The preparation method of tacrolimus 8- propyl analogsTechnical field
The present invention relates to impurity of the drug preparation method fields, and in particular to the preparation side of tacrolimus 8- propyl analogsMethod.
Background technology
Tacrolimus (Tacrolimus) also known as FK-506 are in 1984 by Japanese Teng Ze drugmakers from soil unwrapping wireA kind of macrolide antibiotics extracted in bacterium is a kind of neotype immunosuppressant of strength, mainly by inhibiting interleukin-The release of 2 (IL-2) inhibits the effect of T lymphocytes comprehensively, and more common cyclosporine (CsA) is 100 times strong, therefore substantially reducesClinical practice dosage and medical expense.The patient of this external application this product, bacterium and viral infection rate are also compared with cyclosporin therapy personLow, especially the drug has stronger close liver property.FK-506 and cyclosporin are shared with apparent synergistic effect, and effect is moreIt is good.Tacrolimus cannot be only used for preventing the generation of immune response, it may also be used for the immune response and itself exempt from that treatment has occurredEpidemic disease disease, to preventing the immune response of organ transplant and the treatment of autoimmune disease to be of great significance.
In order to ensure drug safety, in the development and production of imitation medicine, research should be detected to impurity of the drug, fromAnd drug quality is improved, reduce adverse drug reaction.Since the main production process of tacrolimus is fermentation, so in its productMiddle there are more impurity, and tacrolimus 8- propyl analogs are one of major impurities of tacrolimus, and chemical formula isC44H71NO12, structural formula is：
The distribution situation that each impurity of tacrolimus is elaborated in United States Pharmacopeia USP35 is shown in attached drawing 1, and retention time existsIt is impurity tacrolimus 8- propyl analogs at 35.457min, but it is without reference to the method for separating and preparing of specific impurity；TextOffer Evaluation, synthesis and characterization of tacrolimus impurities（PatriziaFerraboschi,Diego Colombo,Maria De Mieri and Paride Grisenti,The Journal ofAntibiotics(2012)65,349–354）Impurity Distribution situation according to USP35 records is to tacrolimus 8- propyl analogsChemical synthesis is carried out, but the impurity product purity that chemical synthesis obtains is relatively low, has been detected the sample size of research in gram-gradeMore than, and synthesis device therefor is more, and it is cumbersome.
Invention content
Chromatographic process is prepared the purpose of the present invention is to provide a kind of, separation prepares tacrolimus 8- propyl analogs.
In preparative chromatography, the selection of chromatographic condition is extremely important, it to the peak sequence of each substance in sample solution,Peak shape, separating effect etc. play conclusive effect；Chromatographic condition includes mainly chromatographic column (including filler, internal diameter etc.), flowingPhase (including form and match), column temperature, Detection wavelength etc., selection of each chromatographic condition and combinations thereof is most important.
In order to achieve the object of the present invention, inventor finally obtains following technical solution by a large number of experiments：
The preparation method of tacrolimus impurity is included the following steps using preparative chromatography：
A, dissolution filter：By tacrolimus raw material organic solvent and water dissolution, filtering is configured to sample solution, describedOrganic solvent is selected from one or more of methanol, ethyl alcohol and acetonitrile；
B, prepared by separation：Chromatography retention behavior shown by relative retention time according to impurity determines preparative chromatographyCondition collects the corresponding impurity of retention time with preparative chromatography；
C, drying：By the component being collected into step B vacuum distillation and desivac drying, the impurity after drying carries outHPLC is detected；
D, the impurity product obtained in step C is subjected to LC-MS detections.
The volume ratio of organic solvent and water described in step A is preferably 40~60:60~40, the sample solutionConcentration is preferably 15~25mg/mL.
The condition of preparative chromatography is in step B：Mobile phase：A- purified waters（Second acid for adjusting pH is to 4.0）, B- methanol or secondAlcohol or acetonitrile；Flow velocity：70mL/min；Detection wavelength：220nm；Column temperature：50℃；Prepare column internal diameter：50~300mm；Filler：TenEight alkyl silane bonded silica gels, 5~10 μm of grain size；Sample size：30mL；Eluent gradient elution setting is as follows：
| Time/min | A% | B% | Wavelength/nm |
| 0 | 35~60 | 65~40 | 220 |
| 30 | 10~35 | 90~65 | 220 |
| 35 | 35~60 | 65~40 | 220 |
| 40 | 35~60 | 65~40 | 220 |
Collect at corresponding retention time by separation impurity.
HPLC conditions described in step C are：
Chromatographic column：Agilent polaris3C18-A columns（4.6×150mm,3μm）
Mobile phase A：60mM phosphate aqueous solutions, B：Acetonitrile/t-butyl methyl ether=81/19
C：Mobile phase A/Mobile phase B=4/1D：Mobile phase A/Mobile phase B=1/4
Gradient elution is set as：
| Time/min | C% | D% |
| 0 | 72 | 28 |
| 30 | 72 | 28 |
| 53 | 15 | 85 |
| 54 | 72 | 28 |
| 60 | 72 | 28 |
Detection wavelength：220nm
Sample size：100μL
Flow velocity：1.5mL/min
Column temperature：60.0℃
Liquid phase chromatogram condition is used by LC-MS detection methods described in step D：Chromatographic column：Sepax120-5-C184.6×250mm；Wavelength：220nm；Flow velocity：1.5mL/min；Column temperature：50℃；Collect the dissolving of impurity：It is 1 with volume ratio:1 acetonitrile-aqueous solution dissolving；Mobile phase：A- ultra-pure waters（Acetic acid tune pH to 3.5）, B- acetonitriles, gradient elution is arranged as follows：
| Time/min | 0 | 40 | 45 | 60 |
| A% | 65 | 40 | 30 | 65 |
| B% | 35 | 60 | 70 | 35 |
Compared with prior art, preparation chromatographic process of the present invention, equipment is simple, easy to operate, and separation is quick, instituteIt is high to obtain target product purity（＞ 97%）, the impurity sample size for detecting research only needs milligram grade, to be ground for the follow-up of impurityOffer basis is provided, the raising of tacrolimus quality is conducive to.
Description of the drawings
Fig. 1：The HPLC collection of illustrative plates of tacrolimus Impurity Distribution in USP35.
Specific implementation mode
The following is specific embodiments of the present invention, and technical scheme of the present invention is further described, but of the inventionProtection domain be not limited to these examples.It is every to be included in this hair without departing substantially from the change of present inventive concept or equivalent substituteWithin bright protection domain.
Embodiment 1
A, dissolution filter：It is 1 by tacrolimus raw material volume ratio:The mixed solution dissolving of 1 acetonitrile and water, with 0.45μm filtering with microporous membrane, is configured to the sample solution of a concentration of 20mg/mL of tacrolimus.
B, prepared by separation：The condition of preparative chromatography is：Mobile phase：A- purified waters（Second acid for adjusting pH is to 4.0）, B- secondNitrile；Flow velocity：70mL/min；Detection wavelength：220nm；Column temperature：50℃；Prepare column internal diameter：50mm；Filler：Octadecylsilane keyClose silica gel, 5 μm of grain size；Sample size：30mL；Eluent gradient elution setting is as follows：
| Time/min | A% | B% | Wavelength/nm |
| 0 | 50 | 50 | 220 |
| 30 | 20 | 80 | 220 |
| 35 | 50 | 50 | 220 |
| 40 | 50 | 50 | 220 |
Collection retention time is at 28.71min by separation impurity.
C, drying：The component being collected into step B is evaporated under reduced pressure with Rotary Evaporators and is concentrated, sample after concentration is frozenDry, the impurity sample after drying carries out HPLC detections, and principal component content is 98.3%, and the HPLC conditions are：
Chromatographic column：Agilent polaris3C18-A columns（4.6×150mm,3μm）
Mobile phase A：60mM phosphate aqueous solutions, B：Acetonitrile/t-butyl methyl ether=81/19
C：Mobile phase A/Mobile phase B=4/1D：Mobile phase A/Mobile phase B=1/4
Gradient elution is set as：
| Time/min | C% | D% |
| 0 | 72 | 28 |
| 30 | 72 | 28 |
| 53 | 15 | 85 |
| 54 | 72 | 28 |
| 60 | 72 | 28 |
Detection wavelength：220nm, sample size：100 μ L, flow velocity：1.5mL/min, column temperature：60.0℃.
D, the impurity product obtained in step C is subjected to LC-MS detections, [M+K]+Mass-to-charge ratio is 844.5, chemical formulaFor C44H71NO12, it is the tacrolimus 8- propyl analogs described in USP.Liquid phase used by the LC-MS detection methodsChromatographic condition is：Chromatographic column：Sepax120-5-C184.6×250mm；Wavelength：220nm；Flow velocity：1.5mL/min；Column temperature：50℃；Collect the dissolving of impurity：It is 1 with volume ratio:1 acetonitrile-aqueous solution dissolving；Mobile phase：A- ultra-pure waters（Acetic acid tune pH is extremely3.5）, B- acetonitriles, gradient elution is arranged as follows：
| Time/min | 0 | 40 | 45 | 60 |
| A% | 65 | 40 | 30 | 65 |
| B% | 35 | 60 | 70 | 35 |
High resolution mass spec condition is electron spray ionisation source cation (ESI+).
Embodiment 2
A, dissolution filter：It is 40 by tacrolimus raw material volume ratio:The mixed solution dissolving of 60 acetonitrile and water, is used0.45 μm of filtering with microporous membrane, is configured to the sample solution of a concentration of 15mg/mL of tacrolimus.
B, prepared by separation：The condition of preparative chromatography is：Mobile phase：A- purified waters（Second acid for adjusting pH is to 4.0）, B- secondNitrile；Flow velocity：70mL/min；Detection wavelength：220nm；Column temperature：50℃；Prepare column internal diameter：150mm；Filler：OctadecylsilaneBonded silica gel, 8 μm of grain size；Sample size：30mL；Eluent gradient elution setting is as follows：
| Time/min | A% | B% | Wavelength/nm |
| 0 | 35 | 65 | 220 |
| 30 | 10 | 90 | 220 |
| 35 | 35 | 65 | 220 |
| 40 | 35 | 65 | 220 |
Collection retention time is at 26.51min by separation impurity.
C, drying：The component being collected into step B is evaporated under reduced pressure with Rotary Evaporators and is concentrated, sample after concentration is frozenDry, the impurity sample after drying carries out HPLC detections, and principal component content is 97.8%, in the HPLC conditions and embodiment 1It is identical.
D, the impurity product obtained in step C is subjected to LC-MS detections, [M+K]+Mass-to-charge ratio is 844.5, and chemical formula isC44H71NO12, it is the tacrolimus 8- propyl analogs described in USP.Condition used by the LC-MS detection methods withIt is identical in embodiment 1.
Embodiment 3
A, dissolution filter：It is 60 by tacrolimus raw material volume ratio:The mixed solution dissolving of 40 acetonitrile and water, is used0.45 μm of filtering with microporous membrane, is configured to the sample solution of a concentration of 25mg/mL of tacrolimus.
B, prepared by separation：The condition of preparative chromatography is：Mobile phase：A- purified waters（Second acid for adjusting pH is to 4.0）, B- secondNitrile；Flow velocity：70mL/min；Detection wavelength：220nm；Column temperature：50℃；Prepare column internal diameter：300mm；Filler：OctadecylsilaneBonded silica gel, 10 μm of grain size；Sample size：30mL；Eluent gradient elution setting is as follows：
| Time/min | A% | B% | Wavelength/nm |
| 0 | 60 | 40 | 220 |
| 30 | 35 | 65 | 220 |
| 35 | 60 | 40 | 220 |
| 40 | 60 | 40 | 220 |
Collection retention time is at 30.34min by separation impurity.
C, drying：The component being collected into step B is evaporated under reduced pressure with Rotary Evaporators and is concentrated, sample after concentration is frozenDry, the impurity sample after drying carries out HPLC detections, and principal component content is 97.6%, in the HPLC conditions and embodiment 1It is identical.
D, the impurity product obtained in step C is subjected to LC-MS detections, [M+K]+Mass-to-charge ratio is 844.5, and chemical formula isC44H71NO12, it is the tacrolimus 8- propyl analogs described in USP.Condition used by the LC-MS detection methods withIt is identical in embodiment 1.
Embodiment 4
A, dissolution filter：It is 40 by tacrolimus raw material volume ratio:The mixed solution of 60 first alcohol and water dissolves, and uses0.45 μm of filtering with microporous membrane, is configured to the sample solution of a concentration of 20mg/mL of tacrolimus.
B, prepared by separation：The condition of preparative chromatography is：Mobile phase：A- purified waters（Second acid for adjusting pH is to 4.0）, B- firstAlcohol；Flow velocity：70mL/min；Detection wavelength：220nm；Column temperature：50℃；Prepare column internal diameter：50mm；Filler：Octadecylsilane keyClose silica gel, 8 μm of grain size；Sample size：30mL；Eluent gradient elution setting is as follows：
| Time/min | A% | B% | Wavelength/nm |
| 0 | 45 | 55 | 220 |
| 30 | 15 | 85 | 220 |
| 35 | 45 | 55 | 220 |
| 40 | 45 | 55 | 220 |
Collection retention time is at 31.16min by separation impurity.
C, drying：The component being collected into step B is evaporated under reduced pressure with Rotary Evaporators and is concentrated, sample after concentration is frozenDry, the impurity sample after drying carries out HPLC detections, and principal component content is 97.5%, in the HPLC conditions and embodiment 1It is identical.
D, the impurity product obtained in step C is subjected to LC-MS detections, [M+K]+Mass-to-charge ratio is 844.5, chemical formulaFor C44H71NO12, it is the tacrolimus 8- propyl analogs described in USP.Condition used by the LC-MS detection methodsIt is identical as in embodiment 1.
Embodiment 5
A, dissolution filter：It is 60 by tacrolimus raw material volume ratio:The mixed solution of 40 second alcohol and water dissolves, and uses0.45 μm of filtering with microporous membrane, is configured to the sample solution of a concentration of 20mg/mL of tacrolimus.
B, prepared by separation：The condition of preparative chromatography is：Mobile phase：A- purified waters（Second acid for adjusting pH is to 4.0）, B- secondAlcohol；Flow velocity：70mL/min；Detection wavelength：220nm；Column temperature：50℃；Prepare column internal diameter：50mm；Filler：Octadecylsilane keyClose silica gel, 5 μm of grain size；Sample size：30mL；Eluent gradient elution setting is as follows：
| Time/min | A% | B% | Wavelength/nm |
| 0 | 55 | 45 | 220 |
| 30 | 35 | 65 | 220 |
| 35 | 55 | 45 | 220 |
| 40 | 55 | 45 | 220 |
Collection retention time is at 33.65min by separation impurity.
C, drying：The component being collected into step B is evaporated under reduced pressure with Rotary Evaporators and is concentrated, sample after concentration is frozenDry, the impurity sample after drying carries out HPLC detections, and principal component content is 97.3%, in the HPLC conditions and embodiment 1It is identical.
D, the impurity product obtained in step C is subjected to LC-MS detections, [M+K]+Mass-to-charge ratio is 844.5, and chemical formula isC44H71NO12, it is the tacrolimus 8- propyl analogs described in USP.Condition used by the LC-MS detection methods withIt is identical in embodiment 1.