CN106153797A

CN106153797A - One replaces Buddhist nun and according to Shandong for Buddhist nun's preparation Related substance method according to Shandong

Info

Publication number: CN106153797A
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: 2016-11-23
Anticipated expiration: 2035-04-20
Also published as: CN106153797B

Abstract

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

Description

One replaces Buddhist nun and according to Shandong for Buddhist nun's preparation Related substance method according to Shandong

Technical field

The invention belongs to pharmaceutical synthesis field.In particular it relates to according to Shandong for Buddhist nun (1-[(3R)-3-[4- Amino-3-(4-Phenoxyphenyl)-1H pyrazolo [3,4-d] pyrimidine-1-base]-piperidino]-2-propylene-1- Ketone) impurity that occurs in preparation process and to the method being analyzed for Buddhist nun's preparation according to Shandong.

Background technology

First Ibrutinib is developed in 2007 by Celera Genomics company of the U.S., after transfer The Pharmacyclics company exploitation of California, subsidiary's Janssen Pharmaceutica (Jassen) of Johson & Johnson in 2011 Participate in cooperative development.There is no the Chinese translation of standard at present, therefore its transliteration at this is by the applicant " according to Shandong for Buddhist nun ".According to Shandong for the chemical name of Buddhist nun it is: 1-[(3R)-3-[4-amino-3-(4-phenoxy group benzene Base)-1H pyrazolo [3,4-d] pyrimidine-1-base]-piperidino]-2-propylene-1-ketone, structural formula is:

Patent WO201384572A discloses divides having carried out HPLC according to Shandong for the chemical purity of Buddhist nun Analysis.Enter for Buddhist nun's crude product (preparation in embodiment 1) according to Shandong according to the method for this patent Example 8 Row is analyzed, and the high performance liquid chromatograph (HPLC) used is Shimadzu LC-20A, PDA Detector (purchased from Shimadzu Corporation of Japan), result as it is shown in figure 1, wherein the summit in Fig. 1 be The peak of Buddhist nun is replaced according to Shandong.As shown in Figure 1, there is following drawback in the analysis method of this patent:

(1) according to Shandong, for the impurity contained in Buddhist nun's crude product, (under the method, retention time is about: 19.554min) Having interference to according to Shandong for Ni Zhuchengfenfeng, the two separating degree does not meets in ICH Q3A for having Requirement to specificity under related substance Method validation item, i.e. between impurity and main constituent point From degree less than 2.0；

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

(3) computational methods of the method impurity are area normalization method, owing to each impurity structure is different, Under the detection wavelength drafted, uv absorption situation is different, therefore, and area normalization method Each impurity real content in the final product can not be gone out by Accurate Determining.

(4) qualitative analysis is not carried out to according to Shandong for the specific impurities in Buddhist nun. In sum, the testing result of prior art can not the quality of actual response medicine.

Summary of the invention

The invention provides new, interchangeable method, for having for Buddhist nun's preparation for Buddhist nun and Yi Lu according to Shandong The qualitative and quantitative analysis of related substance, and carry out miscellaneous with these impurity as reference standard or reference substance The qualitatively and quantitatively analysis of matter.It is a further object to provide reference standard or reference substance, use Replace the impurity being referred to as A～H formed during Buddhist nun according to Shandong in preparation in detection.

The analysis that is used for of present invention offer replaces Buddhist nun or according to Shandong for the HPLC having related substance in Buddhist nun's preparation according to Shandong Method, wherein, flowing includes two or more liquid mutually, and the relative concentration of liquid is with predetermined Gradient and change.Inventor utilizes 8 kinds of impurity prepared and identify structure (claiming compound A～H) to use Yu Yilu for Buddhist nun or containing according to Shandong for Buddhist nun pharmaceutical preparation Related substance method reference standard or Reference substance.Prior art not yet discloses the structure of these impurity and impurity.

Therefore, the first aspect of the invention provides compound A, and it has a chemical name: (R)-1-(3-(4- Amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base] piperidin-1-yl)-3-chloropropyl-1- Ketone, and have a structure that

Second aspect provides compound B, and it has a chemical name: (R)-8-(1-acryloylpiperidine-3- Base)-10-(4-Phenoxyphenyl)-3,4-dihydro-pyrazolo [4,3-e] pyrimido [1,2-c] pyrimidine-2 (8H)-one, And have a structure that

3rd aspect provides compound C, and it has a chemical name: (R)-1-3-(4-amino-3-(4-benzene oxygen Base phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base] piperidin-1-yl)-ethyl ketone, and have a structure that

4th aspect provides compound D, and it has a chemical name: (R)-4-(3-(4-amino-3-(4-benzene oxygen Base phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base)-piperidines-1-carbonyl) amylene-4-acid, and have following Structure:

5th aspect provides compound E, and it has chemical name: 1-((R)-1-acryloylpiperidine-3- Base)-4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-oxide, and have following Structure:

6th aspect provides compound F, and it has chemical name: 1,3-bis-((R)-3-(4-amino-3-(4- Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) piperidin-1-yl) propane-1-ketone, and there is following knot Structure:

7th aspect provides compound G, and it has chemical name: 1-((R)-3-(4-((3-((R)-3-(4- Amino-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) piperidin-1-yl)-3-oxopropyl) Ammonia)-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) piperidin-1-yl) propyl group-2-alkene-1-ketone, And have a structure that

8th aspect provides compound H, and it has a chemical name: (R)-1-(3-(4-amino-3-(4-benzene oxygen Base phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base)-piperidin-1-yl)-propyl group-1-ketone, and there is following knot Structure:

Compound A～H is for intermediate, by-product or the degradation impurity in Buddhist nun's building-up process according to Shandong, It is suitable as reference standard or reference substance, for the quality control of this 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 former, in this work Never detect during skill is multiple batches of.

Impurity B: in the synthesis replacing Buddhist nun according to Shandong of Chinese Patent Application No. 201510165869.6 report In method second step amidation process, amidatioon occurs (be expressed as Buddhist nun's intermediate-1 according to Shandong YLTN-1) two positions, the nitrogen on piperidines nuclear nitrogen and pyrimidine ring, produce bisamideization and produce Thing impurity B-1, due to Thermodynamic effect, there is intramolecular cyclization in the latter, generates more stable pair Pyrimidine ring impurity B.

Impurity C: in the synthesis replacing Buddhist nun according to Shandong of Chinese Patent Application No. 201510165869.6 report In method second step amidation process, containing a small amount of acetic anhydride impurity in initiation material acrylic anhydride, with YLTN-1 occurs amidation process to generate impurity C.

Impurity D: in the synthesis replacing Buddhist nun according to Shandong of Chinese Patent Application No. 201510165869.6 report In method second step amidation process, according to Shandong for Buddhist nun (YLTN) and by-product acrylic acid urging at alkali (it is probably DIPEA, is likely to as replacing the nitrogen heterocyclic ring of Buddhist nun self according to Shandong) under change and occur Baylis-Hillman reacts and produces impurity D.

Impurity E, F, G are or to comprise according to Shandong for pharmaceutical preparation strong of Buddhist nun for Buddhist nun's active component according to Shandong Principal degradation impurity in system degraded sample, wherein impurity E is to replace Buddhist nun or according to Shandong for Buddhist nun's preparation system according to Shandong The primary product of oxidative degradation during Bei, impurity F, G are to replace Buddhist nun or according to Shandong for Buddhist nun's preparation system according to Shandong Catabolite during Bei, is also for forcing degraded or the main fall of hydrolysis under the conditions of Buddhist nun's alkalescence according to Shandong Hydrolysis products.

Impurity H: in the synthesis replacing Buddhist nun according to Shandong of Chinese Patent Application No. 201510165869.6 report In method second step amidation process, initiation material acrylic anhydride contains a small amount of propionic andydride impurity, can be with Intermediate-1 reacts and generates impurity H:

In a particularly preferred embodiment, it is singulation according to compound A～H of the present invention Compound, the purest form, preferably purity greater than about 95%, preferred purity greater than about 98%, Most preferably purity greater than about 99%, is preferably measured by HPLC.

A kind of detection is provided or to contain according to Shandong for Buddhist nun's for Buddhist nun according to Shandong according to the ninth aspect of the present invention The method of the sample purity of pharmaceutical dosage form, the method includes measuring according to Shandong for containing the present invention in Buddhist nun's sample Compound A～H in one or more.In the method for the invention, described compound is used as miscellaneous The reference standard of matter or reference substance.

According to the tenth aspect of the present invention, it is provided that a kind of method for characterization compound A～H, The method utilizes HPLC method to analyze according to Shandong for the described impurity A in Buddhist nun～H.Preferably this HPLC Method is the method that LC-MS is compatible.

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

On the other hand, the present invention also provides for a kind of chromatograph side replacing Buddhist nun's sample purity for detection according to Shandong Method, described method includes: by utilizing the reference standard according to the present invention or reference substance replacing enforcement Mode utilizes the reference standard according to the present invention or reference substance, measures compound A～H in sample In the existence of one or more.

Another aspect, it is provided that a kind of by measure containing according to Shandong for Buddhist nun sample in compound A～H Any one of or multiple existence detect according to Shandong for the chromatographic process of purity of Buddhist nun's sample, described side Method includes:

(1) Buddhist nun will be replaced according to Shandong or dissolve in a solvent to prepare sample for the formulation samples of Buddhist nun containing according to Shandong Product solution；

(2) sample of any one of compound A～H or multiple is dissolved in a solvent to prepare ginseng Than standard solution or reference substance solution；

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

(4) (a kind of or many by referring to known compound A-H present in this reference standard solution Kind) existence, measure the existence of any one of compound A～H or multiple in sample.

In one embodiment, this chromatographic process is liquid chromatography, as HPLC, UPLC, LC-MS；Preferably this chromatography is HPLC method, preferred gradient HPLC method.

Present invention preferably uses is fixing mutually for anti-phase.Suitably fix and include octadecylsilane key mutually Close silica gel or octyl group silane group silica gel.

In a preferred embodiment of the invention, it is provided that a kind of gradient HPLC method, wherein, flowing Include mutually containing buffer solution (A) and the combination of organic solvent (B).Preferably buffer solution (A) is for containing Water buffer, preferably acetate, formates, phosphate, trifluoroacetic acid, formic acid or theirs is mixed The aqueous solution of compound.Preferred buffer solution (A) is formates, most preferably ammonium formate and formic acid Or the aqueous solution formic acid of ammonium formate regulated the aqueous solution of pH.In particularly preferred embodiments The concentration that ammonium formate exists is about 0.01M to 0.1M, preferably 0.03-0.08M, more preferably 0.04-0.06M, most preferably 0.05M.Preferably organic solvent (B) is polar aprotic solvent, as methanol, Ethanol or isopropanol；Or dipolar aprotic solvent, such as acetonitrile.Preferably organic solvent (B) is selected from including first In the group of alcohol, acetonitrile, ethanol, isopropanol or their mixture, most preferably acetonitrile and methanol Mixture.

Flowing specifically preferred according to the invention comprise mutually ammonium formate and first aqueous acid (A) and acetonitrile and The combination of methanol (B).

Further provide for the gradient HPLC method according to the present invention, wherein flow and comprise following ladder mutually Degree 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, buffer solution (A) ammonium formate in HPLC method and formic acid The pH of aqueous solution be 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, this HPLC analyzes method and carries out at a temperature of about 30～60 DEG C, It is preferably 35～55 DEG C, 40～50 DEG C, most preferably 40～50 DEG C.

In other embodiments, this HPLC analyzes method under the flow velocity of about 0.4～1.2ml/min Carry out, preferably 0.5～1.1ml/min, 0.6～1.0ml/min, most preferably 0.6～1.0ml/min.

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

HPLC method according to the present invention effectively detects with single operation and quantitatively includes that those are selected from All impurity of the compound in following compound:

Compound A:(R)-1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1- Base] piperidin-1-yl)-3-chloropropyl-1-ketone.

Compound B:(R)-8-(1-acryloylpiperidine-3-base)-10-(4-Phenoxyphenyl)-3,4-dihydro pyrrole Azoles also [4,3-e] pyrimido [1,2-c] pyrimidine-2 (8H)-one.

Compound C:(R)-1-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1- Base] piperidin-1-yl)-ethyl ketone.

Compound D:(R)-4-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1- Base)-piperidines-1-carbonyl) amylene-4-acid.

Compound E:1-((R)-1-acryloylpiperidine-3-base)-4-amino-3-(4-Phenoxyphenyl)-1H- Pyrazolo [3,4-d] pyrimidine-1-oxide.

Compound F:1,3-bis-((R)-3-(4-amino-3-(4-Phenoxyphenyl)-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-Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) piperidin-1-yl)-3-oxopropyl) ammonia)-3-(4-Phenoxyphenyl)-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-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1- Base)-piperidin-1-yl)-propyl group-1-ketone.

The present invention can be used for analyzing as active pharmaceutical ingredient (API) according to Shandong for Buddhist nun and/or medicine group In compound according to Shandong for Buddhist nun.The pharmaceutical composition that the present invention can analyze includes solid or liquid combination Thing, and include one or more pharmaceutically acceptable excipient alternatively.The compositions of solid form Including powder, tablet, capsule, pill and dispersible granule etc..Fluid composition includes Solution or suspension, it can be by being administered orally, inject or instil administration.Description and claim The term " according to Shandong for Buddhist nun " that book is used in the whole text refers to according to Shandong for Buddhist nun and/or solvate (such as hydrate). Term " impurity " or " having related substance " that description is used in the whole text can refer to manufacturing API or medicine group The impurity formed in compound, and/or the impurity that formed of API degraded or medicine group in storage The impurity formed in compound or preparation.

As it has been described above, the HPLC method reported in prior art, be not suitable for analyzing and replace according to Shandong Buddhist nun and containing according to Shandong for the preparation of Buddhist nun or pharmaceutical composition.

But, the method for the present invention solves this problem, and effectively detects also with single operation Quantitatively, this specific building-up process of qualitative analysis or preparation preparation in formed all impurity and centre Body.Present invention have an advantage that to disclose for the first time and replace the impurity in Buddhist nun's preparation according to Shandong for Buddhist nun and Yi Lu Concrete structure, and use gradient HPLC method polar impurity and non polar impurities to be eluted out simultaneously, And carry out qualitative and quantitative analysis.

The invention is particularly suited to measure and one or more in compound A～H in quantitative sample Exist.Unless otherwise described, term " impurity " the most herein and " compound " with compound Under the scope that A～H is relevant, use can be exchanged.

It is also an advantage of the invention that this method is for replacing Buddhist nun and containing in the preparation replacing Buddhist nun according to Shandong according to Shandong The analysis having related substance to have specificity strong, the features such as accuracy, precision are high, ruggedness is good. Additionally, the present invention has height sensitivity, and allow detection and quantitatively replace Buddhist nun API or medicine group according to Shandong The acceptable limits of regulation during level amount is substantially less than Yao Jian department and ICH guideline in compound There is related substance.

Additionally, the method for the present invention can be used for detection and quantitatively stores up for Buddhist nun's sample or pharmaceutical composition according to Shandong The all degradation impurity formed during depositing.By carrying out forcing degraded to be ground according to ICH Q1A guide Study carefully to determine the method, and verify according to ICH Q2A guide, this checking covering following items: Specificity, linearity and range, precision, accuracy, detection limit, quantitative limit, ruggedness and system Adaptability.

The novel gradient HPLC method that the present inventor develops eight kinds of impurity As of qualitative determination～H.Institute The method of stating can disposably be analyzed and replace in Buddhist nun preparation technology according to Shandong and city according to prepared by the present embodiment Sell according to Shandong for Buddhist nun and store during produce by-product, degradation impurity isopolarity differ greatly miscellaneous Matter, 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 The fixing phase of octyl group silane group silica gel is the most favourable.Particularly preferred fixing contains Agilent mutually Poroshell EC-C₁₈120 (150mm × 4.6mm), 2.7 μm posts.

The inventive method preferably includes gradient design so that the relative concentration of mobile phase A and B exists 10～60 minutes are typically changed to the gradient of 100%A: 0%B to 0%A: 100%B.Excellent Selection of land, through 15 to 55 minutes, gradient is 95%A: 5%B to 5%A: 95%B, It is highly preferred that through 20 to 50 minutes, gradient is 90%A: 10%B to 10%A: 90%B, Most preferably, through 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 is, it is possible to will be according to Shandong for Buddhist nun API or according to Shandong The impurity the most close for opposed polarities various in Buddhist nun's pharmaceutical composition or polarity is totally separated out, it is simple to The most qualitative and quantitative.

The flowing used is preferably selected from one or more buffer solution (A) mutually and one or more are organic molten The combination of agent (B).

Buffer solution be preferably selected from including phosphate, acetate, formates, trifluoroacetic acid, formic acid or The aqueous solution combination of their mixture of acetic acid.

The concentration of buffer solution can be 0.01M to 0.1M, and preferred concentration is 0.01M to 0.08M, More preferably concentration is 0.03M to 0.08M, further preferred 0.04-0.06M, most preferably 0.05M. Particularly preferred flowing comprises ammonium formate and first aqueous acid mutually, or ammonium formate solution formic acid regulates The combination of the mixture of the aqueous solution (A) of pH and acetonitrile (B) or acetonitrile and methanol (B).

According in the particularly preferred embodiment of the present invention, it is further provided a kind of gradient HPLC Method, wherein flow and include following gradient design mutually:

The particularly preferred HPLC gradient method that the present invention also provides for, wherein, flowing comprises formic acid mutually Ammonium and/or formic acid are as buffer solution (A).In other particularly preferred embodiment, flow phase Comprise acetonitrile as organic solvent (B), and/or acetonitrile-methanol mixed solvent is as organic solvent (B). Inventor finds when flowing comprises ammonium formate and/or formic acid (A) and acetonitrile and carbinol mixture (B) mutually Time gradient design be particularly effective.

Buffer solution (A) can contain one or more added solvent, these added solvent can be methanol, Ethanol, isopropanol or their mixture are as organic solvent.Additional molten in buffer solution (A) Agent may or may not be the solvent identical with organic solvent (B).Adding in buffer solution (A) Solvent is preferably the mixture of acetonitrile or acetonitrile and methanol.

The pH of buffer solution (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 method of the present invention is carried out at a temperature of about 30～60 DEG C, preferably 35～55 DEG C, 40～50 DEG C, most preferably 40～50 DEG C.

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

Organic solvent (B) but a kind of organic solvent, this organic solvent can be methanol, ethanol, Acetonitrile, isopropanol.Organic solvent (B) can also be methanol and the mixed solvent of acetonitrile or acetonitrile With the mixed solution of ethanol, inventor finds that the mixed solvent of methanol and acetonitrile is as organic facies (B) Optimum effect.The mixed volume ratio of methanol and acetonitrile is about 40: 60, and 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.This solution comprise be dissolved in the most molten One or more compounds A～H in agent (such as acetonitrile).Described reference standard solution can be used for measuring Utilize any compound as impurity in the sample being analyzed according to the chromatographic technique of the present invention The existence of A～H.

According to a further aspect in the invention, it is provided that a kind of reference standard solution, it is known that the one of amount Plant or multiple compounds A～H is dissolved in suitable solvent (such as acetonitrile).Described reference standard solution can For measuring appointing as impurity in utilizing the sample being analyzed according to the chromatographic technique of the present invention What compound A's～H is qualitative and quantitative.Important and square for technical staff of the method for described analysis It is obvious.

The present inventor the most extensively verifies the method for the present invention, and the result shows the specificity of this method By force, accuracy, precision and highly sensitive, ruggedness is good.

Accompanying drawing illustrates:

Fig. 1 is for replacing Buddhist nun's crude product (real according to method disclosed in WO201384572A embodiment 8 to according to Shandong Execute in example 2 preparation without recrystallisation from isopropanol according to Shandong for Buddhist nun) carry out HPLC and analyze collection of illustrative plates.

-3-(4-Phenoxyphenyl)-1-(piperidines-3-base)-1H-pyrazolo [3,4-d] Fig. 2: intermediate-1:(R) Pyrimidine-4-amine¹H-NMR；

-3-(4-Phenoxyphenyl)-1-(piperidines-3-base)-1H-pyrazolo [3,4-d] Fig. 3: intermediate-1:(R) The high resolution mass spectrum of pyrimidine-4-amine；

Fig. 4: according to Shandong for Buddhist nun: 1-[(3R)-3-[4-amino-3-(4-Phenoxyphenyl)-1H pyrazolo [3,4-d] Pyrimidine-1-base]-piperidino]-2-propylene-1-ketone¹H-NMR；

Fig. 5: according to Shandong for Buddhist nun: 1-[(3R)-3-[4-amino-3-(4-Phenoxyphenyl)-1H pyrazolo [3,4-d] Pyrimidine-1-base]-piperidino] high resolution mass spectrum of-2-propylene-1-ketone.

Fig. 6: impurity A: ((4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] is phonetic for 3-for (R)-1- Pyridine-1-base] piperidin-1-yl)-3-chloropropyl-1-ketone¹H-NMR；

Fig. 7: impurity A: ((4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] is phonetic for 3-for (R)-1- Pyridine-1-base] piperidin-1-yl) high resolution mass spectrum of-3-chloropropyl-1-ketone；

Fig. 8: impurity B: (R)-8-(1-acryloylpiperidine-3-base)-10-(4-Phenoxyphenyl)-3,4-bis- Hydrogen pyrazolo [4,3-e] pyrimido [1,2-c] pyrimidine-2 (8H)-one¹H-NMR；

Fig. 9: impurity B: (R)-8-(1-acryloylpiperidine-3-base)-10-(4-Phenoxyphenyl)-3,4-bis- The high resolution mass spectrum of hydrogen pyrazolo [4,3-e] pyrimido [1,2-c] pyrimidine-2 (8H)-one；

-1-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] phonetic Figure 10: impurity C:(R) Pyridine-1-base] piperidin-1-yl)-ethyl ketone¹H-NMR；

-1-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] phonetic Figure 11: impurity C:(R) Pyridine-1-base] piperidin-1-yl) high resolution mass spectrum of-ethyl ketone.

-4-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] Figure 12: impurity D:(R) Pyrimidine-1-base)-piperidines-1-carbonyl) amylene-4-acid¹H-NMR；

-4-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] Figure 13: impurity D:(R) Pyrimidine-1-base)-piperidines-1-carbonyl) amylene-4-acid high resolution mass spectrum；

-1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] Figure 14: impurity H:((R) Pyrimidine-1-base)-piperidin-1-yl)-propyl group-1-ketone¹H-NMR；

-1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] Figure 15: impurity H:(R) Pyrimidine-1-base)-piperidin-1-yl) high resolution mass spectrum of-propyl group-1-ketone；

Figure 16: impurity E: 1-((R)-1-acryloylpiperidine-3-base)-4-amino-3-(4-phenoxy group benzene Base)-1H-pyrazolo [3,4-d] pyrimidine-1-oxide¹H-NMR。

Figure 17: impurity E: 1-((R)-1-acryloylpiperidine-3-base)-4-amino-3-(4-phenoxy group benzene Base) high resolution mass spectrum of-1H-pyrazolo [3,4-d] pyrimidine-1-oxide.

Figure 18: impurity F: 1,3-bis-((R)-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] Pyrimidine-1-base) piperidin-1-yl) propane-1-ketone¹H-NMR。

Figure 19: impurity F: 1,3-bis-((R)-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] Pyrimidine-1-base) 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-Phenoxyphenyl)-1H-pyrrole Azoles [3,4-d] pyrimidine-1-base) piperidin-1-yl)-3-oxopropyl) ammonia)-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) 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-Phenoxyphenyl)-1H-pyrrole Azoles [3,4-d] pyrimidine-1-base) piperidin-1-yl)-3-oxopropyl) ammonia)-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) piperidin-1-yl) high resolution mass spectrum of propyl group-2-alkene-1-ketone；

Figure 22. the HPLC spectrogram of mark-on mixed solution.

Figure 23. the HPLC spectrogram replacing Buddhist nun's need testing solution according to Shandong of embodiment 2 preparation.

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

Detailed description of the invention

Although its detailed description of the invention is described by the present invention, but some amendment and equivalent pair It is apparent from those skilled in the art, and within being intended to be included in the scope of the present invention.

Being illustrated the present invention by following example, following example limit this never in any form Invention.

Embodiment 1

Intermediate-1:(R)-3-(4-Phenoxyphenyl)-1-(piperidines-3-base)-1H-pyrazolo [3,4-d] pyrimidine-4- The synthesis of amine

In the reaction bulb of 500mL, add 155mL oxolane, under stirring, be sequentially added into 3-(4- Phenoxyphenyl)-1H-pyrazoles [3,4-D] pyrimidine-4-amine (SM1) (5g, 1eq), (S)-1-tertiary fourth oxygen Carbonyl-3-hydroxy piperidine (SM2) (4.97g, 1.5eq), triphenylphosphine (13g, 3.0eq).Temperature control Under the conditions of 25 DEG C, in 30 minutes, the tetrahydrofuran solution of dropping diisopropyl azodiformate (will 10g, 3.0eq diisopropyl azodiformate is dissolved in the oxolane of 10mL).After being added dropwise to complete, Temperature control 25 DEG C, continues reaction 5 hours (TLC monitors: ethyl acetate: methanol=10: 1).Stirring Lower decompression distillation.Temperature control 15 DEG C, drips 30mL concentrated hydrochloric acid, time for adding 30 points in residue Clock, after being added dropwise to complete, temperature control 25 DEG C, continues reaction 2 hours.By dichloromethane aqueous phase extracted three Secondary (each 75mL), retains aqueous phase.Extract once by 50mL ethyl acetate.Residue aqueous phase fills Divide stirring, temperature control 15 DEG C, drip the sodium hydrate aqueous solution of about 45g 20%, use pH reagent paper prison Measured reaction liquid, pH=5～6 (time for adding about 30 minutes) obtains light yellow solid.Above-mentioned solid is led to Cross ethyl alcohol recrystallization twice, obtain off-white powder 2.87g, yield: 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 spectrogram shows Molecular ion peak m/z=387.19449 [M+H]+, the structural formula reason of corresponding molecular weight and offer Opinion value of calculation (387.19279) is consistent.Absolute error is 4.41ppm, at high resolution mass spectrum error model Within enclosing.(referring to Fig. 3)

Embodiment 2: replace the preparation of Buddhist nun according to Shandong

Under nitrogen protection, in 100mL there-necked flask, it is sequentially added into 50mL dichloromethane, intermediate -1 (5g, 1eq), DIPEA (2g, 1.2eq).Under the conditions of temperature control-10 DEG C, start The dichloromethane solution of dropping acrylic anhydride (1.96g, 1.2eq), time for adding 30 minutes, dropping After completing, solution is become clarification by muddiness, is incubated-10 DEG C, is stirred well to raw material reaction (TLC completely Detection, methanol: ethyl acetate: triethylamine=1: 5: 0.05).Reactant liquor is with 200mL 5% Fructus Citri Limoniae Aqueous acid washs, and removes aqueous phase, concentrates and dichloromethane is evaporated off.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), 2.128 (m, 1H), 1.903～1.937 (m, 1H), 1.577～1.605 (m, 1H) (referring to Fig. 4) ESI-HRMS spectrogram display molecular ion peak: 441.20366 [M+H]⁺, according to Shandong Calculated value for Buddhist nun's molecular ion peak is: 441.20335 [M+H]⁺, absolute error is 0.71 Ppm, meets high resolution mass spectrum range of error, and measured value is consistent with theoretical value.(referring to Fig. 5).

Embodiment 3: impurity A: ((4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] is phonetic for 3-for (R)-1- Pyridine-1-base] piperidin-1-yl) synthesis of-3-chloropropyl-1-ketone.

Under nitrogen protection, in the there-necked flask of 100mL, add 50m1 dichloromethane, depend under stirring Secondary addition intermediate-1:(R)-3-(4-Phenoxyphenyl)-1-(piperidines-3-base)-1H-pyrazoles [3,4-d] pyrimidine -4-amine (YLTN-1) (1.00g, 1eq), DIPEA (0.40g, 1.2eq), fall Temperature, to-20～-10 DEG C, starts to drip 3-chlorpromazine chloride (1.46g, 1eq), drips complete, solution by Muddy change is clarified, and continues stirring 20～30 minutes, and LC-MS detects, and raw material disappears, distillation of reducing pressure, Dichloromethane being evaporated off extremely steam without fraction, obtain crude product column chromatography method and purify, eluting ratio is: Methanol: ethyl acetate=1: 10, collects eluent 400mL altogether, decompression distillation, solvent is evaporated off to nothing Fraction steams.Obtaining off-white powder 830mg is (R)-1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H- Pyrazolo [3,4-d] pyrimidine-1-base] piperidin-1-yl)-3-chloropropyl-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 spectrogram display 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 range of error. (referring to Fig. 7).Impurity A replaces when relatively retaining of Buddhist nun relative to main constituent according to Shandong on HPLC spectrogram Between (RRT) be about 1.06.

Embodiment 4: impurity B: (R)-8-(1-acryloylpiperidine-3-base)-10-(4-Phenoxyphenyl)-3,4-bis- The synthesis of hydrogen pyrazolo [4,3-e] pyrimido [1,2-c] pyrimidine-2 (8H)-one.

Nitrogen is protected, and adds 100ml dichloromethane, under stirring successively in the there-necked flask of 250mL Add intermediate-1:(R)-3-(4-Phenoxyphenyl)-1-(piperidines-3-base)-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 to drip acrylic anhydride (1.33g, 2eq), dropping process temperature control-10 DEG C with Under；Dripping and finish, continue stirring 20～30 minutes, LC-MS detects, and has target product to generate, temperature control 30～40 DEG C, vacuum :-0.08MPa, decompression distillation, dichloromethane is evaporated off to steaming without fraction； Obtaining crude product column chromatography method to purify, eluting ratio is: methanol: ethyl acetate=1: 5, collects eluting Liquid, temperature control 30～40 DEG C, vacuum :-0.08MPa, decompression distillation, solvent is evaporated off and steams to without fraction Go out.Obtaining off-white powder 531mg is (R)-8-(1-acryloylpiperidine-3-base)-10-(4-phenoxy group benzene Base)-3,4-dihydro-pyrazolo [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) (referring to Fig. 8).ESI-HRMS spectrogram display molecule Quasi-molecular ions m/z=495.21580 [MW+H]⁺, corresponding molecular weight is theoretical with the structural formula of offer Value of calculation (494.20664) is consistent.Absolute error is 3.82ppm, in high resolution mass spectrum range of error Within (referring to Fig. 9).Impurity B is protected for the relative of Buddhist nun according to Shandong relative to main constituent on HPLC spectrogram The time (RRT) is stayed to be about 0.97.

Embodiment 5: impurity C:(R) (4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] is phonetic for-1-3- Pyridine-1-base] piperidin-1-yl) synthesis of-ethyl ketone

By intermediate-1:(R)-3-(4-Phenoxyphenyl)-1-(piperidines-3-base)-1H-pyrazoles [3,4-d] pyrimidine -4-amine (YLTN-1) (2.00g, 1eq) is dissolved in dichloromethane (50ml), adds N, N- Diisopropyl ethyl amine (0.80g, 2eq), is cooled to-10 DEG C, and nitrogen is protected, and drips acetic anhydride (0.53 G, 1eq), finish, react 30 minutes, LC-MS monitors, and reaction completes, by system concentrating under reduced pressure To dry, residue passes through silica gel column chromatography, eluent: ethyl acetate: methanol=10: 1, obtains off-white color Powder 1.38g be (R)-1-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base] Piperidin-1-yl)-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 spectrogram Display molecular ion peak m/z=429.20416 [MW+H]⁺, corresponding molecular weight and the knot of offer Structure formula calculated value (428.19607) is consistent.Absolute error is 1.89ppm, at high resolution mass spectrum (Figure 11 is referred to) within range of error.Impurity C relative main constituent on HPLC spectrogram replaces Buddhist nun according to Shandong Relative retention time (RRT) be about 0.96.

Embodiment 6: impurity D:(R) ((4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] is phonetic for 3-for-4- Pyridine-1-base)-piperidines-1-carbonyl) amylene-4-acid synthesis

Under nitrogen protection, will be according to Shandong for Buddhist nun 1-[(3R)-3-[4-amino-3-(4-Phenoxyphenyl)-1H pyrrole Azoles also [3,4-d] pyrimidine-1-base]-piperidino]-2-propylene-1-ketone (2g, 1eq) is dissolved in dichloromethane (50ml), in, it is sequentially added into N, N-diisopropyl ethyl amine (0.70g, 2eq), acrylic acid (0.98 G, 3eq), reaction is warming up to 35 DEG C, stirs 36 hours, is evaporated to do by system, residue By silica gel column chromatography, eluent: ethyl acetate: methanol=8: 1, obtain off-white powder 103mg For (R)-4-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base)-piperidines-1- Carbonyl) amylene-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 spectrogram shows Show molecular ion peak m/z=513.22606 [MW+H]⁺, corresponding molecular weight and the structure of offer Formula calculated value (512.21720) is consistent.Absolute error is 3.09ppm, at high resolution mass spectrum by mistake (Figure 13 is referred to) within the scope of difference.Impurity D replaces Buddhist nun's relative to main constituent according to Shandong on HPLC spectrogram Relative retention time (RRT) is about 1.02.

Embodiment 7: impurity H:(R) ((4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] is phonetic for 3-for-1- Pyridine-1-base)-piperidin-1-yl) synthesis of-propyl group-1-ketone

Under nitrogen protection, by intermediate-1:(R)-3-(4-Phenoxyphenyl)-1-(piperidines-3-base)-1H-pyrrole Azoles [3,4-d] pyrimidine-4-amine (YLTN-1) (2.00g, 1eq) dissolves in dichloromethane (50mL), Adding N, N-diisopropyl ethyl amine (0.80g, 2eq), be cooled to-10 DEG C, nitrogen is protected, and drips third Anhydride (0.67g, 1eq), finishes, and reacts 30 minutes, and LC-MS monitors, and reaction completes, by system Be evaporated to do, residue pass through silica gel column chromatography, eluent: ethyl acetate: methanol=10: 1, Obtaining lightpink powder 1.72g is (R)-1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] Pyrimidine-1-base)-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 Spectrogram display molecular ion peak m/z=443.25380 [MW+H]⁺, corresponding molecular weight and offer Structural formula calculated value (442.21172) be consistent.Absolute error is 3.66ppm, at high-resolution (Figure 15 is referred to) within mass spectrum range of error.Impurity H on HPLC spectrogram relative main constituent according to Shandong Relative retention time (RRT) for Buddhist nun is about 1.04.

Embodiment 8: impurity E: 1-((R)-1-acryloylpiperidine-3-base)-4-amino-3-(4-phenoxy group benzene Base) preparation of-1H-pyrazolo [3,4-d] pyrimidine-1-oxide

(1) preparation of impurity E

Example 2 preparation without three recrystallization of isopropanol according to Shandong for Buddhist nun about 5.17g, put In 500ml reaction bulb, add 150ml 80% acetonitrile-water and dissolve, then add 30% hydrogen peroxide 30ml, room Temperature is stirred overnight, and reaction terminates, and adds sodium thiosulfate, produce to bubble-free, 35 DEG C in reactant liquor It is evaporated to no liquid flow out, then adds the ethyl acetate of 3 times of aqueous phase volumes, extraction, collect organic Phase, merges, and 35 DEG C are evaporated to do, and get Yi Lu replaces Buddhist nun impurity E crude product 2.65g.LC-MS examines Surveying crude product purity is 51.26%, and liquid chromatograph relative to the relative retention time replacing Ni Feng according to Shandong is 0.98, mass-to-charge ratio [MW+H]⁺It is 457.2.

(2) purification of impurity E crude product

The impurity E crude product DMSO of above-mentioned for 2.95g preparation is dissolved into concentration and is about 150mg/ml Solution, use following preparative chromatography to be further purified:

Chromatographic column (purchased from Ai Jieer science and technology): use the C18 filler of particle diameter 10 μm, use isopropanol Post (50mm × 250mm) is filled after homogenate；

Flowing 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

Collect the fraction corresponding to peak that retention time is 30～37min；LC-MS or HPLC detects The purity of impurity E.

Fraction collected by 35 DEG C of concentrating under reduced pressure flows out to no liquid, adds the ethyl acetate of 3 times of volumes Extraction, collects organic facies, merges, adds anhydrous sodium sulfate and be dried, and filters, 30 DEG C of concentrating under reduced pressure of filtrate To dry, obtaining 2.06g light pink solid, measure through HPLC, purity is 98.97%, and total recovery is 36.6%.

(3) Structural Identification of impurity E

Impurity E¹H-NMR figure sees Figure 16, and high resolution mass spectrum sees Figure 17.

The high resolution mass spectrum (ESI source) of impurity E shows its mass-to-charge ratio [M+H]⁺It is 457.19930, Corresponding molecular weight is 2.27 with the absolute error of the structural formula calculated value 456.19099 of offer Ppm, meets the range of error of high resolution mass spectrum.The structure of Buddhist nun is replaced according to Shandong, in conjunction with miscellaneous with reference to formula (2) Matter E¹H-NMR、¹³C-NMR spectrum identifies that its structural formula is as shown in following formula E.

Impurity E high resolution mass spectrum shows its molecular weight and is consistent completely with the molecular formula provided；

The nuclear magnetic resonance, NMR of table 1 impurity E¹H-NMR、¹³C-NMR analysis result

Information shown by the proton nmr spectra of impurity E, carbon spectrum can be to the formula E chemical combination provided On thing molecular structural formula, hydrogen, carbon all belong to.Therefore, according to Shandong for Buddhist nun's impurity E and formula E provided Molecular structural formula is consistent.

Embodiment 9: impurity F: 1,3-bis-((R)-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] Pyrimidine-1-base) piperidin-1-yl) propane-1-ketone and impurity G:1-((R)-3-(4-((3-((R)-3-(4-amino-3-(4- Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) piperidin-1-yl)-3-oxopropyl) ammonia)-3-(4-benzene Phenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) piperidin-1-yl) preparation of propyl group-2-alkene-1-ketone.

(1) impurity F and the preparation of impurity G

Heavily tying for three times without isopropanol of about 20g embodiment 2 preparation is added in 1000ml reaction bulb Brilliant replaces Buddhist nun according to Shandong, adds 250ml acetonitrile and 200ml 5M sodium hydrate aqueous solution, 80 DEG C of water-baths Reacting by heating 40min, is cooled to room temperature, adds 5M HCl and is neutralized to neutrality, and 35 DEG C are evaporated to nothing Liquid reserves, and adds the ethyl acetate extraction of 5 times of aqueous phase volumes, collects upper organic phase, merge, 35 DEG C are evaporated to do, and obtain according to Shandong for Buddhist nun's impurity F, G crude product 18.72g, and LC-MS detection is thick Product purity, according to corresponding MS quasi-molecular ions, liquid chromatograph replaces when relatively retaining of Ni Feng relative to according to Shandong Between to be about the peak of 0.94 (impurity F) and 1.15 (impurity G) be object, the content in its crude product It is respectively 16.9% (impurity F) and 18.4% (impurity G).

(2) impurity F and the purification of impurity G crude product

The 18.72g impurity F of above-mentioned preparation, the crude product of G are added DMSO and are dissolved into the solution of 10ml, Loading:

Middle compacting is for post (purchased from Agela company): Agela XBP C18 filler, 480g, filler grain Footpath: 20～35 μm

Flowing 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

Collecting the fraction at all peaks respectively, LC-MS or HPLC confirms target product.Collect The fraction (impurity F) of 30～33min and the fraction (impurity G) of 40～44min, merge identical fraction, Concentrating under reduced pressure respectively, flows out to no liquid, adds the ethyl acetate extraction of 3 times of volumes, receive in aqueous phase Collection organic facies, adds anhydrous sodium sulfate and is dried, and filters, and collects filtrate.It is evaporated to do, respectively Buddhist nun impurity G secondary separation crude product 2.57g is replaced for Buddhist nun's impurity F sterling 2.21g and Yi Lu according to Shandong.Warp LC-MS or HPLC detects analysis, and the purity replacing Buddhist nun's impurity F according to Shandong is 98.15%, according to Shandong for Buddhist nun The purity of impurity G secondary separation crude product is 85.32%.

(3) secondary separation of impurity G

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

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

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

Flowing 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/ time every time；

Collect retention time 26min～the fraction of 30min, and confirm object purity with LC-MS. Merge identical fraction, 35 DEG C be evaporated to without acetonitrile flow out, add 3 times of volumes ethyl acetate extraction, Merge organic facies, add anhydrous sodium sulfate and be dried, filter, be evaporated to do, obtain according to Shandong miscellaneous for Buddhist nun Matter G sterling 1.64g, off-white color pressed powder, LC-MS detection purity is 95.13%.

(4) Structural Identification of impurity F

Impurity F¹H-NMR figure sees Figure 18, and high resolution mass spectrum sees Figure 19.

The high resolution mass spectrum (ESI source) of impurity F shows its mass-to-charge ratio [M+H]⁺It is 827.39065, Corresponding molecular weight with the absolute error of the structural formula calculated value 826.34520 of offer is 2.16ppm, meets the range of error of high resolution mass spectrum.The high resolution mass spectrum of sample shows its molecular weight It is consistent completely with the molecular formula provided.Reference replaces the structure of Buddhist nun according to Shandong, in conjunction with this product¹H-NMR、¹³C-NMR, H-HCOSY, HMBC, HSQC, DEPT spectrum identifies its structure Formula is as shown in following formula F.

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

Table 3 impurity F¹³C-NMR, DEPT, HSQC, HMBC data parsing

By sample¹H-NMR, H-HCOSY and HMBC spectrum understands, Hydrogen Proton in its structure Number is consistent with the structure of formula F compound.By sample¹³C-NMR, DEPT, HSQC and HMBC Spectrum understands, and the carbon number in its structure is consistent with the structure of formula F compound.Nucleus magnetic hydrogen spectrum, carbon spectrum with And the hydrogen on structural formula can rationally be belonged to carbon and associate by two-dimensional spectrum data.

(5) Structural Identification of impurity G

Impurity G's¹H-NMR figure sees Figure 20, and high resolution mass spectrum sees Figure 21.

The high resolution mass spectrum (ESI source) of impurity G shows its mass-to-charge ratio [M+H]⁺It is 881.40140, Corresponding molecular weight with the absolute error of the structural formula calculated value 880.39215 of offer is 1.43ppm, meets the range of error of high resolution mass spectrum.The high resolution mass spectrum of sample shows its molecular weight It is consistent completely with the molecular formula provided.In conjunction with according to Shandong for the structure of Buddhist nun, in conjunction with 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 composes, combines knowable to H-HCOSY, HMBC two-dimensional spectrum, its knot In structure, Hydrogen Proton number is consistent with the structure of formula G compound.By sample¹³C-NMR、DEPT、 HSQC and HMBC spectrum understands, and the carbon number in its structure is consistent with the structure of formula G compound.

Embodiment 10: have the HPLC of related substance to analyze for Buddhist nun according to Shandong

Utilize impurity external standard method (version Chinese Pharmacopoeia in 2010), analyze and have related substance according to Shandong in Buddhist nun, And by external standard method, each impurity is carried out quantitatively.The quantitative of impurity calculates according to external standard method, specifically joins See 2010 editions two annex V D of Chinese Pharmacopoeia.According to the method carrying out described analysis this Bright gradient HPLC method.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

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

Detection wavelength: 260nm

Diluent: 80% acetonitrile-water

Column temperature: 50 DEG C

Flow velocity: 0.8ml/min

Gradient design is as described below.

Sample preparation:

Prepare impurity location solution

(1) compound A～H standard setting solution: take compound A～H 10mg respectively, accurately weighed, Put respectively in 100ml volumetric flask, add that acetonitrile is ultrasonic makes dissolving, and by dilution in acetonitrile to scale, shake up, Make the solution that concentration is about 100 μ g/ml, as compound A～H stock solution.

(2) mark-on mixed solution: take above-mentioned preparation replaces Buddhist nun crude drug 100mg according to Shandong, accurately weighed, Put in 100mg measuring bottle, more accurate add compound A～each 1ml of H stock solution, add 80% acetonitrile- Water is appropriate, ultrasonic makes dissolving, then is diluted to scale with 80% acetonitrile-water, shakes up, makes every 1ml Containing according to Shandong for Buddhist nun about 1mg, mixed solution containing compound A～H the most about 1 μ g, mix as mark-on Close solution.

(3) compound A～H reference standard solution or reference substance solution are prepared: precision measures compound A～H Stock solution 1.0ml, puts in 100ml volumetric flask, is diluted to scale with 80% acetonitrile-water, shakes up, Make every 1ml and be respectively the solution of 1 μ g containing about compound A～H, as the reference of compound A～H Standard solution or reference substance solution.

(4) preparation replaces Buddhist nun's need testing solution according to Shandong: take above-mentioned preparation replaces Buddhist nun crude drug 10mg, essence according to Shandong Close weighed, put in 10ml volumetric flask, add 80% acetonitrile-water appropriate, ultrasonic make dissolving, and with 80% Acetonitrile-water is diluted to scale, shakes up, both.The concentration of Buddhist nun's crude drug need testing solution is replaced about according to Shandong For 1.0mg/ml.

(5) according to Shandong for the preparation of Buddhist nun's capsule need testing solution: take according to Shandong that (Imbruvica is purchased from for Buddhist nun's capsule Pharmacyclics Janssen company, 140mg, lot number: L0404951) the finely ground powder of content fits Amount (containing about according to Shandong for Buddhist nun 10mg), accurately weighed, put in 10ml volumetric flask, add 80% acetonitrile- Water is appropriate, ultrasonic makes dissolving, and is diluted to scale with 80% acetonitrile-water, shakes up, uses 0.22 μm Organic syringe filter filters, and discards just filtrate 2ml, takes subsequent filtrate, supplies for Buddhist nun's capsule as according to Shandong Test sample solution.The concentration replacing Buddhist nun's capsule need testing solution according to Shandong is about 1.0mg/ml.

Embodiment 11:

Replacing the preparation of Buddhist nun with embodiment 2 according to Shandong, sample preparation is with embodiment 10, and gradient design is with implementing Example 10.

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

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

Detection wavelength: 260nm

Diluent: 80% acetonitrile-water

Column temperature: 50 DEG C

Flow velocity: 0.8ml/min

Embodiment 12:

Replacing the preparation of Buddhist nun with embodiment 2 according to Shandong, sample preparation is with embodiment 10, and gradient design is with embodiment 10.

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 DEG C

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

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

Detection wavelength: 260nm

Diluent: 80% acetonitrile-water

Column temperature: 40 DEG C

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

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

Detection wavelength: 260nm

Diluent: 80% acetonitrile-water

Column temperature: 50 DEG C

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 DEG C

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

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

Detection wavelength: 260nm

Diluent: 80% acetonitrile-water

Column temperature: 45 DEG C

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

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

Detection wavelength: 260nm

Diluent: 80% acetonitrile-water

Column temperature: 45 DEG C

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

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

Detection wavelength: 260nm

Diluent: 80% acetonitrile-water

Column temperature: 45 DEG C

Flow velocity: 0.6ml/min

Test procedure:

(1) under gradient HPLC method of the present invention, at Agilent 1260HPLC instrument (U.S. Agilent Company) on, according to the chromatographic condition of embodiment 16, take compound A～H location solution, mark-on mix Close solution, compound A～H reference solution, according to Shandong for Buddhist nun's need testing solution, supply for Buddhist nun's capsule according to Shandong The each 10 μ l of test sample solution, inject chromatograph of liquid, record chromatogram.

(2) under gradient HPLC method of the present invention, according to the chromatographic condition of embodiment 10～18, take and add Mark mixed solution 10 μ l, injects chromatograph of liquid, records chromatogram.

Result of the test:

Compound A～H and main constituent replace Buddhist nun under gradient HPLC method in the present embodiment 16 according to Shandong Positioning result is shown in Table 1；Under the chromatographic condition of the embodiment of the present invention 10～18, in mark-on mixed solution Separating degree between each impurity, each impurity are shown in Table 2 with replacing the relative retention time of Ni Feng according to Shandong.

Utilize the chromatographic condition of embodiment 16, calculate the change contained in need testing solution according to external standard method The content of compound A～H and total impurities amount, the results are shown in Table 3, the HPLC spectrogram of mark-on mixed solution, Replace the HPLC spectrogram of Buddhist nun's need testing solution according to Shandong, compose for the HPLC of Buddhist nun's capsule need testing solution according to Shandong Figure is shown in Figure 22～Figure 24 respectively, and the most as shown in figure 22, the summit in Figure 23-24 is for replace according to Shandong The peak of Buddhist nun.

Replace Buddhist nun capsule relevant for Buddhist nun with Yi Lu according to compound A～H and main constituent positioning result and according to Shandong The analysis result of material, according in embodiment according to Shandong for the preparation method of Buddhist nun prepare former for Buddhist nun according to Shandong That expects has related substance as follows: replaces according to Shandong in the chromatogram of Buddhist nun's need testing solution, replaces the reservation of Ni Feng according to Shandong Time is about 16.48min, and retention time is about 13.75min, the impurity of relative retention time 0.83 For compound C；Retention time about 15.85min, the impurity of relative retention time 0.96 are compound D；Retention time about 18.55min, the impurity of relative retention time 1.13 are compound H；Retain Time about 18.55min, the impurity of relative retention time 1.38 are compound F；Retention time is about 33.50min, the impurity of relative retention time 2.04 are compound G；According to Shandong in Buddhist nun's need testing solution Do not detect compound A, B, E.

According to Shandong for Buddhist nun's capsule (Imbruvica) need testing solution has detected compound A, B, C, F, G and H, wherein the content of compound A, F is all more than 0.1%.

Prepared by table 1 compound A～H and the embodiment of the present invention 2 replaces Buddhist nun in embodiment 16 according to Shandong The separating degree between location and each peak in method

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

Prepared by table 3 embodiment 2 replaces in Buddhist nun's capsule (Imbruvica) relevant according to Shandong for Buddhist nun's crude drug and Yi Lu Material is according to the testing result of the chromatographic condition of embodiment 16

It should be noted that all documents mentioned in the present invention are incorporated as reference in this application, It is individually recited as with reference to like that just as each document.In addition, it is to be understood that above-described it is The present invention is embodied as row and the know-why used, after having read present disclosure, this Skilled person the present invention can be made various change or amendment without departing from the present invention spirit with Scope, these equivalent form of values also fall within the scope of the present invention.

Claims

1. that detects Formulas I replaces Buddhist nun or its solvate (such as hydrate) according to Shandong, or replaces Buddhist nun containing according to Shandong The method of sample purity of medicine, including by the impurity in chromatography determination sample, described impurity One or more in compound A-H,

Wherein Buddhist nun is replaced to be 1-[(3R)-3-[4-amino-3-(4-Phenoxyphenyl)-1H pyrazolo [3,4-d] according to Shandong Pyrimidine-1-base]-piperidino]-2-propylene-1-ketone, structure shown in formula I:

Compound A is (R)-1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine -1-base] piperidin-1-yl)-3-chloropropyl-1-ketone, and have a structure that

Compound B is (R)-8-(1-acryloylpiperidine-3-base)-10-(4-Phenoxyphenyl)-3,4-dihydro Pyrazolo [4,3-e] pyrimido [1,2-c] pyrimidine-2 (8H)-one, and have a structure that

Compound C is (R)-1-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1- Base] piperidin-1-yl)-ethyl ketone, and have a structure that

Compound D is (R)-4-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine -1-base)-piperidines-1-carbonyl) amylene-4-acid, and have a structure that

Compound E is 1-((R)-1-acryloylpiperidine-3-base)-4-amino-3-(4-phenoxy group benzene Base)-1H-pyrazolo [3,4-d1 pyrimidine-1-oxides, and have a structure that

Compound F is 1,3-bis-((R)-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine -1-base) piperidin-1-yl) propane-1-ketone, and have a structure that

Compound G is 1-((R)-3-(4-((3-((R)-3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) piperidin-1-yl)-3-oxopropyl) ammonia)-3-(4-Phenoxyphenyl)-1H-pyrazoles [3,4-d] pyrimidine-1-base) piperidin-1-yl) propyl group-2-alkene-1-ketone, and have a structure that

Compound H is (R)-1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine -1-base)-piperidin-1-yl)-propyl group-1-ketone, and have a structure that

Wherein said method comprises the steps:

(1) for Buddhist nun or will contain according to Shandong molten to prepare sample in a solvent for the medicine dissolution of Buddhist nun according to Shandong Liquid；

(4) by referring in this reference standard solution compound A-H one or more, mensuration depends on Shandong is replaced Buddhist nun or contains the existence of any one of compound A～H or multiple in the medicine replacing Buddhist nun according to Shandong.

2. the process of claim 1 wherein that described chromatography is liquid chromatography, as HPLC, UPLC、LC-MS；The most described chromatography is HPLC method, more preferably gradient HPLC method.? It is preferably the HPLC method compatible with LC-MS.

3. the method for claim 2, fixing that wherein said chromatography uses is anti-phase mutually, preferably ten Eight alkyl silane bonded silica gels or octyl group silane group silica gel.

4. the method for claim 3, in described gradient HPLC method, flowing includes mutually containing buffering Solution (A) and the combination of organic solvent (B), described buffer solution (A) is water-containing buffering liquid, excellent Elect acetate, formates, phosphate, trifluoroacetic acid, formic acid or the aqueous solution of their mixture as, Preferred buffer solution (A) is the water-soluble of formates, most preferably ammonium formate and formic acid or ammonium formate Liquid formic acid regulated the aqueous solution of pH, and described organic solvent (B) is polar aprotic solvent, as Methanol, ethanol or isopropanol；Or dipolar aprotic solvent, as acetonitrile, preferably organic solvent (B) are selected from In group including methanol, acetonitrile, ethanol, isopropanol or their mixture, most preferably acetonitrile Mixture with methanol.

5. the method for claim 4, wherein said buffer solution (A) is the water-soluble of ammonium formate and formic acid Liquid, described organic solvent (B) is the combination of acetonitrile and methanol.

6. the method for claim 5, wherein the concentration of ammonium formate is about 0.01M to 0.1M, preferably 0.03-0.08M, more preferably 0.04-0.06M, most preferably 0.05M；Methanol and the mixed volume of acetonitrile Ratio is about 40: 60, and preferably 45: 55,55: 45, or 50: 50, most preferably 50: 50.

7. the method for claim 5, wherein the pH of ammonium formate and first aqueous acid is about 2.7～4.8, Preferably from about 2.8～4.7,2.8～4.5,2.8～4.0 or 2.8～3.5, most preferably 2.8～3.5.

8. the method for claim 2, wherein said HPLC method is entered at a temperature of about 30～60 DEG C OK, preferably 35～55 DEG C or 40～50 DEG C, carry out at a temperature of most preferably 40～50 DEG C.

9. the method for claim 2, wherein said HPLC method is about 0.4～the stream of 1.2ml/min Carry out under speed, preferably 0.5～1.1ml/min or 0.6～1.0ml/min, most preferably 0.6～1.0ml/min Flow velocity under carry out.

10. the method for any one of claim 2-9, wherein in gradient HPLC method, mobile phase A 100%A:0%B to 0%A typically it is changed at 10～60 minutes with the relative concentration of B: The gradient of 100%B；Preferably, through 15 to 55 minutes, gradient is 95%A:5%B To 5%A:95%B；It is highly preferred that through 20 to 50 minutes, gradient is 90%A:10%B To 10%A:90%B；Most preferably, through about 30 minutes, gradient be 65%A:35%B extremely 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 method of 11. claim 10, wherein in gradient HPLC method, flowing comprises following mutually 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

Method described in 12. any one of claim 1-12, wherein or comprises according to Shandong for Buddhist nun's for Buddhist nun according to Shandong Pharmaceutical composition is solid form or liquid form, and described solid form is selected from powder, tablet, capsule Agent, pill and dispersible granule, described liquid form is selected from solution or suspension.