CN107305202A

CN107305202A - The HPLC methods and impurity that analysis methanesulfonic acid pleasure is cut down for Buddhist nun and its preparation impurity make the purposes of reference standard

Info

Publication number: CN107305202A
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: 2017-10-31
Anticipated expiration: 2036-04-22
Also published as: CN107305202B

Abstract

It is related to and pleasure is cut down for Buddhist nun's (carboxylic acid amide of 4 (3 chlorine 4 (N ' cyclopropylureidos) phenoxy group) 7 methoxy quinoline 6) or the method analyzed for the preparation of Buddhist nun is cut down containing pleasure, including passing through the impurity in chromatography determination sample, one or more of the impurity in compound A I and LVTN 1, methods described comprises the following steps：(1) methanesulfonic acid is found pleasure in cut down for Buddhist nun or find pleasure in cut down containing methanesulfonic acid and dissolved in a solvent for the medicine of Buddhist nun to prepare sample solution；(2) sample any one or more of in compound A~I and intermediate -1 (LVTN-1) is dissolved in a solvent to prepare reference standard solution or reference substance solution；(3) chromatographic technique is implemented to sample solution and reference standard solution；And (4) are by referring to compound A-I in the reference standard solution and intermediate -1 (LVTN-1) one or more, determine methanesulfonic acid pleasure and cut down the presence that any one or more of compound A~I and intermediate -1 (LVTN-1) in the medicine for Buddhist nun are cut down for Buddhist nun or containing methanesulfonic acid pleasure.

Description

Analysis methanesulfonic acid pleasure is cut down makees reference for the HPLC methods and impurity of Buddhist nun and its preparation impurity The purposes of standard

Technical field

The invention belongs to pharmaceutical synthesis field.In particular it relates to which pleasure is cut down for Buddhist nun (4- (the chloro- 4- of 3- (N '-cyclopropyl Urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides) impurity that occurs in preparation process and to being cut down containing pleasure for Buddhist nun's preparation The method analyzed.

Background technology

What Lenvatinib was developed and developed by Wei Cai companies, the Chinese translation of standard, therefore the applicant are there is no at present It is herein " pleasure is cut down for Buddhist nun " by its transliteration.The medicine obtained U.S. FDA approval in 2 months 2015, partly recurs or turns for having Move, progressivity, the treatment of the intractable patients with differentiated thyroid carcinoma of radioiodine, trade name LENVIMA.In March, 2015, pleasure is cut down Ratified to list by Japanese health ministry for Buddhist nun, as treatment irresectability thyroid cancer (including differentiated thyroid carcinoma, thyroid gland Cephaloma, undifferentiated type thyroid cancer) first molecular targeted therapy；In May, 2015, by European Food drug surveilance pipe The approval listing of reason office.LENVIMA is a kind of molecular targeted preparation of oral administration.The Chinese translation of standard is there is no at present, therefore Its transliteration is herein " methanesulfonic acid pleasure is cut down for Buddhist nun " by the applicant.

Pleasure is cut down is for the chemical name of Buddhist nun：4- (the chloro- 4- of 3- (N '-cyclopropylureido) phenoxy group) -7- methoxy quinolines -6- Carboxylic acid amide, the structural formula of its mesylate is：

Disclosed in patent CN200480036184.1 and happy cut down is carried out for Buddhist nun or happy cut down for the chemical purity of the different salt of Buddhist nun The HPLC methods of analysis.Happy cut down (is prepared not for Buddhist nun's crude product in this patent embodiment 2 according to the method for test example 3 of the patent Through ethyl alcohol recrystallization) to be analyzed, used high performance liquid chromatograph (HPLC) is Agilent 1260, PDA detectors (being purchased from Agilent company), as a result as shown in figure 1, the top in wherein Fig. 1 is the happy peak cut down for Buddhist nun.As shown in Figure 1, this is special There is following drawback in the analysis method of profit：

(1) it is happy to cut down for the impurity contained in Buddhist nun's crude product that (retention time is about under this method：5.063min, 5.180min and Baseline separation can not 5.273min) be realized each other, it is impossible to carry out accurate quantitative analysis；Separating degree between impurity does not meet ICH Verify that the requirement under item to specificity, i.e. separating degree between impurity are less than 1.5 for relevant substance method in Q3A；

(2) the clear and definite solvent for use of this method be water-methanol (v/v, 3: 1), the concentration of need testing solution is 0.1mg/ml, this The low need testing solution concentration of sample, certainly will cause under some specified wavelengths the weak defects inspecting of UV absorption less than causing impurity Underestimate., can not be completely molten using the solvent in patent when the concentration of need testing solution under the conditions of by this is improved to 0.5mg/ml Solution, plus after dimethyl sulfoxide (DMSO) (DMSO) hydrotropy, sample introduction is analyzed under this methodology, multiple spectral peaks are bifurcated peak, are specifically shown in Fig. 2.

(3) computational methods of this method impurity are area normalization method, because each impurity structure is different, in the detection drafted Under wavelength, UV absorption situation be it is different, therefore, area normalization method can not Accurate Determining go out each impurity in marketed products In real content.

(4) methanesulfonic acid is not found pleasure in cut down and carries out qualitative analysis for the specific impurities in Buddhist nun.

In summary, the testing result of prior art can not truly reflect the quality of medicine.

The content of the invention

The invention provides new, interchangeable method, cut down for pleasure for Buddhist nun or cut down relevant thing in the preparation for Buddhist nun containing happy The qualitative and quantitative analysis of matter, and the qualitatively and quantitatively analysis of impurity is carried out with these impurity as reference standard or reference substance. It is a further object to provide reference standard or reference substance, for detecting during happy cut down for Buddhist nun is prepared or preparation What is formed in technique and storage is referred to as A~I and LVTN-1 impurity.

Cut down provided by the present invention for analysis is happy for Buddhist nun or the happy HPLC methods cut down in the preparation for Buddhist nun about material.Invention People is prepared and identified that the impurity (claiming compound A~I and LVTN-1) of structure is used for pleasure and cuts down for Buddhist nun or cut down containing pleasure for Buddhist nun using 9 kinds Pharmaceutical preparation Related substance method reference standard or reference substance.Including disclosed six kinds in the prior art Impurity and structure and the three kinds of impurity and structure not yet disclosed in the prior art.

Therefore, analysis method of the invention is used for analysis of compounds A, and it has chemical name：4, the 4 '-(((double (ureas two of carbonyl Base)) double (chloro- 4, the 1- phenyl of 3-)) double (epoxides)) double (7- methoxy quinoline -6- formamides), and with following structure：

Second aspect provides compound B, and it has chemical name：4- (the chloro- 4- of 3- (3,3- dimethyl urea groups) phenoxy group)- 7- methoxy quinoline -6- carboxylic acid amides, and with following structure：

3rd aspect provides compound C, and it has chemical name：4- ethyoxyl -7- methoxy quinoline -6- formamides, and With following structure：

4th aspect provides compound D, and it has chemical name：4- hydroxyl -7- methoxy quinoline -6- carboxylic acid amides, and With following structure：

5th aspect provides compound E, and it has chemical name：1- (2- chloro-4-hydroxyls phenyl) -3- cyclopropylamino ureas, And with following structure：

6th aspect provides compound F, and it has chemical name：4- (the chloro- 4- of 3- (3- cyclopropylureidos) phenoxy group) -7- Methoxy quinoline -6- carboxylic acids, and with following structure：

7th aspect provides compound G, and it has chemical name：4- (4- amino -3- chlorophenoxies) -7- methoxyl group quinolines Quinoline -6- carboxylate methyl esters, and with following structure：

8th aspect provides compound H, and it has chemical name：4- (4- (3- cyclopropylureidos) phenoxy group) -7- methoxies Base QUINOLINE-6-CARBOXYLIC ACID's acid amides, and with following structure：

9th aspect provides compound I, and it has chemical name：4- (the chloro- 4- of 3- (3- methyl urea groups) phenoxy group) -7- first Phenoxyl quinoline -6- carboxylic acid amides, and with following structure：

Tenth aspect offer methanesulfonic acid pleasure is cut down and prepares intermediate -1 (LVTN-1) for Buddhist nun, and it has chemical name：4- (4- ammonia Base -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylic acid amides, and with following structure.

Compound A~I and intermediate -1 (LVTN-1) are that methanesulfonic acid pleasure is cut down for the intermediate or by-product in Buddhist nun's building-up process Thing or methanesulfonic acid pleasure are cut down for Buddhist nun and cut down containing methanesulfonic acid pleasure for the catabolite in Buddhist nun's preparation compositions storage process, it is adaptable to make Reference standard or reference substance.In a particularly preferred embodiment, according to the compound A~I and intermediate -1 of the present invention (LVTN-1) it is monomeric compound, most preferably pure form, preferably purity is greater than about 95%, preferably purity and is greater than about 98%th, most preferably purity is greater than about 99%, is preferably measured by HPLC.

A kind of detection methanesulfonic acid pleasure is provided according to the tenth aspect of the present invention to cut down for Buddhist nun or cut down for Buddhist nun containing methanesulfonic acid pleasure Pharmaceutical dosage form sample purity method, this method include determination sample according to the present invention compound A~I and centre One or more presence in body -1 (LVTN-1).In the method for the invention, the compound is used as reference standard or right According to product.

It is used to characterize compound A~I and intermediate -1 (LVTN-1) there is provided one kind according to the eleventh aspect of the present invention Method, this method analyzed using HPLC methods pleasure cut down for the impurity A~I and intermediate -1 (LVTN-1) in Buddhist nun.It is excellent It is the HPLC method compatible with LC-MS to select the HPLC methods.

On the other hand provide a kind of for detecting happy cut down for Buddhist nun or containing the happy chromatogram side for cutting down the formulation samples purity for Buddhist nun Method, methods described includes：By using the reference standard or reference substance according to the present invention, come in determination sample compound A~I and One or more presence in intermediate -1 (LVTN-1).Compound A~I and intermediate -1 (LVTN-1) are that methanesulfonic acid pleasure is cut down For the process contaminants in Buddhist nun's building-up process, accessory substance or degradation impurity, it is adaptable to as reference standard or reference substance, for this The quality control of product.

The way of production of above impurity is as follows：

Impurity A：This impurity is impurity in commercially available capsule, it should which finding pleasure in for bulk drug methanesulfonic acid, it is miscellaneous for the technique of Buddhist nun's introducing to cut down Matter, i.e. methanesulfonic acid pleasure cut down the impurity generated for Buddhist nun preparation technology second step.

Impurity B：The step of synthetic method the 3rd for Buddhist nun is cut down in the Chinese Patent Application No. CN101024627 methanesulfonic acid pleasures reported With in the reaction of cyclopropylamine, because the solvent used is DMF, being given birth to wherein a small amount of dimethylamine may be contained Into impurity B.

Impurity C：The methanesulfonic acid pleasure of Chinese Patent Application No. CN101024627 reports cut down the step of synthetic method the 4th for Buddhist nun into In reactant salt, under acid condition, intermediate -3 (LVTN-3, i.e. pleasure are cut down for Buddhist nun's free alkali) generates impurity C by etoh solvent attack.

Impurity D and impurity E：Cut down in the Chinese Patent Application No. CN101024627 methanesulfonic acid pleasures reported for the synthesis side of Buddhist nun The step of method the 4th is into the reaction of salt, and in acid condition, happy cut down is decomposed for Buddhist nun's free alkali (LVTN-3), generation impurity D and Impurity E, the two is process contaminants, is also degradation impurity.

Impurity F (LVTN-ZZ-10)：Methanesulfonic acid pleasure is cut down to contain amide group in Buddhist nun, can be with water under conditions of acid or alkali Solve as carboxylic acid, be degradation impurity.

Impurity G：Methanesulfonic acid pleasure cuts down the generation degradation impurity G that degraded for Buddhist nun under acid, alkali, wet heat condition.

Acid degradation mechanism：

Impurity H：In the methanesulfonic acid pleasure that Chinese Patent Application No. CN101024627 is reported cuts down the synthetic method for Buddhist nun, rise The dehalogenation impurity subsequent reactions of beginning material 2 are generated.

Impurity I：The 3rd in the methanesulfonic acid pleasure that Chinese Patent Application No. CN101024627 is reported cuts down the synthetic method for Buddhist nun In step reaction, solvent N-methyl pyrilidone competes with cyclopropylamine formation with that may contain a small amount of methylamine in cyclopropylamine, participates in taking Generation reaction.Mechanism of production is as follows：

Intermediate -1 (LVTN-1)：The conjunction for Buddhist nun is cut down in the Chinese Patent Application No. CN101024627 methanesulfonic acid pleasures reported The first step intermediate into method, is also that methanesulfonic acid pleasure is cut down for the degradation impurity in Buddhist nun's storage or subsequent process steps.It is produced Mechanism is as follows：

(1) second in the methanesulfonic acid pleasure that Chinese Patent Application No. CN101024627 is reported cuts down the synthetic method for Buddhist nun Step, LVTN-1 is excessive, remaining.

(2) the 3rd step of the synthetic method for Buddhist nun is cut down in the Chinese Patent Application No. CN101024627 methanesulfonic acid pleasures reported During into salt, LVTN-3 meets acid and temperature action degraded.

Acid degradation mechanism：

Impurity D, E, F, G, LVTN-1 are that methanesulfonic acid pleasure is cut down for Buddhist nun or cut down comprising methanesulfonic acid pleasure in the pharmaceutical preparation for Buddhist nun Principal degradation impurity.

It is monomer chemical combination according to the compound A~I and LVTN-1 of the present invention in a particularly preferred embodiment Thing, most preferably pure form, preferably purity is greater than about 95%, preferably purity and is greater than about 98%, most preferably purity and is greater than about 99%, preferably measured by HPLC.

A kind of detection pleasure is provided according to the tenth aspect of the present invention to cut down for Buddhist nun or containing the happy pharmaceutical dosage form cut down for Buddhist nun The method of sample purity, this method is included in determination sample containing one kind or many in compound A of the invention~I and LVTN-1 Kind.In the method for the invention, the compound is used as the reference standard or reference substance of impurity.

There is provided a kind of method for characterizing compound A~I and LVTN-1, the party according to the tenth aspect of the present invention Method is analyzed pleasure using HPLC methods and cut down for the impurity A~I and LVTN-1 in Buddhist nun.It is preferred that the HPLC methods are and LC-MS Compatible HPLC methods.

It thus provides according to the compound A~I and LVTN-1 (one or more) of the present invention detection find pleasure in cut down for Buddhist nun or Contain the happy purposes cut down in the sample purity for the pharmaceutical preparation of Buddhist nun as reference standard or reference substance.

On the other hand, the present invention also provides a kind of for detecting that pleasure is cut down for the chromatographic process of Buddhist nun's sample purity, methods described Including：By using the reference standard or reference substance according to the present invention, come in determination sample in compound A~I and LVTN-1 One or more presence.

Another aspect is appointed by determining there is provided a kind of containing happy cut down in the sample for Buddhist nun in compound A~I and LVTN-1 What one or more presence cuts down the chromatographic process of the purity for Buddhist nun's sample to detect that methanesulfonic acid is found pleasure in, and methods described includes：

(1) pleasure is cut down for Buddhist nun or dissolved in a solvent for the formulation samples of Buddhist nun to prepare sample solution containing happy cut down；

(2) sample any one or more of in compound A~I and LVTN-1 is dissolved in a solvent to prepare reference mark Quasi- solution or reference substance solution；

(3) chromatographic technique is implemented to sample solution and reference standard solution；And

(4) by referring to known compound A-I and LVTN-1 (one or more) present in the reference standard solution, survey Any one or more of compound A~I presence in random sample product.

In one embodiment, the chromatographic process is liquid chromatography, such as HPLC, UPLC, LC-MS；It is preferred that the chromatogram Method is HPLC methods, preferred gradient HPLC methods.

Present invention preferably uses stationary phase to be anti-phase.Suitable stationary phase includes octadecylsilane chemically bonded silica or pungent Base silane bonded silica gel.

In a preferred embodiment of the invention there is provided a kind of gradient HPLC methods, wherein, mobile phase includes molten containing buffering The combination of liquid (A) and organic solvent (B).It is preferred that cushioning liquid (A) is water-containing buffering liquid, preferably acetate, formates, phosphoric acid Salt, trifluoracetic acid, the aqueous solution of formic acid or their mixture.Preferred cushioning liquid (A) is acetate, most preferably vinegar The aqueous solution of sour ammonium and acetic acid or ammonium acetate adjusted the pH aqueous solution with acetic acid.Ammonium acetate in particularly preferred embodiments The concentration of presence is about 0.001M to 1.0M, most preferably preferably 0.02M to 0.08M, more preferably 0.02M to 0.05M, 0.03M.It is excellent It is polar aprotic solvent, such as methanol, ethanol or isopropanol to select organic solvent (B)；Or dipolar aprotic solvent, such as acetonitrile.It is preferred that Organic solvent (B) in the group including methanol, acetonitrile, ethanol, isopropanol or their mixture, preferably methanol and The combination of acetonitrile, wherein in terms of mobile phase A and B percent of total, the percentage of methanol is 25 ± 5%.

The combination of the aqueous solution (A) and acetonitrile (B) of the mobile phase specifically preferred according to the invention comprising ammonium acetate and acetic acid.

The gradient HPLC methods according to the present invention are further provided for, wherein mobile phase includes following gradient design：

Time (minute)	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 further preferred embodiment, the aqueous solution of cushioning liquid (A) ammonium acetate and acetic acid in HPLC methods PH is about 3.5~6.5, preferably from about 4.0~6.5,4.0~6.0,4.0~5.5, most preferably 4.5~5.5.

In other embodiments, the HPLC analysis methods are carried out at a temperature of about 20~40 DEG C, and preferably 20~35 DEG C, most preferably 25~35 DEG C.

In other embodiments, the analysis method is carried out under about 0.3~1.0ml/min flow velocity, preferably 0.3~ 0.9ml/min, 0.3~0.8ml/min, 0.3~0.7ml/min, 0.3~0.6ml/min, most preferably 0.3~0.5ml/ min。

Effectively detected with single operation according to the HPLC methods of the present invention and quantitatively include those and be selected from following compound In compound all impurity：

Compound A：4,4 '-(((carbonyl is double (urea diyl)) double (chloro- 4, the 1- phenyl of 3-)) double (epoxides)) double (7- methoxyl groups Quinoline -6- formamides).

Compound B：4- (the chloro- 4- of 3- (3,3- dimethyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides.

Compound C：4- ethyoxyl -7- methoxy quinoline -6- formamides.

Compound D：4- hydroxyl -7- methoxy quinoline -6- carboxylic acid amides.

Compound E：1- (2- chloro-4-hydroxyls phenyl) -3- cyclopropylamino ureas.

Compound F：4- (the chloro- 4- of 3- (3- cyclopropylureidos) phenoxy group) -7- methoxy quinoline -6- carboxylic acids.

Compound G：4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylate methyl esters.

Compound H：4- (4- (3- cyclopropylureidos) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides.

Compound I：4- (the chloro- 4- of 3- (3- methyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides.

LVTN-1：4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylic acid amides

The present invention can be used for analysis to be cut down as the pleasure of active pharmaceutical ingredient (API) for the pleasure in Buddhist nun and/or pharmaceutical composition Cut down for Buddhist nun.The pharmaceutical composition that the present invention can be analyzed includes solid or fluid composition, and alternatively includes one or more Pharmaceutically acceptable excipient.The composition of solid form includes pulvis, tablet, capsule, pill and dispersible Granula etc..Fluid composition includes solution or suspension, and it can be administered by oral, injection or instillation approach.Specification and power Used term " pleasure is cut down for Buddhist nun " refers to 4- (the chloro- 4- of 3- (N '-cyclopropylureido) phenoxy group) -7- methoxies to sharp claim in the whole text Base QUINOLINE-6-CARBOXYLIC ACID acid amides or its solvate (such as hydrate) and/or its salt, including mesylate.Specification is made in the whole text Term " impurity " or " about material " can refer to the impurity formed in manufacture API or pharmaceutical composition, and/or API degrades the impurity to be formed or the impurity formed in pharmaceutical composition or preparation in storage.

As described above, the HPLC methods reported in the prior art, are not suitable for analysis pleasure and cut down for Buddhist nun and cut down containing pleasure For the preparation or pharmaceutical composition of Buddhist nun.

However, the method for the present invention solves this problem, and effectively detected with single operation and it is quantitative, qualitative point Analyse all impurity formed in this specific building-up process or preparation preparation.Advantage of the invention is that：Pleasure is disclosed to cut down for Buddhist nun Cut down for the concrete structure of the impurity in Buddhist nun's preparation, and simultaneously wash polar impurity and non polar impurities with pleasure with gradient HPLC method Take off, and carry out qualitative and quantitative analysis.

One or more deposited the invention is particularly suited to determine and quantify in sample in compound A~I and LVTN-1 .Unless otherwise described, otherwise term " impurity " herein and " compound " related to compound A~I and LVTN-1 , can be with used interchangeably under scope.

It is also an advantage of the invention that this method is cut down for Buddhist nun and containing the relevant material in the happy preparation cut down for Buddhist nun for happy Analysis have specificity strong, the degree of accuracy, the features such as precision is high, durability is good.In addition, the present invention has height sensitivity, And allow to detect and quantitatively happy cut down that be substantially less than for level amount in Buddhist nun API or pharmaceutical composition can specified in ICH guidelines Receive the relevant material of limit value.

In addition, the method for the present invention can be used for detection and quantitative pleasure to cut down for shape during Buddhist nun's sample or pharmaceutical composition storage Into all degradation impurities.This method is determined by carrying out forced degradation research according to ICH Q1A guides, and according to ICH Q2A guides are verified that the checking covers following items：It is specificity, linearity and range, precision, the degree of accuracy, test limit, fixed Amount limit, durability and system suitability.

Nine kinds of impurity A~I and LVTN-1 of novel gradient HPLC methods qualitative determination that the present inventor develops.The side Method can be analyzed disposably to be cut down in Buddhist nun preparation technology and commercially available pleasure is cut down for Buddhist nun and stored according to pleasure manufactured in the present embodiment The impurity that the degradation impurity isopolarity produced in journey differs greatly, therefore, inventors believe that gradient design is the most suitable.

In the embodiment of this invention, the inventors found that containing octadecylsilane chemically bonded silica or octyl group silane key The stationary phase for closing silica gel is the most favourable.Particularly preferred stationary phase contains Waters XBridge BEH Shield RP18 4.6 × 150mm, 2.5 μm or Agilent Poroshell HPH-C184.6 × 150mm, 2.7 μm.

The inventive method preferably includes gradient design so that mobile phase A and B relative concentration are in 10~60 minutes allusion quotations Become type and turn to 100%A：0%B to 0%A：100%B gradient.Preferably, by 30 to 55 minutes, gradient is 95% A：5%B to 5%A：95%B, it is highly preferred that by 20 to 50 minutes, gradient is 90%A：10%B to 15%A：85% B, most preferably, by about 30 minutes, gradient was 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 advantage of this gradient method exists In, can by it is happy cut down cut down for Buddhist nun API or happy it is complete for the very close impurity of various opposed polarities or polarity in Buddhist nun's pharmaceutical composition Part is left, and is easy to accurately qualitative and quantitative.

The mobile phase used is preferably selected from the group of one or more cushioning liquid (A) and one or more organic solvents (B) Close.

Cushioning liquid (A) be preferably selected from including phosphate, acetate, formates, trifluoracetic acid, formic acid or acetic acid they The aqueous solution combination of mixture.

The concentration of cushioning liquid (A) can be 0.001M to 1.0M, and preferred concentration is 0.02M to 0.08M, more preferably concentration For 0.02M to 0.05M, most preferably 0.03M.The aqueous solution of the particularly preferred mobile phase comprising ammonium acetate and acetic acid, or ammonium acetate The combination of the solution aqueous solution (A) and acetonitrile (B) of vinegar acid for adjusting pH.

In the particularly preferred embodiment according to the present invention, it is further provided a kind of gradient HPLC method, wherein Mobile phase includes following gradient design：

The particularly preferred HPLC gradient methods that the present invention is also provided, wherein, mobile phase is made comprising ammonium acetate and/or acetic acid For cushioning liquid (A).In other particularly preferred embodiment, mobile phase includes acetonitrile as organic solvent (B), and/or Acetonitrile-methanol mixed solvent is used as organic solvent (B).Inventor is had found when mobile phase includes ammonium acetate and/or acetic acid (A) and second Gradient design is particularly effective during nitrile (B).

Cushioning liquid (A) can be containing one or more of added solvents, and these added solvents can be methanol, ethanol, isopropyl Alcohol, acetonitrile or their mixture are used as organic solvent.Added solvent in cushioning liquid (A) may or may not be with it is organic molten Agent (B) identical solvent.Added solvent in cushioning liquid (A) is preferably acetonitrile.

The pH of cushioning liquid (A) is about 3.5~6.5, preferably from about 4.0~6.5,4.0~6.0,4.0~5.5, most preferably For 4.5~5.5.

The HPLC methods of the present invention are carried out at a temperature of about 20~40 DEG C, preferably 20~35 DEG C, most preferably 25~ 35℃。

The analysis method is carried out under about 0.3~1.0ml/min flow velocity, preferably 0.3~0.9ml/min, 0.3~ 0.8ml/min, 0.3~0.7ml/min, 0.3~0.6ml/min, most preferably 0.3~0.5ml/min.

Another aspect of the present invention provides a kind of reference standard solution.The solution includes and is dissolved in suitable solvent (such as second Nitrile) in one or more compound A~I and LVTN-1.The reference standard solution can be used for determining using according to this hair Any compound A~I as impurity and LVTN-1 presence in the sample that bright chromatographic technique is analyzed.

According to another aspect of the present invention there is provided a kind of reference standard solution, it is known that one or more chemical combination of amount Thing A~I and LVTN-1 are dissolved in suitable solvent (such as acetonitrile or dimethyl sulfoxide (DMSO) or the mixture of the two).The reference mark Quasi- solution can be used for determining any chemical combination in using the sample analyzed according to the chromatographic technique of the present invention as impurity Thing A~I's and LVTN-1 is qualitative and quantitative.The method of the analysis is for the important of technical staff and is conveniently apparent 's.

The method that the present inventor has verified the present invention extensively, the result shows the strong specificity of this method, the degree of accuracy, essence Density and sensitivity are high, and durability is good.

Brief description of the drawings：

Fig. 1 is to carry out HPLC analysis collection of illustrative plates to happy cut down for Buddhist nun according to method disclosed in CN200480036184.1 test examples 3；

Fig. 2：For according to method disclosed in CN200480036184.1 test examples 3, increasing pleasure is cut down dense for Buddhist nun's need testing solution HPLC analysis collection of illustrative plates is carried out after degree；

Fig. 3：Intermediate -1 (LVTN-1)：4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylic acid amides¹H-NMR；

Fig. 4：Intermediate -1 (LVTN-1)：4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylic acid amides High resolution mass spectrum (ESI-HRMS)；

Fig. 5：Methanesulfonic acid pleasure is cut down for Buddhist nun：4- (the chloro- 4- of 3- (N '-cyclopropylureido) phenoxy group) -7- methoxy quinoline -6- carboxylics Sour amide mesylate¹H-NMR；

Fig. 6：4- (the chloro- 4- of 3- (N '-cyclopropylureido) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amide mesylates High resolution mass spectrum；

Fig. 7：Impurity A：4,4 '-(((carbonyl is double (urea diyl)) double (chloro- 4, the 1- phenyl of 3-)) double (epoxides)) double (7- methoxies Base quinoline -6- formamides)¹H-NMR；

Fig. 8：Impurity A：4,4 '-(((carbonyl is double (urea diyl)) double (chloro- 4, the 1- phenyl of 3-)) double (epoxides)) double (7- methoxies Base quinoline -6- formamides) high resolution mass spectrum；

Fig. 9：Impurity B：4- (the chloro- 4- of 3- (3,3- dimethyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides 's¹H-NMR；

Figure 10：Impurity B：4- (the chloro- 4- of 3- (3,3- dimethyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides High resolution mass spectrum；

Figure 11：Impurity C：4- ethyoxyl -7- methoxy quinoline -6- formamides¹H-NMR；

Figure 12：Impurity C：The high resolution mass spectrum of 4- ethyoxyl -7- methoxy quinoline -6- formamides；

Figure 13：Impurity D：4- hydroxyl -7- methoxy quinoline -6- carboxylic acid amides¹H-NMR；

Figure 14：Impurity D：The high resolution mass spectrum of 4- hydroxyl -7- methoxy quinoline -6- carboxylic acid amides；

Figure 15：Impurity E：1- (2- chloro-4-hydroxyls phenyl) -3- cyclopropylamino ureas¹H-NMR；

Figure 16：Impurity E：The high resolution mass spectrum of 1- (2- chloro-4-hydroxyls phenyl) -3- cyclopropylamino ureas；

Figure 17：Impurity F：4- (the chloro- 4- of 3- (3- cyclopropylureidos) phenoxy group) -7- methoxy quinoline -6- carboxylic acids¹H- NMR；

Figure 18：Impurity F：The high score of 4- (the chloro- 4- of 3- (3- cyclopropylureidos) phenoxy group) -7- methoxy quinoline -6- carboxylic acids Distinguish mass spectrum；

Figure 19：Impurity G：4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylate methyl esters¹H-NMR；

Figure 20：Impurity G：The high resolution mass spectrum of 4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylate methyl esters；

Figure 21：Impurity H：4- (4- (3- cyclopropylureidos) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides¹H- NMR；

Figure 22：Impurity H：The high-resolution of 4- (4- (3- cyclopropylureidos) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides Mass spectrum；

Figure 23：Impurity I：4- (the chloro- 4- of 3- (3- methyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides¹H- NMR。

Figure 24：Impurity I：The height of 4- (the chloro- 4- of 3- (3- methyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides Resolution Mass Spectrometry；

Figure 25：The HPLC spectrograms of test sample mark-on mixed solution；

Figure 26：Methanesulfonic acid pleasure prepared by patent Example 3 of the present invention cuts down the HPLC spectrograms for Buddhist nun's need testing solution；

Figure 27：Homemade methanesulfonic acid pleasure is cut down for the HPLC spectrograms of Buddhist nun's capsule -10mg need testing solutions；

Figure 28：Commercially available methanesulfonic acid pleasure is cut down for the HPLC spectrograms of Buddhist nun's capsule -10mg need testing solutions；

Embodiment

Although its embodiment is described by the present invention, but some modifications and equivalent are for this area skill Art personnel are it will be apparent that and being intended to be included within the scope of the present invention.

The present invention is illustrated by following examples, following examples do not limit the present invention in any way.

Embodiment 1：The synthesis of 4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylic acid amides (LVTN-1).

Under nitrogen protection, into 500mL there-necked flask, 200mL dimethyl sulfoxide (DMSO)s are added, stirring is opened, sequentially adds The chloro- 7- methoxy quinolines -6- carboxylic acid amides (SM1) of 20.00g 4-, 18.20g 4- amino -3- chlorophenols (SM2) and 14.22g Potassium tert-butoxide.Charging is finished, and is warming up to 65 DEG C, insulated and stirred 19 hours.Above-mentioned reaction system is poured into pure water, there is big Measure solid to separate out, filtering, the pure water wash of filter cake, under the conditions of 60 DEG C, forced air drying 1~2 hour obtains brown solid 25.07g 4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylic acid amides (LVTN-1), yield：86.32%.In Mesosome -1¹H-NMR and high resolution mass spectrum are respectively referring to Fig. 3 and Fig. 4.

¹H-NMR (400Mz, DMSO) δ：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 spectrograms are shown Molecular ion peak m/z=344.08046 [M+H]⁺, corresponding molecular weight and the structural formula calculated value of offer (344.07237) it is consistent.Absolute error is 2.38ppm, within high resolution mass spectrum error range.

Embodiment 2：4- (the chloro- 4- of 3- (N '-cyclopropylureido) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides (LVTN-3) the happy synthesis cut down for Buddhist nun

250mL 1-METHYLPYRROLIDONEs are added into 500mL there-necked flasks, stirring is opened, sequentially adds 11.50g pyridines With 25.00g 4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylic acid amides (LVTN-1).Under ice-water bath, Xiang Ti It is added dropwise in system after 4.94g phenyl chloroformates, completion of dropping, system is warming up to room temperature.Pure water is added dropwise into reaction system, drips 1~2 hour between added-time, filter, elution, forced air drying 2 hours under the conditions of 60 DEG C obtain 33.74g (4- ((6- carboxylic acid formyls Amido -7- methoxy quinoline -4- bases) oxygen) -2- chlorphenyls) phenyl carbamate (LVTN-2) brown ceramic powder.Yield： 81.26%.

300mL 1-METHYLPYRROLIDONEs are added into 1000mL there-necked flasks, stirring is opened, adds 30.00g (4- ((6- acid methylamide base -7- methoxy quinoline -4- bases) oxygen) -2- chlorphenyls) phenyl carbamate (LVTN-2).At room temperature, 4.43g cyclopropylamines, time for adding 1 hour are added dropwise into system.After completion of dropping, continue insulated and stirred and stay overnight.To reaction system Middle addition 300mL pure water, stirring continues to stir 1 hour, filtered to there is a large amount of solids to separate out (about 1~2 hour), elution, in Forced air drying 12 hours under the conditions of 60 DEG C, obtain 27.50g crude products, and 21.08g off-white powder 4- (3- are obtained through ethyl alcohol recrystallization Chloro- 4- (N '-cyclopropylureido) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides (LVTN-3), yield：76.35%

Embodiment 3：Methanesulfonic acid pleasure cuts down the preparation for Buddhist nun, 4- (the chloro- 4- of 3- (N '-cyclopropylureido) phenoxy group) -7- methoxies The preparation of base QUINOLINE-6-CARBOXYLIC ACID's amide mesylate and methanesulfonic acid pleasure are cut down for the preparation of Buddhist nun's capsule

Under nitrogen protection, 100mL acetic acid and 2.70g methanesulfonic acids, 40 DEG C of conditions of temperature control are sequentially added into 250mL there-necked flasks Under, then into system 10.00g LVTN-3 are added, stir 1~2 hour, mother liquor is collected in filtering, by tri- mouthfuls of mother liquor transferase 45 00mL In bottle, under nitrogen protection, 170mL normal propyl alcohols, control time for adding 1~2 hour are added dropwise into system for 40 DEG C of temperature control.Filtering, filter Cake 70mL normal propyl alcohols, elution, obtain methanesulfonic acid pleasure and cut down acetic acid compound for Buddhist nun.Under nitrogen protection, add into 250mL there-necked flasks Enter 100mL ethanol, open under the conditions of stirring, 40 DEG C of temperature control, add methanesulfonic acid pleasure and cut down for Buddhist nun's acetic acid compound, it is small that continuation stirs 36 When, filtering, filter cake ethanol rinse, under the conditions of 60 DEG C, forced air drying 12 hours obtains methanesulfonic acid pleasure and cut down for Buddhist nun's finished product 10.23g, is off-white powder, yield：83.49%.

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), 2.572~2.578 (m, 1H), 2.401 (s, 1H), 0.651~0.665 (m, 2H), 0.429 (m, 2H). ESI-HRMS spectrograms show molecular ion peak m/z=427.11803 [M+H]⁺, corresponding molecular weight and the structural formula provided are managed It is consistent by calculated value (427.10948).Absolute error is 2.98ppm, within high resolution mass spectrum error range.Methanesulfonic acid pleasure is cut down For Buddhist nun¹H-NMR spectrum and high resolution mass spectrum (ESI-HRMS) figure are shown in Fig. 5~6.

The methanesulfonic acid pleasure for weighing following recipe quantity is cut down for Buddhist nun, PEARLITOL 25C, precipitated calcium carbonate, hydroxypropyl cellulose, low taken For hydroxypropyl cellulose and microcrystalline cellulose PH101, put in high-speed stirred mixer-granulator and mix, plus Purified Water q. s system is soft Material, crosses the granulation of 18 mesh sieves, and fluidized bed drying to moisture is less than 2.0%, puts dry particl Fastgranulatemachine whole grain, sieve mesh number Particle adds microcrystalline cellulose PH102, talcum powder mixing after 14 mesh, whole grain, fills No. 4 HPMC capsules, thus Methanesulfonic acid pleasure is prepared to cut down for Buddhist nun's capsule.

Composition	Consumption (mg/)
		Methanesulfonic acid pleasure is cut down for Buddhist nun	12.5
Winnofil	33
		PEARLITOL 25C	8.5
Microcrystalline cellulose PH101	10
		Low-substituted hydroxypropyl cellulose	25
HPC-L	3
		Microcrystalline cellulose PH102	5
Talcum powder	3

Embodiment 4：The synthesis of impurity A

Impurity A, 4,4 '-(((carbonyl is double (urea diyl)) double (chloro- 4, the 1- phenyl of 3-)) double (epoxides)) double (7- methoxyl group quinolines Quinoline -6- formamides) synthesis

20ml 1-METHYLPYRROLIDONEs are added into 100mL there-necked flask, 1.00g LVTN-1 are sequentially added under stirring, 0.92g pyridines, nitrogen protection, are cooled to 0~10 DEG C, start that 1.46g phenyl chloroformates are added dropwise, drop finishes, continue to stir 20~30 Minute, 60 DEG C are warming up to, is stirred overnight；Sample TLC detections (methanol: dichloromethane=1: 10) raw material disappears.Add water 20ml, has A large amount of solids are separated out, and continue to stir 30 minutes.Filtering, is drained, 40 DEG C of forced air dryings 30 minutes, is obtained solid crude product, will be obtained Crude product carries out column chromatography purifying, and elution ratio is：Methanol: dichloromethane=1: 50, the collection common 160mL of eluent, temperature control 30~ 40 DEG C, vacuum：- 0.08MPa, vacuum distillation is evaporated off solvent to being steamed without cut, obtains faint yellow solid 105mg, yield： 5.05%, that is, obtain impurity A：4,4 '-(((carbonyl is double (urea diyl)) double (chloro- 4, the 1- phenyl of 3-)) double (epoxides)) double (7- first Phenoxyl quinoline -6- formamides).

The structural identification of impurity A is as follows：

The impurity A of table 1¹H-NMR and¹³C-NMR test datas (DMSO-d₆) and ownership

The ESI-HRMS spectrograms of impurity A show molecular ion peak [M+H]⁺Mass-to-charge ratio be 713.13146, corresponding point Son amount is consistent with the structural formula calculated value (713.12400) provided.Absolute error is 0.26ppm, in defined error model Within enclosing.

Impurity A¹H NMR and ESI-HRMS spectrogram are shown in accompanying drawing 7-8 respectively.

Embodiment 5：The synthesis of impurity B

The conjunction of impurity B, i.e. 4- (the chloro- 4- of 3- (3,3- dimethyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides Into.

463.87mg methanesulfonic acids pleasure is added into 50ml there-necked flask to cut down for Buddhist nun's intermediate LVTN-2 and 9mLN- methylpyrrole Alkanone.Controlling reaction temperature adds 99.18mg dimethylamine into reaction system at 0~10 DEG C, continues to stir 30 minutes, TLC prisons Survey (dichloromethane: methanol=10: 1), reaction terminates.80% acetone/water (V/V) mixed solvent is added into reaction system 18mL, has a large amount of solids to separate out, after stirring 30 minutes, and filtering, 60 DEG C of forced air dryings obtain impurity B crude product.The crude product is carried out Column chromatography is purified, and solvent and ratio are：Methanol: dichloromethane=1: 20 (V/V), the collection common 150mL of eluent, temperature control 30~ 40 DEG C, vacuum：- 0.08MPa, vacuum distillation, to being steamed without cut, obtains pale pink solid 153.04mg, yield： 36.89%, that is, obtain impurity B：4- (the chloro- 4- of 3- (3,3- dimethyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acyloxies Amine.

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 is composed Figure shows molecular ion peak m/z=415.11742 [M+H]⁺, corresponding molecular weight and the structural formula calculated value of offer (414.84226) it is consistent.Absolute error is 1.60ppm, within high resolution mass spectrum error range.

Impurity B¹H NMR and ESI-HRMS spectrogram are shown in accompanying drawing 9-10 respectively.

Embodiment 6：Impurity C synthesis

Impurity C, i.e. 4- ethyoxyls -7- methoxy quinolines -6- formamides synthesis

2.00g methanesulfonic acids pleasure is added into 100ml there-necked flask to cut down for Buddhist nun, 50mL ethanol.Return stirring 72 hours, TLC Monitoring (methanol: dichloromethane=1: 10, there is new point to produce).Remove solvent under reduced pressure, crude product will be obtained and carry out column chromatography purifying, washed De- ratio is：Methanol: dichloromethane=1: 20, collect the common 60mL of eluent, 30~40 DEG C of temperature control, vacuum：- 0.08MPa, subtracts Pressure distillation, is evaporated off solvent to being steamed without cut, obtains pale pink solid 122mg, yield：6.10%, that is, obtain impurity C：4- ethoxies Base -7- methoxy quinoline -6- formamides.

Impurity C structural identification see the table below：

The impurity C's of table 2¹H-NMR and¹³C-NMR test datas (DMSO-d₆) and ownership

Impurity C ESI-HRMS spectrograms show molecular ion peak [M+H]⁺Mass-to-charge ratio be 247.10872, [2M+Na+H]⁺ Mass-to-charge ratio be 515.19080, corresponding molecular weight is consistent with the structural formula calculated value (247.10044) of offer.Absolutely It is 4.08ppm to error, within defined error range.

Impurity C's¹H NMR and ESI-HRMS spectrogram are shown in accompanying drawing 11-12 respectively.

Embodiment 7：Impurity D synthesis

10.00g LVTN-3,50ml acetonitriles, 80 DEG C of temperature control, stirring, plus 6% hydrogen peroxide are added into 250ml there-necked flasks 3ml, continues to heat 15 minutes, is quenched, concentrate.The crude product is subjected to column chromatography purifying, elution ratio is：Methanol: dichloromethane =1: 10, collect the common 720mL of eluent, 30~40 DEG C of temperature control, vacuum：- 0.08MPa, vacuum distillation is evaporated off solvent to nothing and evaporated Divide and steam, solvent is evaporated off to being steamed without cut in vacuum distillation, obtains compound D1.12g, and compound D is yellow powder, and HPLC is pure Degree：99.19%.

Impurity D's¹H NMR and ESI-HRMS spectrogram are shown in accompanying drawing 13-14 respectively.

Embodiment 8：Be purchased from outside impurity E Nanjing very can medication chemistry Co., Ltd, HPLC purity be 99.06%.

Impurity E¹H NMR and ESI-HRMS spectrogram are shown in accompanying drawing 15-16 respectively.

Embodiment 9：The synthesis of impurity F

LVTN-3 about 2.00g, plus acetonitrile 20ml, 2mol/L hydrochloric acid solution are added into 100ml there-necked flasks, ultrasound makes to disperse Uniformly, in 80 DEG C of heating water baths 30 minutes, concentration.The crude product is subjected to column chromatography purifying, elution ratio is：Methanol: dichloromethane Alkane=1: 10, collect the common 620mL of eluent, 30~40 DEG C of temperature control, vacuum：Solvent is evaporated off to nothing in -0.08MPa, vacuum distillation Cut is steamed, vacuum distillation, and solvent is evaporated off to being steamed without cut, pale yellow powder 0.66g, HPLC purity is obtained：98.15%.It is miscellaneous Matter F's¹H NMR and ESI-HRMS spectrogram are shown in accompanying drawing 17-18 respectively.

Embodiment 10：Impurity G synthesis

5.00gLVTN-2,50mLN- methyl pyrrolidones are added into 250ml there-necked flask.0~10 DEG C of temperature control, is added 7.56g ammoniacal liquor, continues to stir 30 minutes, TLC monitoring (dichloromethane: methanol=10: 1), reaction terminates.Add 33% acetone/ Water 75mL, has a large amount of solids to produce, after stirring 30 minutes, and filtering, 60 DEG C of forced air dryings obtain compound G crude products.By the crude product Column chromatography purifying is carried out, elution ratio is：Methanol: dichloromethane=1: 20, the collection common 630mL of eluent, 30~40 DEG C of temperature control, Vacuum：- 0.08MPa, vacuum distillation is evaporated off solvent to being steamed without cut, obtains faint yellow solid 0.86g, yield：20.63%, Obtain compound G, HPLC purity：99.60%.Impurity G's¹H NMR and ESI-HRMS spectrogram are shown in accompanying drawing 19-20 respectively.

Embodiment 11：Impurity H synthesis

(1) 15ml dimethyl sulfoxide (DMSO)s are added into 250mL there-necked flask, under stirring, the chloro- 7- first of 3.00g 4- are sequentially added Phenoxyl quinoline -6- carboxylic acid amides (SM1) and 1.66g PAPs (SM2-ZZ-02).20~30 DEG C of temperature control, adds 1.71g Potassium tert-butoxide, continues to stir 14 hours, and TLC monitoring (dichloromethane: methanol=10: 1), reaction terminates is done in sampling.Add 30mL pure water, has a large amount of solids to separate out, and filtering obtains crude product and purified with column chromatography method, elution ratio is：Dichloromethane: Methanol=30: 1, collect the common 820mL of eluent, 30~40 DEG C of temperature control, vacuum：Solvent is evaporated off extremely in -0.08MPa, vacuum distillation Steamed without cut.Obtain pale pink solid 2.31g, yield：58.89%, that is, obtain compound H intermediates -1.

(2) 40ml 1-METHYLPYRROLIDONEs are added into 250mL there-necked flask, 2.00g compounds are sequentially added under stirring H intermediate -1,2.05g pyridines, nitrogen protection, are cooled to 0~10 DEG C, start that 3.04g phenyl chloroformates are added dropwise, drop finishes, and continues Stirring 20~30 minutes；Sample TLC detections (methanol: dichloromethane=1: 10) raw material disappears.Add water 40ml, has a large amount of solids to analyse Go out, continue to stir 30 minutes.Filtering, is drained, 60 DEG C of forced air dryings 30 minutes, obtains brownish-yellow powder 2.21g, yield： 79.54%, it is directly used in next step compound H synthesis.

(3) 25ml 1-METHYLPYRROLIDONEs are added into 100mL there-necked flask, stirring is lower to add the above-mentioned centres of 2.21g Body.Nitrogen is protected, and is cooled to 0~10 DEG C, is started that 0.65g cyclopropylamines are added dropwise, drop finishes, is continued to stir 20~30 minutes；Sample TLC Detection (methanol: dichloromethane=1: 10) raw material disappears.Add water 50ml, has a large amount of solids to separate out, and continues to stir 30 minutes.Cross Filter, is drained, 40 DEG C of forced air dryings 30 minutes, is obtained crude product and is purified with column chromatography method, elution ratio is：：Methanol: dichloromethane =1: 20, collect the common 530mL of eluent, 30~40 DEG C of temperature control, vacuum：- 0.08MPa, vacuum distillation is evaporated off solvent to nothing and evaporated Divide and steam.Pale pink solid 0.89g is obtained, that is, obtains compound H, yield：44.04%, HPLC purity：99.60%.

Impurity H's¹H NMR and ESI-HRMS spectrogram are shown in accompanying drawing 21-22 respectively.

Embodiment 12：Impurity I synthesis

3.00gLVTN-2,60mLN- methyl pyrrolidones are added into 250ml there-necked flask.0~10 DEG C of temperature control, is added 1.47g methylamine methanol solutions, continue to stir 30 minutes, TLC monitoring (dichloromethane: methanol=10: 1), reaction terminates.Add 33% acetone water 90mL, has a large amount of solids to produce, after stirring 30 minutes, and filtering, 60 DEG C of forced air dryings obtain compound I crude products. The crude product is subjected to column chromatography purifying, elution ratio is：Methanol: dichloromethane=1: 20, collect the common 760mL of eluent, temperature control 30~40 DEG C, vacuum：- 0.08MPa, vacuum distillation is evaporated off solvent to being steamed without cut, obtains pale pink solid 0.90g, receives Rate：34.70%, that is, obtain compound I, HPLC purity：98.77%.Impurity I's¹H NMR and ESI-HRMS spectrograms are shown in respectively Accompanying drawing 23-24.

Embodiment 13：Methanesulfonic acid pleasure is cut down to be analyzed for Buddhist nun or happy cut down for Buddhist nun about the HPLC of material

Using impurity external standard method (version Chinese Pharmacopoeia in 2010), analysis methanesulfonic acid pleasure is cut down to be cut down for relevant thing in Buddhist nun for Buddhist nun or pleasure Matter, and each impurity is quantified with external standard method.The quantitative of impurity is calculated according to external standard method, referring specifically to Chinese Pharmacopoeia 2010 editions two annex V D.Method for carrying out the analysis is the gradient HPLC methods according to the present invention.The chromatogram used Condition is as follows：

Chromatographic condition：

Chromatographic column：Waters XBridge BEH Shield RP184.6 × 150mm, 2.5 μm or

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

The concentration of need testing solution：0.5mg/ml

The concentration of each impurity of reference standard solution：1000ppm

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

Detection wavelength：252nm

Diluent：Dimethyl sulfoxide (DMSO): acetonitrile: A (2: 4: 4)

Column temperature：20℃

Flow velocity：0.3ml/min

Gradient design is as described below：

Sample preparation：

Prepare impurity positioning solution

(1) compound A~I, LVTN-1 standard setting solution：Each about 5mg of compound A~I and LVTN-1 are taken respectively, it is accurate It is weighed, put respectively in 100ml volumetric flasks, plus acetonitrile or DMSO ultrasounds make dissolving, and shaken up with dilution in acetonitrile to scale, it is made Concentration is about 50 μ g/ml solution, is used as compound A~I and LVTN-1 stock solution.

(2) mark-on mixed solution：Take the methanesulfonic acid pleasure of above-mentioned preparation to cut down (to cut down for Buddhist nun containing about pleasure for Buddhist nun's bulk drug is appropriate 25mg), it is accurately weighed, put in 50ml measuring bottles, then accurate addition compound A~each 0.5ml of I, LVTN-1 stock solution, plus dilution Appropriate agent, ultrasound makes dissolving, then uses diluent constant volume, shakes up, be made every 1ml containing it is happy cut down for Buddhist nun about 0.5mg, A containing compound~ I, LVTN-1 about 0.5 μ g respectively mixed solution, are used as mark-on mixed solution.

(3) prepare compound A~I, LVTN-1 reference standard solution or reference substance solution：Precision measures compound A~I storages Standby solution 0.5ml, puts in 50ml volumetric flasks, uses diluent constant volume, shake up, it is respectively 0.5 that every 1ml, which is made, containing about compound A~I μ g solution, is used as compound A~I reference standard solution or reference substance solution.

(4) pleasure is prepared to cut down for Buddhist nun's need testing solution：Methanesulfonic acid pleasure prepared by Example 3 is cut down for Buddhist nun's bulk drug in right amount, about Cut down containing pleasure for Buddhist nun 5mg, it is accurately weighed, put in 10ml volumetric flasks, plus appropriate diluent, ultrasound makes dissolving, and use diluent constant volume, Shake up, both.The happy concentration about 0.5mg/ml cut down for Buddhist nun's bulk drug need testing solution.

(5) the methanesulfonic acid pleasure prepared according to patent Example 3 of the present invention is cut down to be cut down for Buddhist nun's glue for Buddhist nun and the former methanesulfonic acid pleasure ground The preparation of capsule need testing solution：Taking methanesulfonic acid pleasure to cut down for Buddhist nun's capsule, (Lenvima is purchased from Eisai Inc., 10mg, lot number： L15044) the finely ground powder of content is appropriate (being cut down containing about pleasure for Buddhist nun 5mg), accurately weighed, puts in 10ml volumetric flasks, plus diluent is suitable Amount, ultrasound makes dissolving, and uses diluent constant volume, shakes up, and is filtered with 0.22 μm of organic syringe filter, discards primary filtrate 2ml, take Subsequent filtrate, cuts down for Buddhist nun's capsule need testing solution as methanesulfonic acid pleasure.Methanesulfonic acid pleasure cut down for Buddhist nun's capsule need testing solution concentration about For 0.5mg/ml.

Embodiment 14：

Methanesulfonic acid pleasure cuts down the preparation be the same as Example 3 for Buddhist nun, sample preparation be the same as Example 13, gradient design be the same as Example 13.

Chromatographic condition：

Chromatographic column：Waters XBridge BEH Shield RP184.6 × 150mm, 2.5 μm or

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

The concentration of need testing solution：0.5mg/ml

The concentration of each impurity of reference standard solution：1000ppm

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

Detection wavelength：252nm

Diluent：Dimethyl sulfoxide (DMSO): acetonitrile: A (2: 4: 4)

Column temperature：25℃

Flow velocity：0.9ml/min

Embodiment 15：

Chromatographic condition：

Chromatographic column：Waters XBridge BEH Shield RP184.6 × 150mm, 2.5 μm or

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

The concentration of need testing solution：0.5mg/ml

The concentration of each impurity of reference standard solution：1000ppm

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

Detection wavelength：252nm

Diluent：Dimethyl sulfoxide (DMSO): acetonitrile: A (2: 4: 4)

Column temperature：35℃

Flow velocity：0.5ml/min

Embodiment 16：

Chromatographic condition：

Chromatographic column：Waters XBridge BEH Shield RP184.6 × 150mm, 2.5 μm or

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

The concentration of need testing solution：0.5mg/ml

The concentration of each impurity of reference standard solution：1000ppm

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

Detection wavelength：252nm

Diluent：Dimethyl sulfoxide (DMSO): acetonitrile: A (2: 4: 4)

Column temperature：25℃

Flow velocity：0.4ml/min

Embodiment 17：

Chromatographic condition：

Chromatographic column：Waters XBridge BEH Shield RP184.6 × 150mm, 2.5 μm or

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

The concentration of need testing solution：0.5mg/ml

The concentration of each impurity of reference standard solution：1000ppm

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

Detection wavelength：252nm

Diluent：Dimethyl sulfoxide (DMSO): acetonitrile: A (2: 4: 4)

Column temperature：35℃

Flow velocity：0.3ml/min

Embodiment 18：

Chromatographic condition：

Chromatographic column：Waters XBridge BEH Shield RP184.6 × 150mm, 2.5 μm or

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

The concentration of need testing solution：0.5mg/ml

The concentration of each impurity of reference standard solution：1000ppm

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

Detection wavelength：252nm

Diluent：Dimethyl sulfoxide (DMSO): acetonitrile: A (2: 4: 4)

Column temperature：30℃

Flow velocity：0.4ml/min

Embodiment 19：

Chromatographic condition：

Chromatographic column：Waters XBridge BEH Shield RP184.6 × 150mm, 2.5 μm or

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

The concentration of need testing solution：0.5mg/ml

The concentration of each impurity of reference standard solution：1000ppm

Detection wavelength：252nm

Diluent：Dimethyl sulfoxide (DMSO): acetonitrile: A (2: 4: 4)

Column temperature：35℃

Flow velocity：0.3ml/min

Embodiment 20：

Chromatographic condition：

Chromatographic column：Waters XBridge BEH Shield RP184.6 × 150mm, 2.5 μm or

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

The concentration of need testing solution：0.5mg/ml

The concentration of each impurity of reference standard solution：1000ppm

Mobile phase：0.03M ammonium acetate solutions-(A) (acetic acid adjusts pH to 4.5), acetonitrile (B)

Detection wavelength：252nm

Diluent：Dimethyl sulfoxide (DMSO): acetonitrile: A (2: 4: 4)

Column temperature：30℃

Flow velocity：0.5ml/min

Embodiment 21：

Chromatographic condition：

Chromatographic column：Waters XBridge BEH Shield RP184.6 × 150mm, 2.5 μm or

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

The concentration of need testing solution：0.5mg/ml

The concentration of each impurity of reference standard solution：1000ppm

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

Detection wavelength：252nm

Diluent：Dimethyl sulfoxide (DMSO): acetonitrile: A (2: 4: 4)

Column temperature：25℃

Flow velocity：1.0ml/min

Test procedure：

(1) under gradient HPLC methods of the present invention, on Agilent1260HPLC instruments (Agilent companies of the U.S.), press According to the chromatographic condition of embodiment 13, take compound A~I, LVTN-1 positioning solution, mark-on mixed solution, compound A~I, LVTN-1 reference solutions, methanesulfonic acid pleasure are cut down to be cut down for each 5 μ l of Buddhist nun's capsule need testing solution, note for Buddhist nun's need testing solution, methanesulfonic acid pleasure Enter liquid chromatograph, record chromatogram.

(2) under gradient HPLC methods of the present invention, according to the chromatographic condition of embodiment 13~21, the μ of mark-on mixed solution 5 is taken L, injects liquid chromatograph, records chromatogram.

Result of the test：

Compound A~I, LVTN-1 principal component methanesulfonic acid pleasure cuts down for Buddhist nun the determining under gradient HPLC methods in the present embodiment 13 Position the results are shown in Table 3；Under the chromatographic condition of the embodiment of the present invention 13~21, the separation in mark-on mixed solution between each impurity Degree, each impurity and methanesulfonic acid pleasure are cut down and are shown in Table 4 for Ni Feng relative retention time.

Using the chromatographic condition of embodiment 16, calculated according to external standard method contain in need testing solution compound A~I, LVTN-1 content and total impurities amount, the results are shown in Table 5, the HPLC spectrograms of mark-on mixed solution, methanesulfonic acid pleasure are cut down for Buddhist nun's test sample The HPLC spectrograms of solution, methanesulfonic acid pleasure are cut down for Buddhist nun's capsule, the former need testing solution for grinding control formulation Lenvima-10mg specifications HPLC spectrograms are shown in that the top in Figure 25~Figure 28, Figure 25-28 is the happy peak cut down for Buddhist nun respectively, are represented by LVTN.

Cut down and cut down for Buddhist nun and methanesulfonic acid pleasure for Buddhist nun according to compound A~I, LVTN-1 and principal component positioning result and methanesulfonic acid pleasure Capsule is about the analysis result of material, and methanesulfonic acid pleasure cuts down for compound C has been detected in Buddhist nun's need testing solution that (relative retention time is about For 0.47), (relative retention time is about by G (relative retention time is about 0.61), H (relative retention time is about 0.78), I 0.82), B (relative retention time is about 0.96) and impurity A (relative retention time is about 1.12)；Methanesulfonic acid pleasure is cut down for Buddhist nun's capsule Compound C, G, H, I, B have been detected in need testing solution and 4 unknown impurities (normalize impurity of the content below 0.01% to neglect Slightly disregard).Impurity C, G, I, LVTN-1 (relative retention time is about 0.94), B, A have been detected in former triturate Lenvima capsules With 7 unknown impurities (impurity of the normalization content below 0.01% is ignored).

The compound A of table 3~I, LVTN-1 and methanesulfonic acid pleasure are cut down between positioning of the Buddhist nun in the method for embodiment 13 and each peak Separating degree

The relative retention time of each impurity under the conditions of the embodiment 13~21 of table 4

Note：When chromatographic condition changes, the retention time of each impurity peaks can change, but relative retention time is basic It is constant.The worst separating degree of impurity C and impurity E separating degree under the chromatographic condition of embodiment 18 is 1.21, still above hardly possible point Separating degree from impurity requirement is not less than 1.2.

Methanesulfonic acid pleasure prepared by the embodiment 3 of table 5 cut down for relevant material in Buddhist nun's bulk drug and commercially available Lenvima capsules according to The testing result of the chromatographic condition of embodiment 16

N.D. " not detecting " is represented.

It should be noted that all documents referred in the present invention are incorporated as reference in this application, just as each Piece document is individually recited as with reference to such.In addition, it is to be understood that above-described is that the specific implementation of the present invention is arranged and transported Technical principle, after present disclosure has been read, those skilled in the art can do various changes or repair to the present invention Change without departing from spirit and scope of the invention, these equivalent form of values are also fallen within the scope of the present invention.

Claims

Cut down 1. detection is happy for Buddhist nun or its solvate or its salt or containing the happy medicine cut down for Buddhist nun or its solvate or its salt The method of sample purity, including by the impurity in chromatography determination sample, the impurity is in compound A-I and LVTN-1 One or more, wherein happy cut down for Buddhist nun is 4- (the chloro- 4- of 3- (N '-cyclopropylureido) phenoxy group) -7- methoxy quinoline -6- carboxylics Sour acid amides, structure is as shown in following formula LVTN-3：

Compound A is 4,4 '-(((carbonyl is double (urea diyl)) double (chloro- 4, the 1- phenyl of 3-)) double (epoxides)) double (7- methoxyl group quinolines Quinoline -6- formamides), and with following structure：

Compound B is 4- (the chloro- 4- of 3- (3,3- dimethyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides, and is had Following structure：

Compound C is 4- ethyoxyl -7- methoxy quinoline -6- formamides, and with following structure：

Compound D is 4- hydroxyl -7- methoxy quinoline -6- carboxylic acid amides, and with following structure：

Compound E is 1- (2- chloro-4-hydroxyls phenyl) -3- cyclopropylamino ureas, and with following structure：

Compound F is 4- (the chloro- 4- of 3- (3- cyclopropylureidos) phenoxy group) -7- methoxy quinoline -6- carboxylic acids, and with following knot Structure：

Compound G is 4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylate methyl esters, and with following structure：

Compound H is 4- (4- (3- cyclopropylureidos) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides, and with following knot Structure：

Compound I is 4- (the chloro- 4- of 3- (3- methyl urea groups) phenoxy group) -7- methoxy quinoline -6- carboxylic acid amides, and with following Structure：

Intermediate -1 is represented by LVTN-1, is 4- (4- amino -3- chlorophenoxies) -7- methoxy quinoline -6- carboxylic acid amides, and have There is following structure：

Wherein methods described comprises the following steps：

(1) cut down happy for Buddhist nun or its solvate or its salt or containing the happy medicine dissolving cut down for Buddhist nun or its solvate or its salt In a solvent to prepare sample solution；

(2) sample any one or more of in compound A~I and intermediate -1 is dissolved in a solvent to prepare reference standard Solution or reference substance solution；

(3) chromatographic technique is implemented to sample solution and reference standard solution；And

(4) by referring to the one or more of compound A-I and intermediate -1 in the reference standard solution, determine it is happy cut down for Buddhist nun or Its solvate or its salt cut down compound A~I and intermediate -1 in the medicine for Buddhist nun or its solvate or its salt containing pleasure Any one or more of (LVTN-1) presence,

Wherein described chromatography is gradient HPLC method, in the gradient HPLC methods, mobile phase include containing cushioning liquid (A) and The combination of organic solvent (B), the cushioning liquid (A) adjusted pH for the aqueous solution of ammonium acetate and acetic acid or ammonium acetate with acetic acid The aqueous solution, the organic solvent (B) be acetonitrile or the mixed solvent containing acetonitrile.
2. the method for claim 1 wherein the pleasure cut down the salt for Buddhist nun for it is happy cut down for Buddhist nun's hydrobromate, it is happy cut down for Buddhist nun's hydrochloride, Happy cut down is cut down for Buddhist nun for Buddhist nun's fumarate, happy cut down for Buddhist nun's maleate or methanesulfonic acid pleasure, and wherein methanesulfonic acid pleasure is cut down for Buddhist nun's structural formula such as Shown in following formula 1：
3. the stationary phase used the method for claim 1 wherein the chromatography is anti-phase, preferably octadecylsilane bonded silica Glue or octyl group silane group silica gel.
4. any one of claim 1-3 method, wherein the gradient HPLC is the HPLC method compatible with LC-MS.
5. the method for claim 1 wherein the concentration that ammonium acetate is present is about 0.001M to 1.0M, preferably 0.02M to 0.08M, More preferably 0.02M to 0.05M, most preferably 0.03M.
6. the pH of the aqueous solution of the method for claim 5, wherein ammonium acetate and acetic acid is about 3.5~6.5, preferably from about 4.0~ 6.5,4.0~6.0,4.0~5.0, most preferably 4.5~5.5.
7. any one of claim 1-6 method, wherein the HPLC methods are carried out at a temperature of about 20~40 DEG C, is preferably Carried out at a temperature of 20~35 DEG C, most preferably 25~35 DEG C.
8. any one of claim 1-7 method, wherein flow velocity of the HPLC analysis methods in about 0.3~1.0ml/min It is lower to carry out, preferably 0.3~0.9ml/min, 0.3~0.8ml/min, 0.3~0.7ml/min, preferably 0.3~0.6ml/ Carried out under min, most preferably 0.3~0.5ml/min flow velocity.
9. in any one of claim 1-8 method, wherein gradient HPLC methods, mobile phase A and B relative concentration are 10~60 Minutes, which typically become, turns to 100%A：0%B to 0%A：100%B gradient.Preferably, by 30 to 55 minutes, Gradient is 95%A：5%B to 5%A：95%B, it is highly preferred that by 20 to 50 minutes, gradient is 90%A：10%B is extremely 15%A：85%B, most preferably, by about 30 minutes, gradient was 90%A：10%B to 5%A：95%B or 85%A：15%B To 20%A：80%B or：80%A：20%B to 30%A：70%B or 70%A：30%B to 40%A to 60%B.
10. in any one of claim 1-9 method, wherein gradient HPLC methods, mobile phase includes following gradient design：

Time (minute) 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

。
11. the method for claim 1 wherein organic solvent (B) is acetonitrile：The mixed solvent of methanol, wherein with mobile phase A and B Percent of total meter, the percentage of methanol is 25 ± 5%.
12. in any one of claim 1-9 method, wherein gradient HPLC methods, mobile phase includes following gradient design：
13. the method described in claim any one of 1-10, wherein happy cut down for Buddhist nun or its solvate or its salt or cut down comprising pleasure For Buddhist nun or its solvate or its salt pharmaceutical composition be solid form or liquid form, the solid form be selected from pulvis, Tablet, capsule, pill and dispersible granule, the liquid form are selected from solution or suspension.