CN106153797B

CN106153797B - It is a kind of to replace Buddhist nun's preparation Related substance method according to Shandong for Buddhist nun and according to Shandong

Info

Publication number: CN106153797B
Application number: CN201510188803.9A
Authority: CN
Inventors: 贾慧娟; 何学敏; 王艳鑫; 李衍; 李厚全
Original assignee: Beijing Creatron Institute Of Pharmaceutical Research Co Ltd; Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Current assignee: Beijing Creatron Institute Of Pharmaceutical Research Co Ltd; Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Priority date: 2015-04-20
Filing date: 2015-04-20
Publication date: 2017-08-29
Anticipated expiration: 2035-04-20
Also published as: CN106153797A

Abstract

The invention belongs to pharmaceutical synthesis field.Specifically, the present invention relates to the method according to Shandong for the impurity occurred in Buddhist nun (ketone of 1 [(3R) 3 [base of 4 amino 3 (4 Phenoxyphenyl) 1H pyrazolos [3,4 d] pyrimidine 1] 1 piperidyl] 2 propylene 1) preparation process and to being analyzed according to Shandong for Buddhist nun's preparation.

Description

It is a kind of to replace Buddhist nun's preparation Related substance method according to Shandong for Buddhist nun and according to Shandong

Technical field

The invention belongs to pharmaceutical synthesis field.In particular it relates to replace Buddhist nun (1- [(3R) -3- [4- amino -3- according to Shandong (4- Phenoxyphenyls) -1H pyrazolos [3,4-d] pyrimidine -1- bases] -1- piperidyls] -2- propylene -1- ketone) occur in preparation process Impurity and to replacing the method analyzed of Buddhist nun's preparation according to Shandong.

Background technology

Ibrutinib was developed first by Celera Genomics companies of the U.S. in 2007, after transfer California Pharmacyclics companies develop, and subsidiary's Janssen Pharmaceutica (Jassen) of Johson ＆ Johnson in 2011 participates in cooperative development.At present The Chinese translation of standard is there is no, therefore its transliteration is herein " replacing Buddhist nun according to Shandong " by the applicant.It is for the chemical name of Buddhist nun according to Shandong： 1- [(3R) -3- [4- amino -3- (4- Phenoxyphenyls) -1H pyrazolos [3,4-d] pyrimidine -1- bases] -1- piperidyls] -2- third Alkene -1- ketone, structural formula is：

Patent WO201384572A is disclosed to having carried out HPLC analyses according to Shandong for the chemical purity of Buddhist nun.It is real according to the patent The method of example 8 is applied to being analyzed according to Shandong for Buddhist nun's crude product (being prepared in embodiment 1), used high performance liquid chromatograph (HPLC) it is Shimadzu LC-20A, PDA detector (purchased from Japanese Shimadzu Corporation), as a result as shown in figure 1, in wherein Fig. 1 Top is the peak that Buddhist nun is replaced according to Shandong.As shown in Figure 1, there is following drawback in the analysis method of the patent：

(1) according to Shandong, for the impurity contained in Buddhist nun's crude product, (retention time is about under this method：19.554min) to replacing Buddhist nun according to Shandong There is interference at principal component peak, and the two separating degree does not meet in ICH Q3A and verified for relevant substance method under item to specificity It is required that, i.e., the separating degree between impurity and principal component is less than 2.0；

(2) the analysis method run time is longer, and single needle run time is 60min；

(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 final products In real content.

(4) not to replacing the specific impurities in Buddhist nun to carry out qualitative analysis according to Shandong.In summary, the detection knot of prior art Fruit can not actual response medicine quality.

The content of the invention

The invention provides new, interchangeable method, for replacing determining for the relevant material of Buddhist nun's preparation with Yi Lu according to Shandong for Buddhist nun Property, quantitative analysis, and the qualitatively and quantitatively analysis of impurity is carried out with these impurity as reference standard or reference substance.The present invention Another purpose be to provide reference standard or reference substance, for detect prepare according to Shandong replace Buddhist nun during formed be referred to as A ~H impurity.

Provided by the present invention for analyzing according to Shandong for Buddhist nun or replacing the HPLC methods in Buddhist nun's preparation about material according to Shandong, wherein, Mobile phase includes two or more liquid, and the relative concentration of liquid changes with predetermined gradient.Inventor utilizes 8 kinds of systems Standby and identification structure impurity (claiming compound A~H) is used for according to Shandong for Buddhist nun or containing the relevant material of pharmaceutical preparation that Buddhist nun is replaced according to Shandong The reference standard or reference substance of analysis method.The structure of these impurity and impurity is not yet disclosed in the prior art.

Therefore, the first aspect of the invention provides compound A, and it has chemical name：(R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases] piperidin-1-yl) -3- chloropropyl -1- ketone, and with following Structure：

Second aspect provides compound B, and it has chemical name：(R) -8- (1- acryloylpiperidine -3- bases) -10- (4- Phenoxyphenyl) -3,4- dihydro-pyrazolos [4,3-e] pyrimido [1,2-c] pyrimidine -2 (8H) -one, and with following structure：

3rd aspect provides compound C, and it has chemical name：(R) -1-3- (4- amino -3- (4- Phenoxyphenyls) - 1H- pyrazolos [3,4-d] pyrimidine -1- bases] piperidin-1-yl)-ethyl ketone, and with following structure：

4th aspect provides compound D, and it has chemical name：(R) -4- (3- (4- amino -3- (4- Phenoxyphenyls) - 1H- pyrazolos [3,4-d] pyrimidine -1- bases)-piperidines -1- carbonyls) amylene -4- acid, and with following structure：

5th aspect provides compound E, and it has chemical name：1- ((R) -1- acryloylpiperidine -3- bases) -4- ammonia Base -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- oxides, and with following structure：

6th aspect provides compound F, and it has chemical name：1,3- bis- ((R) -3- (4- amino -3- (4- phenoxy group benzene Base) -1H- pyrazoles [3,4-d] pyrimidine -1- bases) piperidin-1-yl) propane -1- ketone, and with following structure：

7th aspect provides compound G, and it has chemical name：1- ((R) -3- (4- ((3- ((R) -3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] pyrimidine -1- bases) piperidin-1-yl) -3- oxopropyls) ammonia) -3- (4- phenoxy groups Phenyl) -1H- pyrazoles [3,4-d] pyrimidine -1- bases) piperidin-1-yl) propyl group -2- alkene -1- ketone, and with following structure：

8th aspect provides compound H, and it has chemical name：(R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) - 1H- pyrazolos [3,4-d] pyrimidine -1- bases)-piperidin-1-yl)-propyl group -1- ketone, and with following structure：

Compound A~H is that intermediate, accessory substance or the degradation impurity in Buddhist nun's building-up process are replaced according to Shandong, it is adaptable to be used as ginseng Than standard or reference substance, the quality control for the product.

The way of production of above impurity is as follows：

Impurity A：This impurity is impurity in commercially available capsule, it should grind process contaminants for original, in this technique is multiple batches of from Do not detect.

Impurity B：The synthetic method second step acyl that Buddhist nun is replaced according to Shandong reported in Chinese Patent Application No. 201510165869.6 In aminating reaction, amidatioon occurs replacing two positions of Buddhist nun's intermediate -1 (being expressed as YLTN-1), piperidines nuclear nitrogen according to Shandong And the nitrogen on pyrimidine ring, bisamide impurity in products B-1 is produced, due to Thermodynamic effect intramolecular cyclization occurs for the latter, raw Into more stable Sulfadiazine Compound ring impurity B.

Impurity C：The synthetic method second step acyl that Buddhist nun is replaced according to Shandong reported in Chinese Patent Application No. 201510165869.6 In aminating reaction, a small amount of acetic anhydride impurity is contained in initiation material acrylic anhydride, it is miscellaneous to occur amidation process generation with YLTN-1 Matter C.

Impurity D：The synthetic method second step acyl that Buddhist nun is replaced according to Shandong reported in Chinese Patent Application No. 201510165869.6 In aminating reaction, according to Shandong for Buddhist nun (YLTN) and accessory substance acrylic acid under the catalysis of alkali (be probably DIPEA, May for according to Shandong replace Buddhist nun itself nitrogen heterocyclic ring) occur Baylis-Hillman reaction produce impurity D.

Impurity E, F, G are for Buddhist nun's active component or comprising in the forced degradation sample according to Shandong for the pharmaceutical preparation of Buddhist nun according to Shandong Principal degradation impurity, wherein impurity E are to replace Buddhist nun according to Shandong or the primary product of oxidative degradation in Buddhist nun's formulation process is replaced according to Shandong, miscellaneous Matter F, G is to replace the catabolite in Buddhist nun's formulation process according to Shandong for Buddhist nun or according to Shandong, is also to replace to force under Buddhist nun's alkalescence condition according to Shandong Degraded or the main degradation products of hydrolysis.

Impurity H：The synthetic method second step acyl that Buddhist nun is replaced according to Shandong reported in Chinese Patent Application No. 201510165869.6 In aminating reaction, initiation material acrylic anhydride contains a small amount of propionic andydride impurity, and can react generation impurity H with intermediate -1：

It is monomeric compound according to compound A~H of the present invention, most preferably in a particularly preferred embodiment Be pure form, preferably purity is greater than about 95%, preferably purity is greater than about 98%, most preferably purity and is greater than about 99%, preferably Measured by HPLC.

A kind of detect according to Shandong for Buddhist nun or containing the pharmaceutical dosage form that Buddhist nun is replaced according to Shandong is provided according to the ninth aspect of the present invention The method of sample purity, this method includes determining one kind or many in the compound A~H replaced according to Shandong containing the present invention in Buddhist nun's sample Kind.In the method for the invention, the compound is used as the reference standard or reference substance of impurity.

According to the tenth aspect of the present invention there is provided a kind of method for characterizing compound A~H, this method is utilized HPLC methods come analyze according to Shandong replace Buddhist nun in the impurity A~H.It is preferred that the HPLC methods are the compatible methods of LC-MS.

It thus provides replacing Buddhist nun or containing according to Shandong according to Shandong in detection according to compound A~H (one or more) of the present invention For the purposes in the sample purity of the pharmaceutical preparation of Buddhist nun as reference standard or reference substance.

On the other hand, the present invention also provides a kind of chromatographic process for detecting according to Shandong for Buddhist nun's sample purity, methods described Including：Utilize the reference according to the present invention hi an alternative embodiment by using the reference standard or reference substance according to the present invention Standard or reference substance, carry out one or more presence in compound A~H in determination sample.

Another aspect there is provided it is a kind of by determine containing according to Shandong in the sample of Buddhist nun in compound A~H it is any or A variety of presence detects the chromatographic process according to Shandong for the purity of Buddhist nun's sample, and methods described includes：

(1) by according to Shandong for Buddhist nun or containing according to Shandong for Buddhist nun formulation samples dissolving in a solvent to prepare sample solution；

(2) by any one or more of sample dissolving in compound A~H in a solvent with 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 presence of known compound A-H (one or more) present in the reference standard solution, determine Any one or more of compound A~H presence in sample.

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, trifluoroacetic acid, the aqueous solution of formic acid or their mixture.Preferred cushioning liquid (A) is formates, most preferably first The aqueous solution of sour ammonium and formic acid or ammonium formate adjusted the pH aqueous solution with formic acid.Ammonium formate in particularly preferred embodiments The concentration of presence is about 0.01M to 0.1M, most preferably preferably 0.03-0.08M, more preferably 0.04-0.06M, 0.05M.It is preferred that having Machine solvent (B) is polar aprotic solvent, such as methanol, ethanol or isopropanol；Or dipolar aprotic solvent, such as acetonitrile.It is preferred that organic Solvent (B) is in the group including methanol, acetonitrile, ethanol, isopropanol or their mixture, most preferably acetonitrile and methanol Mixture.

The aqueous solution (A) and acetonitrile and the group of methanol (B) of the mobile phase specifically preferred according to the invention comprising ammonium formate and formic acid Close.

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	50	50
25	35	65
			30	10	90
35	10	90
			35.01	50	50
40	50	50

In further preferred embodiment, the aqueous solution of cushioning liquid (A) ammonium formate and formic acid in HPLC methods PH is about 2.7~4.8, preferably from about 2.8~4.7,2.8~4.5,2.8~4.0,2.8~3.5, most preferably 2.8~3.5.

In other embodiments, the HPLC analysis methods are carried out at a temperature of about 30~60 DEG C, and preferably 35~55 DEG C, 40~50 DEG C, most preferably 40~50 DEG C.

In other embodiments, the HPLC analysis methods are carried out under about 0.4~1.2ml/min flow velocity, are preferably 0.5~1.1ml/min, 0.6~1.0ml/min, most preferably 0.6~1.0ml/min.

In other embodiments, in the HPLC analysis methods, organic phase B is the mixture of methanol and acetonitrile, methanol and The mixed volume ratio of acetonitrile is about 40: 60, preferably 45: 55,55: 45, or 50: 50, most preferably 50: 50.

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：(R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases] Piperidin-1-yl) -3- chloropropyl -1- ketone.

Compound B：(R) -8- (1- acryloylpiperidine -3- bases) -10- (4- Phenoxyphenyls) -3,4- dihydro-pyrazolos [4,3-e] pyrimido [1,2-c] pyrimidine -2 (8H) -one.

Compound C：(R) -1-3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases] piperazine Pyridine -1- bases)-ethyl ketone.

Compound D：(R) -4- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) - Piperidines -1- carbonyls) amylene -4- acid.

Compound E：1- ((R) -1- acryloylpiperidine -3- bases) -4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- oxides.

Compound F：1,3- bis- ((R) -3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] pyrimidine -1- Base) piperidin-1-yl) propane -1- ketone.

Compound G：1- ((R) -3- (4- ((3- ((R) -3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4- D] pyrimidine -1- bases) piperidin-1-yl) -3- oxopropyls) ammonia) -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] pyrimidine -1- Base) piperidin-1-yl) propyl group -2- alkene -1- ketone.

Compound H：(R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) - Piperidin-1-yl)-propyl group -1- ketone.

The present invention can be used for analysis as active pharmaceutical ingredient (API) according to Shandong in Buddhist nun and/or pharmaceutical composition according to Replace Buddhist nun in Shandong.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 " replacing Buddhist nun according to Shandong " refers to replace Buddhist nun and/or solvate (such as hydrate) according to Shandong sharp claim in the whole text.Specification Used term " impurity " or " about material " can refer to the impurity formed in manufacture API or pharmaceutical composition in the whole text, with And/or person 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 according to Shandong for Buddhist nun and containing according to Shandong 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 and intermediate formed in this specific building-up process or preparation preparation.Advantage of the invention is that：For the first time The concrete structure for replacing the impurity in Buddhist nun's preparation for Buddhist nun and Yi Lu according to Shandong is disclosed, and with gradient HPLC method simultaneously by polar impurity It is eluted out with non polar impurities, and carries out qualitative and quantitative analysis.

The invention is particularly suited to determine and quantify one or more presence in sample in compound A~H.Unless another It is described, otherwise term herein " impurity " and " compound " are under the scope related to compound A~H, and can exchange makes With.

It is also an advantage of the invention that this method according to Shandong for replacing Buddhist nun and containing the relevant material in the preparation that Buddhist nun is replaced according to Shandong 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 quantify according to Shandong and be substantially less than drug administration department and ICH guidelines for level amount in Buddhist nun API or pharmaceutical composition Specified in acceptable limits relevant material.

In addition, the method for the present invention can be used for detection and quantify according to Shandong 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.

Eight kinds of impurity A~H of novel gradient HPLC methods qualitative determination that the present inventor develops.Methods described can one The analysis of secondary property according to it is manufactured in the present embodiment replaced according to Shandong in Buddhist nun preparation technology and it is commercially available according to Shandong for being produced during Buddhist nun and storage Accessory substance, the impurity that differs greatly of degradation impurity isopolarity, 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 Agilent Poroshell EC-C₁₈120(150mm× 4.6mm), 2.7 μm of posts.

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 turn to 100%A: 0%B to 0%A: 100%B gradient.Preferably, by 15 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 10%A: 90% B, most preferably, by about 30 minutes, gradient was 65%A: 35%B to 25%A: 75%B or 60%A: 40%B to 20%A: 80%B or 55%A: 45%B to 15%A: 85%B or 50%A: 50%B to 10%A: 90%B.The advantage of this gradient method exists According to Shandong for Buddhist nun API or according to Shandong the very close impurity of various opposed polarities or polarity in Buddhist nun's pharmaceutical composition being replaced complete 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 is preferably selected from including phosphate, acetate, formates, trifluoroacetic acid, formic acid or acetic acid they mixed The aqueous solution combination of compound.

The concentration of cushioning liquid can be 0.01M to 0.1M, and preferred concentration is 0.01M to 0.08M, and more preferably concentration is 0.03M is to 0.08M, further preferred 0.04-0.06M, most preferably 0.05M.Particularly preferred mobile phase includes ammonium formate and first The aqueous solution of acid, or the ammonium formate solution aqueous solution (A) and acetonitrile (B) or acetonitrile of first acid for adjusting pH and the mixing of methanol (B) The combination of thing.

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 formate and/or formic 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 formate and/or formic acid (A) and second Nitrile and gradient design is particularly effective during carbinol mixture (B).

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

The pH of cushioning liquid (A) is about 2.7~4.8, preferably from about 2.8~4.7,2.8~4.5,2.8~4.0,2.8~ 3.5, most preferably 2.8~3.5.

The HPLC methods of the present invention are carried out at a temperature of about 30~60 DEG C, preferably 35~55 DEG C, 40~50 DEG C, optimal Elect 40~50 DEG C as.

The analysis method is carried out under about 0.4~1.2ml/min flow velocity, preferably 0.5~1.1ml/min, 0.6~ 1.0ml/min, most preferably 0.6~1.0ml/min.

Organic solvent (B) can be a kind of organic solvent, and this organic solvent can be methanol, ethanol, acetonitrile, isopropanol. Organic solvent (B) can also be the mixed solvent or acetonitrile and the mixed solution of ethanol of methanol and acetonitrile, and inventor has found methanol Organic phase (B) optimal effect is used as with the mixed solvent of acetonitrile.The mixed volume ratio of methanol and acetonitrile is about 40: 60, is preferably 45: 55,55: 45, or 50: 50, most preferably 50: 50.

Another aspect of the present invention provides a kind of reference standard solution.The solution includes and is dissolved in suitable solvent (such as acetonitrile) In one or more compound A~H.The reference standard solution, which can be used for determining, is utilizing the chromatographic technique according to the present invention In the sample analyzed as impurity any compound A~H presence.

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~H is dissolved in suitable solvent (such as acetonitrile).The reference standard solution can be used for determining using according to the present invention's Any compound A~H qualitative of impurity and quantitative is used as in the sample that chromatographic technique is analyzed.The method pair of the analysis In the important of technical staff and be conveniently obvious.

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 according to method disclosed in WO201384572A embodiments 8 to (being prepared according to Shandong for Buddhist nun's crude product in embodiment 2 Without recrystallisation from isopropanol Buddhist nun is replaced according to Shandong) carry out HPLC analysis collection of illustrative plates.

Fig. 2：Intermediate -1：(R) -3- (4- Phenoxyphenyls) -1- (piperidines -3- bases) -1H- pyrazolos [3,4-d] pyrimidine - 4- amine¹H-NMR；

Fig. 3：Intermediate -1：(R) -3- (4- Phenoxyphenyls) -1- (piperidines -3- bases) -1H- pyrazolos [3,4-d] pyrimidine - The high resolution mass spectrum of 4- amine；

Fig. 4：Buddhist nun is replaced according to Shandong：1- [(3R) -3- [4- amino -3- (4- Phenoxyphenyls) -1H pyrazolos [3,4-d] pyrimidine - 1- yls] -1- piperidyls] -2- propylene -1- ketone¹H-NMR；

Fig. 5：Buddhist nun is replaced according to Shandong：1- [(3R) -3- [4- amino -3- (4- Phenoxyphenyls) -1H pyrazolos [3,4-d] pyrimidine - 1- yls] -1- piperidyls] -2- propylene -1- ketone high resolution mass spectrum.

Fig. 6：Impurity A：(R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- Base] piperidin-1-yl) -3- chloropropyl -1- ketone¹H-NMR；

Fig. 7：Impurity A：(R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- Base] piperidin-1-yl) -3- chloropropyl -1- ketone high resolution mass spectrum；

Fig. 8：Impurity B：(R) -8- (1- acryloylpiperidine -3- bases) -10- (4- Phenoxyphenyls) -3,4- pyrazolines And [4,3-e] pyrimido [1,2-c] pyrimidine -2 (8H) -one¹H-NMR；

Fig. 9：Impurity B：(R) -8- (1- acryloylpiperidine -3- bases) -10- (4- Phenoxyphenyls) -3,4- pyrazolines And the high resolution mass spectrum of [4,3-e] pyrimido [1,2-c] pyrimidine -2 (8H) -one；

Figure 10：Impurity C：(R) -1-3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- Base] piperidin-1-yl)-ethyl ketone¹H-NMR；

Figure 11：Impurity C：(R) -1-3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- Base] piperidin-1-yl)-ethyl ketone high resolution mass spectrum.

Figure 12：Impurity D：(R) -4- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- Base)-piperidines -1- carbonyls) amylene -4- acid¹H-NMR；

Figure 13：Impurity D：(R) -4- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- Base)-piperidines -1- carbonyls) amylene -4- acid high resolution mass spectrum；

Figure 14：Impurity H：((R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- Base)-piperidin-1-yl)-propyl group -1- ketone¹H-NMR；

Figure 15：Impurity H：(R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- Base)-piperidin-1-yl)-propyl group -1- ketone high resolution mass spectrum；

Figure 16：Impurity E：1- ((R) -1- acryloylpiperidine -3- bases) -4- amino -3- (4- Phenoxyphenyls) -1H- pyrroles Azoles simultaneously [3,4-d] pyrimidine -1- oxides¹H-NMR。

Figure 17：Impurity E：1- ((R) -1- acryloylpiperidine -3- bases) -4- amino -3- (4- Phenoxyphenyls) -1H- pyrroles The high resolution mass spectrum of azoles simultaneously [3,4-d] pyrimidine -1- oxides.

Figure 18：Impurity F：1,3- bis- ((R) -3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] pyrimidine - 1- yls) piperidin-1-yl) propane -1- ketone¹H-NMR。

Figure 19：Impurity F：1,3- bis- ((R) -3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] pyrimidine - 1- yls) piperidin-1-yl) propane -1- ketone high resolution mass spectrum.

Figure 20：Impurity G：1- ((R) -3- (4- ((3- ((R) -3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3, 4-d] pyrimidine -1- bases) piperidin-1-yl) -3- oxopropyls) ammonia) -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] pyrimidine - 1- yls) piperidin-1-yl) propyl group -2- alkene -1- ketone¹H-NMR。

Figure 21：Impurity G：1- ((R) -3- (4- ((3- ((R) -3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3, 4-d] pyrimidine -1- bases) piperidin-1-yl) -3- oxopropyls) ammonia) -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] pyrimidine - 1- yls) piperidin-1-yl) propyl group -2- alkene -1- ketone high resolution mass spectrum；

The HPLC spectrograms of Figure 22 mark-on mixed solutions.

The HPLC spectrograms that Buddhist nun's need testing solution is replaced according to Shandong prepared by Figure 23 embodiments 2.

Figure 24 are commercially available according to HPLC spectrogram of the Shandong for Buddhist nun's capsule need testing solution.

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

Intermediate -1：(R) -3- (4- Phenoxyphenyls) -1- (piperidines -3- bases) -1H- pyrazolos [3,4-d] pyrimidine -4- amine Synthesis

155mL tetrahydrofurans are added into 500mL reaction bulb, 3- (4- Phenoxyphenyls) -1H- is sequentially added under stirring Pyrazoles [3,4-D] pyrimidine -4- amine (SM1) (5g, 1eq), (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines (SM2) (4.97g, 1.5eq), triphenylphosphine (13g, 3.0eq).Under the conditions of 25 DEG C of temperature control, in 30 minutes, diisopropyl azodiformate is added dropwise Tetrahydrofuran solution (by 10g, 3.0eq diisopropyl azodiformates are dissolved in 10mL tetrahydrofuran).After being added dropwise to complete, control 25 DEG C of temperature, continues to react (TLC monitorings in 5 hours：Ethyl acetate: methanol=10: 1).The lower vacuum distillation of stirring.15 DEG C of temperature control, to Dropwise addition 30mL concentrated hydrochloric acids in residue, time for adding 30 minutes, after being added dropwise to complete, 25 DEG C of temperature control continues to react 2 hours.With two Chloromethanes aqueous phase extracted three times (each 75mL), retains aqueous phase.With the extraction of 50mL ethyl acetate once.Remaining aqueous phase is fully stirred Mix, 15 DEG C of temperature control, about 45g 20% sodium hydrate aqueous solution is added dropwise, reaction solution is monitored using pH test paper, pH=5~6 (are added dropwise About 30 minutes time) obtain light yellow solid.By above-mentioned solid by ethyl alcohol recrystallization twice, obtain off-white powder 2.87g, receive Rate：45%.¹H-NMR (400Mz, DMSO-d₆)δ：8.226 (s, 1H), 7.656~7.634 (m, 2H), 7.435~7.395 (m, 2H), 7.185~7.094 (m, 5H), 4.689~4.635 (m, 1H), 3.081~3.043 (m, 1H), 2.957~2.930 (m, 1H), 2.901~2.873 (m, 1H), 2.490~2.457 (m, 1H), 2.125~2.015 (m, 2H), 1.750~1.717 (m, 1H), 1.589~1.518 (m, 1H) (referring to Fig. 2)；ESI-HRMS spectrograms show molecular ion peak m/z=387.19449 [M+H] +, corresponding molecular weight is consistent with the structural formula calculated value (387.19279) provided.Absolute error is 4.41ppm, Within high resolution mass spectrum error range.(referring to Fig. 3)

Embodiment 2：The preparation of Buddhist nun is replaced according to Shandong

Under nitrogen protection, 50mL dichloromethane, intermediate -1 (5g, 1eq), N, N- are sequentially added into 100mL there-necked flasks Diisopropylethylamine (2g, 1.2eq).Under the conditions of -10 DEG C of temperature control, start that the dichloromethane of acrylic anhydride (1.96g, 1.2eq) is added dropwise Alkane solution, time for adding 30 minutes, after being added dropwise to complete, solution is become by muddiness to be clarified, and is incubated -10 DEG C, is stirred well to raw material anti- Should (TLC detections, methanol: ethyl acetate: triethylamine=1: 5: 0.05) completely.The reaction solution aqueous citric acid solutions of 200mL 5% Washing, removes aqueous phase, and dichloromethane is evaporated off in concentration.To residue recrystallisation from isopropanol three times, get Yi Lu replaces Buddhist nun's finished product： 3.63g。¹H-NMR (400Mz, DMSO-d₆)δ：8.259 (s, 1H), 7.654~7.674 (m, 2H), 7.405~7.444 (m, 2H), 7.109~7.195 (m, 5H), 6.676~6.743 (m, 1H), 6.046~6.152 (m, 1H), 5.570~5.713 (m, 1H), 4.690~4.716 (m, 1H), 4.554~4.583 (m, 0.5H), 4.208 (m, 1H), 4.052~4.085 (m, 0.5H), 3.674~3.731 (m, 0.5H), 3.184~3.214 (m, 1H), 2.972~3.027 (m, 0.5H), 2.245~2.282 (m, 1H), (m, 1H) (the referring to Fig. 4) ESI-HRMS of 2.128 (m, 1H), 1.903~1.937 (m, 1H), 1.577~1.605 spectrograms show Show molecular ion peak：441.20366[M+H]⁺, it is for the calculated value of Buddhist nun's molecular ion peak according to Shandong：441.20335[M+H]⁺, Absolute error is 0.71ppm, meets high resolution mass spectrum error range, and measured value is consistent with theoretical value.(referring to Fig. 5).

Embodiment 3：Impurity A：(R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls] piperidin-1-yl) -3- chloropropyl -1- ketone synthesis.

Under nitrogen protection, 50m1 dichloromethane is added into 100mL there-necked flask, intermediate -1 is sequentially added under stirring： (R) -3- (4- Phenoxyphenyls) -1- (piperidines -3- bases) -1H- pyrazoles [3,4-d] pyrimidine -4- amine (YLTN-1) (1.00g, 1eq), DIPEA (0.40g, 1.2eq), is cooled to -20~-10 DEG C, start to be added dropwise 3- chlorpromazine chlorides (1.46g, 1eq), completion of dropping, solution is become by muddiness to be clarified, and continues to stir 20~30 minutes, and LC-MS detections, raw material is disappeared, and decompression is steamed Evaporate, dichloromethane is evaporated off to being steamed without cut, obtains crude product and is purified with column chromatography method, elution ratio is：Methanol: ethyl acetate =1: 10, the common 400mL of eluent is collected, vacuum distillation is evaporated off solvent and extremely steamed without cut.Obtaining off-white powder 830mg is (R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases] piperidin-1-yl) -3- chlorine third Base -1- ketone.¹H-NMR (400Mz, CDCl₃)δ：8.382 (d, J=13.2Hz, 1H), 7.660~7.638 (m, 2H), 7.419~ 7.380 (m, 2H), 7.200~7.083 (m, 5H), 5.751 (m, 2H), 4.891~4.853 (m, 1H), 4.842~4.812 (m, 0.5H), 4.558 (m, 0.5H), 4.066~4.033 (m, 0.5H), 3.844~3.833 (m, 1H), 3.773~3.746 (m, 0.5H), 3.355 (m, 0.5H), 3.191 (m, 0.5H), 2.877~2.843 (m, 2H), 2.812~2.795 (m, 1H), 2.434 ~2.270 (m, 2H), 2.048~1.955 (m, 1H), 1.756~1.689 (m, 1H).(referring to Fig. 6) ESI-HRMS spectrograms are shown Molecular ion peak m/z=477.17930 [MW+H]+, corresponding molecular weight and the structural formula calculated value of offer (476.17275) it is consistent.Absolute error is 1.52ppm, within high resolution mass spectrum error range.(referring to Fig. 7).Impurity A exists The relative retention time (RRT) of Buddhist nun is replaced to be about 1.06 according to Shandong with respect to principal component on HPLC spectrograms.

Embodiment 4：Impurity B：(R) -8- (1- acryloylpiperidine -3- bases) -10- (4- Phenoxyphenyls) -3,4- dihydros The synthesis of pyrazolo [4,3-e] pyrimido [1,2-c] pyrimidine -2 (8H) -one.

Nitrogen is protected, and 100ml dichloromethane is added into 250mL there-necked flask, intermediate -1 is sequentially added under stirring： (R) -3- (4- Phenoxyphenyls) -1- (piperidines -3- bases) -1H- pyrazoles [3,4-d] pyrimidine -4- amine (YLTN-1) (2.00g, 1eq), DIPEA (1.30g, 2.0eq) is cooled to -20 DEG C~-10 DEG C.Start be added dropwise acrylic anhydride (1.33g, 2eq), it is added dropwise below -10 DEG C of process temperature control；Drop finishes, and continues to stir 20~30 minutes, and LC-MS detections have target product generation, 30~40 DEG C of temperature control, vacuum：- 0.08MPa, vacuum distillation is evaporated off dichloromethane and extremely steamed without cut；Obtain crude product post layer Methods For Purification is analysed, elution ratio is：Methanol: ethyl acetate=1: 5, collect eluent, 30~40 DEG C of temperature control, vacuum：- 0.08MPa, vacuum distillation is evaporated off solvent and extremely steamed without cut.It is (R) -8- (1- acryloyl group piperazines to obtain off-white powder 531mg Pyridine -3- bases) -10- (4- Phenoxyphenyls) -3,4- dihydro-pyrazolos [4,3-e] pyrimido [1,2-c] pyrimidine -2 (8H) -one.¹H-NMR (400Mz, CDCl₃)δ：8.352 (m, 2H), 7.874 (m, 2H), 7.367~7.328 (m, 2H), 7.144~7.050 (m, 5H), 6.591~6.558 (m, 1H), 6.335~6.293 (m, 1H), 5.747~5.658 (m, 1H), 4.803 (m, 1H), 4.649~4.622 (m, 0.5H), 4.300 (m, 0.5H), 4.333~4.300 (m, 2H), 4.153 (m, 0.5H), 4.051~ 4.019 (m, 0.5H), 3.751~3.680 (m, 0.5H), 3.354~3.217 (m, 1H), 2.902 (m, 0.5H), 2.789~ 2.770 (m, 2H), 2.397~2.223 (m, 2H), 2.194~2.022 (m, 1H), 2.005~1.718 (m, 1H) (refer to figure 8).ESI-HRMS spectrograms show molecular ion peak m/z=495.21580 [MW+H]⁺, corresponding molecular weight and the structure provided Formula calculated value (494.20664) is consistent.Absolute error is 3.82ppm, and (figure is referred within high resolution mass spectrum error range 9).Impurity B replaces the relative retention time (RRT) of Buddhist nun to be about 0.97 with respect to principal component on HPLC spectrograms according to Shandong.

Embodiment 5：Impurity C：(R) -1-3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls] piperidin-1-yl)-ethyl ketone synthesis

By intermediate -1：(R) -3- (4- Phenoxyphenyls) -1- (piperidines -3- bases) -1H- pyrazoles [3,4-d] pyrimidine -4- amine (YLTN-1) (2.00g, 1eq) is dissolved in dichloromethane (50ml), addition N, N- diisopropyl ethyl amines (0.80g, 2eq), - 10 DEG C are cooled to, nitrogen protection is added dropwise acetic anhydride (0.53g, 1eq), finished, reacts 30 minutes, LC-MS monitoring has been reacted Into system being concentrated under reduced pressure into dry, residue passes through silica gel column chromatography, eluent：Ethyl acetate: methanol=10: 1, obtain class white Color powder 1.38g be (R) -1-3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases] piperidines - 1- yls)-ethyl ketone.¹H-NMR (400Mz, CDCl₃)δ：8.352 (d, J=16Hz, 1H), 7.657~7.623 (m, 2H), 7.402~ 7.364 (m, 2H), 7.187~7.069 (m, 5H), 5.971 (m, 2H), 4.849~4.841 (m, 0.5H), 4.841~4.821 (m, 1H), 4.574~4.541 (m, 0.5H), 4.045~4.005 (m, 0.5H), 3.864~3.831 (m, 0.5H), 3.744~ 3.685 (m, 0.5H), 3.318~3.253 (m, 0.5H), 3.201~3.141 (m, 0.5H), 2.802~2.745 (m, 0.5H), 2.403~2.249 (m, 2H), 1.962~1.697 (m, 2H) (referring to Figure 10)；ESI-HRMS spectrograms show molecular ion peak m/z =429.20416 [MW+H]⁺, corresponding molecular weight is consistent with the structural formula calculated value (428.19607) provided.Definitely Error is 1.89ppm, and (Figure 11 is referred to) within high resolution mass spectrum error range.Impurity C relative principal components on HPLC spectrograms The relative retention time (RRT) that Buddhist nun is replaced according to Shandong is about 0.96.

Embodiment 6：Impurity D：(R) -4- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls)-piperidines -1- carbonyls) amylene -4- acid synthesis

Under nitrogen protection, Buddhist nun 1- [(3R) -3- [4- amino -3- (4- Phenoxyphenyls) -1H pyrazolos [3,4- will be replaced according to Shandong D] pyrimidine -1- bases] -1- piperidyls] -2- propylene -1- ketone (2g, 1eq) is dissolved in dichloromethane (50ml), sequentially adds N, N- Diisopropyl ethyl amine (0.70g, 2eq), acrylic acid (0.98g, 3eq), reaction is warming up to 35 DEG C, stirs 36 hours, by system It is concentrated under reduced pressure into dry, residue passes through silica gel column chromatography, eluent：Ethyl acetate: methanol=8: 1, obtain off-white powder 103mg For (R) -4- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-piperidines -1- carbonyls) penta Alkene -4- acid,¹H-NMR (400Mz, DMSO-d₆)δ：12.309 (br, 1H), 8.263 (S, 1H), 7.687~7.666 (m, 2H), 7.422~7.461 (m, 2H), 7.122~7.211 (m, 5H), 6.009~6.072 (m, 0.5H), 5.570~5.660 (m, 0.5H), 4.647~4.772 (m, 1H), 4.516~4.547 (m, 0.5H), 4.200-4.232 (m, 0.5H), 4.069-4.101 (m, 0.5H), 3.913~3.945 (m, 0.5H), 3.629-3.655 (m, 0.5H), 3.129~3.157 (m, 1H), 2.878 (m, 0.5H), 2.445-2.513 (m, 4H), 2.206~2.264 (m, 1H), 2.085~2.125 (m, 1H), 1.861~1.936 (m, 1H), 1.522~1.666 (m, 1H) (referring to Figure 12)；ESI-HRMS spectrograms show molecular ion peak m/z=513.22606 [MW+ H]⁺, corresponding molecular weight is consistent with the structural formula calculated value (512.21720) provided.Absolute error is 3.09ppm, (Figure 13 is referred within high resolution mass spectrum error range).Impurity D replaces Buddhist nun's relative with respect to principal component on HPLC spectrograms according to Shandong Retention time (RRT) is about 1.02.

Embodiment 7：Impurity H：(R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls)-piperidin-1-yl)-propyl group -1- ketone synthesis

Under nitrogen protection, by intermediate -1：(R) -3- (4- Phenoxyphenyls) -1- (piperidines -3- bases) -1H- pyrazoles [3,4- D] in pyrimidine -4- amine (YLTN-1) (2.00g, 1eq) dissolving dichloromethane (50mL), add N, N- diisopropyl ethyl amines (0.80g, 2eq), is cooled to -10 DEG C, nitrogen protection is added dropwise propionic andydride (0.67g, 1eq), finished, reacts 30 minutes, LC-MS Monitoring, reaction is completed, and system is concentrated under reduced pressure into dry, and residue passes through silica gel column chromatography, eluent：Ethyl acetate: methanol= 10: 1, obtaining lightpink powder 1.72g, ((4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] are phonetic by 3- for (R) -1- Pyridine -1- bases)-piperidin-1-yl)-propyl group -1- ketone.¹H-NMR (400Mz, CDCl₃)δ：8.338~8.302 (m, 2H), 7.658~ 7.627 (m, 2H), 7.440~7.400 (m, 2H), 7.224~7.097 (m, 5H), 4.881~4.634 (m, 1H), 4.881~ 4.826 (m, 1H), 4.108~3.094 (m, 1H), 2.762 (m, 1H), 3.310~3.167 (m, 1H), 2.452~2.315 (m, 4H), 1.213~1.157 (m, 3H), 2.069~1.691 (m, 2H) (referring to Figure 14)；ESI-HRMS spectrograms show molecular ion Peak m/z=443.25380 [MW+H]⁺, corresponding molecular weight and structural formula calculated value (442.21172) phase provided Symbol.Absolute error is 3.66ppm, and (Figure 15 is referred to) within high resolution mass spectrum error range.Impurity H is relative on HPLC spectrograms Principal component is about 1.04 for the relative retention time (RRT) of Buddhist nun according to Shandong.

Embodiment 8：Impurity E：1- ((R) -1- acryloylpiperidine -3- bases) -4- amino -3- (4- Phenoxyphenyls) -1H- The preparation of pyrazolo [3,4-d] pyrimidine -1- oxides

(1) preparation of impurity E

Prepared by Example 2 replaces Buddhist nun about 5.17g without three recrystallizations of isopropanol according to Shandong, puts in 500ml reaction bulbs, Plus the dissolving of the acetonitrile-waters of 150ml 80%, then add 30% hydrogen peroxide 30ml, it is stirred overnight at room temperature, reaction terminates, adds into reaction solution Enter sodium thiosulfate, to bubble-free produce, 35 DEG C be concentrated under reduced pressure into no liquid outflow, then add 3 times of aqueous phase volumes ethyl acetate, Extraction, collect organic phase, merge, 35 DEG C be concentrated under reduced pressure into it is dry, get Yi Lu replace Buddhist nun's impurity E crude product 2.65g.LC-MS detects crude product Purity is 51.26%, replaces Ni Feng relative retention time to be 0.98, mass-to-charge ratio [MW+H] relative to according to Shandong in liquid chromatogram⁺For 457.2。

(2) purification of impurity E crude product

The impurity E crude product of the above-mentioned preparations of 2.95g is dissolved into the solution that concentration is about 150mg/ml with DMSO, using following Preparative chromatography is further purified：

Chromatographic column (purchased from Ai Jieer science and technology)：Using 10 μm of C18 fillers of particle diameter, post (50mm is filled after being homogenized with isopropanol ×250mm)；

Mobile phase：Water and acetonitrile

Flow velocity：120ml/min；

Detection wavelength：254nm；

Sample size：10ml

Gradient elution, elution program is：

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	65	35
10	65	35
			30	45	55
50	45	55
			50.01	20	80
55	20	80
			55.01	65	35
60	65	35

It is the cut corresponding to 30~37min peak to collect retention time；LC-MS or HPLC checked for impurities E purity.

35 DEG C of collected cut to no liquids that are concentrated under reduced pressure flow out, and add the ethyl acetate extraction of 3 times of volumes, and collection has Machine phase, merges, plus anhydrous sodium sulfate drying, filtering, and 30 DEG C of filtrate is concentrated under reduced pressure into dry, obtains 2.06g light pink solids, passes through HPLC is determined, and purity is 98.97%, and total recovery is 36.6%.

(3) Structural Identification of impurity E

Impurity E¹H-NMR figures are referring to Figure 16, and high resolution mass spectrum is referring to Figure 17.

The high resolution mass spectrum (ESI sources) of impurity E shows its mass-to-charge ratio [M+H]⁺For 457.19930, corresponding molecular weight Absolute error with the structural formula calculated value 456.19099 of offer is 2.27ppm, meets the error model of high resolution mass spectrum Enclose.The structure of Buddhist nun is replaced according to Shandong with reference to formula (2), with reference to impurity E¹H-NMR、¹³C-NMR spectrums identify its structural formula as shown in following formula E.

Impurity E high resolution mass spectrum shows that molecular formula of its molecular weight with being provided is consistent completely；

The nuclear magnetic resonance of the impurity E of table 1¹H-NMR、¹³C-NMR analysis results

The shown information of the proton nmr spectra of impurity E, carbon spectrum can be to the formula E molecular structure of compounds formulas that are provided Upper hydrogen, carbon all belong to.Therefore, it is consistent according to Shandong for Buddhist nun's impurity E with the formula E molecular structural formulas provided.

Embodiment 9：Impurity F：1,3- bis- ((4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] is phonetic by (R) -3- Pyridine -1- bases) piperidin-1-yl) propane -1- ketone and impurity G：1- ((R) -3- (4- ((3- ((R) -3- (4- amino -3- (4- phenoxy groups Phenyl) -1H- pyrazoles [3,4-d] pyrimidine -1- bases) piperidin-1-yl) -3- oxopropyls) ammonia) -3- (4- Phenoxyphenyls) -1H- Pyrazoles [3,4-d] pyrimidine -1- bases) piperidin-1-yl) propyl group -2- alkene -1- ketone preparation.

(1) impurity F and impurity G preparation

The preparation of about 20g embodiments 2 is added in 1000ml reaction bulbs replaces Buddhist nun without three recrystallizations of isopropanol according to Shandong, 250ml acetonitriles and 200ml 5M sodium hydrate aqueous solutions, 80 DEG C of heating in water bath for reaction 40min are added, room temperature, plus 5M is cooled to HCl is neutralized to neutrality, and 35 DEG C are concentrated under reduced pressure into no liquid and reserve, and adds the ethyl acetate extraction of 5 times of aqueous phase volumes, collects upper strata Organic phase, merges, and 35 DEG C are concentrated under reduced pressure into dry, obtain according to Shandong for Buddhist nun's impurity F, G crude products 18.72g, LC-MS detection crude product purity, According to correspondence MS quasi-molecular ions, the relative relative retention time for replacing Ni Feng according to Shandong is about 0.94 (impurity F) and 1.15 in liquid chromatogram The peak of (impurity G) is that the content in object, its crude product is respectively 16.9% (impurity F) and 18.4% (impurity G).

(2) purification of impurity F and impurity G crude products

DMSO is added to be dissolved into 10ml solution, loading the crude product of the 18.72g impurity Fs of above-mentioned preparation, G：

The middle standby post of compacting (being purchased from Agela companies)：Agela XBP C18 fillers, 480g, packing material size：20~35 μm

Mobile phase：Water (0.05%TFA) and pure acetonitrile

Flow velocity：40ml/min

Detection wavelength：254nm

Applied sample amount：~5g

Gradient elution, elution program is：

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	75	25
5	75	25
			30	65	35
40	65	35
			50	50	50
55	50	50
			55.01	75	25
60	75	25

The cut at all peaks is collected respectively, and LC-MS or HPLC confirm target product.Collect 30~33min cut (impurity F) and 40~44min cut (impurity G), merge identical cut, be concentrated under reduced pressure respectively, to no liquid outflow, 3 are added in aqueous phase The ethyl acetate extraction of times volume, collects organic phase, plus anhydrous sodium sulfate drying, and filtrate is collected in filtering.Be concentrated under reduced pressure into it is dry, Get Yi Lu replaces Buddhist nun's impurity G secondary separation crude products 2.57g for Buddhist nun's impurity F sterling 2.21g and Yi Lu respectively.Examined through LC-MS or HPLC Analysis is surveyed, replaces the purity of Buddhist nun's impurity F to be 98.15% according to Shandong, replaces the purity of Buddhist nun's impurity G secondary separation crude products to be 85.32% according to Shandong.

(3) impurity G secondary separation

The 2.57g of above-mentioned preparation adds 10ml DMSO to dissolve according to Shandong for Buddhist nun's impurity G secondary separation crude products

The condition used according to Shandong for Buddhist nun's impurity G secondary separations for：

Chromatographic column：Waters XBridge C8 30mm × 100mm, 5 μm

Mobile phase：Water and acetonitrile

Detection wavelength：254nm

Flow velocity：25ml/min

Gradient elution, elution program is as follows：

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	75	25
5	75	25
			15	35	65

25	35	65
			25.01	75	25
30	75	25

Sampling volume：2ml/ times every time；

Retention time 26min~30min cut is collected, and confirms object purity with LC-MS.Merge identical cut, 35 DEG C are concentrated under reduced pressure into no acetonitrile outflow, plus the ethyl acetate of 3 times of volumes is extracted, merging organic phase, plus anhydrous sodium sulfate drying, Filtering, be concentrated under reduced pressure into it is dry, obtain according to Shandong replace Buddhist nun impurity G sterling 1.64g, white solid powder, LC-MS detection purity be 95.13%.

(4) Structural Identification of impurity F

Impurity F¹H-NMR figures are referring to Figure 18, and high resolution mass spectrum is referring to Figure 19.

The high resolution mass spectrum (ESI sources) of impurity F shows its mass-to-charge ratio [M+H]⁺For 827.39065, corresponding molecular weight Absolute error with the structural formula calculated value 826.34520 of offer is 2.16ppm, meets the error model of high resolution mass spectrum Enclose.The high resolution mass spectrum of sample shows that molecular formula of its molecular weight with being provided is consistent completely.With reference to the knot that Buddhist nun is replaced according to Shandong Structure, with reference to this product¹H-NMR、¹³C-NMR, H-HCOSY, HMBC, HSQC, DEPT spectrum identify its structural formula as shown in following formula F.

Table 2 is impurity F¹H-NMR, H-HCOSY and HMBC parse data

The impurity F of table 3¹³C-NMR, DEPT, HSQC, HMBC data are parsed

By sample¹Knowable to H-NMR, H-HCOSY and HMBC spectrum, the knot of Hydrogen Proton number and formula F compounds in its structure Structure is consistent.By sample¹³Knowable to C-NMR, DEPT, HSQC and HMBC spectrum, carbon number and the structure of formula F compounds in its structure It is consistent.Nucleus magnetic hydrogen spectrum, carbon spectrum and two-dimentional modal data can carry out rationally belonging to and associating to the hydrogen on structural formula and carbon.

(5) impurity G Structural Identification

Impurity G's¹H-NMR figures are referring to Figure 20, and high resolution mass spectrum is referring to Figure 21.

Impurity G high resolution mass spectrum (ESI sources) shows its mass-to-charge ratio [M+H]⁺For 881.40140, corresponding molecular weight Absolute error with the structural formula calculated value 880.39215 of offer is 1.43ppm, meets the error model of high resolution mass spectrum Enclose.The high resolution mass spectrum of sample shows that molecular formula of its molecular weight with being provided is consistent completely.With reference to the knot that Buddhist nun is replaced according to Shandong Structure, with reference to this product¹H-NMR、¹³C-NMR, DEPT, H-HCOSY, HMBC and hsqc spectrum identify its structure as shown in following formula G.

Table 4 is impurity G's¹H-NMR, H-HCOSY and HMBC data and hydrogen ownership

Table 5 is impurity G's¹³C-NMR, DEPT, HSQC and HMBC data and carbon ownership

According to sample¹H-NMR is composed, with reference to knowable to H-HCOSY, HMBC two-dimensional spectrum, Hydrogen Proton number and formula G in its structure The structure of compound is consistent.By sample¹³Knowable to C-NMR, DEPT, HSQC and HMBC spectrum, carbon number and formula Gization in its structure The structure of compound is consistent.

Embodiment 10：Analyzed according to Shandong for Buddhist nun about the HPLC of material

Using impurity external standard method (version Chinese Pharmacopoeia in 2010), analysis replaces relevant material in Buddhist nun according to Shandong, and with external standard method pair Each impurity is quantified.The quantitative of impurity is calculated according to external standard method, referring specifically to two annex V of Chinese Pharmacopoeia 2010 edition D.Method for carrying out the analysis is the gradient HPLC methods according to the present invention.The chromatographic condition used is as follows：

Chromatographic condition：

Chromatographic column：Agilent Poroshell EC C₁₈Post 4.6 × 150mm, 2.7 μm

The concentration of need testing solution：1mg/ml

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

Mobile phase：0.06M ammonium formate solutions (A) (formic acid adjusts pH to 3.5), acetonitrile-methanol, 45: 55 (B)

Detection wavelength：260nm

Diluent：80% acetonitrile-water

Column temperature：50℃

Flow velocity：0.8ml/min

Gradient design is as described below.

Sample preparation：

Prepare impurity positioning solution

(1) compound A~H standard setting solution：Compound A~H 10mg is taken respectively, it is accurately weighed, 100ml is put respectively In volumetric flask, plus acetonitrile ultrasound makes dissolving, and is shaken up with dilution in acetonitrile to scale, and the solution that concentration is about 100 μ g/ml is made, It is used as compound A~H stock solutions.

(2) mark-on mixed solution：Take above-mentioned preparation according to Shandong for Buddhist nun's bulk drug 100mg, it is accurately weighed, put 100mg measuring bottles In, then accurate addition compound A~each 1ml of H stock solutions, plus 80% acetonitrile-water is in right amount, ultrasound makes dissolving, then uses 80% second Nitrile-water is diluted to scale, shakes up, and every 1ml is made and contains molten for Buddhist nun about 1mg, A containing compound~H about 1 μ g respectively mixing according to Shandong Liquid, is used as mark-on mixed solution.

(3) prepare compound A~H reference standard solutions or reference substance solution：Precision measures compound A~H stock solutions 1.0ml, puts in 100ml volumetric flasks, is diluted to scale with 80% acetonitrile-water, shakes up, and every 1ml is made containing about compound A~H points Not Wei 1 μ g solution, be used as compound A~H reference standard solution or reference substance solution.

(4) prepare and replace Buddhist nun's need testing solution according to Shandong：Take above-mentioned preparation according to Shandong for Buddhist nun's bulk drug 10mg, it is accurately weighed, put In 10ml volumetric flasks, plus 80% acetonitrile-water is appropriate, and ultrasound makes dissolving, and is diluted to scale with 80% acetonitrile-water, shakes up, both .The concentration that Buddhist nun's bulk drug need testing solution is replaced according to Shandong is about 1.0mg/ml.

(5) preparation of Buddhist nun's capsule need testing solution is replaced according to Shandong：Take according to Shandong that (Imbruvica is purchased from for Buddhist nun's capsule Pharmacyclics Janssen companies, 140mg, lot number：L0404951) the finely ground powder of content is appropriate (replaces Buddhist nun containing about according to Shandong 10mg), it is accurately weighed, put in 10ml volumetric flasks, plus 80% acetonitrile-water is appropriate, ultrasound makes dissolving, and dilute with 80% acetonitrile-water Release to scale, shake up, filtered with 0.22 μm of organic syringe filter, discard primary filtrate 2ml, take subsequent filtrate, Buddhist nun is replaced as according to Shandong Capsule need testing solution.The concentration that Buddhist nun's capsule need testing solution is replaced according to Shandong is about 1.0mg/ml.

Embodiment 11：

The preparation be the same as Example 2 of Buddhist nun, sample preparation be the same as Example 10, gradient design be the same as Example 10 are replaced according to Shandong.

Chromatographic condition：

Chromatographic column：Agilent Poroshell EC C₁₈Post 4.6 × 150mm, 2.7 μm

The concentration of need testing solution：1mg/ml

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

Mobile phase：0.06M ammonium formate solutions (A) (formic acid adjusts pH to 2.8), acetonitrile-methanol, 50: 50 (B)

Detection wavelength：260nm

Diluent：80% acetonitrile-water

Column temperature：50℃

Flow velocity：0.8ml/min

Embodiment 12：

Chromatographic condition：

Chromatographic column：Agilent Poroshell EC C₁₈Post 4.6 × 150mm, 2.7 μm

The concentration of need testing solution：1mg/ml

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

Detection wavelength：260nm

Diluent：80% acetonitrile-water

Column temperature：40℃

Flow velocity：0.8ml/min

Embodiment 13：

Chromatographic condition：

Chromatographic column：Agilent Poroshell EC C₁₈Post 4.6 × 150mm, 2.7 μm

The concentration of need testing solution：1mg/ml

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

Mobile phase：0.06M ammonium formate solutions (A) (formic acid adjusts pH to 2.8), acetonitrile-methanol, 45: 55 (B)

Detection wavelength：260nm

Diluent：80% acetonitrile-water

Column temperature：40℃

Flow velocity：0.8ml/min

Embodiment 14：

Chromatographic condition：

Chromatographic column：Agilent Poroshell EC C₁₈Post 4.6 × 150mm, 2.7 μm

The concentration of need testing solution：1mg/ml

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

Mobile phase：0.06M ammonium formate solutions (A) (formic acid adjusts pH to 3.5), acetonitrile-methanol, 55: 45 (B)

Detection wavelength：260nm

Diluent：80% acetonitrile-water

Column temperature：50℃

Flow velocity：0.6ml/min

Embodiment 15：

Chromatographic condition：

Chromatographic column：Agilent Poroshell EC C₁₈Post 4.6 × 150mm, 2.7 μm

The concentration of need testing solution：1mg/ml

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

Detection wavelength：260nm

Diluent：80% acetonitrile-water

Column temperature：50℃

Flow velocity：1.0ml/min

Embodiment 16：

Chromatographic condition：

Chromatographic column：Agilent Poroshell EC C₁₈Post 4.6 × 150mm, 2.7 μm

The concentration of need testing solution：1mg/ml

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

Mobile phase：0.05M ammonium formate solutions (A) (formic acid adjusts pH to 3.0), acetonitrile-methanol, 50: 50 (B)

Detection wavelength：260nm

Diluent：80% acetonitrile-water

Column temperature：45℃

Flow velocity：0.8ml/min

Embodiment 17：

Chromatographic condition：

Chromatographic column：Agilent Poroshell EC C₁₈Post 4.6 × 150mm, 2.7 μm

The concentration of need testing solution：1mg/ml

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

Mobile phase：0.03M ammonium formate solutions (A) (formic acid adjusts pH to 3.5), acetonitrile-methanol, 45: 55 (B)

Detection wavelength：260nm

Diluent：80% acetonitrile-water

Column temperature：45℃

Flow velocity：0.8ml/min

Embodiment 18：

Chromatographic condition：

Chromatographic column：Agilent Poroshell EC C₁₈Post 4.6 × 150mm, 2.7 μm

The concentration of need testing solution：1mg/ml

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

Mobile phase：0.1M ammonium formate solutions (A) (formic acid adjusts pH to 3.5), acetonitrile-methanol, 45: 55 (B)

Detection wavelength：260nm

Diluent：80% acetonitrile-water

Column temperature：45℃

Flow velocity：0.6ml/min

Test procedure：

(1) under gradient HPLC methods of the present invention, on Agilent 1260HPLC instruments (Agilent companies of the U.S.), According to the chromatographic condition of embodiment 16, take compound A~H positioning solution, mark-on mixed solution, compound A~H reference solutions, Each 10 μ l of Buddhist nun's capsule need testing solution are replaced for Buddhist nun's need testing solution, according to Shandong according to Shandong, liquid chromatograph is injected, records chromatogram.

(2) under gradient HPLC methods of the present invention, according to the chromatographic condition of embodiment 10~18, mark-on mixed solution 10 is taken μ l, inject liquid chromatograph, record chromatogram.

Result of the test：

Compound A~H and principal component are shown in Table according to Shandong for positioning result of the Buddhist nun in the present embodiment 16 under gradient HPLC methods 1；Under the chromatographic condition of the embodiment of the present invention 10~18, separating degree, each impurity in mark-on mixed solution between each impurity with According to Shandong 2 are shown in Table for Ni Feng relative retention time.

Using the chromatographic condition of embodiment 16, according to containing for the compound A~H contained in external standard method calculating need testing solution Amount and total impurities amount, the results are shown in Table 3, the HPLC spectrograms of mark-on mixed solution, according to Shandong for Buddhist nun's need testing solution HPLC spectrograms, according to Figure 22~Figure 24 is seen in Shandong respectively for the HPLC spectrograms of Buddhist nun's capsule need testing solution, wherein as shown in figure 22, the highest in Figure 23-24 Peak is the peak that Buddhist nun is replaced according to Shandong.

Buddhist nun's capsule is replaced about the analysis of material according to compound A~H and principal component positioning result and according to Shandong for Buddhist nun and Yi Lu As a result, according in embodiment according to Shandong for Buddhist nun preparation method prepare according to Shandong for Buddhist nun's raw material relevant material it is as follows：Replaced according to Shandong For Ni Feng retention time be about 16.48min according to Shandong in the chromatogram of Buddhist nun's need testing solution, retention time be about 13.75min, The impurity of relative retention time 0.83 is compound C；Retention time is about that 15.85min, the impurity of relative retention time 0.96 are Compound D；Retention time be about 18.55min, relative retention time 1.13 impurity be compound H；Retention time is about 18.55min, the impurity of relative retention time 1.38 are compound F；Retention time is about 33.50min, relative retention time 2.04 impurity be compound G；Replaced according to Shandong in Buddhist nun's need testing solution and do not detect compound A, B, E.

Replaced according to Shandong in Buddhist nun's capsule (Imbruvica) need testing solution and detected compound A, B, C, F, G and H, wherein chemical combination Thing A, F content are more than 0.1%.

The compound A of table 1~H and the embodiment of the present invention 2 prepare according to Shandong for positioning of the Buddhist nun in the method for embodiment 16 and each Separating degree between peak

The relative retention time of each impurity under the conditions of the embodiment 10~18 of table 2

The embodiment 2 of table 3 prepare according to Shandong for Buddhist nun's bulk drug and Yi Lu replace in Buddhist nun's capsule (Imbruvica) relevant material according to The testing result of the chromatographic condition of embodiment 16

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

1. detect Formulas I according to Shandong replace Buddhist nun or its solvate, or containing according to Shandong for Buddhist nun medicine sample purity method, including By the impurity in chromatography determination sample, one or more of the impurity in compound A-H,

Wherein Buddhist nun is replaced to be 1- [(3R) -3- [4- amino -3- (4- Phenoxyphenyls) -1H pyrazolos [3,4-d] pyrimidine -1- according to Shandong Base] -1- piperidyls] -2- propylene -1- ketone, structure is shown in formula I：

Compound A be (R) -1- (3- (and 4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases] piperazine Pyridine -1- bases) -3- chloropropyl -1- ketone, and with following structure：

Compound B be (R) -8- (1- acryloylpiperidine -3- bases) -10- (4- Phenoxyphenyls) -3,4- dihydro-pyrazolos [4, 3-e] pyrimido [1,2-c] pyrimidine -2 (8H) -one, and with following structure：

Compound C be (R) -1-3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases] piperidines - 1- yls)-ethyl ketone, and with following structure：

Compound D is (R) -4- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-piperazines Pyridine -1- carbonyls) amylene -4- acid, and with following structure：

Compound E be 1- ((R) -1- acryloylpiperidine -3- bases) -4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3, 4-d] pyrimidine -1- oxides, and with following structure：

Compound F is ((R) -3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3, the 4-d] pyrimidine -1- bases) piperazines of 1,3- bis- Pyridine -1- bases) propane -1- ketone, and with following structure：

Compound G is that ((((((4- amino -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] is phonetic by (R) -3- by 3- by 4- by (R) -3- by 1- Pyridine -1- bases) piperidin-1-yl) -3- oxopropyls) ammonia) -3- (4- Phenoxyphenyls) -1H- pyrazoles [3,4-d] pyrimidine -1- bases) piperazine Pyridine -1- bases) propyl group -2- alkene -1- ketone, and with following structure：

Compound H is (R) -1- (3- (4- amino -3- (4- Phenoxyphenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-piperazines Pyridine -1- bases)-propyl group -1- ketone, and with following structure：

Wherein methods described comprises the following steps：

(1) by according to Shandong for Buddhist nun or its solvate or containing according to Shandong for the medicine dissolving of Buddhist nun in a solvent to prepare sample solution；

(2) dissolved any one or more of in compound A~H in a solvent to prepare reference standard solution；

(4) by referring to the one or more of compound A-H in the reference standard solution, determine and replace Buddhist nun or its solvate according to Shandong Or containing according to presence of the Shandong for any one or more of compound A~H in the medicine of Buddhist nun, wherein the chromatography is gradient HPLC, wherein the stationary phase that the chromatography is used is octadecylsilane chemically bonded silica, in the gradient HPLC methods, stream Dynamic mutually to include the combination of A containing cushioning liquid and organic solvent B, wherein in gradient HPLC methods, mobile phase includes following gradient Design：

Wherein described cushioning liquid A is the aqueous solution of ammonium formate and formic acid, and the organic solvent B is the combination of acetonitrile and methanol,

Wherein the concentration of ammonium formate is 0.01M to 0.1M, and the pH of the aqueous solution of ammonium formate and formic acid is 2.7-4.8, methanol and second The mixed volume ratio of nitrile is 40: 60,45: 55,55: 45, or 50: 50.

2. the method for claim 1 wherein the solvate that Buddhist nun is replaced according to Shandong is the hydrate according to Shandong for Buddhist nun.

3. the method for claim 1 wherein the gradient HPLC is the HPLC method compatible with LC-MS.

4. the method for claim 1 wherein the concentration of ammonium formate is 0.03-0.08M, 0.04-0.06M or 0.05M.

5. the method for claim 1 wherein the pH of ammonium formate and the aqueous solution of formic acid be 2.8~4.7,2.8~4.5,2.8~ 4.0 or 2.8~3.5.

6. the method for claim 1 wherein the HPLC methods are entered at a temperature of 30~60 DEG C, 35~55 DEG C or 40~50 DEG C OK.

7. the method for claim 1 wherein the HPLC methods are in 0.4~1.2ml/min, 0.5~1.1ml/min or 0.6~ Carried out under 1.0ml/min flow velocity.

8. the method described in claim any one of 1-7, wherein comprising for the medicine of Buddhist nun being solid form or liquid form according to Shandong, The solid form is selected from pulvis, tablet, capsule, pill and dispersible granule, and the liquid form is selected from solution Or suspension.