CN104086564A - Method for preparing high-purity temsirolimus - Google Patents
Method for preparing high-purity temsirolimus Download PDFInfo
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- CN104086564A CN104086564A CN201410370625.7A CN201410370625A CN104086564A CN 104086564 A CN104086564 A CN 104086564A CN 201410370625 A CN201410370625 A CN 201410370625A CN 104086564 A CN104086564 A CN 104086564A
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- ethyl acetate
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- CBPNZQVSJQDFBE-FUXHJELOSA-N Temsirolimus Chemical compound C1C[C@@H](OC(=O)C(C)(CO)CO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 CBPNZQVSJQDFBE-FUXHJELOSA-N 0.000 title claims abstract description 133
- 229960000235 temsirolimus Drugs 0.000 title claims abstract description 133
- 238000000034 method Methods 0.000 title claims abstract description 62
- QFJCIRLUMZQUOT-UHFFFAOYSA-N temsirolimus Natural products C1CC(O)C(OC)CC1CC(C)C1OC(=O)C2CCCCN2C(=O)C(=O)C(O)(O2)C(C)CCC2CC(OC)C(C)=CC=CC=CC(C)CC(C)C(=O)C(OC)C(O)C(C)=CC(C)C(=O)C1 QFJCIRLUMZQUOT-UHFFFAOYSA-N 0.000 title abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 188
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 132
- 239000007864 aqueous solution Substances 0.000 claims abstract description 40
- 238000010828 elution Methods 0.000 claims abstract description 31
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims abstract description 30
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 18
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000945 filler Substances 0.000 claims abstract description 14
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 14
- 239000010703 silicon Substances 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 8
- 239000012043 crude product Substances 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 10
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 10
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 9
- 229910019142 PO4 Inorganic materials 0.000 claims description 8
- 239000010452 phosphate Substances 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 6
- CHKVPAROMQMJNQ-UHFFFAOYSA-M potassium bisulfate Chemical compound [K+].OS([O-])(=O)=O CHKVPAROMQMJNQ-UHFFFAOYSA-M 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- 238000011049 filling Methods 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- JXAZAUKOWVKTLO-UHFFFAOYSA-L sodium pyrosulfate Chemical compound [Na+].[Na+].[O-]S(=O)(=O)OS([O-])(=O)=O JXAZAUKOWVKTLO-UHFFFAOYSA-L 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 230000002378 acidificating effect Effects 0.000 claims description 4
- 230000001186 cumulative effect Effects 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- 235000019260 propionic acid Nutrition 0.000 claims description 3
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- 235000011054 acetic acid Nutrition 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- BIGPRXCJEDHCLP-UHFFFAOYSA-N ammonium bisulfate Chemical compound [NH4+].OS([O-])(=O)=O BIGPRXCJEDHCLP-UHFFFAOYSA-N 0.000 claims description 2
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 2
- 239000011707 mineral Substances 0.000 claims description 2
- 229940045641 monobasic sodium phosphate Drugs 0.000 claims description 2
- 150000007524 organic acids Chemical class 0.000 claims description 2
- 235000011007 phosphoric acid Nutrition 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 2
- 229940093916 potassium phosphate Drugs 0.000 claims description 2
- 235000011009 potassium phosphates Nutrition 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 2
- 238000013375 chromatographic separation Methods 0.000 abstract 2
- 238000004305 normal phase HPLC Methods 0.000 abstract 1
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 38
- 229960002930 sirolimus Drugs 0.000 description 38
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 37
- 239000012071 phase Substances 0.000 description 32
- 238000002360 preparation method Methods 0.000 description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Natural products OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 25
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 24
- 239000012046 mixed solvent Substances 0.000 description 21
- 238000005406 washing Methods 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 15
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 14
- 239000012074 organic phase Substances 0.000 description 14
- 239000012535 impurity Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 9
- 238000004090 dissolution Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 7
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000002243 precursor Substances 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 238000010898 silica gel chromatography Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000011260 aqueous acid Substances 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- 238000010606 normalization Methods 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 229910000343 potassium bisulfate Inorganic materials 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- -1 CCI-779 compound Chemical class 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 206010027336 Menstruation delayed Diseases 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical class [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010050513 Metastatic renal cell carcinoma Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 241000187391 Streptomyces hygroscopicus Species 0.000 description 1
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 description 1
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000011242 molecular targeted therapy Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229940100411 torisel Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
- C07D498/18—Bridged systems
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a high-purity temsirolimus represented by the formula (I), the HPLC purity of the temsirolimus can be above 99.5%. The invention also provides a method for preparing high-purity temsirolimus by virtue of a preparative high performance liquid chromatographic separation system. The method comprises, but not limited to the sequence of the steps, the following steps of A) normal-phase high performance liquid chromatography and B) reverse phase high performance liquid chromatographic separation, wherein in the step A), gradient elution is carried out by taking a silicon ball filler as a stationary phase, ethyl acetate of which the concentration is 60-100v/v% and n-hexane or n-heptane of which the concentration is 40-0v/v% as mobile phases; in step B), gradient elution is carried out by taking C4, C6, C8 or C18 fillers as stationary phases and acetonitrile and aqueous solution of which the pH value is 3.5-6.0 as mobile phases; and the temperature of the system is 10-30 DEG C. The process is simple and environmentally friendly and high-purity and high-quality temsirolimus can be obtained by virtue of the process.
Description
Technical field
The present invention relates to pharmaceutical chemistry technical field, be specifically related to a kind of preparation method of high purity CCI-779.
Background technology
CCI-779 (temsirolimus), has another name called CCI-779, trade(brand)name Torisel, chemistry 42-[3-hydroxyl-2-(methylol)-2 Methylpropionic acid ester by name]-rapamycin, molecular formula C
56h
87nO
16, structural formula is as follows:
CCI-779 is a kind of target mTOR kinases inhibitor, is the derivative by the rapamycin (sirolimus) of streptomyces hygroscopicus FC904 fermentation generation.In May, 2007, U.S. FDA approval CCI-779 is for the first-line treatment of renal cell carcinoma in intractable late period, and November in the same year, Europe EMBA also ratified its listing.CCI-779 is as molecular targeted therapy medicine, for excision, radiotherapy, chemotherapy, the undesirable metastatic renal cell carcinoma patient in late period of immunotherapy texts have brought dawn.
Because the commercially available formulation of CCI-779 is injection with small volume, therefore high to the specification of quality of CCI-779 bulk drug, especially to residual sirolimus raw material, isomer and other related degradation impurity, clarity of solution, the aspect controls such as weight loss on drying are tighter.Preparing in CCI-779 process, due to the structural unstable reason of CCI-779 itself, especially itself be very easy to change into isomer impurities, cause the related substance detection in bulk drug finished product to be difficult to up to standard.If it is improper to store, as hot and humid condition or strong illumination, the open loop that is easy to degrade generates impurity.
The purification process of existing CCI-779 (being CCI-779) is typically implemented in the aftertreatment for preparing crude product.CN103664999A discloses a kind of method of purification: CCI-779 precursor is placed in 5mL THF, drips 2N HCl, and 0 DEG C is stirred 12h, and TLC judges reaction end.After having reacted, add the dilution of 10mL ethyl acetate, add 10mL water, use 20mL ethyl acetate extracted organic phase three times, merge organic phase.Organic phase saturated sodium carbonate solution washing one time, water washing three times, saturated common salt water washing one time, anhydrous magnesium sulfate drying.Silica gel column chromatography after underpressure distillation, ethyl acetate: normal hexane (2:1～4:1) is wash-out fast, obtains CCI-779.
CN103421023A discloses a kind of method of preparing CCI-779 from CCI-779 precursor, in 1L round-bottomed flask, drop into 75g CCI-779 precursor, 1g tosic acid is dissolved in the tetrahydrofuran (THF) of 400mL, at 0～5 DEG C, drip 5mL ethylene glycol, in 10min, drip off, stir after 1h, reaction finishes, add the water of 1L, be extracted with ethyl acetate three times (500mL*3), gained ethyl acetate layer is washed (200mL*3) three times with saturated sodium-chloride again, after anhydrous sodium sulfate drying, evaporated under reduced pressure obtains crude product 72g, gained crude product is through silica gel column chromatography, PE:AC=4:1 wash-out obtains 45g CCI-779.
Above-mentioned method of purification is all silica gel column chromatography, and the product purity making is not high, poor to Impurity removal effect, and starting raw material sirolimus content is not controlled, and is difficult to meet the specification of quality of injection type to bulk drug.Therefore, in order effectively to remove impurity, obtain other CCI-779 of more highly purified injection stage, need the effectively reliable method of exploitation to carry out purification to it.
Summary of the invention
The object of the present invention is to provide a kind of high purity CCI-779 and preparation method thereof.Described method is removed sirolimus residual in crude product and other organic impuritys by highly effective liquid phase chromatographic system, simple and safe, environmentally friendly, effectively improves the purity of product, meets the specification of quality of injection formulations to bulk drug.
For achieving the above object, the invention provides following technical scheme:
According to a first aspect of the invention, provide a kind of high purity CCI-779, its chemical structure is suc as formula shown in I, and the HPLC purity of described high purity CCI-779 is greater than 98%,
One preferred embodiment in, the HPLC purity of described high purity CCI-779 is greater than 99%.
In further preferred embodiment, the HPLC purity of described high purity CCI-779 is greater than 99.5%, and even 99.9%.
According to a second aspect of the invention, provide a kind of method of preparing above-mentioned high purity CCI-779, comprised step:
A) positive high performance liquid chromatography: by CCI-779 crude product through performance liquid chromatographic column purification system, taking positive phase filling as stationary phase, mixture taking ethyl acetate and normal heptane or ethyl acetate and normal hexane carries out wash-out as moving phase, obtains CCI-779 work in-process;
B) RPLC: CCI-779 work in-process, through performance liquid chromatographic column purification system, taking reverse phase filler as stationary phase, are carried out to wash-out taking the mixture of the aqueous solution of acetonitrile and pH3.5～6.0 as moving phase, obtain high purity CCI-779.
Described steps A) in, positive phase filling is silicon ball filler.The particle diameter of silicon ball filler can be any number or the interval within the scope of 5～45 μ m, for example 5,10,15,20,25,20～45 μ m, etc., preferably 5 μ m, 10 μ m, more preferably 10 μ m.
Described steps A) in, CCI-779 crude product can be dissolved in and in ethyl acetate, carry out upper prop.The weightmeasurement ratio of CCI-779 crude product and ethyl acetate can be 1g/3～10ml, preferably 1g/5ml.
Described steps A) in, in the cumulative volume of described moving phase, in moving phase, contain ethyl acetate 60～100v/v%, contain normal hexane or normal heptane 40～0v/v%.
Described steps A) in, type of elution is preferably gradient elution, at 0～10min of wash-out, moving phase contains ethyl acetate 60v/v%, contain normal hexane or normal heptane 40v/v%, at 10～100min of wash-out, in moving phase, the content of ethyl acetate changes to 100v/v% from 60v/v%, and the content of normal heptane or normal hexane changes to 0v/v% from 40v/v% simultaneously.
Described steps A) in, the system temperature of performance liquid chromatographic column is 10～30 DEG C, for example 10,15,20,25,30 DEG C, and preferably 15～25 DEG C, more preferably 20 DEG C.
Described steps A) in, gradient elution Fractional Collections obtains the elutriant that contains CCI-779, by elutriant concentrating under reduced pressure below 30 DEG C of collecting, obtains CCI-779 work in-process.Preferably concentrating under reduced pressure carries out at 20～30 DEG C.
Described step B) in, reverse phase filler is selected from C4, C6, C8 or C18 filler, preferably C18 filler.The particle diameter of reverse phase filler can be any number or the interval within the scope of 5～45 μ m, for example 5,10,15,20,25,20～45 μ m etc., preferably 5 μ m, 10 μ m, more preferably 10 μ m.
Described step B) in, CCI-779 work in-process can be dissolved in and in acetonitrile, carry out upper prop.The weightmeasurement ratio of CCI-779 work in-process and acetonitrile is 1g/3～10ml, preferably 1g/5ml.
Described step B) in, the aqueous solution is in water, to add acidic substance formulated.Acidic substance can be any acid or the acid-salts etc. that those skilled in the art know, comprise organic acid, mineral acid, hydrosulfate, hydrophosphate etc., for example, preferably have formic acid, acetic acid, propionic acid, phosphoric acid, sulfuric acid, sal enixum, sodium pyrosulfate, monoammonium sulfate, potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, primary ammonium phosphate, potassiumphosphate etc.Those skilled in the art's amount that easily adjustment of acidity material adds, making the pH of the described aqueous solution is 3.5～6.0, being preferably pH is 4～5.5.
Described step B) in, in the cumulative volume of described moving phase, in moving phase, contain acetonitrile 50～80v/v%, the aqueous solution 50～20v/v% that contains pH3.5～6.0.
Described step B) in, type of elution is preferably gradient elution, at 0～10min of wash-out, moving phase contains acetonitrile 50v/v%, the aqueous solution 50v/v% that contains pH3.5～6.0, at 10～100min of wash-out, in moving phase, the content of acetonitrile changes to 80v/v% from 50v/v%, and the content of the aqueous solution of pH3.5～6.0 changes to 20v/v% from 50v/v% simultaneously.
Described step B) in, the system temperature of performance liquid chromatographic column is 10～30 DEG C, for example 10,15,20,25,30 DEG C, and preferably 15～25 DEG C, more preferably 20 DEG C.
Described step B) in, gradient elution Fractional Collections obtains the elutriant that contains CCI-779.By elutriant concentrating under reduced pressure below 30 DEG C of collecting, dry, obtain high purity CCI-779.Preferably, the elutriant of collection can first be evaporated on a small quantity, be extracted with ethyl acetate, and saturated nacl aqueous solution washing, dry, filter, be evaporated to dryly, obtain high purity CCI-779.Preferably concentrating under reduced pressure carries out at 20～30 DEG C.
In addition, the inventor also studies discovery, and the sequencing of positive high performance liquid chromatography and RPLC can exchange, and prepares equally high purity CCI-779, i.e. the present invention also provides the another kind of method of preparing high purity CCI-779, comprises step:
1) RPLC: CCI-779 crude product loading, to performance liquid chromatographic column, taking reverse phase filler as stationary phase, is carried out to wash-out taking the mixture of the aqueous solution of acetonitrile and pH3.5～6.0 as moving phase, obtain CCI-779 work in-process;
2) positive high performance liquid chromatography: by CCI-779 work in-process loading to performance liquid chromatographic column, taking positive phase filling as stationary phase, mixture taking ethyl acetate and normal heptane or ethyl acetate and normal hexane carries out wash-out as moving phase, obtains high purity CCI-779.
Wherein, step 1) with 2) carrying out respectively with step B in aforesaid method) and A) consistent, in a kind of upper method to steps A) and step B) description can quote at this be considered as respectively step 1) and 2) restriction.
According to preparation method provided by the invention, CCI-779 crude product is after positive and anti-phase two step high performance liquid chromatography processing, more than the HPLC purity to 99.5% of the CCI-779 of gained, single impurity is less than 0.1% (except isomer), meet the standard of the ICH of European Union, meet the specification of quality of injection to bulk drug.
Method of the present invention can be carried out in full-automatic preparative high performance liquid chromatography column separating purification system, and technique is simple, and repeatability is high, product purity is high, steady quality, and preparation process is not used toxic reagent, less to operator and environmental influence, there is good prospects for commercial application.
Embodiment
Further illustrate the features and advantages of the invention below by preferred embodiment.The method in following embodiment of it should be understood that is only for the present invention is described, instead of limitation of the present invention.
The purity of CCI-779 of the present invention refers to according to HPLC and detects the percentage ratio that peak area that collection of illustrative plates carries out the CCI-779 compound that area normalization method obtains occupies in all peak area summations.The content of sirolimus etc. refers to according to HPLC and detects the percentage ratio that peak area that collection of illustrative plates carries out the respective substance such as sirolimus that area normalization method obtains occupies in all peak area summations.
Embodiment 1
1) the half-finished preparation of CCI-779
By 8.3g CCI-779 crude product (purity 75.68%, sirolimus 3.47%, isomer 16.48%, area normalization method, lower same) with after 25mL acetic acid ethyl dissolution, (biological chromatography Technology Co., Ltd. is matched in Nanjing hundred to be splined on positive silicon ball filled column, lower same, particle diameter 5 μ m), by ethyl acetate: normal heptane mixed solvent system carries out gradient elution, 0～10min, ethyl acetate/normal heptane (v/v, lower same)=60:40, at 10～100min, ethyl acetate/normal heptane tapers to 100:0 from 60:40, system temperature is 20 DEG C, collect component, at 30 DEG C, be evaporated to dry, obtain 6.8g CCI-779 work in-process (purity 84.78%, sirolimus 0.02%, isomer 13.23%).
2) preparation of high purity CCI-779
After above-mentioned 6.8g CCI-779 work in-process are dissolved with 21mL acetonitrile, be splined on anti-phase C18 filled column (Japanese DAISO Co., Ltd, lower same, particle diameter 5 μ m), carry out gradient elution with aqueous formic acid (pH3.5) mixed solvent system of acetonitrile: 1mmol/L, 0～10min, acetonitrile/aqueous formic acid=50:50, 10～100min, acetonitrile/aqueous formic acid tapers to 80:20 from 50:50, system temperature is 20 DEG C, collect component, 30 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, separate organic phase, dry, filter, at 30 DEG C, be evaporated to dry, obtain 4.9g high purity CCI-779 (purity 99.94%, sirolimus does not detect, isomer 0.08%, the equal <0.1% of other single impurity).
Embodiment 2
1) the half-finished preparation of CCI-779
By 56g CCI-779 crude product (purity 79.03%, sirolimus 2.54%, isomer 15.63%) with after 560mL acetic acid ethyl dissolution, (10 μ m) to be splined on positive silicon ball filled column, carry out gradient elution with the mixed solvent system of ethyl acetate and normal hexane, 0～10min, ethyl acetate/normal hexane=60:40, 10～100min, ethyl acetate/normal hexane tapers to 100:0 from 60:40, system temperature is 10 DEG C, collect component, at 20 DEG C, be evaporated to dry, obtain 46g CCI-779 work in-process (purity 86.17%, sirolimus does not detect, isomer 11.65%).
2) preparation of high purity CCI-779
After above-mentioned 46g CCI-779 work in-process are dissolved with 460mL acetonitrile, (10 μ m) to be splined on anti-phase C6 filled column, carry out gradient elution with the mixed solvent system of the potassium dihydrogen phosphate aqueous solution (pH5) of acetonitrile and 20mmol/L, 0～10min, acetonitrile/potassium dihydrogen phosphate aqueous solution=50:50, 10～100min, acetonitrile/potassium dihydrogen phosphate aqueous solution tapers to 80:20 from 50:50, system temperature is 10 DEG C, collect component, 25 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, separate organic phase, dry, filter, at 20 DEG C, be evaporated to dry, obtain 35g high purity CCI-779 (purity 99.81%, sirolimus does not detect, isomer 0.12%, the equal <0.1% of other single impurity).
Embodiment 3
1) the half-finished preparation of CCI-779
By 100g CCI-779 crude product (purity 76.89%, sirolimus 1.95%, isomer 15.92%) with after 500mL acetic acid ethyl dissolution, (20-45 μ m) to be splined on positive silicon ball filled column, by ethyl acetate: normal heptane mixed solvent system carries out gradient elution, 0～10min, ethyl acetate/normal heptane=60:40, 10～100min, ethyl acetate/normal heptane tapers to 100:0 from 60:40, system temperature is 15 DEG C, collect component, at 30 DEG C, be evaporated to dry, obtain 84.4g CCI-779 work in-process (purity 84.23%, sirolimus 0.01%, isomer: 13.47%).
2) preparation of CCI-779 finished product
After 84.4g CCI-779 work in-process are dissolved with 422mL acetonitrile, (20-45 μ m) to be splined on anti-phase C8 filled column, with acetonitrile: the sodium pyrosulfate aqueous solution (pH5.5) mixed solvent system of 3.5 μ mol/L carries out gradient elution, 0～10min, acetonitrile/sodium pyrosulfate the aqueous solution=50:50, 10～100min, acetonitrile/sodium pyrosulfate aqueous solution tapers to 80:20 from 50:50, system temperature is 15 DEG C, collect component, 30 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, separate organic phase, dry, filter, at 30 DEG C, be evaporated to dry, obtain 59.4g high purity CCI-779 (purity 99.75%, sirolimus 0.01%, isomer 0.16%, the equal <0.1% of other single impurity).
Embodiment 4
1) the half-finished preparation of CCI-779
By 230g CCI-779 crude product (purity 75.32%, isomer 11.23%, sirolimus 2.14%) with after the dissolving of 690mL acetonitrile, (5 μ m) to be splined on anti-phase C18 filled column, carry out gradient elution with acetic acid aqueous solution (pH4.5) mixed solvent system of acetonitrile: 0.1mmol/L, 0～10min, acetonitrile/acetic acid aqueous solution=50:50, 10～100min, acetonitrile/containing second aqueous acid=80:20, system temperature is 20 DEG C, collect component, 30 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, separate organic phase, dry, filter, at 30 DEG C, be evaporated to dry, obtain 158g CCI-779 work in-process (purity 98.23%, isomer 0.15%, sirolimus 1.46%).
2) preparation of CCI-779 finished product
Above-mentioned 158g CCI-779 work in-process are used after 475mL acetic acid ethyl dissolution, (5 μ m) to be splined on positive silicon ball filled column, by ethyl acetate: normal heptane mixed solvent system carries out gradient elution, 0～10min, ethyl acetate/normal heptane=60:40, 10～100min, ethyl acetate/normal heptane tapers to 100:0 from 60:40, system temperature is 20 DEG C, collect component, at 30 DEG C, be evaporated to dry, obtain 128g high purity CCI-779 (purity 99.80%, isomer 0.15%, sirolimus 0.01%, the equal <0.1% of other single impurity).
Embodiment 5
1) the half-finished preparation of CCI-779
By CCI-779 crude product 75g (purity 76.93%, isomer 12.01%, sirolimus 1.89%) with after the dissolving of 750mL acetonitrile, (10 μ m) to be splined on anti-phase C8 filled column, with acetonitrile: aqueous potassium hydrogen sulfate (pH5) mixed solvent system of 15 μ mol/L carries out gradient elution, 0～10min, acetonitrile/aqueous potassium hydrogen sulfate=50:50, 10～100min, acetonitrile/aqueous potassium hydrogen sulfate tapers to 80:20 from 50:50, system temperature is 10 DEG C, Fractional Collections, liquid phase monitoring, merge component, 30 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, layering, dry filter, at 30 DEG C, be evaporated to dry, obtain 53g CCI-779 work in-process (purity 97.64%, isomer 0.14%, sirolimus 1.37%).
(2) preparation of high purity CCI-779
Above-mentioned 53g CCI-779 work in-process are used after 530mL acetic acid ethyl dissolution, (20-45 μ m) to be splined on positive silicon ball filled column, by ethyl acetate: normal heptane mixed solvent system carries out gradient elution, 0～10min, ethyl acetate/normal heptane=60:40, 10～100min, ethyl acetate/normal heptane tapers to 100:0 from 60:40, system temperature is 10 DEG C, collect component, at 30 DEG C, be evaporated to dry, obtain 43g high purity CCI-779 (purity 99.81%, isomer 0.16%, sirolimus does not detect, the equal <0.1% of other single impurity).
Embodiment 6
1) the half-finished preparation of CCI-779
By CCI-779 crude product 320g (purity 78.34%, isomer 11.05%, sirolimus 2.52%) with after the dissolving of 1600mL acetonitrile, on enter anti-phase C6 filled column (20-45 μ m), with acetonitrile: Sodium phosphate dibasic/biphosphate sodium water solution is (in phosphate radical 0.03mol/L, pH6) mixed solvent system carries out gradient elution, 0～10min, acetonitrile/water solution=50:50, 10～100min, acetonitrile/water solution tapers to 80:20 from 50:50, system temperature is 15 DEG C, collect component, 30 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, separate organic phase, dry, filter, at 30 DEG C, be evaporated to dry, obtain 228g CCI-779 work in-process (purity 97.50%, isomer 0.18%, sirolimus 2.06%, the equal <0.1% of other single impurity).
2) preparation of high purity CCI-779
Above-mentioned CCI-779 work in-process 228g is used after 1140mL acetic acid ethyl dissolution, on enter positive silicon ball filled column (10 μ m), by ethyl acetate: normal hexane mixed solvent system carries out gradient elution, 0～10min, ethyl acetate/normal hexane=60:40, 10～100min, ethyl acetate/normal hexane changes to 100:0 from 60:40, system temperature is 20 DEG C, collect component, at 30 DEG C, be evaporated to dry, obtain 182g high purity CCI-779 (purity 99.76%, isomer 0.20%, sirolimus: 0.01%, the equal <0.1% of other single impurity).
Reference examples 1
CCI-779 method of purification with reference to CN103664999A is tested: CCI-779 precursor (5g, 4.38mmol) be placed in the tetrahydrofuran (THF) of 25mL, drip 2N HCl22ml (44mmol), 0 DEG C of stir about 12 hours, primitive reaction is complete for TLC point plate reaction raw materials.After having reacted, add the dilution of 200mL ethyl acetate, add 200mL water, use 100mL ethyl acetate extracted organic phase three times, merge organic phase.Saturated sodium carbonate solution washing one time for organic phase, water washing three times, saturated common salt water washing one time, anhydrous magnesium sulfate drying.At 30 DEG C underpressure distillation after dry crude product, crude product is through 200-300 order silica gel column chromatography, ethyl acetate: normal hexane (4:1 → 2:1) is wash-out fast, obtains CCI-779 2.4g (purity 88.75%, sirolimus 2.5%, isomer 8.56%).
Reference examples 2
Prepare the method for CCI-779 tests with reference to the CCI-779 precursor of CN103421023A: in the round-bottomed flask of 1L, drop into CCI-779 precursor (7.5g, 6.56mmol) and tosic acid (0.1g, 0.58mmol) be dissolved in the tetrahydrofuran (THF) of 400mL, at 0 DEG C, drip the ethylene glycol of 5mL, about 5 minutes, drip off, drip and finish, stir after 1 hour, TLC detects and finds that raw material reacts completely, add the water of 1L, be extracted with ethyl acetate three times (500mL*3), gained ethyl acetate layer is washed (200mL*3) three times with saturated sodium-chloride again, after anhydrous sodium sulfate drying, evaporated under reduced pressure must about 7g crude product, gained crude product is through 200-300 order silica gel column chromatography, use the quick wash-out of mixed solvent PE:AC=4:1, obtain 4.2g CCI-779 (purity 85.43%, sirolimus 1.2%, isomer 12.24%).
Reference examples 3
1) the half-finished preparation of CCI-779
By CCI-779 crude product 85.1g (purity 74.65%, sirolimus 3.04%, isomer 16.88%) with after 255mL acetone solution, (20-45 μ m) to be splined on positive silicon ball filled column, with acetone: normal hexane mixed solvent system carries out gradient elution, 0～10min, acetone/normal hexane=90:10, 10～100min, acetone/normal hexane tapers to 100:0 from 90:10, system temperature is 20 DEG C, collect component, at 30 DEG C, be evaporated to dry, obtain 60.4g CCI-779 work in-process (purity 84.78%, sirolimus 0.84%, isomer 11.89%).
2) preparation of CCI-779 finished product
Above-mentioned 60.4g CCI-779 work in-process are used after 286mL dissolve with methanol, (20-45 μ m) to be splined on anti-phase C18 filled column, with methyl alcohol: 1 μ mol/L aqueous sulfuric acid (pH5) mixed solvent system carries out gradient elution, 0～10min, methyl alcohol/aqueous sulfuric acid=60:40, 10～100min, methyl alcohol/aqueous sulfuric acid tapers to 90:10 from 60:40, system temperature is 20 DEG C, collect component, 30 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, separate organic phase, dry filter, at 30 DEG C, be evaporated to dry, obtain 47.7g CCI-779 (purity 97.25%, sirolimus 0.83%, isomer 1.24%).
Reference examples 4
1) the half-finished preparation of CCI-779
By CCI-779 crude product 117g (purity 77.63%, sirolimus 1.58%, isomer 15.69%) with after 585mL acetic acid ethyl dissolution, on enter positive silicon ball filled column (10 μ m), by ethyl acetate: normal heptane mixed solvent system carries out gradient elution, 0～10min, ethyl acetate/normal heptane=60:40, 10～100min, ethyl acetate/normal heptane=100:0, system temperature is 20 DEG C, collect component, at 30 DEG C, be evaporated to dry, obtain the CCI-779 work in-process (purity 82.43% of 89.6g, sirolimus 0.65%, isomer 12.45%).
2) preparation of high purity CCI-779
Above-mentioned 89.6g CCI-779 work in-process are dissolved with 473mL acetonitrile, (10 μ m) to be splined on anti-phase C18 filled column, with acetonitrile: 0.05mol/L acetic acid aqueous solution (pH3) mixed solvent system carries out gradient elution, 0～10min, acetonitrile/phosphate aqueous solution=50:50, 10～100min, acetonitrile/phosphate aqueous solution tapers to 80:20 from 50:50, system temperature is 20 DEG C, collect component, 30 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, separate organic phase, dry, filter, at 30 DEG C, be evaporated to dry, obtain 56.8g CCI-779 (purity 97.50%, sirolimus 0.64%, isomer 1.58%).
Reference examples 5
1) the half-finished preparation of CCI-779
By CCI-779 crude product 5.6g (purity 76.38%, isomer 12.39%, sirolimus 2.86%) with after 22.4mL dissolve with methanol, (20-45 μ m) to be splined on anti-phase C18 filled column, with methyl alcohol: 0.9 μ mol/L phosphate aqueous solution (pH6) mixed solvent system carries out gradient elution, 0～10min, methyl alcohol/phosphate aqueous solution=60:40, 10～100min, methyl alcohol/phosphate aqueous solution tapers to 90:10 from 60:40, system temperature is 20 DEG C, collect component, 30 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, separate organic phase, dry, filter, at 30 DEG C, be evaporated to dry, obtain 3.89g CCI-779 work in-process (purity 96.12%, isomer 1.31%, sirolimus 2.38%).
(2) preparation of high purity CCI-779
Above-mentioned 3.89g CCI-779 work in-process are used after 23.3mL acetone solution, (20-45 μ m) to be splined on positive silicon ball filled column, with acetone: normal hexane mixed solvent system carries out gradient elution, 0～10min, acetone/normal hexane=70:30,10～100min, acetone/normal hexane tapers to 100:0 from 70:30, system temperature is 20 DEG C, collect component, be evaporated at 30 DEG C dryly, obtain the CCI-779 finished product (purity 97.48% of 3.03g, isomer 1.35%, sirolimus 0.42%).
Reference examples 6
1) the half-finished preparation of CCI-779
By CCI-779 crude product 10.6g (purity 76.98%, isomer 12.15%, sirolimus 1.06%) with after the dissolving of 85mL acetonitrile, (10 μ m) to be splined on anti-phase C18 filled column, with acetonitrile: the 0.06mo/L propionic acid aqueous solution (pH3) mixed solvent system carries out gradient elution, 0～10min, acetonitrile/containing the third aqueous acid=50:50, 10～100min, acetonitrile/containing the third aqueous acid=80:20, system temperature is 20 DEG C, collect component, 30 DEG C are evaporated to a small amount of, be extracted with ethyl acetate, saturated nacl aqueous solution washing, layering, dry filter, at 30 DEG C, be evaporated to dry, obtain the CCI-779 work in-process (purity 95.41% of 7.3g, isomer 1.30%, sirolimus 0.78%).
(2) preparation of high purity CCI-779
Above-mentioned CCI-779 work in-process 7.3g is used after 58mL acetic acid ethyl dissolution, on enter positive silicon ball filled column (10 μ m), by ethyl acetate: normal heptane mixed solvent system carries out gradient elution, 0～10min, ethyl acetate/normal heptane=60:40,10～100min, ethyl acetate/normal heptane tapers to 100:0 from 60:40, system temperature is 20 DEG C, collect component, be evaporated at 30 DEG C dryly, obtain 5.0g CCI-779 finished product (purity 96.69%, isomer 1.32%, sirolimus 0.32%).
The above-mentioned minority embodiment that only provides, wherein can find out that the composition of moving phase in positive and RPLC and type of elution are crucial factors.The order of positive high performance liquid chromatography and RPLC can be exchanged, first the work in-process after RPLC separates are in the time separating through positive high performance liquid chromatography, the content of isomer may have increased slightly, but this isomer does not affect the drug effect of CCI-779.Two kinds of methods of the present invention can prepare the high purity CCI-779 that meets injection formulations requirement.Operational condition not specified in the present invention can be carried out according to this area ordinary method.Some operating process; the for example preparation of the aqueous solution of pH3.5～6.0 in moving phase; collection and the concentration etc. of elutriant also can be carried out according to other ordinary methods known in the art, only otherwise departing from the improvement made under essence of the present invention and spiritual prerequisite or equivalence changes and all belong to the scope of protection of the invention.
Claims (10)
1. suc as formula the high purity CCI-779 shown in (I), the HPLC purity of described high purity CCI-779 is greater than 98%,
2. high purity CCI-779 as claimed in claim 1, the HPLC purity of wherein said high purity CCI-779 is greater than 99%, is preferably more than 99.5%.
3. a method of preparing high purity CCI-779, comprises step:
A) positive high performance liquid chromatography: by CCI-779 crude product through performance liquid chromatographic column purification system, taking positive phase filling as stationary phase, mixture taking ethyl acetate and normal heptane or ethyl acetate and normal hexane carries out wash-out as moving phase, obtains CCI-779 work in-process;
B) RPLC: CCI-779 work in-process, through performance liquid chromatographic column purification system, taking reverse phase filler as stationary phase, are carried out to wash-out taking the mixture of the aqueous solution of acetonitrile and pH3.5～6.0 as moving phase, obtain high purity CCI-779.
4. method as claimed in claim 3, wherein said positive phase filling is silicon ball filler, described reverse phase filler is selected from C4, C6, C8 or C18 filler.
5. method as claimed in claim 3, wherein said steps A) in, in the cumulative volume of described moving phase, in moving phase, contain ethyl acetate 60～100v/v%, contain normal hexane or normal heptane 40～0v/v%.
6. method as claimed in claim 5, wherein said steps A) in, wash-out is gradient elution, at 0～10min of wash-out, moving phase contains ethyl acetate 60v/v%, contains normal hexane or normal heptane 40v/v%, at 10～100min of wash-out, in moving phase, the content of ethyl acetate changes to 100v/v% from 60v/v%, and the content of normal heptane or normal hexane changes to 0v/v% from 40v/v% simultaneously.
7. method as claimed in claim 3, wherein said step B) in, the aqueous solution is in water, to add acidic substance formulated, described acidic substance are selected from one or more in organic acid, mineral acid, hydrosulfate, hydrophosphate, one or more in preferable formic acid, acetic acid, propionic acid, phosphoric acid, sulfuric acid, sal enixum, sodium pyrosulfate, monoammonium sulfate, potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, primary ammonium phosphate and potassiumphosphate.
8. method as claimed in claim 3, wherein said step B) in, in the cumulative volume of described moving phase, in moving phase, contain acetonitrile 50～80v/v%, the aqueous solution 50～20v/v% that contains pH3.5～6.0.
9. method as claimed in claim 8, wherein said step B) in, wash-out is gradient elution, at 0～10min of wash-out, moving phase contains acetonitrile 50v/v%, and the aqueous solution 50v/v% that contains pH3.5～6.0, at 10～100min of wash-out, in moving phase, the content of acetonitrile changes to 80v/v% from 50v/v%, and the content of the aqueous solution of pH3.5～6.0 changes to 20v/v% from 50v/v% simultaneously.
10. the method as described in claim 3～9 any one, wherein said step is for carrying out successively positive high performance liquid chromatography and RPLC; Or carry out successively RPLC and positive high performance liquid chromatography.
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