral peaks are branched peaks, and the specific figure is 2.

(3) The method for calculating the impurities is an area normalization method, and because the structures of all the impurities are different, the ultraviolet absorption conditions are different under the proposed detection wavelength, the area normalization method cannot accurately determine the real content of all the impurities in the medicines on the market.

(4) No qualitative analysis was performed on specific impurities in the levovatinib mesylate.

In summary, the detection result of the prior art does not truly reflect the quality of the drug.

Disclosure of Invention

The present invention provides a new and alternative method for the qualitative and quantitative analysis of related substances in lenvatinib or formulations containing lenvatinib and the use of these impurities as reference standards or controls for the quantitative and qualitative analysis of impurities. It is another object of the present invention to provide a reference standard or control for detecting impurities known as A-I and LVTN-1 formed during the preparation of lenvatinib or during formulation processing and storage.

The invention provides an HPLC method for analyzing lenvatinib or related substances in a lenvatinib preparation. The inventor utilizes 9 impurities (named compounds A-I and LVTN-1) with prepared and identified structures to be used as reference standards or reference substances of related substance analysis methods of lenvatinib or pharmaceutical preparations containing the lenvatinib. Including six impurities and structures that have been disclosed in the prior art and three impurities and structures that have not been disclosed in the prior art.

Thus, the assay of the invention is used to assay compound a, which has the chemical name: 4, 4' - (((carbonylbis (uretidioyl)) bis (3-chloro-4, 1-phenyl)) bis (oxy)) bis (7-methoxyquinoline-6-carboxamide) and having the following structure:

a second aspect provides compound B having the chemical name: 4- (3-chloro-4- (3, 3-dimethylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide and having the following structure:

a third aspect provides compound C having the chemical name: 4-ethoxy-7-methoxyquinoline-6-carboxamide, and having the following structure:

a fourth aspect provides compound D having the chemical name: 4-hydroxy-7-methoxyquinoline-6-carboxylic acid amide and having the following structure:

a fifth aspect provides compound E, having the chemical name: 1- (2-chloro-4-hydroxyphenyl) -3-cyclopropylaminourea, and has the following structure:

a sixth aspect provides compound F, having the chemical name: 4- (3-chloro-4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid and having the structure:

a seventh aspect provides compound G having the chemical name: 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid methyl ester and has the following structure:

an eighth aspect provides compound H, having the chemical name: 4- (4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide and has the following structure:

a ninth aspect provides compound I having the chemical name: 4- (3-chloro-4- (3-methylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide having the structure:

a tenth aspect provides a preparation intermediate of lenvatinib mesylate-1 (LVTN-1) having the chemical name: 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid amide, and has the following structure.

The compounds A to I and the intermediate-1 (LVTN-1) are intermediates or byproducts in the synthesis process of the mevalontinib mesylate or degradation products in the storage process of the mevalontinib mesylate and compositions containing the mevalontinib mesylate, and are suitable for being used as reference standards or reference substances. In a particularly preferred embodiment, compounds a to I and intermediate-1 (LVTN-1) according to the invention are monomeric compounds, most preferably in pure form, preferably with a purity of more than about 95%, preferably with a purity of more than about 98%, most preferably with a purity of more than about 99%, preferably as measured by HPLC.

According to a tenth aspect of the present invention there is provided a method of detecting the purity of a sample of lenvatinib mesylate or a pharmaceutical dosage form containing lenvatinib mesylate, the method comprising determining the presence of one or more of compounds a to I according to the present invention and intermediate-1 (LVTN-1) in the sample. In the methods of the invention, the compounds are used as reference standards or controls.

According to an eleventh aspect of the present invention, there is provided a method for characterizing compounds a to I and intermediate-1 (LVTN-1), which utilizes an HPLC method for analyzing said impurities a to I and intermediate-1 (LVTN-1) in lenvatinib. Preferably, the HPLC method is an LC-MS compatible HPLC method.

Another aspect provides a chromatographic method for detecting the purity of a sample of lenvatinib or a lenvatinib-containing formulation, said method comprising: the presence of one or more of compounds a-I and intermediate-1 (LVTN-1) in the sample is determined by using a reference standard or control according to the present invention. The compounds A to I and the intermediate-1 (LVTN-1) are process impurities, byproducts or degradation impurities in the synthesis process of the mevalontinib mesylate, are suitable to be used as reference standards or reference substances and are used for quality control of the product.

The above impurities are produced by the following routes:

impurity A: the impurities are impurities in commercially available capsules, and are process impurities introduced into the raw material medicine of the levocarnitine mesylate, namely impurities generated in the second step of the preparation process of the levocarnitine mesylate.

Impurity B: in the reaction between the third step of the synthesis method of lervatinib mesylate reported in chinese patent application No. CN101024627 and cyclopropylamine, N-dimethylformamide is used as a solvent, which may contain a small amount of impurity B generated from dimethylamine.

Impurity C: in the synthesis method of lervatinib mesylate reported in Chinese patent application No. CN101024627, in the salt forming reaction of step 4, under an acidic condition, an intermediate-3 (LVTN-3, namely, lervatinib free base) is attacked by solvent ethanol to generate an impurity C.

Impurity D and impurity E: in the salt forming reaction of the fourth step of the synthesis method of the lenvatinib mesylate reported in the Chinese patent application No. CN101024627, under an acidic condition, the lenvatinib free alkali (LVTN-3) is decomposed to generate an impurity D and an impurity E, and the impurity D and the impurity E are process impurities and degradation impurities.

Impurity F (LVTN-ZZ-10): levatinib mesylate contains an amide group and can be hydrolyzed to a carboxylic acid under acid or base conditions to degrade impurities.

Impurity G: levatinib mesylate is degraded under acid, alkali and damp heat conditions to produce degradation impurity G.

The mechanism of acid degradation:

impurity H: in the synthesis method of the lervatinib mesylate reported in Chinese patent application No. CN101024627, the dehalogenation impurity of the starting material 2 is generated by the subsequent reaction.

Impurity I: in the third step of the synthesis method of the lervatinib mesylate reported in the chinese patent application No. CN101024627, the solvent N-methylpyrrolidone and cyclopropylamine may contain a small amount of methylamine, and compete with the cyclopropylamine to participate in the substitution reaction. The mechanism of generation is as follows:

intermediate-1 (LVTN-1): the intermediate in the first step of the synthetic method of the lervatinib mesylate reported in Chinese patent application No. CN101024627 is also a degradation impurity in the storage or subsequent process steps of the lervatinib mesylate. The mechanism of generation is as follows:

(1) in the second step of the synthetic method of mevastanib mesylate reported in chinese patent application No. CN101024627, LVTN-1 was in excess, remaining.

(2) When the third step of the synthesis method of the lervatinib mesylate reported in the Chinese patent application number CN101024627 is salified, LVTN-3 is degraded under the action of acid and temperature.

The mechanism of acid degradation:

impurity D, E, F, G, LVTN-1 is the major degradation impurity in lenvatinib mesylate or pharmaceutical formulations comprising lenvatinib mesylate.

In a particularly preferred embodiment, the compounds a to I and LVTN-1 according to the invention are monomeric compounds, most preferably in pure form, preferably with a purity of more than about 95%, preferably with a purity of more than about 98%, most preferably with a purity of more than about 99%, preferably measured by HPLC.

According to a tenth aspect of the present invention there is provided a method of determining the purity of a sample of lenvatinib or a pharmaceutical dosage form containing lenvatinib, which method comprises determining the presence in the sample of one or more of compounds a to I and LVTN-1 of the present invention. In the methods of the invention, the compounds are used as reference standards or controls for impurities.

According to a tenth aspect of the present invention, there is provided a method for characterizing compounds a to I and LVTN-1, which method utilizes an HPLC method for analyzing said impurities a to I and LVTN-1 in lenvatinib. Preferably, the HPLC method is an LC-MS compatible HPLC method.

Thus, there is provided the use of compounds a to I and LVTN-1 (one or more) according to the invention as reference standards or controls in the detection of sample purity of lenvatinib or a pharmaceutical preparation containing lenvatinib.

In another aspect, the present invention also provides a chromatographic method for detecting the purity of a sample of lenvatinib, the method comprising: the presence of one or more of compounds a-I and LVTN-1 in the sample is determined by using a reference standard or control according to the present invention.

In yet another aspect, there is provided a chromatographic method for detecting the purity of a sample of lenvatinib mesylate by determining the presence of any one or more of compounds a to I and LVTN-1 in a sample containing lenvatinib, the method comprising:

(1) dissolving a sample of lenvatinib or a lenvatinib-containing formulation in a solvent to prepare a sample solution;

(2) dissolving a sample of any one or more of compounds a-I and LVTN-1 in a solvent to prepare a reference standard solution or a control solution;

(3) subjecting the sample solution and the reference standard solution to a chromatographic technique; and

(4) the presence of any one or more of compounds A-I in the sample is determined by reference to known compound A-I and LVTN-1 (one or more) present in the reference standard solution.

In one embodiment, the chromatographic method is liquid chromatography, such as HPLC, UPLC, LC-MS; preferably the chromatography is HPLC, preferably gradient HPLC.

The stationary phase preferably used in the present invention is reversed phase. Suitable stationary phases include octadecylsilane bonded silica or octylsilane bonded silica.

In a preferred embodiment of the present invention, there is provided a gradient HPLC method wherein the mobile phase comprises a combination comprising a buffer solution (a) and an organic solvent (B). Preferably, the buffer solution (a) is an aqueous buffer, preferably an aqueous solution of acetate, formate, phosphate, trifluoroacetic acid, formic acid or a mixture thereof. More preferably, the buffer solution (a) is an aqueous solution of acetate, most preferably ammonium acetate and acetic acid or an aqueous solution of ammonium acetate adjusted in pH with acetic acid. In a particularly preferred embodiment ammonium acetate is present at a concentration of about 0.001M to 1.0M, preferably 0.02M to 0.08M, more preferably 0.02M to 0.05M, most preferably 0.03M. Preferably, the organic solvent (B) is a polar protic solvent, such as methanol, ethanol or isopropanol; or a dipolar aprotic solvent, such as acetonitrile. Preferably, the organic solvent (B) is selected from the group comprising methanol, acetonitrile, ethanol, isopropanol or mixtures thereof, preferably a combination of methanol and acetonitrile, wherein the percentage of methanol is 25 ± 5% based on the total percentage of mobile phases a and B.

Particularly preferred mobile phases of the present invention comprise a combination of ammonium acetate and an aqueous solution of acetic acid (a) and acetonitrile (B).

Further provided is a gradient HPLC method according to the invention, wherein the mobile phase comprises a gradient design as follows:

time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	90	10
8	75	25
			30	40	60
35	5	95
			37	5	95
37.01	90	10
			45	90	10

In a further preferred embodiment, the pH of the aqueous solution of ammonium acetate and acetic acid in the buffer solution (A) in the HPLC method is about 3.5 to 6.5, preferably about 4.0 to 6.5, 4.0 to 6.0, 4.0 to 5.5, and most preferably 4.5 to 5.5.

In other embodiments, the HPLC analysis is performed at a temperature of about 20-40 deg.C, preferably 20-35 deg.C, and most preferably 25-35 deg.C.

In other embodiments, the analysis is performed at a flow rate of about 0.3 to 1.0ml/min, preferably 0.3 to 0.9ml/min, 0.3 to 0.8ml/min, 0.3 to 0.7ml/min, 0.3 to 0.6ml/min, and most preferably 0.3 to 0.5 ml/min.

The HPLC method according to the invention effectively detects and quantifies all impurities including those selected from the following compounds in a single operation:

a compound A: 4, 4' - (((carbonylbis (uretidioyl)) bis (3-chloro-4, 1-phenyl)) bis (oxy)) bis (7-methoxyquinoline-6-carboxamide).

Compound B: 4- (3-chloro-4- (3, 3-dimethylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide.

Compound C: 4-ethoxy-7-methoxyquinoline-6-carboxamide.

Compound D: 4-hydroxy-7-methoxyquinoline-6-carboxylic acid amide.

Compound E: 1- (2-chloro-4-hydroxyphenyl) -3-cyclopropylaminourea.

Compound F: 4- (3-chloro-4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid.

Compound G: 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid methyl ester.

Compound H: 4- (4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide.

A compound I: 4- (3-chloro-4- (3-methylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide.

LVTN-1: 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid amide.

The present invention may be used to analyze lenvatinib as an Active Pharmaceutical Ingredient (API) and/or lenvatinib in a pharmaceutical composition. The pharmaceutical compositions that can be analyzed according to the present invention include solid or liquid compositions and optionally include one or more pharmaceutically acceptable excipients. Solid form compositions include powders, tablets, capsules, pills, dispersible granules, and the like. Liquid compositions include solutions or suspensions, which may be administered by the oral, injectable or instillation routes. The term "lenvatinib" as used throughout the specification and claims refers to 4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide or solvates (e.g. hydrates) and/or salts thereof, including mesylate. The term "impurities" or "related substances" as used throughout the specification may mean impurities formed in the manufacture of an API or a pharmaceutical composition, and/or impurities formed by degradation of an API or impurities formed in a pharmaceutical composition or formulation in storage.

As mentioned above, the HPLC methods reported in the prior art are not suitable for the analysis of lenvatinib and formulations or pharmaceutical compositions containing lenvatinib.

However, the method of the present invention solves this problem and effectively detects and quantitatively, qualitatively analyzes all impurities formed in this particular synthetic process or preparation in a single operation. The invention has the advantages that: specific structures of impurities in lenvatinib and lenvatinib preparations are disclosed, and polar impurities and non-polar impurities are eluted simultaneously by a gradient HPLC method and qualitatively and quantitatively analyzed.

The invention is particularly suitable for determining and quantifying the presence of one or more of compounds A-I and LVTN-1 in a sample. Unless otherwise stated, the terms "impurities" and "compounds" herein are used interchangeably in the context of compounds A through I and LVTN-1.

The method has the advantages that the method has the characteristics of strong specificity, high accuracy and precision, good durability and the like for analyzing the related substances in the lenvatinib and the preparation containing the lenvatinib. Furthermore, the present invention has a high sensitivity and allows the detection and quantification of related substances in the lenvatinib API or pharmaceutical composition in amounts significantly below the acceptable limits specified in the ICH guidelines.

Furthermore, the method of the present invention can be used to detect and quantify all degradation impurities formed during storage of a sample of lenvatinib or a pharmaceutical composition. The method was determined by conducting a forced degradation study according to ICH Q1A guidelines and verified according to ICHQ2A guidelines, covering the following items: specificity, linearity and range, precision, accuracy, detection limit, quantitation limit, durability, and system adaptability.

The novel gradient HPLC method developed by the present inventors has qualitatively determined nine impurities A to I and LVTN-1. The method can analyze impurities with large polarity difference, such as the Levatinib preparation process prepared according to the embodiment, commercially available Levatinib and degraded impurities generated in the storage process, at one time, and therefore, the inventor considers that the gradient design is most suitable.

In practicing the present invention, the inventors of the present invention have found that a stationary phase containing octadecylsilane-bonded silica or octylsilane-bonded silica is most advantageous. Particularly preferred stationary phases contain Waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or Agilent Poroshell HPH-C184.6X 150mm, 2.7. mu.m.

The process of the invention preferably comprises a gradient design such that the relative concentrations of mobile phases a and B typically vary from 100% a: 0% B to 0% a: gradient of 100% B. Preferably, over a period of 30 to 55 minutes, the gradient is 95% a: 5% B to 5% A: 95% B, more preferably, over a period of 20 to 50 minutes, a gradient of 90% a: 10% B to 15% a: 85% B, most preferably, over about 30 minutes, the gradient is 90% a: 10% B to 15% a: 85% B or 85% a: 15% B to 20% a: 80% B or 80% A: 20% B to 30A%: 70% B or 70% A: 30% B to 40% A to 60% B. The gradient method has the advantages that various impurities with different polarities or very similar polarities in the lenvatinib API or the lenvatinib pharmaceutical composition can be completely separated, and accurate qualitative and quantitative determination is facilitated.

The mobile phase used is preferably selected from the group consisting of one or more buffer solutions (A) and one or more organic solvents (B).

The buffer solution (a) is preferably selected from the group consisting of aqueous solutions of combinations comprising phosphates, acetates, formates, trifluoroacetic acid, formic acid or acetic acid mixtures thereof.

The concentration of the buffer solution (a) may be 0.001M to 1.0M, preferably 0.02M to 0.08M, more preferably 0.02M to 0.05M, most preferably 0.03M. Particularly preferred mobile phases comprise an aqueous solution of ammonium acetate and acetic acid, or a combination of an aqueous solution (a) of ammonium acetate solution adjusted in pH with acetic acid and acetonitrile (B).

In a particularly preferred embodiment according to the present invention, there is further provided a method of gradient HPLC, wherein the mobile phase comprises a gradient design as follows:

the invention also provides a particularly preferred HPLC gradient method, wherein the mobile phase comprises ammonium acetate and/or acetic acid as buffer solution (a). In a further particularly preferred embodiment, the mobile phase comprises acetonitrile as organic solvent (B) and/or an acetonitrile-methanol mixed solvent as organic solvent (B). The inventors have found that the gradient design is particularly effective when the mobile phase comprises ammonium acetate and/or acetic acid (a) and acetonitrile (B).

The buffer solution (a) may contain one or several additional solvents, which may be methanol, ethanol, isopropanol, acetonitrile or a mixture thereof as organic solvent. The additional solvent in the buffer solution (a) may or may not be the same solvent as the organic solvent (B). The additional solvent in the buffer solution (a) is preferably acetonitrile.

The pH of the buffer solution (A) is about 3.5 to 6.5, preferably about 4.0 to 6.5, 4.0 to 6.0, 4.0 to 5.5, and most preferably 4.5 to 5.5.

The HPLC process of the present invention is carried out at a temperature of about 20 to 40 ℃, preferably 20 to 35 ℃, most preferably 25 to 35 ℃.

The analytical method is carried out at a flow rate of about 0.3 to 1.0ml/min, preferably 0.3 to 0.9ml/min, 0.3 to 0.8ml/min, 0.3 to 0.7ml/min, 0.3 to 0.6ml/min, most preferably 0.3 to 0.5 ml/min.

Another aspect of the invention provides a reference standard solution. The solution comprises one or more compounds A-I and LVTN-1 dissolved in a suitable solvent, such as acetonitrile. The reference standard solution can be used to determine the presence of any of compounds a to I and LVTN-1 as impurities in a sample analyzed using the chromatographic technique according to the invention.

According to another aspect of the present invention there is provided a reference standard solution wherein a known amount of one or more of compounds a to I and LVTN-1 is dissolved in a suitable solvent such as acetonitrile or dimethylsulfoxide or a mixture of both. The reference standard solution can be used to determine the qualitative and quantitative nature of any of compounds a to I and LVTN-1 as impurities in a sample analyzed using the chromatographic technique according to the invention. The method of analysis is obvious to the skilled person in terms of importance and convenience.

The inventor has extensively verified the method of the invention, and the verification result shows that the method has strong specificity, high accuracy, precision and sensitivity and good durability.

Description of the drawings:

FIG. 1 is a HPLC analysis of lenvatinib according to the method disclosed in test example 3 of CN 200480036184.1;

FIG. 2: according to the method disclosed in CN200480036184.1 test example 3, HPLC analysis spectrum is carried out after the concentration of a test solution of lenvatinib is increased;

FIG. 3: intermediate-1 (LVTN-1): process for preparing 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid amide¹H-NMR；

FIG. 4: intermediate-1 (LVTN-1): high resolution mass spectrometry (ESI-HRMS) of 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid amide;

FIG. 5: levatinib mesylate: process for preparing 4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide methanesulfonate¹H-NMR；

FIG. 6: high resolution mass spectra of 4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide mesylate;

FIG. 7: impurity A: process for preparing 4, 4' - (((carbonylbis (uretidioyl)) bis (3-chloro-4, 1-phenyl)) bis (oxy)) bis (7-methoxyquinoline-6-carboxamide)¹H-NMR；

FIG. 8: impurity A: high resolution mass spectrometry of 4, 4' - (((carbonylbis (uretidioyl)) bis (3-chloro-4, 1-phenyl)) bis (oxy)) bis (7-methoxyquinoline-6-carboxamide);

FIG. 9: impurity B: process for preparing 4- (3-chloro-4- (3, 3-dimethylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide¹H-NMR；

FIG. 10: impurity B: high resolution mass spectra of 4- (3-chloro-4- (3, 3-dimethylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide;

FIG. 11: impurity C: process for preparing 4-ethoxy-7-methoxyquinoline-6-carboxamide¹H-NMR；

FIG. 12: impurity C: high resolution mass spectra of 4-ethoxy-7-methoxyquinoline-6-carboxamide;

FIG. 13: impurity D: process for preparing 4-hydroxy-7-methoxyquinoline-6-carboxylic acid amides¹H-NMR；

FIG. 14: impurity D: high resolution mass spectra of 4-hydroxy-7-methoxyquinoline-6-carboxylic acid amide;

FIG. 15: impurity E: process for preparing 1- (2-chloro-4-hydroxyphenyl) -3-cyclopropylaminourea¹H-NMR；

FIG. 16: impurity E: high resolution mass spectra of 1- (2-chloro-4-hydroxyphenyl) -3-cyclopropylaminourea;

FIG. 17: impurity F: process for preparing 4- (3-chloro-4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid¹H-NMR；

FIG. 18: impurity F: high resolution mass spectra of 4- (3-chloro-4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid;

FIG. 19: impurity G: process for preparing methyl 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylate¹H-NMR；

FIG. 20: impurity G: high resolution mass spectrum of 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid methyl ester;

FIG. 21: impurity H:process for preparing 4- (4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide¹H-NMR；

FIG. 22: impurity H: high resolution mass spectra of 4- (4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide;

FIG. 23: impurity I: process for preparing 4- (3-chloro-4- (3-methylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide¹H-NMR。

FIG. 24: impurity I: high resolution mass spectra of 4- (3-chloro-4- (3-methylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide;

FIG. 25: HPLC spectrogram of the sample added with the standard mixed solution;

FIG. 26: an HPLC spectrogram of a test solution of the lervatinib mesylate prepared in patent example 3;

FIG. 27 is a schematic view showing: HPLC spectrogram of a self-made lervatinib mesylate capsule-10 mg test solution;

FIG. 28: HPLC chromatogram of commercial Levatinib mesylate capsule-10 mg test solution;

Detailed Description

While the invention has been described with respect to specific embodiments thereof, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the invention.

The invention is illustrated by the following examples, which are not intended to limit the invention in any way.

Example 1: synthesis of 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid amide (LVTN-1).

To a 500mL three-necked flask, 200mL of dimethyl sulfoxide was added under nitrogen protection, and stirring was started, and 20.00g of 4-chloro-7-methoxyquinoline-6-carboxylic acid amide (SM1), 18.20g of 4-amino-3-chlorophenol (SM2), and 14.22g of potassium tert-butoxide were added in this order. After the addition, the temperature is raised to 65 ℃, and the mixture is stirred for 19 hours under the condition of heat preservation. Pouring the reaction system into purified water, precipitating a large amount of solid, filtering, leaching a filter cake with purified water, and drying by blowing air at 60 ℃ for 1-2 hours to obtain 25.07g of brown solid 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid amide (LVTN)-1), yield: 86.32 percent. Of intermediate-1¹See FIG. 3 and FIG. 4 for H-NMR and high resolution mass spectra, respectively.

¹H-NMR (400Mz, DMSO) delta: 8.685(s, 1H), 8.653(d, J ═ 3.6Hz, 1H), 7.754-7.870(br, s, 1H), 7.510(s, 1H), 7.263(d, J ═ 2.0Hz, 1H), 7.023(dd, J ═ 2.0Hz, J ═ 6.0Hz 1H), 6.931(d, J ═ 6.0Hz, 1H), 6.472(d, J ═ 3.2Hz, 1H), 5.479(s, 2H), 4.042(s, 3H). ESI-HRMS spectrum shows molecular ion peak M/z 344.08046[ M + H ]]⁺The corresponding molecular weights are in accordance with the theoretical calculations (344.07237) for the formula provided. The absolute error is 2.38ppm, and is within the error range of high-resolution mass spectrometry.

Example 2: synthesis of 4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide (LVTN-3) lenvatinib

250mL of N-methylpyrrolidone was added to a 500mL three-necked flask, stirring was turned on, and 11.50g of pyridine and 25.00g of 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid amide (LVTN-1) were successively added thereto. 4.94g of phenyl chloroformate was dropped in the system in an ice-water bath, and after the dropping was completed, the system was warmed to room temperature. And (3) dropwise adding purified water into the reaction system for 1-2 hours, filtering, leaching, and drying for 2 hours at 60 ℃ by blowing to obtain 33.74g of (4- ((6-carboxylic acid formamido-7-methoxyquinoline-4-yl) oxy) -2-chlorophenyl) carbamic acid phenyl ester (LVTN-2) brown powder. Yield: 81.26 percent.

To a 1000mL three-necked flask was added 300mL of N-methylpyrrolidone, stirring was turned on, and 30.00g of phenyl (4- ((6-carboxamido-7-methoxyquinolin-4-yl) oxy) -2-chlorophenyl) carbamate (LVTN-2) was added. To the system was added dropwise 4.43g of cyclopropylamine at room temperature over a period of 1 hour. After the dropwise addition, the mixture is kept under heat and stirred overnight. Adding 300mL of purified water into the reaction system, stirring until a large amount of solid is separated out (about 1-2 hours), continuing stirring for 1 hour, filtering, leaching, drying by blowing air at 60 ℃ for 12 hours to obtain 27.50g of a crude product, and recrystallizing by using ethanol to obtain 21.08g of an off-white solid, namely 4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide (LVTN-3), wherein the yield is as follows: 76.35 percent

Example 3: preparation of lervatinib mesylate, preparation of mesylate salt of 4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide and preparation of lervatinib mesylate capsule

Under the protection of nitrogen, sequentially adding 100mL of acetic acid and 2.70g of methanesulfonic acid into a 250mL three-necked bottle, controlling the temperature to be 40 ℃, then adding 10.00g of LVTN-3 into the system, stirring for 1-2 hours, filtering, collecting mother liquor, transferring the mother liquor into a 500mL three-necked bottle, controlling the temperature to be 40 ℃ under the protection of nitrogen, dropwise adding 170mL of n-propanol into the system, and controlling the dropwise adding time to be 1-2 hours. Filtering, and leaching a filter cake by using 70mL of n-propanol to obtain the acetic acid compound of the levocarnitine mesylate. Adding 100mL of ethanol into a 250mL three-necked bottle under the protection of nitrogen, starting stirring, controlling the temperature to be 40 ℃, adding a mevastatin mesylate acetic acid compound, continuing stirring for 36 hours, filtering, leaching a filter cake with ethanol, and drying by air blowing for 12 hours at 60 ℃ to obtain 10.23g of a mevastatin mesylate finished product as off-white powder with the yield: 83.49 percent.

¹H-NMR(400Mz，CDCl₃) δ: 8.975(d, J ═ 6.8Hz, 1H), 8.715(s, 1H), 8.354(d, J ═ 9.2Hz, 1H), 8.079(s, 1H), 7.971(br, s, 1H), 7.909(br, s, 1H), 7.699(s, 1H), 7.638(d, J ═ 2.4Hz, 1H), 7.359(dd, J ═ 2.4Hz, J ═ 9.2Hz, 1H), 7.274(s, 1H), 6.963(d, J ═ 6.8Hz, 1H), 4.081(s, 3H), 2.572 to 2.578(m, 1H), 2.401(s, 1H), 0.651 to 0.665(m, 2H), 0.429(m, 2H). ESI-HRMS spectrum shows molecular ion peak M/z 427.11803[ M + H ]]⁺The corresponding molecular weights are in accordance with the theoretical calculations (427.10948) for the formula provided. The absolute error is 2.98ppm, and is within the error range of high-resolution mass spectrometry. Levatinib mesylate¹The H-NMR spectrum and the high resolution mass spectrum (ESI-HRMS) are shown in the figures 5-6.

Weighing the following prescription amounts of the lervatinib mesylate, the D-mannitol, the precipitated calcium carbonate, the hydroxypropyl cellulose, the low-substituted hydroxypropyl cellulose and the microcrystalline cellulose PH101, mixing in a high-speed stirring mixing granulator, adding a proper amount of purified water to prepare a soft material, sieving with a 18-mesh sieve for granulation, drying by a fluidized bed until the moisture content is lower than 2.0%, placing in a dry particle rapid granulator for granulation, sieving with a sieve mesh number of 14 meshes, adding the microcrystalline cellulose PH102 and the talcum powder into the granules after granulation for mixing, and filling a No. 4 hydroxypropyl methyl cellulose capsule, thereby preparing the lervatinib mesylate capsule.

Prescription composition	Dosage (mg/granule)
		Levatinib mesylate	12.5
Precipitated calcium carbonate	33
		D-mannitol	8.5
Microcrystalline cellulose PH101	10
		Low-substituted hydroxypropyl cellulose	25
Hydroxypropyl cellulose-L	3
		Microcrystalline cellulose PH102	5
Talcum powder	3

Example 4: synthesis of impurity A

Synthesis of impurity A, 4, 4' - (((carbonylbis (uretidioyl)) bis (3-chloro-4, 1-phenyl)) bis (oxy)) bis (7-methoxyquinoline-6-carboxamide)

Adding 20mL of N-methyl pyrrolidone into a 100mL three-necked bottle, sequentially adding 1.00g of LVTN-1 and 0.92g of pyridine under stirring, cooling to 0-10 ℃ under the protection of nitrogen, starting to dropwise add 1.46g of phenyl chloroformate, continuing to stir for 20-30 minutes after dropwise adding, heating to 60 ℃, and stirring overnight; TLC sampling (methanol: dichloromethane 1: 10) the disappearance of starting material. 20ml of water was added to precipitate a large amount of solid, and the stirring was continued for 30 minutes. Filtering, pumping, and drying by air blast at 40 ℃ for 30 minutes to obtain a solid crude product, and carrying out column chromatography purification on the obtained crude product, wherein the elution ratio is as follows: and (3) collecting 160mL of eluent at the ratio of methanol to dichloromethane of 1: 50, controlling the temperature to be 30-40 ℃, and controlling the vacuum degree: -0.08MPa, vacuum distillation, solvent distillation until no distillate is distilled off, yielding 105mg of a pale yellow solid, yield: 5.05%, to obtain impurity A: 4, 4' - (((carbonylbis (uretidioyl)) bis (3-chloro-4, 1-phenyl)) bis (oxy)) bis (7-methoxyquinoline-6-carboxamide).

The structure of impurity a is confirmed as follows:

TABLE 1 of impurity A¹H-NMR and¹³C-NMR test data (DMSO-d)₆) And attribution

ESI-HRMS spectrogram of impurity A shows molecular ion peak [ M + H ]]⁺The mass to charge ratio of 713.13146 corresponds to a molecular weight consistent with the theoretical calculation of the formula provided (713.12400). The absolute error was 0.26ppm, which was within the specified error range.

Of impurity A¹The H NMR and ESI-HRMS spectra are shown in FIGS. 7-8, respectively.

Example 5: synthesis of impurity B

Synthesis of impurity B, 4- (3-chloro-4- (3, 3-dimethylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide.

To a 50ml three-necked flask was added 463.87mg of lenvatinib mesylate intermediate LVTN-2 and 9ml of N-methylpyrrolidone. The reaction temperature was controlled at 0 to 10 ℃, 99.18mg of dimethylamine was added to the reaction system, the mixture was stirred for 30 minutes, and the reaction was terminated by TLC monitoring (dichloromethane: methanol 10: 1). Adding 18mL of 80% acetone/water (V/V) mixed solvent into the reaction system, separating out a large amount of solid, stirring for 30 minutes, filtering, and drying by blowing at 60 ℃ to obtain crude product of impurity B. The crude product is purified by column chromatography, and the developing agent and the proportion are as follows: and (3) methanol and dichloromethane are 1: 20(V/V), a total of 150mL of eluent is collected, the temperature is controlled to be 30-40 ℃, and the vacuum degree is as follows: -0.08MPa, distillation under reduced pressure until no distillate is distilled off, obtaining 153.04mg of light pink solid, yield: 36.89%, thus obtaining impurity B: 4- (3-chloro-4- (3, 3-dimethylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide.

¹H-NMR(400Mz，CDCl₃) δ: 8.709(d, J ═ 3.2Hz, 1H), 8.678(s, 1H), 7.981(s, 2H), 7.874(s, 1H), 7.724(d, J ═ 6.0Hz, 1H), 7.544(s, 1H), 7.274(dd, J ═ 2.0Hz, J ═ 6.0Hz, 1H), 7.266(d, J ═ 2.0Hz, 1H), 6.559(d, J ═ 3.2Hz, 1H), 4.051(s, 3H), 2.978(s, 6H). ESI-HRMS spectrum shows molecular ion peak M/z 415.11742[ M + H ]]⁺The corresponding molecular weights are in accordance with the theoretical calculations (414.84226) for the formula provided. The absolute error is 1.60ppm, and is within the error range of high-resolution mass spectrometry.

Of impurities B¹The H NMR and ESI-HRMS spectra are shown in FIGS. 9-10, respectively.

Example 6: synthesis of impurity C

Synthesis of impurity C, i.e. 4-ethoxy-7-methoxyquinoline-6-carboxamide

To a 100mL three-necked flask was added 2.00g of lenvatinib mesylate, 50mL of ethanol. Reflux stirred for 72 h and monitored by TLC (methanol: dichloromethane ═ 1: 10 with new spots). And (3) evaporating the solvent under reduced pressure, and performing column chromatography purification on the obtained crude product, wherein the elution ratio is as follows: and (3) collecting 60mL of eluent with the ratio of methanol to dichloromethane being 1: 20, controlling the temperature to be 30-40 ℃, and controlling the vacuum degree: -0.08MPa, vacuum distillation, solvent distillation until no distillate is distilled off, yielding 122mg of a pale pink solid, yield: 6.10%, obtaining impurity C: 4-ethoxy-7-methoxyquinoline-6-carboxamide.

The structure of impurity C is confirmed in the following table:

TABLE 2 preparation of impurity C¹H-NMR and¹³C-NMR test data (DMSO-d)₆) And attribution

ESI-HRMS spectrogram of impurity C shows molecular ion peak [ M + H ]]⁺Has a mass-to-charge ratio of 247.10872, [2M + Na + H ]]⁺The mass to charge ratio of 515.19080 corresponds to a molecular weight consistent with the theoretical calculation of the formula provided (247.10044). The absolute error was 4.08ppm and was within the specified error range.

Of impurity C¹The H NMR and ESI-HRMS spectra are shown in FIGS. 11-12, respectively.

Example 7: synthesis of impurity D

Adding 10.00g of LVTN-3 and 50ml of acetonitrile into a 250ml three-necked bottle, controlling the temperature at 80 ℃, stirring, adding 3ml of 6% hydrogen peroxide, continuing to heat for 15 minutes, quenching and concentrating. And (3) carrying out column chromatography purification on the crude product, wherein the elution ratio is as follows: collecting 720mL of eluent at the ratio of methanol to dichloromethane of 1: 10, controlling the temperature to be 30-40 ℃, and controlling the vacuum degree: -0.08MPa, vacuum distillation, distilling off the solvent until no fraction is distilled off, and obtaining compound d1.12g, compound D is yellow powder, HPLC purity: 99.19 percent.

Of impurity D¹The H NMR and ESI-HRMS spectra are shown in FIGS. 13-14, respectively.

Example 8:impurity E was purchased from Nanjing QiCan be prepared by pharmaceutical chemical company Limited, and the HPLC purity is 99.06%.

Of impurity E¹The H NMR and ESI-HRMS spectra are shown in FIGS. 15-16, respectively.

Example 9: synthesis of impurity F

Adding about 2.00g of LVTN-3 into a 100ml three-necked flask, adding 20ml of acetonitrile and 2mol/L hydrochloric acid solution, performing ultrasonic treatment to uniformly disperse, heating in a water bath at 80 ℃ for 30 minutes, and concentrating. And (3) carrying out column chromatography purification on the crude product, wherein the elution ratio is as follows: collecting 620mL of eluent at the ratio of methanol to dichloromethane of 1: 10, controlling the temperature to be 30-40 ℃, and controlling the vacuum degree: -0.08MPa, vacuum distillation, distilling off solvent until no distillate is distilled off, obtaining light yellow powder 0.66g, HPLC purity: 98.15 percent. Of impurity F¹The H NMR and ESI-HRMS spectra are shown in FIGS. 17-18, respectively.

Example 10: synthesis of impurity G

To a 250ml three-necked flask was added 5.00g of LVTN-2, 50ml of N-methylpyrrolidone. Controlling the temperature to be 0-10 ℃, adding 7.56g of ammonia water, continuing stirring for 30 minutes, monitoring by TLC (dichloromethane: methanol is 10: 1), and finishing the reaction. 75mL of 33% acetone/water was added, a large amount of solid was produced, and after stirring for 30 minutes, the mixture was filtered and dried by blowing at 60 ℃ to obtain a crude compound G. And (3) carrying out column chromatography purification on the crude product, wherein the elution ratio is as follows: and (3) collecting 630mL of eluent with the ratio of methanol to dichloromethane being 1: 20, controlling the temperature to be 30-40 ℃, and controlling the vacuum degree: -0.08MPa, vacuum distillation, solvent distillation until no distillate is distilled off, to obtain a pale yellow solid 0.86g, yield: 20.63%, compound G was obtained, HPLC purity: 99.60 percent. Of impurity G¹The H NMR and ESI-HRMS spectra are shown in FIGS. 19-20, respectively.

Example 11: synthesis of impurity H

(1) To a 250mL three-necked flask was added 15mL of dimethyl sulfoxide, and 3.00g of 4-chloro-7-methoxyquinoline-6-carboxylic acid amide (SM1) and 1.66g of 4-aminophenol (SM2-ZZ-02) were successively added with stirring. Controlling the temperature to be 20-30 ℃, adding 1.71g of potassium tert-butoxide, continuing stirring for 14 hours, sampling and monitoring by TLC (dichloromethane: methanol is 10: 1), and finishing the reaction. Adding 30mL of purified water, separating out a large amount of solids, filtering, and purifying the obtained crude product by a column chromatography method, wherein the elution ratio is as follows: collecting 820mL of eluent at the ratio of dichloromethane to methanol of 30: 1, controlling the temperature to be 30-40 ℃, and controlling the vacuum degree: -0.08MPa, distilling under reduced pressure, and distilling off the solvent until no fraction is distilled off. Yield 2.31g of a pale pink solid: 58.89%, to obtain the intermediate-1 of the compound H.

(2) Adding 40mL of N-methyl pyrrolidone into a 250mL three-necked bottle, sequentially adding 2.00g of compound H intermediate-1 and 2.05g of pyridine under stirring, cooling to 0-10 ℃ under the protection of nitrogen, starting to dropwise add 3.04g of phenyl chloroformate, and continuing to stir for 20-30 minutes after dropwise addition; TLC sampling (methanol: dichloromethane 1: 10) the disappearance of starting material. 40ml of water was added to precipitate a large amount of solid, and the stirring was continued for 30 minutes. Filtration, suction drying, forced air drying at 60 ℃ for 30 minutes gave 2.21g of a tan powder, yield: 79.54 percent and is directly used for the next synthesis of the compound H.

(3) To a 100mL three-necked flask, 25mL of N-methylpyrrolidone was added, and 2.21g of the above intermediate was added with stirring. Under the protection of nitrogen, cooling to 0-10 ℃, starting to dropwise add 0.65g of cyclopropylamine, and continuing to stir for 20-30 minutes after dropwise adding is finished; TLC sampling (methanol: dichloromethane 1: 10) the disappearance of starting material. 50ml of water was added to precipitate a large amount of solid, and the stirring was continued for 30 minutes. Filtering, pumping, blowing and drying at 40 ℃ for 30 minutes to obtain a crude product, and purifying the crude product by a column chromatography method, wherein the elution ratio is as follows: : collecting 530mL of eluent at the ratio of methanol to dichloromethane of 1: 20, controlling the temperature to be 30-40 ℃, and controlling the vacuum degree: -0.08MPa, distilling under reduced pressure, and distilling off the solvent until no fraction is distilled off. 0.89g of a pale pink solid was obtained, giving compound H, yield: 44.04%, HPLC purity: 99.60 percent.

Of impurity H¹The H NMR and ESI-HRMS spectra are shown in FIGS. 21-22, respectively.

Example 12: synthesis of impurity I

To a 250ml three-necked flask was added 3.00g of LVTN-2, 60ml of N-methylpyrrolidone. And controlling the temperature to be 0-10 ℃, adding 1.47g of methylamine methanol solution, continuing stirring for 30 minutes, monitoring by TLC (dichloromethane: methanol is 10: 1), and finishing the reaction. Adding 90mL of 33% acetone water to generate a large amount of solid, stirring for 30 minutes, filtering, and drying by blowing at 60 ℃ to obtain a crude product of the compound I. Will be provided withThe crude product is purified by column chromatography, and the elution ratio is as follows: and (3) collecting 760mL of eluent at the ratio of methanol to dichloromethane of 1: 20, controlling the temperature to be 30-40 ℃, and controlling the vacuum degree: -0.08MPa, vacuum distillation, solvent distillation until no distillate is distilled off, obtaining 0.90g of light pink solid, yield: 34.70%, to obtain compound I with HPLC purity: 98.77 percent. Of impurity I¹The H NMR and ESI-HRMS spectra are shown in FIGS. 23-24, respectively.

Example 13: HPLC analysis of Levatinib mesylate or Levatinib-related substances

The impurities in the mevastatin mesylate or the relative substances in the mevastatin mesylate are analyzed by an external standard method (2010 version of the Chinese pharmacopoeia), and the impurities are quantified by the external standard method. The quantitative determination of impurities is calculated according to an external standard method, which is specifically referred to appendix V D of the second part of the Chinese pharmacopoeia 2010 edition. The method used to perform the analysis is a gradient HPLC method according to the invention. The chromatographic conditions used were as follows:

chromatographic conditions are as follows:

a chromatographic column: waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or

Agilent Poroshell HPH-C184.6×150mm，2.7μm

Concentration of test solution: 0.5mg/ml

Reference standard solution concentration of each impurity: 1000ppm of

Mobile phase: 0.08M ammonium acetate solution (pH adjusted to 3.5 with acetic acid) (A), acetonitrile-methanol (B)

Detection wavelength: 252nm

Diluent agent: dimethyl sulfoxide acetonitrile A (2: 4)

Column temperature: 20 deg.C

Flow rate: 0.3ml/min

The gradient design is as follows:

sample preparation:

preparation of impurity-positioning solution

(1) Standard positioning solution for Compound A-I, LVTN-1: taking about 5mg of each of the compounds A to I and LVTN-1, accurately weighing, respectively placing in a 100ml volumetric flask, adding acetonitrile or DMSO, ultrasonically dissolving, diluting to scale with acetonitrile, shaking up, and preparing into a solution with the concentration of about 50 mu g/ml as a storage solution of the compounds A to I and LVTN-1.

(2) Adding a standard mixed solution: taking a proper amount of the prepared raw material medicaments of the Levatinib mesylate (about 25mg of Levatinib), precisely weighing, placing in a 50ml measuring flask, precisely adding 0.5ml of each of the stock solutions of the compounds A-I, LVTN-1, adding a proper amount of diluent, dissolving by ultrasonic treatment, metering the volume by using the diluent, and shaking up to prepare a mixed solution containing about 0.5mg of Levatinib and about 0.5 mu g of each of the compounds A-I, LVTN-1 as a standard-added mixed solution.

(3) Preparation of reference standard solutions or control solutions for Compounds A-I, LVTN-1: precisely measuring 0.5ml of compound A-I stock solution, placing the stock solution in a 50ml volumetric flask, fixing the volume by using a diluent, and shaking up to prepare solutions containing 0.5 mu g of the compound A-I respectively in each 1ml as reference standard solutions or reference substance solutions of the compound A-I.

(4) Preparing a lenvatinib test solution: taking a proper amount of the levofloxacin mesylate raw material medicine prepared in the embodiment 3, containing 5mg of levofloxacin mesylate, precisely weighing, placing in a 10ml volumetric flask, adding a proper amount of diluent, performing ultrasonic dissolution, fixing the volume with the diluent, and shaking up to obtain the levofloxacin mesylate. The concentration of the lenvatinib raw material drug test solution is about 0.5 mg/ml.

(5) Preparation of test solutions of lenvatinib mesylate prepared according to patent example 3 of the present invention and lenvatinib mesylate capsule of the original research: taking a proper amount of finely ground powder (about 5mg of lenvatinib) of the content of a lenvatinib mesylate capsule (Lenvima purchased from Eisai Inc., 10mg, batch number: L15044), precisely weighing, putting into a 10ml volumetric flask, adding a proper amount of diluent, carrying out ultrasonic dissolution, carrying out volume measurement by using the diluent, shaking up, filtering by using a 0.22 mu m organic needle type filter, discarding 2ml of primary filtrate, and taking secondary filtrate as a sample solution of the lenvatinib mesylate capsule. The concentration of the test solution of the Levatinib mesylate capsule was about 0.5 mg/ml.

Example 14:

levatinib mesylate was prepared as in example 3, the sample was prepared as in example 13, and the gradient design was as in example 13.

Chromatographic conditions are as follows:

a chromatographic column: waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or

Agilent Poroshell HPH-C184.6×150mm，2.7μm

Concentration of test solution: 0.5mg/ml

Reference standard solution concentration of each impurity: 1000ppm of

Mobile phase: 0.02M ammonium acetate solution (A) (pH adjusted to 6.5 with acetic acid), acetonitrile-methanol (B)

Detection wavelength: 252nm

Diluent agent: dimethyl sulfoxide acetonitrile A (2: 4)

Column temperature: 25 deg.C

Flow rate: 0.9ml/min

Example 15:

Chromatographic conditions are as follows:

a chromatographic column: waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or

Agilent Poroshell HPH-C184.6×150mm，2.7μm

Concentration of test solution: 0.5mg/ml

Reference standard solution concentration of each impurity: 1000ppm of

Mobile phase: 0.02M ammonium acetate solution (A) (pH adjusted to 5.0 with acetic acid), acetonitrile-methanol (B)

Detection wavelength: 252nm

Diluent agent: dimethyl sulfoxide acetonitrile A (2: 4)

Column temperature: 35 deg.C

Flow rate: 0.5ml/min

Example 16:

Chromatographic conditions are as follows:

a chromatographic column: waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or

Agilent Poroshell HPH-C184.6×150mm，2.7μm

Concentration of test solution: 0.5mg/ml

Reference standard solution concentration of each impurity: 1000ppm of

Mobile phase: 0.03M ammonium acetate solution (A) (pH adjusted to 5.0 with acetic acid), acetonitrile-methanol (B)

Detection wavelength: 252nm

Diluent agent: dimethyl sulfoxide acetonitrile A (2: 4)

Column temperature: 25 deg.C

Flow rate: 0.4ml/min

Example 17:

Chromatographic conditions are as follows:

a chromatographic column: waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or

Agilent Poroshell HPH-C184.6×150mm，2.7μm

Concentration of test solution: 0.5mg/ml

Reference standard solution concentration of each impurity: 1000ppm of

Mobile phase: 0.08M ammonium acetate solution (A) (pH adjusted to 5.0 with acetic acid), acetonitrile-methanol (B)

Detection wavelength: 252nm

Diluent agent: dimethyl sulfoxide acetonitrile A (2: 4)

Column temperature: 35 deg.C

Flow rate: 0.3ml/min

Example 18:

Chromatographic conditions are as follows:

a chromatographic column: waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or

Agilent Poroshell HPH-C184.6×150mm，2.7μm

Concentration of test solution: 0.5mg/ml

Reference standard solution concentration of each impurity: 1000ppm of

Mobile phase: 0.03M ammonium acetate solution (A) (pH adjusted to 5.5 with acetic acid), acetonitrile-methanol (B)

Detection wavelength: 252nm

Diluent agent: dimethyl sulfoxide acetonitrile A (2: 4)

Column temperature: 30 deg.C

Flow rate: 0.4ml/min

Example 19:

Chromatographic conditions are as follows:

a chromatographic column: waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or

Agilent Poroshell HPH-C184.6×150mm，2.7μm

Concentration of test solution: 0.5mg/ml

Reference standard solution concentration of each impurity: 1000ppm of

Detection wavelength: 252nm

Diluent agent: dimethyl sulfoxide acetonitrile A (2: 4)

Column temperature: 35 deg.C

Flow rate: 0.3ml/min

Example 20:

Chromatographic conditions are as follows:

a chromatographic column: waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or

Agilent Poroshell HPH-C184.6×150mm，2.7μm

Concentration of test solution: 0.5mg/ml

Reference standard solution concentration of each impurity: 1000ppm of

Mobile phase: 0.03M ammonium acetate solution- (A) (pH adjusted to 4.5 with acetic acid), acetonitrile (B)

Detection wavelength: 252nm

Diluent agent: dimethyl sulfoxide acetonitrile A (2: 4)

Column temperature: 30 deg.C

Flow rate: 0.5ml/min

Example 21:

Chromatographic conditions are as follows:

a chromatographic column: waters Xbridge BEH Shield RP 184.6X 150mm, 2.5 μm or

Agilent Poroshell HPH-C184.6×150mm，2.7μm

Concentration of test solution: 0.5mg/ml

Reference standard solution concentration of each impurity: 1000ppm of

Mobile phase: 0.02M ammonium acetate solution (A) (pH adjusted to 3.5 with acetic acid), acetonitrile-methanol (B)

Detection wavelength: 252nm

Diluent agent: dimethyl sulfoxide acetonitrile A (2: 4)

Column temperature: 25 deg.C

Flow rate: 1.0ml/min

The test steps are as follows:

(1) under the gradient HPLC method of the present invention, on an Agilent1260HPLC apparatus (Agilent Corp., USA), according to the chromatographic conditions of example 13, 5. mu.l of each of the compound A-I, LVTN-1 positioning solution, the spiked mixed solution, the compound A-I, LVTN-1 reference solution, the test solution of Levatinib mesylate and the test solution of Levatinib mesylate capsule was injected into a liquid chromatograph, and the chromatogram was recorded.

(2) Under the gradient HPLC method, according to the chromatographic conditions of the embodiments 13-21, 5 μ l of the standard-added mixed solution is taken and injected into a liquid chromatograph, and the chromatogram is recorded.

And (3) test results:

the localization results of the compound A-I, LVTN-1 main ingredient levovatinib mesylate in the gradient HPLC method of this example 13 are shown in Table 3; under the chromatographic conditions of the examples 13-21 of the invention, the degrees of separation between the impurities in the spiked mixed solution and the relative retention times of the impurities and the lenvatinib mesylate peak are shown in table 4.

The content and total impurity amount of the compounds a to I, LVTN-1 contained in the sample solution were calculated by the external standard method using the chromatographic conditions of example 16, and the results are shown in table 5, the HPLC chromatogram of the spiked mixed solution, the HPLC chromatogram of the lenvatinib mesylate sample solution, the HPLC chromatogram of the lenvatinib mesylate capsule, and the HPLC chromatogram of the Lenvima-10mg standard sample solution of the original research control formulation are shown in fig. 25 to 28, respectively, and the highest peak in fig. 25 to 28 is the peak of lenvatinib, which is represented by LVTN.

According to the positioning results of the compounds A-I, LVTN-1 and the main components and the analysis results of related substances of the mevalontinib mesylate and the mevalontinib mesylate capsules, compounds C (relative retention time is about 0.47), G (relative retention time is about 0.61), H (relative retention time is about 0.78), I (relative retention time is about 0.82), B (relative retention time is about 0.96) and impurities A (relative retention time is about 1.12) are detected in a mevalontinib mesylate test solution; c, G, H, I, B and 4 unknown impurities (the impurities with the normalized content of below 0.01 percent are ignored) are detected in the test solution of the Levatinib mesylate capsule. The original Lenvima capsule detects C, G, I, LVTN-1 (relative retention time is about 0.94), B, A and 7 unknown impurities (the impurities with normalized content below 0.01 percent are ignored).

TABLE 3 localization of Compounds A-I, LVTN-1 and Levatinib mesylate in the procedure of example 13 and degree of separation between peaks

TABLE 4 relative retention time of each impurity under the conditions of examples 13 to 21

Note: when chromatographic conditions are changed, the retention time of each impurity peak is changed, but the relative retention time is basically unchanged. The separation degrees of the impurity C and the impurity E were at the worst 1.21 under the chromatographic conditions of example 18, and still more than the separation degree required for the impurity difficult to separate was not less than 1.2.

Table 5 test results of related substances in lenvatinib mesylate bulk drug prepared in example 3 and commercially available Lenvima capsules according to the chromatographic conditions of example 16

N.d. represents "not detected".

It is noted that all references mentioned in this application are incorporated herein by reference as if each reference were individually incorporated by reference. Furthermore, it should be understood that the above description is of the specific embodiments of the present invention and the technical principles employed, and after reading the contents of the present invention, those skilled in the art can make various changes or modifications to the present invention without departing from the spirit and scope of the present invention, and these equivalents also fall within the scope of the present invention.

Claims

1. A method for determining the purity of a sample of lenvatinib or a solvate or salt thereof or a medicament containing lenvatinib or a solvate or salt thereof, comprising chromatographically determining impurities in the sample, said impurities selected from one or more of compounds a-I and LVTN-1, wherein lenvatinib is 4- (3-chloro-4- (N' -cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide having the structure shown in formula LVTN-3 below:

compound a is 4, 4' - ((((carbonylbis (uretidioyl)) bis (3-chloro-4, 1-phenyl)) bis (oxy)) bis (7-methoxyquinoline-6-carboxamide) and has the following structure:

compound B is 4- (3-chloro-4- (3, 3-dimethylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide and has the following structure:

compound C is 4-ethoxy-7-methoxyquinoline-6-carboxamide and has the following structure:

compound D is 4-hydroxy-7-methoxyquinoline-6-carboxylic acid amide and has the following structure:

compound E is 1- (2-chloro-4-hydroxyphenyl) -3-cyclopropylaminourea and has the following structure:

compound F is 4- (3-chloro-4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid and has the following structure:

compound G is 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid methyl ester and has the following structure:

compound H is 4- (4- (3-cyclopropylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide and has the following structure:

compound I is 4- (3-chloro-4- (3-methylureido) phenoxy) -7-methoxyquinoline-6-carboxylic acid amide and has the following structure:

intermediate-1 is represented by LVTN-1, is 4- (4-amino-3-chlorophenoxy) -7-methoxyquinoline-6-carboxylic acid amide, and has the following structure:

wherein the method comprises the steps of:

(1) dissolving lenvatinib or a solvate thereof or a salt thereof or a drug containing lenvatinib or a solvate thereof or a salt thereof in a solvent to prepare a sample solution;

(2) dissolving a sample of any one or more of compounds a-I and intermediate-1 in a solvent to prepare a reference standard solution or a control solution;

(4) determining the presence of any one or more of compound A-I and intermediate-1 (LVTN-1) in lenvatinib or a solvate thereof or a salt thereof or a medicament containing lenvatinib or a solvate thereof or a salt thereof by reference to one or more of compound A-I and intermediate-1 in the reference standard solution,

wherein the chromatography is a gradient HPLC method in which the mobile phase comprises a combination comprising a buffer solution (mobile phase a) being an aqueous solution of ammonium acetate and acetic acid or an aqueous solution of ammonium acetate adjusted in pH with acetic acid and an organic solvent (mobile phase B) being acetonitrile or a mixed solvent comprising acetonitrile, wherein the mobile phase comprises a gradient design as follows:

wherein the organic solvent (B) is acetonitrile: a mixed solvent of methanol, wherein the percentage of methanol is 25 + -5% based on the total percentage of the mobile phases A and B.

2. The method according to claim 1, wherein the salt of lenvatinib is lenvatinib hydrobromide, lenvatinib hydrochloride, lenvatinib fumarate, lenvatinib maleate or lenvatinib mesylate, wherein the lenvatinib mesylate has the formula 1:

3. the process according to claim 1, wherein the stationary phase used for chromatography is octadecylsilane bonded silica or octylsilane bonded silica.

4. The method according to claim 1, wherein the gradient HPLC is an LC-MS compatible HPLC method.

5. The process according to claim 1, wherein ammonium acetate is present in a concentration of 0.001M to 1.0M.

6. The process according to claim 1, wherein ammonium acetate is present in a concentration of 0.02M to 0.08M.

7. The process according to claim 1, wherein ammonium acetate is present in a concentration of 0.02M to 0.05M.

8. The process according to claim 1, wherein ammonium acetate is present at a concentration of 0.03M.

9. A process according to claim 5, wherein the pH of the aqueous solution of ammonium acetate and acetic acid is from 3.5 to 6.5.

10. A process according to claim 5, wherein the pH of the aqueous solution of ammonium acetate and acetic acid is from 4.0 to 6.5.

11. A process according to claim 5, wherein the pH of the aqueous solution of ammonium acetate and acetic acid is from 4.0 to 6.0.

12. A process according to claim 5, wherein the pH of the aqueous solution of ammonium acetate and acetic acid is from 4.0 to 5.0.

13. A process according to claim 5, wherein the pH of the aqueous solution of ammonium acetate and acetic acid is from 4.5 to 5.5.

14. A process according to any one of claims 1 to 13 wherein the HPLC process is carried out at a temperature of from 20 to 40 ℃.

15. A process according to any one of claims 1 to 13 wherein the HPLC process is carried out at a temperature of from 20 to 35 ℃.

16. A process according to any one of claims 1 to 13 wherein the HPLC process is carried out at a temperature of from 25 to 35 ℃.

17. The process according to any one of claims 1 to 13, wherein the HPLC is carried out at a flow rate of 0.3 to 1.0 ml/min.

18. The process according to any one of claims 1 to 13, wherein the HPLC is carried out at a flow rate of 0.3 to 0.9 ml/min.

19. The process according to any one of claims 1 to 13, wherein the HPLC is carried out at a flow rate of 0.3 to 0.8 ml/min.

20. The process according to any one of claims 1 to 13, wherein the HPLC is carried out at a flow rate of 0.3 to 0.7 ml/min.

21. The process according to any one of claims 1 to 13, wherein the HPLC is carried out at a flow rate of 0.3 to 0.6 ml/min.

22. The process according to any one of claims 1 to 13, wherein the HPLC is carried out at a flow rate of 0.3 to 0.5 ml/min.

23. The method of any one of claims 1-13, wherein the lenvatinib or a solvate or salt thereof or the pharmaceutical composition comprising lenvatinib or a solvate or salt thereof is in a solid form selected from the group consisting of powders, tablets, capsules, pills, and dispersible granules or a liquid form selected from the group consisting of a solution or a suspension.