CN107870217B

CN107870217B - Detection method of eltrombopag intermediate I related substances

Info

Publication number: CN107870217B
Application number: CN201611263699.6A
Authority: CN
Inventors: 马超; 李书彬; 管凯林; 周英兰; 刘振; 杜永辉; 范传文
Original assignee: Qilu Pharmaceutical Co Ltd
Current assignee: Qilu Pharmaceutical Co Ltd
Priority date: 2016-09-26
Filing date: 2016-12-30
Publication date: 2022-03-04
Anticipated expiration: 2036-12-30
Also published as: CN107870217A

Abstract

The invention discloses a method for detecting related substances of an eltrombopag intermediate I, belonging to the technical field of pharmaceutical analysis. The invention adopts chromatographic columns which take phenyl and octadecylsilane chemically bonded silica as fillers respectively, phosphate buffer solution and acetonitrile or acetonitrile phosphate solution as mobile phases, and adopts a gradient elution mode, and 2 high performance liquid analysis methods established can respectively and effectively determine the contents of I2 '-hydroxy-3' -amino-biphenyl-3-carboxylic acid as an intermediate in the synthesis of Eltrombopag, II 2- (3, 4-dimethyl-phenyl) -5-methyl-2, 4-dihydro-pyrazole-3-ketone as an intermediate and related impurities of the intermediate. The invention also discloses a novel impurity reference substance compound.

Description

Detection method of eltrombopag intermediate I related substances

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a drug analysis method for determining the content of related impurities of an intermediate I2 '-hydroxy-3' -amino-biphenyl-3-carboxylic acid and an intermediate II 2- (3, 4-dimethyl-phenyl) -5-methyl-2, 4-dihydro-pyrazol-3-one for synthesizing eltrombopag.

The invention also relates to a new impurity reference compound DH used in the analysis method of the eltrombopag intermediate II.

Background

Eltrombopag, chemical name 3' - { (2Z) -2- [1- (3, 4-dimethylphenyl) -3-methyl-5-oxo-1, 5-dihydro-4H-pyrazol-4-ylidene]Hydrazine group } -2' -hydroxy biphenyl-3-carboxylic diethanol amine, molecular formula is C₂₅H₂₂N₄O₄·2C₂H₇NO, molecular weight 564.63, and its structural formula is as follows:

eltrombopag is an oral thrombopoietin drug, and the compound can interact with a thrombopoietin receptor of a transmembrane region to generate a signal cascade amplification effect so as to induce the proliferation and differentiation of bone marrow megakaryocytes. The eltrombopag is developed by the Kuransu Schk company, is approved by the Food and Drug Administration (FDA) of the United states to be on the market in 11 months in 2008, has a trade name of Promacta, is used for treating glucocorticoid medicaments, is used for treating thrombocytopenia of patients with ineffective immunoglobulin treatment or chronic idiopathic thrombocytopenic purpura after splenectomy, and is a small-molecule thrombopoietin receptor agonist with remarkable curative effect.

The synthetic routes of eltrombopag are reported in various documents such as patents WO2010114943, WO2013072921, US20150087845a1 and the like. Despite the different process parameters, the synthetic route is essentially identical, i.e. it is prepared by a condensation coupling reaction between 2 '-hydroxy-3' -amino-biphenyl-3-carboxylic acid (intermediate i) and 2- (3, 4-dimethyl-phenyl) -5-methyl-2, 4-dihydro-pyrazol-3-one (intermediate ii) and the two intermediates.

The quality control of the intermediate has an important role in reaction monitoring and yield improvement, and also influences the quality of the final product. So far, no related documents and patents have reports of an eltrombopag intermediate analysis method, and the quality control of the eltrombopag from a starting raw material to a final product, particularly key intermediates in a synthesis process, is comprehensively and systematically carried out, so that the eltrombopag intermediate analysis method has important guiding significance for the quality research of eltrombopag and the improvement of a synthesis process.

According to the synthetic route of the eltrombopag intermediate I and the intermediate II, impurities which can be generated in the preparation and storage processes comprise raw materials, intermediate products, position isomers, demethylation products, dimers and other byproducts. In order to perfect intermediate process control, effectively analyze the quality of medicines and ensure the safety of medication, an analysis method for conveniently and effectively detecting related substances of an eltrombopag intermediate needs to be developed.

Disclosure of Invention

The invention provides a method for analyzing and measuring the content of 2 '-hydroxy-3' -amino-biphenyl-3-carboxylic acid related impurities in an Eltrombopag intermediate I by using a high performance liquid chromatography.

Through structural analysis of the intermediate I and known impurities thereof, aiming at the conditions that the compound has generally small molecular weight, benzene ring structure, position isomerism and the like, phenyl silane bonded silica gel which has steric hindrance characteristics and unique retention effect on aromatic compounds is selected as a filling agent. In addition, according to the dissociation characteristics of the functional groups of the compound, a phosphate buffer solution with the pH value of 3.0-4.0 is used as a mobile phase A, an acetonitrile phosphate solution is used as a mobile phase B, and the pH value of a mobile phase system is changed in the gradient change process, so that impurities with both amino and carboxyl can show good chromatographic retention behavior and separation effect.

Specifically, the analysis method of the present invention can be implemented as follows:

(1) sample preparation

Test solution: taking a proper amount of the product, adding methanol to dissolve and diluting to prepare a solution containing about 0.2mg of the intermediate I in every 1ml of methanol solution as a test solution.

System applicability solution: taking appropriate amount of intermediate I, impurity AL, impurity BL, impurity CL, impurity SQ, impurity YW, impurity BD, impurity LS and impurity QA, precisely weighing, dissolving with methanol, and diluting to obtain mixed solution containing about 0.02mg of intermediate I and each impurity in 1ml of methanol solution. The structure of each impurity is shown in table 1:

TABLE 1 Structure of related impurities for intermediate I

(2) Chromatographic conditions

The instrument comprises the following steps: high performance liquid chromatograph-ultraviolet detector

A chromatographic column: a chromatographic column using phenyl silane bonded silica gel as a filler; the particle size of the chromatographic column is 5 mu m; the length of the chromatographic column is 250 mm; the inner diameter of the chromatographic column is 4.6 mm.

Mobile phase A: a phosphate buffer solution with a pH value of 3.0-4.0 (preferably pH value of 3.2-3.8, most preferably 3.5); the phosphate buffer solution is a sodium dihydrogen phosphate or disodium hydrogen phosphate aqueous solution with the concentration of 5-15 mmol/L (preferably 8-12 mmol/L, most preferably 10mmol/L), and the pH value is adjusted by using phosphoric acid solution (more than or equal to 85%) or potassium hydroxide aqueous solution (1mol/L) with the concentration conventional in the field.

Mobile phase B: phosphoric acid acetonitrile solution, wherein the volume fraction of phosphoric acid in acetonitrile is 0.05-0.15%; preferably the volume fraction is between 0.08% and 0.12%, most preferably the volume fraction is 0.1%.

A preferred gradient elution procedure of the present invention is performed as follows:

flow rate: 1.0-1.5 ml/min, preferably 1.1-1.3 ml/min, most preferably 1.2 ml/min;

column temperature: 45 ℃ to 55 ℃, preferably 48 ℃ to 52 ℃, most preferably 50 ℃;

detection wavelength: 230 nm;

sample introduction volume: 10 μ l.

Preferred gradient elution procedure

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	87	13
3	87	13
			26	64	36
38	40	60
			39	87	13
45	87	13

(3) Calculation of impurities

And (4) calculating the content of each known impurity and each unknown impurity in the intermediate I according to an area normalization method at the impurity peak in the chromatogram of the test solution.

The second aspect of the invention provides a method for analyzing and determining the content of impurities related to an eltrombopag intermediate II, namely 2- (3, 4-dimethyl-phenyl) -5-methyl-2, 4-dihydro-pyrazol-3-one, by high performance liquid chromatography. Specifically, the above method of the present invention can be implemented as follows:

(1) sample preparation

Test solution: taking a proper amount of the product, adding methanol, ultrasonically dissolving, and diluting to prepare a solution containing about 0.5mg of the intermediate II in every 1ml of methanol solution, wherein the solution is used as a test solution.

System applicability solution: taking appropriate amount of intermediate II, impurity SM1, impurity FJ and impurity DH respectively, precisely weighing, dissolving with methanol, and diluting to obtain mixed solution containing intermediate I and each impurity 0.05mg respectively per 1ml methanol solution. The structure of each impurity is shown in table 2:

TABLE 2 Structure of related impurities for intermediate II

(2) Chromatographic conditions

A chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler; the grain diameter of the chromatographic column is 3.5 mu m; the length of the chromatographic column is 150 mm; the inner diameter of the chromatographic column is 4.6 mm.

Mobile phase A: a phosphate buffer solution having a pH of 2.5 to 3.5 (preferably 2.8 to 3.2, most preferably 3.0); the phosphate buffer solution is a sodium dihydrogen phosphate or disodium hydrogen phosphate aqueous solution with a concentration of 15-25 mmol/L (preferably 18-22 mmol/L, most preferably 20mmol/L), and the pH value is adjusted by using a phosphoric acid solution with a concentration (such as ≥ 85%) or a potassium hydroxide solution (such as 1mol/L) which is conventional in the art.

Mobile phase B: acetonitrile

flow rate: 0.8-1.2 ml/min, preferably 0.9-1.1 ml/min, most preferably 1.0 ml/min;

column temperature: 30 ℃ to 40 ℃, preferably 32 ℃ to 38 ℃, most preferably 35 ℃;

temperature of the sample chamber: 4 ℃ to 8 ℃, preferably 4 ℃ to 6 ℃, and most preferably 4 ℃;

detection wavelength: 243 nm;

sample introduction volume: 10 μ l.

Preferred gradient elution procedure

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	85	15
5	60	40
			22	35	65
23	20	80
			32	20	80
33	85	15
			40	85	15

(3) Calculation of impurities

And calculating the content of each known impurity and each unknown impurity in the intermediate II according to an area normalization method by using the impurity peak in the chromatogram of the test solution.

In summary, the analysis method claimed by the present invention has the following positive effects: the analysis method can effectively separate the eltrombopag intermediate and the related impurity system thereof, has the advantages of good specificity, high sensitivity, convenient operation, proper analysis time and the like, accurately monitors the impurity spectrum from the upstream of the synthesis route according to the concept that the quality is derived from the design (QBD), ensures that the subsequent impurity transfer rule and the process parameter optimization research are more convenient, and provides effective guarantee for the quality control of the eltrombopag final product.

In a third aspect, the present invention provides a novel impurity control compound, DH, and a method for preparing the impurity compound, DH, comprising the steps of:

(1) dissolving SM1, ethyl acetoacetate and sodium bisulfite in ethanol and purified water, stirring and heating to reflux, and reacting for 2-4 hours;

(2) cooling to 35-45 ℃, and stirring for 0.5-1 hour;

(3) cooling to 20-30 ℃, and carrying out heat preservation and crystallization for 4-5 hours;

(4) suction filtering, evaporating the filtrate to dryness, and separating by column chromatography to obtain compound DH;

wherein the mass ratio of SM1, ethyl acetoacetate and sodium bisulfite in the step (1) is 1:0.8: 1.2; the mass-to-volume ratio of SM1 to ethanol was 1:4, and the mass-to-volume ratio of SM1 to purified water was 1: 4.

Drawings

FIG. 1 System suitability solution profile of the assay method described in example 1;

FIG. 2 System suitability solution profile of the assay method described in example 2.

Detailed Description

The invention is further illustrated by the following specific examples. It should be understood that: the examples of the present invention are provided for illustration only and not for limitation of the present invention. The technical scheme obtained by simply improving the invention or equivalently replacing the conventional means or components on the basis of the technical scheme of the invention belongs to the protection scope of the invention.

The reagents or raw materials used in the present invention can be prepared by the existing literature or the existing technology, or can be purchased.

EXAMPLE 1 intermediate I method validation

1. Chromatographic conditions

The instrument comprises the following steps: waters2695-2489-2998 HPLC-UV detector-diode array detector

A chromatographic column: phenyl silane bonded silica gel [ Xbridge Phenyl (4.6X 250mm,5 μm) ] as filler

Mobile phase A: 10mmol/L sodium dihydrogen phosphate (pH adjusted to 3.5 with phosphoric acid)

Mobile phase B: 0.1% acetonitrile phosphate solution

Gradient elution procedure

Flow rate: 1.2 ml/min; column temperature: 50 ℃; detection wavelength: 230 nm; sample introduction volume: 10 μ l

2. Method verification

(1) System applicability

Impurity localization solution: taking appropriate amount of the intermediate I, the impurity AL, the impurity BL, the impurity CL, the impurity SQ, the impurity YW, the impurity BD, the impurity LS and the impurity QA, precisely weighing, respectively placing in different measuring bottles, dissolving with methanol and diluting to prepare a solution containing about 0.2mg of the intermediate I and each impurity in every 1ml of methanol solution.

System applicability solution: precisely measuring appropriate amount of the impurity positioning solution, placing in a measuring flask, and diluting with methanol to obtain mixed solution containing intermediate I and impurities 0.02mg per 1ml methanol solution.

The retention time and the separation degree of each impurity in the solution detected according to the chromatographic conditions of the embodiment of the invention, the impurity locating solution and the system applicability solution are shown in a table 3, and a system applicability map is shown in a figure 1. The results show that the separation degrees between each impurity and the main peak and between each impurity peak are both larger than 1.5, and the purity angles of the main peak and each impurity peak are both smaller than the purity threshold. Therefore, the system suitability is satisfactory.

TABLE 3 intermediate I System suitability results

(2) Limit of quantification

Determination of baseline noise: precisely measuring 10 mu l of methanol, injecting the methanol into a liquid chromatograph, continuously injecting a sample with 3 needles, and detecting the chromatographic condition according to the embodiment of the invention. And recording blank baseline noise in the intermediate I and the peak-off time range of each impurity, and calculating an average value.

Preparation of a quantitative limiting solution: taking a proper amount of the intermediate I, the impurity AL, the impurity BL, the impurity CL, the impurity SQ, the impurity YW, the impurity BD, the impurity LS and the impurity QA, precisely weighing, placing in the same measuring flask, dissolving by using methanol and gradually diluting, detecting the chromatographic conditions according to the embodiment of the invention until the intermediate I and each impurity peak height and the ratio of corresponding noise (namely signal-to-noise ratio, S/N) are about 10, wherein the ratio of the sample concentration to the measured concentration of a sample is the quantitative limit, continuously injecting the sample by using the concentration for 6 needles, and calculating the average value of the peak heights.

Test results show that under the analysis method, the quantitative limit of each impurity is below 0.05 percent, namely, each known impurity and each unknown impurity with the content of more than 0.05 percent in a sample can be detected and accurately quantified. Thus, the sensitivity of the method is satisfactory.

TABLE 4 quantitative Limit results for intermediate I and various impurities

Name (R)	Concentration (μ g/ml)	Average peak height (μ V)	Average noise (μ V)	Signal to noise ratio	Quantitative limit (%)
						Intermediate I	0.0209	131.0	6.4	10.6	0.01
Impurity QA	0.1004	451.3	4.1	14.1	0.05
						Impurity BD	0.0407	127.3	3.5	12.3	0.02
Impurity AL	0.0205	231.7	2.0	11.8	0.01
						Impurity LS	0.0208	294.3	0.6	11.9	0.01
Impurity SQ	0.0214	147.8	2.3	10.8	0.01
						Impurity YW	0.0212	95.2	3.7	11.0	0.01
Impurity BL	0.0207	172.5	0.9	12.6	0.01
						Impurity CL	0.0211	192.2	1.5	13.7	0.01

(3) Stability of solution

Test solution: taking a proper amount of the intermediate I, precisely weighing, dissolving with methanol and diluting to prepare a solution containing about 0.2mg of the intermediate I in each 1 ml.

The solution is placed at room temperature, 10 mu l of the solution is precisely measured and injected into a liquid chromatograph for 0 hour, 1 hour, 2 hours, 5 hours, 8 hours and 13 hours respectively, and the chromatographic condition is detected according to the embodiment of the invention. The rate of change of known impurities, unknown impurities and total impurities was calculated and the results are shown in table 5.

Test results show that the impurity YW and the total impurities of the intermediate I determination solution have a weak increasing trend under the room temperature condition, the change rate of the total impurities is 0.02% within 5 hours and is less than 0.05% of the generally acceptable neglect threshold of pharmacopoeia standards of various countries and ICH, so that the intermediate I solution is stable within 5 hours under the room temperature condition, namely the solution preparation should be performed within 5 hours for determination.

Table 5 stability results for intermediate i solution

Note: ND: not detected.

Example 2 intermediate II method validation

1. Chromatographic conditions

A chromatographic column: octadecylsilane bonded silica [ Xbridge Shield RP-18 (4.6X 150mm, 3.5 μm) ] was used as a filler

Mobile phase A: 20mmol/L sodium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid)

Mobile phase B: acetonitrile

Gradient elution procedure

Flow rate: 1.0 ml/min; column temperature: 35 ℃; temperature of the sample chamber: 4 ℃; detection wavelength: 243 nm; sample introduction volume: 10 μ l

2. Method verification

(1) System applicability

Impurity localization solution: taking a proper amount of the intermediate II, the impurity SM1, the impurity FJ and the impurity DH respectively, precisely weighing, respectively placing in different measuring bottles, dissolving with methanol and diluting to prepare a solution containing about 0.5mg of the intermediate II and each impurity in every 1ml of methanol solution.

System applicability solution: precisely measuring appropriate amount of the impurity locating solution, placing in a measuring flask, and diluting with methanol to obtain mixed solution containing intermediate II and impurities 0.05mg per 1ml methanol solution.

The retention time and the separation degree of each impurity in the solution subjected to chromatographic condition detection according to the embodiment of the invention, the impurity locating solution and the system applicability solution are shown in Table 6, and the system applicability map is shown in FIG. 2. The results show that the separation degrees between each impurity and the main peak and between each impurity peak are both larger than 1.5, and the purity angles of the main peak and each impurity peak are both smaller than the purity threshold. Therefore, the system suitability is satisfactory.

TABLE 6 intermediate II System suitability results

(2) Limit of quantification

Determination of baseline noise: precisely measuring 10 mu l of methanol, injecting the methanol into a liquid chromatograph, continuously injecting a sample with 3 needles, and detecting the chromatographic condition according to the embodiment of the invention. And recording blank baseline noise in the intermediate II and each impurity peak-off time range, and calculating an average value.

Preparation of a quantitative limiting solution: taking a proper amount of each of the intermediate II, the impurity SM1, the impurity FJ and the impurity DH, precisely weighing, placing in the same measuring bottle, dissolving by using methanol and gradually diluting, detecting according to the chromatographic condition of the embodiment of the invention until the intermediate I and each impurity peak height and the corresponding noise ratio (namely signal-to-noise ratio, S/N) are about 10, wherein the ratio of the sample concentration to the measured concentration of a sample is the quantitative limit, continuously injecting 6 needles according to the concentration, and calculating the average value of the peak heights.

Test results show that under the analysis method, the quantitative limit of each impurity is below 0.02 percent, namely, each known impurity and each unknown impurity with the content of more than 0.02 percent in a sample can be detected and accurately quantified. Thus, the sensitivity of the method is satisfactory.

TABLE 7 quantitation limit results for intermediate II and various impurities

Name (R)	Concentration (μ g/ml)	Average peak height (μ V)	Average noise (μ V)	Signal to noise ratio	Quantitative limit (%)
						Intermediate II	0.1048	148.0	14.7	10.1	0.02
Impurity SM1	0.1019	76.8	6.3	12.1	0.02
						Impurity FJ	0.0514	188.3	15.0	12.6	0.01
Impurity DH	0.0511	249.3	21.0	11.9	0.01

(3) Stability of solution

Test solution: taking a proper amount of the intermediate II, accurately weighing, dissolving with methanol and diluting to prepare a solution containing about 0.5mg of the intermediate I in each 1ml of methanol solution.

The solution is precisely measured and injected into a liquid chromatograph for 0 hour, 1 hour, 2 hours, 5 hours, 8 hours and 13 hours at the temperature of 4 ℃, and the chromatographic condition is detected according to the embodiment of the invention. The rate of change of known impurities, unknown impurities and total impurities was calculated and the results are shown in table 8.

Test results show that the intermediate II determination solution is placed at 4 ℃ for 12 hours, the change rate of total impurities is less than 0.02 percent and is lower than 0.05 percent of the generally acceptable neglect threshold of pharmacopoeia standards of various countries and ICH, and therefore, the intermediate II solution is stable within 12 hours at 4 ℃.

TABLE 8 stability test results of intermediate II solution at 4 deg.C

Time/h	SM1％	FJ％	DH％	Other single impurities%	Total impurities%	The change rate of total impurities%
							0	ND	ND	0.17	0.02	0.30	\
2	ND	ND	0.17	0.02	0.29	-0.01
							4	ND	ND	0.17	0.02	0.32	0.02
8	ND	ND	0.17	0.02	0.30	0.00
							12	ND	ND	0.17	0.02	0.31	0.01

Note: ND: not detected.

EXAMPLE 3 preparation of the impurity Compound DH

Dissolving 3.0g of compound SM1, 2.4g of compound ethyl acetoacetate and 3.6g of sodium bisulfite in 12mL of ethanol and 12mL of purified water, stirring, heating to reflux, and reacting for 3 hours; after the reaction is finished, cooling to 40 ℃, and stirring for 1 hour; cooling to 25 ℃, and carrying out heat preservation and crystallization for 5 hours; suction filtering, evaporating filtrate to dryness, and separating by column chromatography to obtain impurity DH 1.1 g.

ESI(+)：m/z 231.20；

[M+1]⁺；¹H NMR：(400MHz,DMSO-d6)δ＝7.40(d,1H),7.39(m,1H),7.13(d,1H),5.63 (s,1H)，4.10(d,2H),2.22(s,3H),2.19(s,3H),2.12(s,3H),1.31(m,3H)。

Claims

1. A method for analyzing and determining the content of related impurities of an Eltrombopag intermediate I by high performance liquid chromatography is disclosed, wherein the intermediate I is 2 '-hydroxy-3' -amino-biphenyl-3-carboxylic acid, and is characterized in that a chromatographic column using phenyl silane bonded silica gel as a filler takes phosphate buffer solution as a mobile phase A and acetonitrile phosphate solution as a mobile phase B, and a gradient elution mode is adopted, wherein:

the chromatographic column has a particle size of 5 μm, a length of 250mm and an inner diameter of 4.6mm,

the pH value of the phosphate buffer solution as the mobile phase A is 3.5, and the phosphate buffer solution is a sodium dihydrogen phosphate or disodium hydrogen phosphate aqueous solution with the concentration of 5-15 mmol/L; in the acetonitrile phosphate solution as the mobile phase B, the volume fraction of phosphoric acid in acetonitrile is 0.1 percent,

the elution gradient was:

time/min Mobile phase A/%) Mobile phase B/%) 0 87 13 3 87 13 26 64 36 38 40 60 39 87 13 45 87 13

The flow rate of the mobile phase is 1.2 ml/min; the column temperature of the chromatographic column is 50 ℃; the detection wavelength is 230 nm; the injection volume was 10. mu.l.

2. The method according to claim 1, wherein the phosphate buffer as the mobile phase A is an aqueous solution of sodium dihydrogen phosphate or disodium hydrogen phosphate having a concentration of 8 to 12 mmol/L.

3. The method according to claim 1, wherein the phosphate buffer as the mobile phase A is an aqueous solution of sodium dihydrogen phosphate or disodium hydrogen phosphate having a concentration of 10 mmol/L.