[0002] the application requires the rights and interests of U.S. Provisional Application series number of submitting on December 22nd, 2,004 60/638,628 and the U.S. Provisional Application series number of submitting on December 23rd, 2,004 60/638,815, and their content is attached to herein by quoting in full.
Summary of the invention
[0008] in one embodiment, the present invention relates to measure content of impurities and be less than about 0.50% (area), preferably be less than the tacrolimus of about 0.16% (area) through HPLC.
[0009] in another embodiment, the present invention relates to measure to have be less than about 0.06% (area), preferably be less than the tacrolimus of the ascosin of about 0.02% (area) through HPLC.
[00010] in another embodiment, the present invention relates to measure to have be less than about 0.07% (area), preferably be less than the tacrolimus of the dihydro tacrolimus of about 0.05% (area) through HPLC.
[00011] in another embodiment, the present invention relates to measure tacrolimus with the impurity RRT1.19 that is less than about 0.02% (area) through HPLC.
[00012] in another embodiment, the present invention relates to measure to have be less than about 0.12% (area), preferably be less than the tacrolimus of the impurity RRT0.60 of about 0.02% (area) through HPLC.
[00013] in another embodiment, the present invention relates to measure to have be less than about 0.12% (area), preferably be less than the tacrolimus of the impurity RRT0.83 of about 0.02% (area) through HPLC.
[00014] in another embodiment, the present invention relates to measure to have be less than about 0.08% (area), preferably be less than the tacrolimus of the impurity RRT1.45 of about 0.02% (area) through HPLC.
[00015] in another embodiment, the present invention relates to measure to have be less than about 0.06% (area), preferably be less than the tacrolimus of any indivedual impurity of about 0.02% (area) through HPLC.
[00016] in one embodiment, the invention provides a kind of method of purification of tacrolimus.This method comprises: the filling material that tacrolimus is provided, tacrolimus filling material is filled on the polymeric adsorbent bed, with the elutriant wash-out filling material and the bed that contain THF and/or acetonitrile and water, to obtain effluent liquid, collect the main flow point of effluent liquid, from main flow point, reclaim tacrolimus, make the tacrolimus crystallization and further make its recrystallize.The resulting tacrolimus of above method preferably has aforesaid impurity level.Tacrolimus can be from any source.
[00017] on the other hand, the present invention relates to according to the prepared tacrolimus of aforesaid method.
Detailed Description Of The Invention
[00018] term " envrionment temperature " that is used for this paper refers to about 0 °-Yue 40 ℃, preferred about 10 ℃-Yue 35 ℃ temperature.
[00019] term " pressure of minimizing " that is used for this paper refers to the pressure less than about 760mm Hg.
[00020] term " anti-solvent " that is used for this paper refers to that tacrolimus is in wherein dissolving minimum material, normally liquid at ambient temperature.
[00021] term " impurity " that is used for this paper refers to, at least according to the chromatographic detection limit that is used for measuring retention time, any compound with the retention time that is different from tacrolimus.For example, can measure different retention time through HPLC method hereinafter described.
[00022] term " impurity RRT1.19 " that is used for this paper refers to come across at the HPLC color atlas impurity of about 1.19RRT.This impurity is the isomer of tacrolimus.
[00023] term " impurity RRT0.60 " that is used for this paper refers to come across at the HPLC color atlas impurity of about 0.60RRT.
[00024] term " impurity RRT0.83 " that is used for this paper refers to come across at the HPLC color atlas impurity of about 0.83RRT.
[00025] term " impurity RRT1.45 " that is used for this paper refers to come across at the HPLC color atlas impurity of about 1.45RRT.
[00026] term ascosin and the dihydro tacrolimus that is used for this paper respectively refers to RRT0.95 and RRT1.25, and they are the impurity in the tacrolimus, and during all HPLC as mentioned below analyzed, with respect to tacrolimus, its retention time was about 0.95 and 1.25.(vol-%) refer to the volume fraction (is example with the substance A) of following calculating with mixtures of liquids and the relevant term that is used for this paper " volume percent " of combination and " percentage by volume ":
vol-％A＝WtA×ρA/(WtA×ρ+WtB×ρB)
Wt whereinAAnd WtBRespectively be the weight (gram) of substance A and B, ρAAnd ρBRespectively be the density (g/ml) of substance A and B.
[00027] in one embodiment, the present invention relates to measure content of impurities and be less than about 0.50% (area), preferably be less than the tacrolimus of about 0.16% (area) through HPLC.
[00028] in another embodiment, the present invention relates to measure to have be less than about 0.06% (area), preferably be less than the tacrolimus of the ascosin of about 0.02% (area) through HPLC.
[00029] in another embodiment, the present invention relates to measure to have be less than about 0.07% (area), preferably be less than the tacrolimus of about 0.05% (area) dihydro tacrolimus through HPLC.
[00030] in another embodiment, the present invention relates to measure tacrolimus with the impurity RRT1.19 that is less than about 0.02% (area) through HPLC.
[00031] in another embodiment, the present invention relates to measure to have be less than about 0.12% (area), preferably be less than the tacrolimus of the impurity RRT0.60 of about 0.02% (area) through HPLC.
[00032] in another embodiment, the present invention relates to measure to have be less than about 0.12% (area), preferably be less than the tacrolimus of the impurity RRT0.83 of about 0.02% (area) through HPLC.
[00033] in another embodiment, the present invention relates to measure to have be less than about 0.08% (area), preferably be less than the tacrolimus of the impurity RRT1.45 of about 0.02% (area) through HPLC.
[00034] in another embodiment, the present invention relates to measure to have be less than about 0.06% (area), preferably be less than the tacrolimus of any indivedual impurity of about 0.02% (area) through HPLC.
[00035] in one embodiment, the invention provides a kind of purification of tacrolimus, promptly reduce the method for foreign matter content in the tacrolimus.This method comprises provides the filling of tacrolimus material, tacrolimus filling material is filled on the polymeric adsorbent bed, with the elutriant wash-out filling material and the bed that contain THF and/or acetonitrile and water, thereby obtain effluent liquid, collect the main flow point of effluent liquid, from main flow point, reclaim tacrolimus, make tacrolimus crystallization and further recrystallize.The impurity level of the tacrolimus that obtains in the above method preferably as mentioned above.Tacrolimus can be from any source.
[00036] in enforcement of the present invention, mainly, obtain effluent liquid by be filled with the polymeric adsorbent bed of tacrolimus filling material with the elutriant wash-out, the minimizing of realization impurity with separate.It is known in the art to be used to implement polymeric adsorbent of the present invention, and is preferably crosslinked non-ionic type vinylbenzene-divinylbenzene material, and it can be through chemically modified.The acrylic type polymeric adsorbent also is known.This polymeric adsorbent has high vesicular structure, and it has adsorbable and the surface of the various chemical substances of desorb again.The influence of intussusception environment (for example used solvent) is conciliate in absorption.In the presence of polar solvent (for example water), polymeric adsorbent shows hydrophobicity.When adopting non-polar solvent (for example hydrocarbon), polymeric adsorbent can show certain polarity.Polymeric adsorbent generally has big reticulated structure, and its surface-area is at least about 300m2/ g.
[00037] is used to implement polymeric adsorbent of the present invention and comprises the AMBERLITE XAD resin that can derive from Rohm and Haas; Only introduce several: XAD4, XAD7 HP, XAD16 HP, XAD761 and XAD1180.The also available Diaion polymeric adsorbent that derives from Mitsubishi; Only introduce several: HP10, HP20, HP21, HP30, HP40, HP50, SP800, SP825, SP850, SP875, SP205, SP206, SP207, HP1MG and HP2MG.AMBERLITE XAD1180 is the example that is used to implement preferred polymeric adsorbent of the present invention.AMBERLITE XAD1180 is big cross-linked network aromatic(based)polymer.It is nonionic, hydrophobic, cross-linked polymer, its absorptivity come from its big reticulated structure of obtaining patent (contain continuous polymer mutually and continuous hole mutually), high surface area and its surperficial aromaticity.Surface-area is 500m2/ g or higher.Porosity is more than the 0.60ml/ml.The single information that further provides about this resin of the product data in PDS0205A-98 January-1/2.
[00038] can organic solvent or with water blended organic solvent in tacrolimus solution, or partly provide the filling material with the filling that is filled with tacrolimus that tacrolimus is adsorbed onto polymeric adsorbent filling part.
[00039] before being loaded into the polymeric adsorbent bed, tacrolimus filling material is adsorbed (deposition) filling department timesharing to polymeric adsorbent, this absorption comprises the optional tacrolimus solution that contains water of preparation in the organic solvent, with this solution and a part of polymeric adsorbent and hydration also.Polymeric adsorbent can be identical with the resin that is used to prepare bed, or can be different polymeric adsorbents.Polymeric adsorbent filling part can be about 50% volume of about 33%-of bed.Tacrolimus separates the filling material from residual solution after the absorption on the polymeric adsorbent is finished substantially.Can separate after filtration.When being equipped with the filling material, from the recycle system, isolate post simply with circulation post legal system.
[00040] is used for preparing the organic solvent that therefrom filling or deposition are loaded the solution of material, preferably from tetrahydrofuran (THF) (THF), acetone, acetonitrile (ACN), methyl alcohol, ethanol, propyl carbinol, n-propyl alcohol, Virahol, ester (for example ethyl acetate) and dipolar aprotic solvent, for example dimethyl formamide (DMF).Organic solvent is THF, acetone or CAN more preferably, most preferably THF and CAN.
[00041] adding of water reduces solvent: the ratio of water, thus increase the absorption of tacrolimus on polymeric adsorbent.
[00042] can in being furnished with any container easily (for example stirred-tank reactor) of agitator, mix filling part and the water that tacrolimus solution loads material, polymeric adsorbent.
[00043] as an example, tacrolimus solution filling material can be about 100g/l, and the volume of water can be at least five times of liquor capacity.The cumulative volume of polymeric adsorbent filling part can approximate the volume of solution.Be to realize the absorption of tacrolimus on polymeric adsorbent filling part, the technician can understand by normal experiment and optimizes this ratio.
[00044] in the subsequent step of this embodiment, will be mounted with the loading station of tacrolimus and place the moistening polymeric adsorbent bed for preparing.This bed is restricted to and is suitable in the container.This preferably is restricted in the post that preferably has circular cross section.Be the preparation bed, the mixture of water or water and solvent (for example THF or ACN) makes the polymeric adsorbent of aequum make pulpous state.When the bed diameter was big, water-solvent combination was favourable.
[00045] by making elutriant pass the filling material, then passes with fluid and filling and expect to be communicated with and juxtaposed polymeric adsorbent bed, separate tacrolimus and impurity, reduce the impurity level in the tacrolimus whereby, obtain pure tacrolimus.Elutriant is optional to contain other organic solvent, and it is selected from the solvent that is used to dissolve tacrolimus in first step of present method.
[00046] the filling material that is provided for organic solvent or with hydration and organic solvent in tacrolimus solution the time, this solution is sprayed into prepared moistening polymeric adsorbent bed, post is contacted with the tacrolimus solution stream, with elutriant introduce flow through and solution stream around polymeric adsorbent filling part in, whereby, the tacrolimus sample is adsorbed onto on the filling part of polymeric adsorbent gradually.
[00047] behind first time wash-out, this can place the fluid that communicates with second bed, so as from first effluent liquid through second bed wash-out.Behind first bed and second bed wash-out, second bed can and preferably separate (promptly destroying fluid is communicated with) with first bed, and continuation is separately through second bed wash-out.Elutriant is optional to be the THF of volume percent about 33 to 37 and the mixture of water.Can collect the elutriant part, and dilute with water, can make it pass through the 3rd bed (post) thereafter.Choose wantonly and extra post can be connected to this system, and dilute, to obtain purer product with the water of additional quantity.The water that preferably adds additional quantity to last post is to improve the absorption of tacrolimus at polymeric adsorbent.
[00048] elutriant comprises water and organic solvent, for example THF, CAN and composition thereof.Preferred elutriant is the mixture of THF and water basically, and it has about 20% volume to about 50% volume, and most preferably from about 31% volume is to the THF of about 40% volume.When organic solvent (for example methyl alcohol, acetonitrile, acetone or propyl carbinol) and THF/ water elution liquid were shared, THF content was less than 38% volume, preferably between about 38% volume of about 4-.Another kind of preferred elutriant is the mixture of acetonitrile and water, and it has about 30% volume-Yue 70% volume, the most preferably from about acetonitrile of 40% volume-Yue 65% volume.When elutriant was the mixture of acetonitrile and water, elutriant also can contain with respect to 1 part of about 0.003 part phosphoric acid of the about 0.0005-of elutriant.
[00049] elutriant with certain speed through filling material and with it juxtaposed polymeric adsorbent bed wash-out go out, this speed depends on total cross-sectional area (measure vertical with the elutriant fluid) of bed.Flow velocity (with respect to cross-sectional area) preferably is lower than about 25cm/ hour, preferably is lower than about 15cm/ hour.Lower elution speed can increase the time, but can improve separation efficiency.Preferred elution speed for the separation efficiency that improves is about 9cm/ hour-Yue 11cm/ hour.
[00050] the available any monitoring of method easily wash-out goes out the content and the composition of flow point.Can carry out the detection of impurity, especially ascosin in the tacrolimus and dihydro tacrolimus and quantitatively by HPLC method hereinafter described.
[00051] composition and the flow velocity of post loading capacity and elutriant especially depended in the collection of main flow point, has about 0.1% (area) or lower ascosin so that final separated products is measured through HPLC.
[00052] as needs, can be by any ordinary method (for example extraction, lyophilize, evaporation, the anti-solvent of adding), separate the tacrolimus that impurity level reduces with impurity thereby isolate from effluent liquid.Water, alkane and naphthenic hydrocarbon are useful anti-solvents, other known in the art.Can be in conjunction with a plurality of separation methods.For example anti-solvent combines with concentrate eluant.
[00053] preferred separation method comprises: preferred below 60 ℃ in below 70 ℃, preferred 760mm Hg or more under the low pressure concentrates main flow point about 50% to its initial volume, obtains spissated tacrolimus flow point whereby.The phosphoric acid that preferably adds every liter of about 10ml of the about 1-of elutriant before concentrating is to stablize tacrolimus.
[00054] chooses wantonly through spissated main flow point and in envrionment temperature, keep one section retention time.When adopting retention time, preferred retention time is about 1-4 days.Water unmixability solvent (for example ethyl acetate or methylene dichloride) and alkali (for example ammonia solution) are added to through concentrating the tacrolimus flow point, divide dried up unmixability solvent phase, and concentrate.Adding alkali is about 9 or lower up to pH.
[00055] can reclaim product and experience several extra processing for example crystallization and recrystallize by making, further reduce impurity.
[00056] crystallization of tacrolimus oily resistates is included in the oily resistates of dissolving tacrolimus in ethyl acetate and the hexanaphthene, adds water to bring out the tacrolimus crystallization, reclaims the crystalline tacrolimus again.Before dissolving step, preferably also be condensed into the oily resistates once more with ethyl acetate dilution oily resistates.Preferably dropwise add entry.Water in the crystallisation process: tacrolimus is generally 0.015kg-0.3kg water to the 1kg tacrolimus.
[00057] recrystallize of tacrolimus comprises and makes tacrolimus be dissolved in ethyl acetate, concentrate this solution up to obtaining the oily resistates, make the oily resistates be dissolved in ethyl acetate, add hexanaphthene to solution, add water to bring out the tacrolimus crystallization, reclaim the crystalline tacrolimus again.But preferred redissolve and enrichment step.Preferably use activated carbon treatment solution, to remove color and fiber.Concentrate as mentioned above.The tacrolimus of preferred further dry gained.
[00058] in enforcement of the present invention, carry out last chromatography step with three columnss in series, this causes ascosin and dihydro tacrolimus significantly to reduce, and in solvent mixture through adding entry and the crystallization time through regulating causes impurity RRT1.19 significantly to reduce with regulating.
[00059] available HPLC method monitoring hereinafter described is by the purifying of the tacrolimus that the inventive method realized.
[00060] in specific embodiments, the level of impurity ascosin and dihydro tacrolimus is lowered at least, obtains highly purified tacrolimus.The level of other impurity also is lowered.This method comprise the following steps: preparation with or without the filling of polymeric adsorbent (especially macroreticular-type resin, for example AMBERLITE XAD1180 and Diaion HP20) part, comprise the tacrolimus filling material of tacrolimus solution; Filling material is loaded into is contained in especially moistening polymeric adsorbent, especially AMBERLITE XAD1180 and the DiaionHP20 in the post of container; With elutriant wash-out filling part and polymeric adsorbent, elutriant is the mixture of tetrahydrofuran (THF) (THF) and water, it has the THF of about 20% volume-Yue 50% volume, especially about 31% volume-Yue 40% volume, or the mixture of acetonitrile (ACN) and water, it has about 30% volume-Yue 70% volume, the most particularly acetonitrile of about 40% volume-Yue 65% volume; At least collect the main flow point (center flow point) of elutriant, it contain surpass about 60%, preferably between the initial tacrolimus (depending on initial purity) of about 60%-about 90%; By for example concentrating main flow point, for example the main flow point of concentrating under reduced pressure in the presence of acid separates the tacrolimus with reduction impurity from main flow point with optional; The product that obtains like this with optional recovery.Products therefrom through further as mentioned above as crystallization and recrystallize.
[00061] tacrolimus of gained preferably is less than 0.50% (area) through HPLC mensuration content of impurities, most preferably is less than 0.16% (area).
[00062] tacrolimus of gained is preferably measured to have through HPLC and is less than about 0.06% (area), most preferably is less than the ascosin of about 0.02% (area).
[00063] tacrolimus of gained is preferably measured to have through HPLC and is less than about 0.07% (area), most preferably is less than the dihydro tacrolimus of about 0.05% (area).
[00064] tacrolimus of gained is preferably measured through HPLC and is had the impurity RRT1.19 that is less than about 0.02% (area).
[00065] tacrolimus of gained is preferably measured to have through HPLC and is less than about 0.12% (area), most preferably is less than the impurity RRT0.60 of about 0.02% (area).
[00066] tacrolimus of gained is preferably measured to have through HPLC and is less than about 0.12% (area), most preferably is less than the impurity RRT0.83 of about 0.02% (area).
[00067] tacrolimus of gained is preferably measured to have through HPLC and is less than about 0.08% (area), most preferably is less than the impurity RRT1.45 of about 0.02% (area).
[00068] tacrolimus of gained is preferably measured to have through HPLC and is less than about 0.06% (area), most preferably is less than any indivedual impurity of about 0.02% (area).
[00069] the invention provides tacrolimus by above method gained.
The chromatographic condition that embodiment adopted
Post: ZORBAX SB-C18 75 * 4.6mm; 3.5 μ m
Pre-column: SymmetryShield RP 183.9 * 20mm; 5 μ m
Elutriant: A: measure the 200ml acetonitrile and join the 2000ml volumetric flask, use the distilled water diluting volume again to the 2000ml cumulative volume.Then, add 100 μ l, 50% acetate.
B: add 100 μ l50% acetate to the 2000ml acetonitrile.
Gradient table
| Time (min) | Elutriant " A " (w/w%) | Elutriant " B " (w/w%) | Flow velocity (ml/min) |
| 0 | 60 | 40 | 2.3 |
| 15 | 55 | 45 | 2.3 |
| 25 | 30 | 70 | 1.8 |
| 25.1 | 60 | 40 | 1.8 |
| 27 | 60 | 40 | 1.8 |
Flow velocity: 2.3ml/min
Detect wavelength: 210nm
Inject volume: 20 μ l
Sample solvent: acetonitrile
Pole unit temperature: 60 ℃
Analysis time: 27min
The retention time of tacrolimus: about 14min
Detection limit: be less than 0.01% (area)
Quantitative limit: be less than 0.02% (area).
The retention time of impurity ascosin (RRT0.95), dihydro tacrolimus (RRT1.25) and impurity RRT1.19 is with respect to the tacrolimus meter, and represents with area % with respect to the area at the whole peaks in the color atlas.
Present available typical HPLC Device Testing and quantitative limit respectively are less than 0.01% (area) and are less than 0.02% (area).
Embodiment
[00070] following non-limiting examples only illustrates the preferred embodiments of the invention, should not be interpreted as limiting the present invention, and scope of the present invention is limited by appended claims.
Embodiment 1
[00071] through chromatography and several crystallisation step purification of tacrolimus raw material.Carry out purity check with the analysis mode HPLC method described in above " chromatographic condition that embodiment adopted ".Raw material contains the dihydro tacrolimus of impurity RRT1.19 and 0.46% (area) of the ascosin, 1.56% (area) of 0.16% (area).The analysis of parent material is obtained the purity of 95% quality.Behind method purifying of the present invention, end product contains the dihydro tacrolimus of impurity RRT1.19 and 0.05% (area) of the ascosin, 0.02% (area) of 0.02% (area).The content of other impurity is no more than 0.02% (area), is 99.84% (area) with the purity of the tacrolimus of the inventive method gained.
Chromatographic step
[00072] AMBERLITE XAD1180 polymeric adsorbent is used for chromatography.Prepare three chromatography columns (40cm diameter, the high and about 100 liters of moistening polymeric adsorbents of 1m post).The tacrolimus raw material (wherein 3623g is an active substance) of 3812g amount is dissolved in 30 liters of acetone.The Resin A MBERLITE XAD1180 of 33 liters of amounts is added to tacrolimus solution.Stir on the limit, and the water that the limit slowly adds 180 liters of amounts is to tacrolimus solution: resin compound.When finishing when adding water, collect polymeric adsorbent filling material after filtration.
[00073] collected filling material is loaded into moistening polymeric adsorbent bed top as one deck.Total resin volume is about 100 liters.Earlier with about 700 liters of tetrahydrofuran (THF)/water (34% volume THF) elutriant wash-out post.Behind the wash-out, second post linked to each other with first post for the first time.Continue with about 1400 liters of THE/ water (34% volume THE) elutriant wash-out.First post and second post are separated, continue with about 1200 liters of THE/ water (34% volume THF) elutriant wash-out.Collect the flow point that 20 liters of volumes are respectively arranged.The water of 0.5 liter of amount is mixed with each flow point, obtain diluted flow point.Make through the dilution flow point and pass through the 3rd post, tacrolimus is adsorbed on the resin of the 3rd post.With about 1800 liters of THE/ water (34% volume THF) elutriant wash-out tacrolimus from the 3rd post.Collect the flow point that 20 liters of volumes are respectively arranged, the usefulness above analysis mode HPLC method described in " chromatographic condition that embodiment adopted " is analyzed several flow points.
[00074] remerges each suitable flow point.Yet, should be noted that, before merging each flow point, can merge the prerun flow point, the 10ml in each suitable flow point for example, and analyze with the HPLC analytical method.Analyze the dihydro tacrolimus concentration that draws the ascosin concentration that is higher than 0.02% (area) and/or be higher than 0.04% (area) if prerun is merged the HPLC of thing, the quantity that merges flow point so should be improved, so that required high purity to be provided, can bring highly purified end product because during merging each flow point, obtain required high purity.
[00075] the main flow point of He Binging (about 500 liters) mixes with 100ml85% phosphoric acid, and concentrating under reduced pressure is to about 200 liters of volumes.Enriched material is cooled to envrionment temperature, adds 50 premium on currency, 100 liters of ethyl acetate and 200ml liquor ammoniae fortis to enriched material.Separating ethyl acetate phase (about 75 liters), concentrating under reduced pressure becomes the oily resistates.
The crystallization of main flow point
[00076] with 10 liters of ethyl acetate dilution oily resistatess, concentrating under reduced pressure becomes the oily resistates once more.Heating temperature is about 60 ℃, estimates that boiling point is 20-40 ℃.Repeat dilution-enrichment step twice.
[00077] through a spot of sample of reduction vaporization, determine the solids content of oily resistates, the solids content that obtains the oily resistates is 1329g.With ethyl acetate the oily resistates is diluted to about 2525g, the 7970ml hexanaphthene is added to solution.To temperature be remained in 25 ℃ with thermocycler.
[00078] in solution, adds the water that 10.6ml measures fast.With the water that in solution, added the 18.6ml amount in 3 hours, start crystallization.Stir after 45 minutes, leach crystal, with the washing of 1600ml hexanaphthene.Descended dry 12 hours at 70 ℃ through washing crystal, obtain the dried crystals that quality is 1250g.
Recrystallize
[00079] tacrolimus with the 1250g amount is dissolved in 7.5 liters of ethyl acetate.Make solution decompression be condensed into the oily resistates.Repeat dilution-enrichment step twice.The oily resistates is dissolved in the 3750ml ethyl acetate, uses the 12.5g activated carbon treatment.Use activated carbon treatment 30 minutes down for 30 ℃.Filter suspension, with 125ml ethyl acetate washing leaching cake.Concentrating under reduced pressure is solution after filtration, is diluted to 2375g with ethyl acetate.
[00080] adds to tacrolimus solution through 1.5 hours hexanaphthenes with 6.25 liters of amounts.The water that added the 27.5ml amount through 2 hours to solution.In solution, add the water of 246ml amount through 2 hours, start crystallization.
[00081] cooling suspension to 8 ℃ added the hexanaphthene of 1.25 liters of amounts under 8 ℃ to suspension through 1 hour.Stirred suspension 12 hours down in 8 ℃ again.Leach crystal, and suspend twice with hexanaphthene.The volume of the hexanaphthene that is used for suspending is 2.5 liters.
Under [00082] 40 ℃, drying under reduced pressure 12 hours, and at about 25 ℃ times dry 24 hours.Use nitrogen inlet in the whole drying process.
[00083] crystallisation step reduces the impurity RRT1.19 content in the product effectively.The quality of end product is 1180g.The purity of gained tacrolimus is determined as 99.84% (area) through HPLC, that is: total impurities content is determined as 0.16% (area) through HPLC.End product contains: the ascosin of 0.02% (area) measured through HPLC, the impurity RRT1.19 of the dihydro tacrolimus of 0.05% (area of measuring through HPLC) and 0.02% (area) measured through HPLC, the RRT0.83 of 0.02% (area) measured through HPLC, the RRT0.60 that is less than 0.02% (area) that measures through HPLC, the RRT1.45 that is less than 0.02% (area) that measures through HPLC, the RRT0.25 of 0.02% (area) measured through HPLC and any indivedual impurity of measuring through HPLC that are less than 0.02% (area).The impurity scope is summarized in table 2.
Table 2
| Impurity | Through the definite approximate per-cent of HPLC |
| Total impurities | 0.16 |
| The dihydro tacrolimus | 0.05 |
| Ascosin | 0.02 |
| RRT0.60 | ＜0.02 |
| RRT0.83 | 0.02 |
| RRT1.45 | ＜0.02 |
| RRT1.19 | 0.02 |
| RRT0.25 | 0.02 |
| Individual indivedual impurity | ＜0.02 |
[00084] if the impurity RRT1.19 concentration after the crystallization in the end product greater than desirable, can carry out one or more extra crystallisation step and remove this impurity.