CN106083892B - Tacrolimus compound and preparation method thereof - Google Patents
Tacrolimus compound and preparation method thereof Download PDFInfo
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- CN106083892B CN106083892B CN201610410561.8A CN201610410561A CN106083892B CN 106083892 B CN106083892 B CN 106083892B CN 201610410561 A CN201610410561 A CN 201610410561A CN 106083892 B CN106083892 B CN 106083892B
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- C07—ORGANIC CHEMISTRY
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- 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
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Abstract
The present invention relates to drug technical field of purification, a kind of high-purity tacrolimus compound and preparation method thereof is disclosed.Pretreated crude tacrolimus is passed through silver ion fluoropolymer resin column chromatographic elution by the present invention, is recrystallized after eluent concentration, obtains high-purity tacrolimus.Method large-scale production provided by the invention is easy to operate, and solvent consumption is few, with short production cycle, at low cost, and friendly environment is detected through HPLC, and isolated ascosin and dihydro tacrolimus are most difficult in crude tacrolimus and is completely removed.It is 99% or more that tacrolimus, which normalizes purity, and the even particle size distribution of obtained tacrolimus crystal, crystal internal stress and heap density are small, and stability, mobility, dispersibility and dissolubility are preferable.Improve the quality and drug safety of tacrolimus.
Description
Technical field
The present invention relates to drug technical field of purification, and in particular to a kind of high-purity tacrolimus compound and its preparation side
Method.
Background technique
Tacrolimus is a kind of macrolide immunosuppressants of novel potent, the entitled general pleasure of Japan's exploitation in 1991
The agent of (Prograf) tacrolimus capsules and injection official listing can be answered, the repulsion for Primary Hepatic transplanting and kidney transplant is anti-
It should treat, it is rear to be approved for bone-marrow transplantation again.Tacrolimus formulations are in more than 80 countries such as China, Japan, U.S. at present
Listing, the anti-repelling treatment after being widely used in the solid organ transplantations such as liver, pancreas, kidney, heart and lung.1999 he gram
Department's ointment does not launch Japanese market, for treating adult atopic dermatitis.
Tacrolimus is the tunning isolated from streptomyces (streptomyces tsukubaensis), by
In tacrolimus, structure is complicated, and chiral radicals are relatively more, and has many isomers and analog in its fermentation liquid, as dihydro he
Ke Mosi, ascosin etc., wherein only than tacrolimus few 1 methylene, dihydro tacrolimus compare tacrolimus to ascosin
More 2 hydrogen atoms, therefore ascosin, dihydro tacrolimus and tacrolimus structure are very much like, dissolubility in a solvent
Close with the affinity to solvent, the presence of these substances makes tacrolimus can not be complete using conventional isolation and purification method
It separates and reaches corresponding purity, to be difficult to meet market needs.
Chinese invention patent CN85109492, disclose a kind of by macroporous absorbent resin HP20, silica gel, and active carbon is made
The method of standby FK506 crude product: a. extraction: the mycelium being obtained by filtration with organic solvent come soaking fermentation liquid filters to obtain leaching liquor,
B. it adsorbs and desorbs: putting into resin HP20 in leaching liquor and adsorb, the mixed solvent elution of organic solvent and water, collection liquid concentration,
C. it extracts: the extraction of the hydrophobic solvents such as ethyl ester being added in concentrate, extract liquor concentration, d. silica gel column chromatography: extraction concentrate passes through
Silicagel column is eluted with ethyl ester n-hexane mixed liquor, is collected eluent and is concentrated to give crude product.
U.S. patent Nos US2012065393 (A1) discloses a kind of method for preparing FK506 crude product: a. extraction: using
Alcohol, ketone, based organic solvent carry out the mycelium that soaking fermentation liquid is obtained by filtration, and filter to obtain leaching liquor, b. back extraction: in leaching liquor
Halogenated alkanes is added to be stripped, c. column chromatography: extract liquor concentration crosses silicagel column after extraction concentrate dilution or alumina column is removed
Pigment and grease, d. crystallization: collecting eluent and be concentrated into certain volume addition deionized water, stir decrease temperature crystalline, e. filtering
Wet crystalline substance, is washed with alkane, dries to obtain crude product, crude product purity is between 92-95%, and yield is between 70-75%.
Chinese invention patent CN200580043951.6 discloses a kind of method of purifying tacrolimus, wherein in big ring
Ester load is set to be placed adjacent with wet absorption resin bed, and load and bed are being greater than 30 DEG C with organic solvent elution, You Jirong
Agent is selected from THF, acetonitrile, normal propyl alcohol, isopropanol, ethyl alcohol and acetone, collects the intermediate fraction of eluent, collects in the big ring of purification
Ester, crystallization use ethyl acetate, the three-phase system of hexamethylene and water.
Chinese invention patent CN200910146378.1 is disclosed using crude tacrolimus as raw material, by organic molten
Agent dissolution realizes that tacrolimus and ascosin, dihydro tacrolimus isolate and purify with C18 reverse phase preparative liquid chromatography.
Chinese invention patent CN201210353577 discloses a kind of purification process of crude tacrolimus, by Ta Kemo
Crude product is taken charge of after the dissolution of the first organic solvent and sonic oscillation, is filtered using fast grade filter paper, is resulting filtrate is filtered later
Sample solution carries out continuous purification by countercurrent chromatography instrument.
Chinese invention patent CN201310534184 discloses a kind of technique for preparing high-purity tacrolimus, utilizes four
The dissolution of hydrogen furans, adds pure water, filters；Followed by C18 reverse-phase chromatography column purification, purity is up to 99.5% or more, single impurity
Content is not more than 0.3%, and total impurities content is not more than 0.5%, and yield is up to 50-80%.
The above purification process is all made of the method for positive or reversed phase column chromatography to obtain crude product, need to special equipment, disappear
A large amount of organic solvent is consumed, a large amount of waste water, waste liquid, disused stuffing material such as silica gel etc. are generated, wherein eluting solvent such as acetonitrile, isopropyl
Alcohol, n-hexane, alkyl halide is big to human toxicity, and time-consuming and complicated for operation dangerous for technique；
Chinese invention patent CN200480008682.5 discloses crystallization and the purification process of a kind of macrolides,
Extracted with polar solvent such as isobutyl acetate, ethyl acetate, gained extract liquor be added water or methanol-water-n-hexane system washing,
Grease concentrate is obtained after concentration, then mix with polar solvent, hydrocarbon solvent and its aqueous solution containing alkali, is recrystallized, acquisition
The crude product of macrocyclic lactone such as tacrolimus, but the purity for obtaining crude product is low, highest purity 85.03%；Although this method does not have
Column chromatographic purifying is used, but needs that the requirement that can be only achieved the drug standards is further purified.
Chinese invention patent CN200580037031.3 discloses a kind of purification process of fujimycin 506, including 4 steps:
1) fermentation liquid extraction-purification on adsorbent resins；2) silica gel purification；3) fujimycin 506-silver-π-complex compound dissolution and purifying；4) acetonitrile/
Water recrystallization obtains the finished product that purity is greater than 99%.But a large amount of organic solvents are used in this method step 2) as eluant, eluent；
Step 3) uses the purifying of C18 reverse phase silica gel, at high cost；Step 4) uses recrystallized from acetonitrile, and toxicity is big；Therefore the technique is unfavorable for
Industrialized production.
Chinese invention patent CN200910146385 is disclosed and a kind of streptomycete fermentation liquid is obtained bacterium with plate compression
Body extracts thallus with the multiple room temperature of organic solvent, obtains leaching liquor, macroreticular resin and silica gel column layer are carried out after leaching liquor is concentrated
Analysis, column chromatography gained tacrolimus is concentrated with mixed solvent, using ethyl acetate/n-hexane or ethyl acetate/hexamethylene
Recrystallization, the purity of tacrolimus is up to 95.8%, and purity is low.
Chinese invention patent CN2005800439516 discloses a kind of purifying tacrolimus process, chromatographs by 3
Column column chromatography, extraction, are recrystallized using ethyl acetate, hexamethylene and aqueous systems, but this method recrystallization time is long, small 12
When more than, 40 DEG C of gained crystal are dried under reduced pressure 12h, and then for 24 hours, drying time is long for 25 DEG C of dryings, the method repeat crystallization 2-4
Secondary, complicated for operation, yield is low, and the product weight content finally obtained is 99.84%.
Therefore it provides a kind of can reduce impurity and production cost, improve the more effective purification process of product quality very
It is necessary.
Summary of the invention
The object of the invention is in order to overcome in the prior art purifies and separates tacrolimus fermentation liquid impurity it is more, separation is difficult
Degree is big, and the deficiency that existing purification process is complicated, time-consuming, purity and yield are low provides a kind of high-purity tacrolimus chemical combination
Object and preparation method thereof, solve in existing purification process ascosin and dihydro tacrolimus separating difficulty are big, yield is low, consumption
Duration, eluting solvent dosage are big, pollution is high, are unfavorable for the problem of industrialized production.
In order to solve the above-mentioned technical problem, the present invention is addressed by following technical proposals:
One of the object of the invention is to provide a kind of high-purity tacrolimus, in the tacrolimus ascosin and dihydro he gram
Department's content is not that 0.0%, other single impurity are respectively smaller than 0.05%.
It is 99% or more that the high-purity tacrolimus, which normalizes purity, and the granularity of the tacrolimus crystal of the high-purity
Be evenly distributed, crystal internal stress and heap density it is small, stability, mobility, dispersibility and dissolubility are preferable, improve him gram
The quality and drug safety that do not take charge of.
Another object of the present invention is to provide a kind of preparation method of high-purity tacrolimus, includes the following steps:
1) crude tacrolimus containing ascosin and dihydro tacrolimus impurity is adsorbed onto silver ion polymer
On resin；
2) it is eluted with moderately polar mixed solvent, collects the fraction containing tacrolimus, obtain tacrolimus after concentration
Concentrate；
3) tacrolimus concentrate is dissolved with middle polarity solvent, water is added after dissolving under stirring condition completely, starts to analyse
Crystalline substance, linear to cool down, crystallization filters afterwards completely, is dry, i.e. acquisition high-purity tacrolimus.
1) refer to the conventional silver ion fluoropolymer resin bought on the market on silver ion fluoropolymer resin described in.
Preferably, moderately polar mixed solvent described in step 2) be methanol/ethyl acetate, ethyl alcohol/ethyl acetate,
The mixing of any one of acetone/ethyl acetate, the volume ratio of the mixed solvent are 35~65:65~35, more preferable ethyl alcohol/second
Acetoacetic ester volume ratio is 45:55.
It is preferred methanol/ethyl acetate in step 2), ethyl alcohol/ethyl acetate, any mixed in acetone/ethyl acetate
It closes, small toxicity, mixed solvent polarity size is just well separated by tacrolimus and ascosin and dihydro tacrolimus impurity
And the mixed liquor intersected is few, reduces the loss of tacrolimus, is conducive to improve yield.
Further, moderately polar mixed solvent elution flow rate described in step 2) is 0.6~1.0BV/h.
In the prior art when separating ascosin and dihydro tacrolimus, reverse-phase chromatographic column is mostly used to chromatograph, or adopt
It is first combined with the aqueous solution containing silver ion with crude tacrolimus, or aqueous solution and ion exchange resin containing silver ion
Exchange, be all purified in aqueous system, but those skilled in the art through experiments, it was found that, tacrolimus is in aqueous solution
In it is unstable, be easy to isomers convert.And the present invention does not use Aquo System in column chromatography whole process of purification
Solvent, conducive to the stability of tacrolimus.
Preferably, middle polarity solvent described in step 3) is any one of methanol, ethyl alcohol, acetone, preferred alcohol；
Tacrolimus concentrate described in step 3) and middle polarity solvent w/v are 150~160:1mg/ml.
Preferably, the mode that water is added described in step 3) is stream plus is added portionwise that water and medium pole is added in first
Property solvent volume ratio be 0.7~0.9:1；It is subsequently added into crystal seed, the crystal seed accounts for the 2~3% of crude tacrolimus weight；It stirs
0.5~1.5h, preferably 1.0h are mixed, it is 0.3~0.5:1 that water and the volume ratio of middle polarity solvent, which is added, in second batch.
Further, time control is at the uniform velocity added in 0.5~1.5h, preferably 1.0h in the stream plus water.
Preferably, beginning crystallization temperature described in step 3) is 30~35 DEG C；It is described to be linearly cooled to 5~10 DEG C, it is excellent
Select 8 DEG C.
Preferably, linear rate of temperature fall described in step 3) is 3~7 DEG C/h, preferably 6 DEG C/h.
Preferably, mixing speed described in step 3) is 70~130rpm；The drying temperature is 40~50 DEG C, dry
4~5h.
The tacrolimus crystalline form particle for the recrystallization method preparation that step 3) of the present invention provides is big and is evenly distributed, in crystal
Stress and heap density are small, and stability, mobility, dispersibility and dissolubility are preferable.The glossiness of crystalline form is good, and space is big, folder
The solvent of slag is few, and drying time is short, by drying 2 days originally, shortens to 4~5 hours, energy saving, avoids dry in long-time
Tacrolimus hydrate crystal forms are converted into the generation of the case where tacrolimus anhydride crystal form in the process.
Compared with prior art, the present invention bring it is unexpected technical effect it is as follows:
1, product purity is high: can be whole with the immediate two impurity ascosins of tacrolimus and dihydro tacrolimus
Removal, reaches 99% purity, improves the drug quality of tacrolimus, so that drug safety performance is higher.
2, it simple process and high income: can easily remove mould with the immediate two impurity ascus of tacrolimus structure
Element and dihydro tacrolimus, and technological operation is simple, solvent consumption is few, and it is with short production cycle, it is at low cost, it can be simultaneously reached
91% or more yield.Therefore this technique is more suitable for industrialized production.
Detailed description of the invention
Fig. 1 is the HPLC spectrogram in the embodiment of the present invention 1 before the removal of impurities of silver ion fluoropolymer resin.
Fig. 2 is the HPLC spectrogram in the embodiment of the present invention 1 after the removal of impurities of silver ion fluoropolymer resin.
Fig. 3 is the crystallographic scanning figure that the embodiment of the present invention 1 disposably adds 1.2BV water.
Fig. 4 is the embodiment of the present invention 2 in two times plus water, first time add water 0.8BV, add the crystallization of water 0.4BV water for the second time
Crystal scanning figure.
Fig. 5 is the particle size distribution figure that tacrolimus sterling is obtained after the embodiment of the present invention 2 recrystallizes.
Fig. 6 is the HPLC spectrogram that tacrolimus sterling is obtained after the embodiment of the present invention 2 recrystallizes.
Specific embodiment
Those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that
All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair
It is bright.
The instrument that the present invention uses is as follows:
Sem analysis is carried out to crystal form using Hitachi, Japan S-4800 type scanning electron microscope, operation voltage is 15.0kV,
Electric current 10 μ A, operating distance 10.3mm.Sample carries out surface metal spraying processing, vacuum degree under vacuum before being observed
10Pa, metal spraying time 60s.
Instrument and equipment model Microtrac S3500 laser particle size instrument used in particle size analyzer (PSD analysis).Detection
Method parameter: zeroing time: 30S；Detection time: 30S；Dispersing agent is water；Dispersing agent flow velocity: 55%, sample refractive index:
1.58, dispersing agent refractive index: 1.33, integral way: volume, laser source wavelength: 780nm.
High performance liquid chromatography (HPLC) spectral data is picked up from high performance liquid chromatograph: Agilent 1260；
Chromatographic column: C8 column (4.6 × 150mm, 5 μm)；
Mobile phase:
Solution A: 0.02mol/L potassium dihydrogen phosphate, with phosphoric acid tune pH to 3.5.
Solution B: acetonitrile；
Ultraviolet detection wavelength: 210nm；
Column temperature: 60 DEG C；
Flow velocity: 1.0ml/min；Sample volume: 20ul.
The silver ion fluoropolymer resin bought on the market is suitable for the invention step 1) and 2), tool of the invention
Body embodiment uses the aperture of silver ion fluoropolymer resin for 15~25nm, and the volume in hole is 1.20~1.35cm3/ g compares table
260~480m of area2/g。
For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.
Embodiment 1
1) silver ion fluoropolymer resin 280ml is taken, before filling column, needs that first resin is placed in glass beaker, is added third
Ketone, infiltration and be more than resin surface layer 2cm or so, immersion 3 hours after, 1/3 volume is added in filter infusion resin in chromatographic column
Acetone adds the resin impregnated, and resin charge finishes, and diameter height is than about 1:7.8；With Mathanol regenerating resin, coutroi velocity
1BV/h, regeneration acetone dosage are the 5BV of column volume, and the mixed solvent that again with methanol and ethyl acetate volume ratio are 49:51 is flat
Weighing apparatus, mixed solvent dosage are the 2BV of column volume.
2) preparation of upper prop liquid and loading: column total hundred million is counted in based on chromatographic resin loading amount volume (on every milliliter of chromatographic resin
9mg), crude tacrolimus 2.8g is weighed, which contains 1.56% ascosin, 4.11% dihydro tacrolimus, weight
Measuring content is 90%, sees Fig. 1, and wherein 2.52g is active constituent.Methanol and ethyl acetate volume ratio are dissolved in for the mixing of 51:49
In solvent dissolution, it is made into the upper prop liquid of 200mg/ml.Before upper prop, chromatography upper end solvent is first moved to resin surface upper end in column
Above-mentioned upper prop liquid is added in solvent about 1cm, opens outlet valve under column, coutroi velocity 0.5BV/h, and the absorption of upper prop liquid finishes.
3) it elutes: being eluted with the mixed solvent that methanol and ethyl acetate volume ratio are 51:49, coutroi velocity 1.0BV/h is received
Collect tacrolimus eluent fraction, eluting solvent dosage 4BV is detected through HPLC, by ascosin and dihydro tacrolimus normalizing
Change the eluent fraction that content is 0.0% to merge, amalgamation liquid volume 410ml, sample detection is shown in Fig. 2, potency: 4924ug/
Ml chromatographs yield up to 80.1%.
4) it is concentrated: the fraction containing tacrolimus component requirements qualification being depressurized under the conditions of bath temperature is 60 DEG C dense
Contracting, obtains its concentrate.
5) it crystallizes: adding methanol to dissolve, be configured to 160mg/ml crystallization solution, be placed in 35 DEG C of crystallization constant temperature low temperature grains, stir
It mixes, mixing speed 80rpm, the 1.2BV pure water of lysate volume is added, 2.0% crystal seed is added, after stirring 1h, with 3 DEG C/h speed
Rate cooling, stirring are linearly cooled to 10 DEG C, and filtering elutes crystal with 15% methanol aqueous solution, leacheate is removed by suction filtration under vacuum,
Crystal vacuum drying, vacuum degree -0.09MPa, 50 DEG C drying 5 hours, obtain 1.83g finished product, finished product taken to detect through HPLC, normalization
Content 97.2%；Crystallization 1 time is repeated, obtains 1.67g finished product, which sees Fig. 3；It takes finished product to detect through HPLC, normalizes
Content 99.2%；Ascosin: 0.00% and dihydro tacrolimus: 0.00%, total impurities normalize content 0.04%.Recrystallization
Yield is up to 91.3%.
Embodiment 2
1) silver ion fluoropolymer resin 280ml (chromatographic column ￠ 3.6cm) is taken, with ethyl alcohol regenerating resin, coutroi velocity
1BV/h, regeneration solvent dosage are the 2BV of column volume, then the mixed solvent for being 45:55 with ethyl alcohol and ethyl acetate volume ratio is put down
Weighing apparatus, mixed solvent dosage are the 2BV of column volume.
2) preparation of upper prop liquid and loading: column total hundred million is counted in based on chromatographic resin loading amount volume (on every milliliter of chromatographic resin
10mg), weigh tacrolimus crude intermediate 3.11g, which contains 1.56% ascosin, 4.11% dihydro he gram
Mo Si, weight content 90%, wherein 2.8g is active constituent, and the volume ratio for being dissolved in ethyl alcohol and ethyl acetate is the mixed of 45:55
It closes in solvent, is made into the upper prop liquid of 250mg/ml.Before upper prop, it is molten that chromatography upper end solvent is first moved to resin surface upper end in column
Above-mentioned upper prop liquid is added in matchmaker about 1-2cm, opens outlet valve under column, coutroi velocity 0.5BV/h, and the absorption of upper prop liquid finishes.
3) elute: use ethyl alcohol: the mixed solvent that the volume ratio of ethyl acetate is 45:55 elutes, coutroi velocity 0.8BV/h,
Tacrolimus eluent fraction is collected, eluting solvent dosage 4BV is detected through HPLC, ascosin and dihydro tacrolimus are returned
One change content is 0.0% eluent fraction merging, amalgamation liquid volume 420ml, sample detection, potency: 5488ug/ml, layer
Yield is analysed up to 82.3%.
4) it is concentrated: the fraction containing tacrolimus component requirements being concentrated under reduced pressure under the conditions of bath temperature is 60 DEG C, is obtained
Obtain its concentrate.
5) it crystallizes: adding ethyl alcohol to dissolve, be configured to 155mg/ml crystallization solution, be placed in 32 DEG C of crystallization constant temperature low temperature grains, stir
It mixes, mixing speed 100rpm, the 0.8BV pure water of lysate volume is added, 2.0% crystal seed is added, after stirring 1h, flow solubilization solution
The 0.4BV pure water of liquid product, 1h are added, and with the cooling of 5 DEG C/h rate, stirring is linearly cooled to 8 DEG C, filtering, with 15% methanol
Aqueous solution elutes crystal, and leacheate is removed by suction filtration under vacuum, and crystal is dried in vacuo, vacuum degree -0.09MPa, and 43 DEG C of dryings 4.5 are small
When, 2.15g finished product is obtained, finished product is taken to detect through HPLC, normalizes content 99.8%；The crystal scanning figure is shown in the grain of Fig. 4, the crystal
Degree distribution map is shown in Fig. 5；See Fig. 6；Ascosin: 0.00% and dihydro tacrolimus: 0.00%, total impurities normalize content
0.00%.Crystallization yield is up to 91.6%.
Embodiment 3
1) silver ion fluoropolymer resin 120L is taken, before filling column (chromatographic column ￠ 30cm), needs first to be placed in resin stainless
In steel drum, be added ethyl alcohol, it is desirable that infiltration and be more than resin surface layer 2cm, immersion 4 hours after, filter infusion resin, in chromatographic column
1/3 moderately polar solvent is added, adds the resin impregnated, resin charge finishes, and diameter height is than about 1:5.7；Again with ethyl alcohol
Green resin, coutroi velocity 1BV/h, regeneration solvent dosage are the 5BV of column volume, then with ethyl alcohol: the volume ratio of ethyl acetate is 45:
55 mixed solvent balance, mixed solvent dosage are the 5BV of column volume.
2) preparation of upper prop liquid and loading: column total hundred million is counted in based on chromatographic resin loading amount volume (on every milliliter of chromatographic resin
9mg), weigh tacrolimus crude intermediate 1220g, which contains 1.15% ascosin, 3.61% dihydro he gram
Mo Si, weight content 88.5%, wherein 1080g is active constituent, is the mixed of 45:55 with ethyl alcohol: the volume ratio of ethyl acetate
It closes solvent and dissolves crude intermediate, be made into the upper prop liquid of 230mg/ml.Before upper prop, first chromatography upper end solvent is moved in column and is set
Above-mentioned upper prop liquid is added in rouge surface upper end solvent about 1-2cm, opens outlet valve under column, the absorption of coutroi velocity 0.5BV/h upper prop liquid
It finishes.
3) elute: use ethyl alcohol: the mixed solvent that the volume ratio of ethyl acetate is 45:55 elutes, coutroi velocity 0.7BV/h,
Opening detector, wavelength: 245nm collects elution fraction, and about every 20L collects one barrel, until there is the fraction of tacrolimus unit, takes
Sample, HPLC detection terminate elution to elution fraction tacrolimus unit < 800ug/ml, and eluting solvent dosage 4BV is mould by ascus
Element and dihydro tacrolimus normalize the eluent fraction that content is 0.0% and merge, amalgamation liquid volume 162L, sample detection,
Potency: 5392ug/ml chromatographs yield up to 80.9%.
4) it is concentrated: the fraction containing tacrolimus component requirements being concentrated under reduced pressure under the conditions of bath temperature is 60, is obtained
Its concentrate.
5) it crystallizes: adding ethyl alcohol to dissolve, be configured to 155mg/ml crystallization solution, be placed in 33 DEG C of crystallization constant temperature low temperature grains, stir
It mixes, mixing speed 110rpm, the 0.8BV pure water of lysate volume is added, 2.5% crystal seed is added, after stirring 1h, flow solubilization solution
The 0.4BV pure water of liquid product, 1.5h are added, and with the cooling of 6 DEG C/h rate, stirring is linearly cooled to 8 DEG C, filtering, with 15% first
Alcohol solution elutes crystal, and leacheate is removed by suction filtration under vacuum, and crystal is dried in vacuo, vacuum degree -0.09MPa, and 45 DEG C of dryings 5 are small
When, 822g finished product is obtained, finished product is taken to detect through HPLC, normalizes content 99.8%；Ascosin: 0.00% and dihydro Ta Kemo
Department: 0.00%, total impurities normalize content 0.00%.Crystallization yield is up to 91.8%.
Embodiment 4
1) 3 used silver ion fluoropolymer resin 280ml (chromatographic column ￠ 3.6cm) of the present embodiment is taken, methanol is used
Regenerating resin, coutroi velocity 1BV/h, regeneration solvent dosage are the 2BV of column volume, and again with methanol is with ethyl acetate volume ratio
The mixed solvent of 35:65 balances, and mixed solvent dosage is the 5BV of column volume.
2) preparation of upper prop liquid and loading: column total hundred million is counted in based on chromatographic resin loading amount volume (on every milliliter of chromatographic resin
12mg), weigh tacrolimus crude intermediate 3.95g, which contains 1.56% ascosin, 4.11% dihydro he gram
Department's weight content is not 85.1%, and wherein 3.36g is active constituent, and the volume ratio for being dissolved in acetone and ethyl acetate is 35:65's
In mixed solvent, it is made into the upper prop liquid of 250mg/ml.Before upper prop, chromatography upper end solvent is first moved to resin surface upper end in column
Above-mentioned upper prop liquid is added in solvent about 1.5cm, opens outlet valve under column, coutroi velocity 0.5BV/h, and the absorption of upper prop liquid finishes.
3) elute: use acetone: the mixed solvent that the volume ratio of ethyl acetate is 35:65 elutes, coutroi velocity 0.7BV/h,
Elution fraction is collected, is detected through HPLC, merges ascosin and dihydro tacrolimus normalization content is 0.0% eluent
Fraction, amalgamation liquid volume 470ml, sample detection, potency: 5756ug/ml chromatographs yield up to 80.5%.
4) it is concentrated: the fraction containing tacrolimus component requirements being concentrated under reduced pressure under the conditions of bath temperature is 60 DEG C, is obtained
Obtain its concentrate.
5) it crystallizes: adding acetone solution, be configured to 150mg/ml crystallization solution, be placed in 30 DEG C of crystallization constant temperature low temperature grains, stir
It mixes, mixing speed 130rpm, the 0.9BV pure water of lysate volume is added, 3% crystal seed is added, after stirring 1h, stream plus lysate
The 0.3BV pure water of volume, 0.5h are added, and with the cooling of 7 DEG C/h rate, stirring is linearly cooled to 5 DEG C, filtering, with 15% methanol
Aqueous solution elutes crystal, and leacheate is removed by suction filtration under vacuum, and crystal is dry, vacuum degree -0.09MPa, 40 DEG C of temperature, 4 hours dry,
2.56g finished product is obtained, finished product is taken to detect through HPLC, normalizes content 99.5%；Ascosin: 0.00% and dihydro tacrolimus:
0.00%, total impurities normalize content 0.05%.Crystallization yield is up to 91.6%.
Comparative example 5
Different chromatography scheme purification of tacrolimus crude products
Concrete outcome is as follows:
Know from upper table, when separating ascosin and dihydro tacrolimus in the prior art, is carried out in Aquo System, and
And in the case where guaranteeing high yield, ascosin and dihydro tacrolimus the two impurity are difficult completely separable.However this hair
The separation method of bright offer is eluted using anhydrous system solvent and is separated, and tacrolimus is avoided to convert to isomers, receives column chromatography
Rate is up to 80% or more, also, the chromatography period is short, and solvent usage is few.The chromatography of column used in the present invention is normal pressure chromatographic column, to setting
It is low for requiring.
Comparative example 6
The crystallophy characteristic concrete outcome of different method for crystallising, acquisition is as follows:
Being known by upper table, after the present invention uses methanol, ethyl alcohol or acetone solution crude tacrolimus, stream adds purified water in batches,
Add crystal seed, linear to cool down, rate of temperature fall preferably 6 DEG C/h, crystallographic granularity is big, is evenly distributed, and heap density is small, good fluidity,
Drying time is short, and product content is high.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise of not departing from the present invention, several improvement can also be made, these improvement also should be regarded as protection of the invention
Range.
Claims (2)
1. a kind of preparation method of tacrolimus, includes the following steps:
1) crude tacrolimus containing ascosin and dihydro tacrolimus impurity is adsorbed onto silver ion fluoropolymer resin
On, applied sample amount 10mg/ml；
2) it is eluted with ethyl alcohol/ethyl acetate mixed solvent that volume ratio is 45:55, collects the fraction containing tacrolimus, concentration
Tacrolimus concentrate is obtained afterwards；
3) tacrolimus concentrate is dissolved with ethyl alcohol, flows plus be added portionwise water after dissolving under stirring condition completely, starts crystallization, line
Property cooling, crystallization filters afterwards completely, is dry, i.e. acquisition tacrolimus；
Wherein, the weight content of the crude tacrolimus is 90%, and the content of ascosin and dihydro tacrolimus is respectively
1.56% and 4.11%；The stream adds the operating method that water is added portionwise are as follows: the volume of water and vehicle of ethanol is added in first
Than for 0.8:1；It is subsequently added into crystal seed, the crystal seed accounts for the 2% of crude tacrolimus weight；Stir 1h, second batch be added water with
The volume ratio of vehicle of ethanol is 0.4:1, and the time controls in 1h；Temperature when starting crystallization is 32 DEG C；Described is linear
The rate of cooling is 5 DEG C/h, is linearly cooled to 8 DEG C.
2. the preparation method of tacrolimus as described in claim 1, it is characterised in that: mixing speed described in step 3) is
100rpm；The drying temperature is 43 DEG C, drying time 4.5h.
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| CN108250216A (en) * | 2016-12-29 | 2018-07-06 | 天津领世生物科技开发有限公司 | The method of tacrolimus, double hydrogen tacrolimus, ascosin in one one-step refining crude tacrolimus |
| CN113912624B (en) * | 2021-12-03 | 2023-03-28 | 北大方正集团有限公司 | Tacrolimus separation and purification method |
| CN116102577A (en) * | 2023-02-06 | 2023-05-12 | 华北制药华胜有限公司 | High-purity tacrolimus and preparation method thereof |
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