CN106153798B - It is a kind of to be used to analyze the purposes for doing reference standard about the HPLC methods and these impurity of material for Buddhist nun's preparation according to Shandong for Buddhist nun and according to Shandong - Google Patents
It is a kind of to be used to analyze the purposes for doing reference standard about the HPLC methods and these impurity of material for Buddhist nun's preparation according to Shandong for Buddhist nun and according to Shandong Download PDFInfo
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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.The method of checked for impurities comprises the following steps：(1) by according to Shandong for Buddhist nun or containing according to Shandong for Buddhist nun medicine dissolving in a solvent to prepare sample solution；(2) sample any one or more of in compound A~H is dissolved in a solvent to prepare reference standard solution or reference substance solution；(3) chromatographic technique is implemented to sample solution and reference standard solution；And (4) are by referring to the one or more of compound A H in the reference standard solution, determine according to Shandong for Buddhist nun or containing according to presence of the Shandong for any one or more of compound A~H in the medicine of Buddhist nun.
Description
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 (prepared in embodiment 2 without recrystallisation from isopropanol), it is used
High performance liquid chromatograph (HPLC) is Shimadzu LC-20A, PDA detector (purchased from Japanese Shimadzu Corporation), as a result such as Fig. 1 institutes
Show, the top in wherein Fig. 1 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 testing result of prior art 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, trifluoracetic acid, the aqueous solution of formic acid or their mixture.Preferred cushioning liquid (A) is acetate, most preferably vinegar
The aqueous solution of sour ammonium and acetic acid or ammonium acetate adjusted the pH aqueous solution with acetic acid.Ammonium acetate in particularly preferred embodiments
The concentration of presence is about 0.001M to 1.0M, most preferably preferably 0.02M to 0.08M, more preferably 0.02M to 0.05M, 0.03M.It is excellent
It is polar aprotic solvent, such as methanol, ethanol or isopropanol to select organic solvent (B)；Or dipolar aprotic solvent, such as acetonitrile.It is preferred that
Organic solvent (B) is in the group including methanol, acetonitrile, ethanol, isopropanol or their mixture, most preferably acetonitrile.
The combination of the aqueous solution (A) and acetonitrile (B) of the mobile phase specifically preferred according to the invention comprising ammonium acetate and acetic acid.
The gradient HPLC methods according to the present invention are further provided for, wherein mobile phase includes following gradient design：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 60 | 40 |
| 12 | 60 | 40 |
| 22 | 20 | 80 |
| 27 | 20 | 80 |
| 27.01 | 60 | 40 |
| 35 | 60 | 40 |
In further preferred embodiment, the aqueous solution of cushioning liquid (A) ammonium acetate and acetic acid in HPLC methods
PH is about 3.8~5.8, preferably from about 4.0~5.5,4.0~5.0,4.3~4.7, most preferably 4.3~4.7.
In other embodiments, the HPLC analysis methods are carried out at a temperature of about 20~40 DEG C, and preferably 25~40
DEG C, 30~40 DEG C, most preferably 30~40 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, 0.7~0.9ml/min, most preferably 0.8ml/min.
Effectively detected with single operation according to the HPLC methods of the present invention and quantitatively include those and be selected from following compound
In compound all impurity：
Compound A：(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：Disclose
The concrete structure of the impurity in Buddhist nun's preparation is replaced for Buddhist nun and Yi Lu according to Shandong, and with gradient HPLC method simultaneously by polar impurity and non-pole
Property Impurity elution comes out, 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 Waters COTRECS C18 (4.6 × 150mm, 2.7 μ
M) post.
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
100%A: 0%B to 0%A: 100%B, it is highly preferred that by 20 to 50 minutes, gradient is 70%A: 30%B to 0%A:
100%B, most preferably, by about 20 minutes, gradient was 65%A: 35%B to 10%A: 90%B or 60%A: 40%B extremely
15%A: 85%B or 60%A: 40%B to 20%A: 80%B.The advantage of this gradient method is that it is possible to that Buddhist nun API will be replaced according to Shandong
Or replace the very close impurity of various opposed polarities or polarity in Buddhist nun's pharmaceutical composition to be totally separated out according to Shandong, it is accurately fixed to be easy to
Property and it is 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, trifluoracetic acid, formic acid or acetic acid they mixed
The aqueous solution combination of compound.
The concentration of cushioning liquid can be 0.001M to 1.0M, and preferred concentration is 0.02M to 0.08M, and more preferably concentration is
0.02M to 0.05M, most preferably 0.03M.The aqueous solution of the particularly preferred mobile phase comprising ammonium acetate and acetic acid, or ammonium acetate are molten
The combination of the liquid aqueous solution (A) and acetonitrile (B) of vinegar acid for adjusting pH.
In the particularly preferred embodiment according to the present invention, it is further provided a kind of gradient HPLC method, wherein
Mobile phase includes following gradient design：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 60 | 40 |
| 12 | 60 | 40 |
| 22 | 20 | 80 |
| 27 | 20 | 80 |
| 27.01 | 60 | 40 |
| 35 | 60 | 40 |
The particularly preferred HPLC gradient methods that the present invention is also provided, wherein, mobile phase is made comprising ammonium acetate and/or acetic acid
For cushioning liquid (A).In other particularly preferred embodiment, mobile phase includes acetonitrile as organic solvent (B), and/or
Acetonitrile-methanol mixed solvent is used as organic solvent (B).Inventor is had found when mobile phase includes ammonium acetate and/or acetic acid (A) and second
Gradient design is particularly effective during nitrile (B).
Cushioning liquid (A) can be containing one or more of added solvents, and these added solvents can be methanol, ethanol, isopropyl
Alcohol 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 acetonitrile.
The pH of cushioning liquid (A) is about 3.8~5.8, preferably from about 4.0~5.5,4.0~5.0,4.3~4.7, most preferably
For 4.3~4.7.
The HPLC methods of the present invention are carried out at a temperature of about 20~40 DEG C, preferably 25~40 DEG C, 30~40 DEG C, optimal
Elect 30~40 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, 0.7~0.9ml/min, most preferably 0.8ml/min.
Another aspect of the present invention provides a kind of reference standard solution.The solution includes and is dissolved in suitable solvent (such as 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 50mL ethyl acetate extractions 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, 50ml 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 asks that (RRT) is about 0.97 when replacing the relative reservation of Buddhist nun according to Shandong with respect to principal component on HPLC spectrograms.
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, 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
The 1H-NMR figures of impurity E 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 the outflow of no acetonitrile, plus 3 times of volumes ethyl acetate extraction, merge 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：Waters COTRECS C18 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：0.02M ammonium acetate solutions (A) (acetic acid adjusts pH to 4.0), acetonitrile (B)
Detection wavelength：260nm
Diluent：80% acetonitrile-water
Column temperature：40℃
Flow velocity：0.8ml/min
Gradient design is as described below.
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 60 | 40 |
| 12 | 60 | 40 |
| 22 | 20 | 80 |
| 27 | 20 | 80 |
| 27.01 | 60 | 40 |
| 35 | 60 | 40 |
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：Waters COTRECS C18 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：0.08M ammonium acetate solutions (A) (acetic acid adjusts pH to 4.0), acetonitrile (B)
Detection wavelength：260nm
Diluent：80% acetonitrile-water
Column temperature：40℃
Flow velocity：0.6ml/min
Embodiment 12：
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：Waters COTRECS C18 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：0.08M ammonium acetate solutions (A) (acetic acid adjusts pH to 5.0), acetonitrile (B)
Detection wavelength：260nm
Diluent：80% acetonitrile-water
Column temperature：40℃
Flow velocity：0.8ml/min
Embodiment 13：
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：Waters COTRECS C18 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：0.02M ammonium acetate solutions (A) (acetic acid adjusts pH to 5.0), acetonitrile (B)
Detection wavelength：260nm
Diluent：80% acetonitrile-water
Column temperature：25℃
Flow velocity：1.0ml/min
Embodiment 14：
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：Waters COTRECS C18 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 acetate solutions (A) (acetic acid adjusts pH to 4.5), acetonitrile (B)
Detection wavelength：260nm
Diluent：80% acetonitrile-water
Column temperature：35℃
Flow velocity：0.6ml/min
Embodiment 15：
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：Waters COTRECS C18 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 acetate solutions (A) (acetic acid adjusts pH to 5.0), acetonitrile (B)
Detection wavelength：260nm
Diluent：80% acetonitrile-water
Column temperature：40℃
Flow velocity：0.6ml/min
Embodiment 16：
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：Waters COTRECS C18 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 acetate solutions (A) (acetic acid adjusts pH to 4.5), acetonitrile (B)
Detection wavelength：260nm
Diluent：80% acetonitrile-water
Column temperature：35℃
Flow velocity：0.8ml/min
Embodiment 17：
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：Waters COTRECS C18 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 acetate solutions (A) (acetic acid adjusts pH to 4.0), acetonitrile (B)
Detection wavelength：260nm
Diluent：80% acetonitrile-water
Column temperature：30℃
Flow velocity：1.0ml/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 10~17, 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 10~17, 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
Shandong sees that the top in Figure 22~Figure 24, Figure 22-24 is for Buddhist nun's according to Shandong respectively for the HPLC spectrograms of Buddhist nun's capsule need testing solution
Peak.
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, replaced according to Shandong in Buddhist nun's need testing solution and detected compound C (relative retention time is about 0.70), (relative retention time is about by B
For 0.76), (relative retention time is about by H (relative retention time is about 1.12), F (relative retention time is about 1.25), G
1.79)；Replaced according to Shandong in Buddhist nun's capsule need testing solution and detected compound C, H, F, A (relative retention time is about 1.52) and G.
The compound A of table 1~H and according to Shandong replace positioning and each peak of the Buddhist nun in the method for embodiment 16 between separating degree
The relative retention time of each impurity under the conditions of the embodiment 10~17 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 (18)
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；
(3) chromatographic technique is implemented to sample solution and reference standard solution；And
(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 methods, in the gradient HPLC methods, mobile phase includes the combination of A containing cushioning liquid and organic solvent B, and the buffering is molten
Liquid A is that pH ammonium acetate aqueous solution was adjusted with acetic acid, and the organic solvent B is acetonitrile, wherein consolidating of using of the chromatography
Fixed is mutually octadecylsilane chemically bonded silica,
Wherein in gradient HPLC methods, mobile phase includes following gradient design：
The concentration that wherein ammonium acetate is present is 0.001M to 1.0M, and cushioning liquid A pH is 3.8~5.8.
2. the method for claim 1 wherein the solvate is hydrate.
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 that ammonium acetate is present is 0.02M to 0.08M.
5. the method for claim 1 wherein the concentration that ammonium acetate is present is 0.02M to 0.05M.
6. the method for claim 1 wherein the concentration that ammonium acetate is present is 0.03M.
7. the method for claim 1 wherein cushioning liquid A pH is 4.0~5.5.
8. the method for claim 1 wherein cushioning liquid A pH is 4.0~5.0.
9. the method for claim 1 wherein cushioning liquid A pH is 4.3~4.7.
10. any one of claim 1-9 method, wherein the HPLC methods are carried out at a temperature of 20~40 DEG C.
11. any one of claim 1-9 method, wherein the HPLC methods are carried out at a temperature of 25~40 DEG C.
12. any one of claim 1-9 method, wherein the HPLC methods are carried out at a temperature of 30~40 DEG C.
13. any one of claim 1-9 method, wherein the HPLC methods are carried out under 0.4~1.2ml/min flow velocity.
14. any one of claim 1-9 method, wherein the HPLC methods are carried out under 0.5~1.1ml/min flow velocity.
15. any one of claim 1-9 method, wherein the HPLC methods are carried out under 0.6~1.0ml/min flow velocity.
16. any one of claim 1-9 method, wherein the HPLC methods are carried out under 0.7~0.9ml/min flow velocity.
17. any one of claim 1-9 method, wherein the HPLC methods are carried out under 0.8ml/min flow velocity.
18. the method described in claim any one of 1-9, wherein comprising for the medicine of Buddhist nun being solid form or liquid shape according to Shandong
Formula, the solid form is selected from pulvis, tablet, capsule, pill and dispersible granule, and the liquid form is selected from molten
Liquid or suspension.
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