CN102924478B - Preparing method of compound FK506-A - Google Patents

Preparing method of compound FK506-A Download PDF

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CN102924478B
CN102924478B CN201210483603.2A CN201210483603A CN102924478B CN 102924478 B CN102924478 B CN 102924478B CN 201210483603 A CN201210483603 A CN 201210483603A CN 102924478 B CN102924478 B CN 102924478B
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silica gel
crude product
dry
ether
compound
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CN102924478A (en
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任风芝
段宝玲
张雪霞
陈书红
李丽红
刘进怀
郑智慧
成晓迅
张艳立
李晓露
李宁
王海燕
孟雅娟
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NCPC New Drug Research and Development Co Ltd
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NCPC New Drug Research and Development Co Ltd
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Abstract

The invention provides a preparing method of a compound FK506-A, which includes the following steps: a) heating FK506 for 2 to 25 minutes under 110 to 150 degrees C, so as to obtain a FK506-A crude product; b) adding organic solvent into the FK506-A crude product, adding silica gel after dissolving, and drying after uniformly stirring, so as to obtain a dry sample; and c) adding the dry sample into a chromatographic column filled with silica gel and eluting by adding eluting solvent, and collecting the eluent with the FK506-A purity determined by HPLC to be no less than 75% and concentrating to be dry solid, so as to obtain FK506-A pure product by crystallizing the solid. The preparing method of compound FK506-A has the advantages that the operation is simple, and the industrial production is easy.

Description

A kind of preparation method of FK506-A compound
Technical field
The invention belongs to technological field of biochemistry, relate in particular to a kind of preparation method of macrolides compound FK506-A compound.
Technical background
FK506(Tacrolimus) be the macrolides compound that streptomyces tsukubaensis (Streptomyces tsukubaensis) produces, it is used to preparation immunosuppression class medicine at present.The molecular formula of this compound is C 44h 69nO 12, molecular weight is 804.02, has the ring texture of 23 yuan in its molecule, and its structural formula is as follows:
According to the literature, FK506 character is unstable, isomerization reaction can occur, and produces isomers (Tetrahedron Letters, Vol31, No.45, pp6477-6480,1990).By structure isomer called after FK506-A compound as the formula (1).
Formula (1)
In current research, FK506-A is considered to the unstable product of FK506, therefore rarely has people to carry out deep research to it.
Summary of the invention
The object of this invention is to provide a kind of preparation method of FK506-A compound, the method is easy and simple to handle, product purity is high, is suitable for suitability for industrialized production application.
For realizing the object of the invention, the invention provides the preparation method of FK506-A compound, this compound structure as the formula (1)
The method comprises the following steps:
A, FK506 is heated 2-25 minute at 110-150 DEG C, obtain FK506-A crude product;
B, in FK506-A crude product, add organic solvent, after dissolving, add silica gel, dry after stirring, obtain dry sample;
C, to be added by dry sample and be equipped with in the chromatography column of silica gel, add eluting solvent and carry out wash-out, collect the elutriant measuring purity >=75% through HPLC, be concentrated into dry, gained solid carries out crystallization and obtains FK506-A sterling.
Heat described in a step of the present invention, its temperature is preferably 120-130 DEG C.At such a temperature, FK506 reaction is more complete, and by product is less.
Organic solvent described in b step of the present invention be in chloroform, ethyl acetate, ether, acetonitrile, butylacetate, acetone, ethanol, methyl alcohol any one.In preferred chloroform, ethyl acetate, acetone, ethanol, methyl alcohol any one.
Silica gel of the present invention, its granularity is preferably 100-400 order.Silica gel adsorption effect under this granularity is excellent, and easier wash-out.
Silica gel described in b step of the present invention, its consumption is preferably the 1-2.5 of FK506-A crude product quality doubly.
Silica gel described in step c of the present invention, its consumption is 8-15 times of FK506-A crude product quality, preferably 10 times.
Eluting solvent described in step c of the present invention is any one or a few mixed solution in sherwood oil, normal hexane, ethyl acetate, ether, isopropyl ether, acetonitrile, butylacetate, acetone, ethanol, methyl alcohol, the mixed solution of any one or two kinds in preferred sherwood oil, normal hexane, ethyl acetate, ether, isopropyl ether, acetonitrile, acetone.
Concentrated described in step c of the present invention, preferably concentrating under reduced pressure at 30-60 DEG C.
Crystallization described in step c of the present invention, its method is in every gram of solid, add 5-15 milliliter recrystallisation solvent, after dissolving, slow cooling crystallization, after crystal is no longer separated out, filter, with vacuum-drying at filter cake being placed in 30-60 DEG C after recrystallisation solvent washing leaching cake 1-2 time, described recrystallisation solvent is normal hexane, ethyl acetate, ether, isopropyl ether, acetonitrile, butylacetate, acetone, ethanol, Virahol, methyl alcohol, any one or a few mixed solution in water, preferred normal hexane, ethyl acetate, ether, acetonitrile, acetone, Virahol, the mixed solution of any one or two kinds in water.
Above-mentioned " add 5-15 milliliter recrystallisation solvent in every gram of solid, dissolve " is so that solid can be made to be dissolved as principle completely.Those skilled in the art can according to the character of selected recrystallisation solvent, uses heating or the assist in dissolving means such as ultrasonic to promote that it dissolves.
FK506-A of the present invention is a kind of macrolides compound, and pharmacological experiment study shows, it has immunosuppressive action, can be used for preparation immunosuppression class medicine.The preparation method of FK506-A of the present invention, make use of the isomerization reaction of FK506, by controlling temperature of reaction and time, improves reaction efficiency, is separated and recrystallization process, has obtained highly purified FK506-A product in conjunction with follow-up silica gel column chromatography.The method of the invention also has advantage easy and simple to handle, with low cost, is suitable for industrial production.
Accompanying drawing explanation
Fig. 1 is the liquid phase-mass spectrum of FK506-A.
Fig. 2 is FK506-A 1hNMR spectrogram.
Fig. 3 is FK506-A 13cNMR spectrogram.
Fig. 4 is the HMQC spectrogram of FK506-A.
Fig. 5 is the HMBC spectrogram of FK506-A.
Fig. 6 is FK506-A 1h- 1h cosy spectrogram.
Embodiment
Further illustrate content of the present invention with specific embodiment below, but and mean never in any form and limit the invention.In the following example, method therefor is ordinary method if no special instructions.
Raw material FK506 used in the present invention is the FK506 crude product that North China Pharmacuetical Group New Drug Research & Development Co., Ltd produces.Silica gel is purchased from Qingdao Marine Chemical Co., Ltd.; High performance liquid chromatograph device comprises 996 type detectors, 515 pumps (production of Waters company); INVOA 500 type nuclear magnetic resonance spectrometer is purchased from Varian company; Acquity uplc system, PDA detector, Xevo TQ MS/MS detector is all purchased from Waters company.
Embodiment 1
A, 10g FK506 is put into furnace pot, heat 15 minutes at 120 DEG C, obtain FK506-A crude product;
B, in FK506-A crude product, add ethyl acetate, after dissolving, add 19g 300-400 object silica gel, dry after stirring, obtain dry sample;
C, dry sample is added in the chromatography column that 80g 300-400 order silica gel is housed, add ether and carry out wash-out, adopt the FK506-A concentration in HPLC method detection elutriant, collect the elutriant measuring FK506-A purity >=75% through HPLC, 30 DEG C are evaporated to dry, obtain 1.6g solid, in every gram of solid, adding 15 milliliters by volume ratio is the acetone of 4:1, the recrystallisation solvent of water composition, after heating for dissolving, slow cooling crystallization, suction filtration after crystal is no longer separated out, after above-mentioned recrystallisation solvent washing leaching cake 2 times, vacuum-drying at filter cake being placed in 60 DEG C, obtain FK506-A sterling 1.2g, the purity adopting HPLC method to measure FK506-A in gained FK506-A sterling is 97.7%.
FK506-A's 1h NMR(500MHz, CD 3cN) and 13c NMR attribution data (125MHz, CD 3cN) in table 1, the liquid phase-mass spectrum of FK506-A, 1hNMR spectrogram, 13cNMR spectrogram, HMQC spectrogram, HMBC spectrogram, 1h- 1h cosy spectrogram is shown in accompanying drawing 1-6.
Table 1
Embodiment 2
A, 10g FK506 is put into furnace pot, heat 5 minutes at 130 DEG C, obtain FK506-A crude product;
B, in FK506-A crude product, add ethanol, after dissolving, add 25g 160-230 object silica gel, dry after stirring, obtain dry sample;
C, dry sample is added in the chromatography column that 150g 160-230 order silica gel is housed, add the isopropyl ether that volume ratio is 8:1, the mixed solution of acetone carries out wash-out, collect the elutriant detecting FK506-A purity >=75% through HPLC, 60 DEG C are evaporated to dry, obtain 2.1g solid, 5 milliliters of ether are added in every gram of solid, after heating for dissolving, slow cooling crystallization, after crystal is no longer separated out, filter, after washed with diethylether filter cake 1 time, vacuum-drying at filter cake being placed in 30 DEG C, obtain FK506-A sterling 1.9g, the purity adopting HPLC method to measure FK506-A in gained FK506-A sterling is 98.0%.
Embodiment 3
A, 10g FK506 is put into furnace pot, heat 25 minutes at 110 DEG C, obtain FK506-A crude product;
B, in FK506-A crude product, add acetone, after dissolving, add 10g 100-200 object silica gel, dry after stirring, obtain dry sample;
C, dry sample is added in the chromatography column that 120g 100-200 order silica gel is housed, add the normal hexane that volume ratio is 10:1, the mixed solution of acetone carries out wash-out, collect the elutriant detecting FK506-A purity >=75% through HPLC, 50 DEG C are evaporated to dry, obtain 1.8g solid, in every gram of solid, adding 13 milliliters by volume ratio is the normal hexane of 10:1, the recrystallisation solvent of acetone composition, after dissolving, slow cooling crystallization, after crystal is no longer separated out, filter, after above-mentioned recrystallisation solvent washing leaching cake 1 time, vacuum-drying at filter cake being placed in 40 DEG C, obtain FK506-A sterling 1.5g, the purity adopting HPLC method to measure FK506-A in gained FK506-A sterling is 98.2%.
Embodiment 4
A, 10g FK506 is put into furnace pot, heat 10 minutes at 115 DEG C, obtain FK506-A crude product;
B, in FK506-A crude product, add chloroform, after dissolving, add 15g 300-400 object silica gel, dry after stirring, obtain dry sample;
C, dry sample is added in the chromatography column that 140g 300-400 order silica gel is housed, add the ether that volume ratio is 8:1, the mixed solution of acetone carries out wash-out, collect the elutriant detecting FK506-A purity >=75% through HPLC, 40 DEG C are evaporated to dry, obtain 1.5g solid, 10 milliliters of Virahols are added in every gram of solid, after heating for dissolving, slow cooling crystallization, suction filtration after crystal is no longer separated out, after washed with isopropyl alcohol filter cake 2 times, vacuum-drying at filter cake being placed in 50 DEG C, obtain FK506-A sterling 1.3g, the purity adopting HPLC method to measure FK506-A in gained FK506-A sterling is 98.4%.
Embodiment 5
A, 10g FK506 is put into furnace pot, heat 15 minutes at 125 DEG C, obtain FK506-A crude product;
B, in FK506-A crude product, add methyl alcohol, after dissolving, add 13g 300-400 object silica gel, dry after stirring, obtain dry sample;
C, dry sample is added in the chromatography column that 100g 300-400 order silica gel is housed, add the sherwood oil that volume ratio is 5:1, the mixed solution of ethyl acetate carries out wash-out, collect the elutriant that HPLC method detects FK506-A purity >=75%, 50 DEG C are evaporated to dry, obtain 2.0g solid, in every gram of solid, adding 10 milliliters by volume ratio is the normal hexane of 1:1, the recrystallisation solvent of ethyl acetate composition, after heating for dissolving, slow cooling crystallization, suction filtration after crystal is no longer separated out, after above-mentioned recrystallisation solvent washing leaching cake 1 time, vacuum-drying at filter cake being placed in 50 DEG C, obtain FK506-A sterling 1.7g, the purity adopting HPLC method to measure FK506-A in gained FK506-A sterling is 98.5%.
Embodiment 6
A, 10g FK506 is put into furnace pot, heat 2 minutes at 150 DEG C, obtain FK506-A crude product;
B, in FK506-A crude product, add ethyl acetate, after dissolving, add 20g 100-200 object silica gel, dry after stirring, obtain dry sample;
C, dry sample added 130g 100-200 order silica gel is housed chromatography column in, add acetonitrile and carry out wash-out, collect the elutriant detecting FK506-A purity >=75% through HPLC, 50 DEG C are evaporated to dry, obtain 2.1g solid, 10 milliliters of acetonitriles are added in every gram of solid, after dissolving, slow cooling crystallization, after crystal is no longer separated out, filter, after acetonitrile wash filter cake 1 time, vacuum-drying at filter cake being placed in 45 DEG C, obtain FK506-A sterling 1.8g, the purity adopting HPLC method to measure FK506-A in gained FK506-A sterling is 98.6%.
Embodiment 7
A, 10g FK506 is put into furnace pot, heat 20 minutes at 135 DEG C, obtain FK506-A crude product;
B, in FK506-A crude product, add butylacetate, after dissolving, add 25g 200-300 object silica gel, dry after stirring, obtain dry sample;
C, dry sample is added in the chromatography column that 150g 200-300 order silica gel is housed, add the sherwood oil that volume ratio is 1:1:1, ethyl acetate, the mixed solution of acetone carries out wash-out, collect concentration detects FK506-A purity >=75% elutriant through HPLC, 40 DEG C are evaporated to dry, obtain 1.6g solid, in every gram of solid, adding 12 milliliters by volume ratio is the normal hexane of 1:1:1.5, ethyl acetate, the recrystallisation solvent of acetone composition, after heating for dissolving, slow cooling crystallization, after crystal is no longer separated out, suction filtration, after above-mentioned recrystallisation solvent washing leaching cake 2 times, vacuum-drying at filter cake being placed in 50 DEG C, obtain FK506-A sterling 1.7g, adopt HPLC method measure FK506-A in gained FK506-A sterling purity be 97.9%.

Claims (3)

  1. The preparation method of 1.FK506-A compound, this compound structure is such as formula shown in (1)
    It is characterized in that comprising the following steps:
    A, FK506 is heated 2-25 minute at 110-150 DEG C, obtain FK506-A crude product;
    B, in FK506-A crude product, add organic solvent, adding granularity after dissolving is 100-400 object silica gel, the consumption of described silica gel be the 1-2.5 of FK506-A crude product quality doubly, dry after stirring, obtain dry sample; Wherein said organic solvent is any one in chloroform, ethyl acetate, ether, acetonitrile, butylacetate, acetone, ethanol, methyl alcohol;
    C, to be added by dry sample and be equipped with in the chromatography column of silica gel, add eluting solvent and carry out wash-out, collect the elutriant measuring FK506-A purity >=75% through HPLC, be concentrated into dry, gained solid carries out crystallization and obtains FK506-A sterling; Wherein said concentrated concentrated condition is concentrating under reduced pressure at 30-60 DEG C; Crystallization method described in it adds 5-15 milliliter recrystallisation solvent in every gram of gained solid, and after dissolving, slow cooling crystallization, treats that crystal is no longer separated out, and filters, with recrystallisation solvent washing leaching cake 1-2 time, and vacuum-drying at filter cake being placed in 30-60 DEG C; Described recrystallisation solvent is any one or a few mixed solution in normal hexane, ethyl acetate, ether, isopropyl ether, acetonitrile, butylacetate, acetone, ethanol, Virahol, methyl alcohol, water; Its consumption of described silica gel is 8-15 times of FK506-A crude product quality.
  2. 2. the preparation method of FK506-A compound according to claim 1, is characterized in that: heating its temperature described in a step is 120-130 DEG C.
  3. 3. the preparation method of FK506-A compound according to claim 1 or 2, is characterized in that: eluting solvent described in step c is any one or a few mixed solution in sherwood oil, normal hexane, ethyl acetate, ether, isopropyl ether, acetonitrile, butylacetate, acetone, ethanol, methyl alcohol.
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CN103819488A (en) * 2014-02-20 2014-05-28 浙江万马药业有限公司 Preparation method for known impurity-tacrolimus position isomer of tacrolimus

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013862A1 (en) * 1991-02-05 1992-08-20 Fujisawa Pharmaceutical Co., Ltd. Lactone compounds
CN1823771A (en) * 2005-02-21 2006-08-30 华北制药集团有限责任公司 FK506 oral medicinal composition

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013862A1 (en) * 1991-02-05 1992-08-20 Fujisawa Pharmaceutical Co., Ltd. Lactone compounds
CN1823771A (en) * 2005-02-21 2006-08-30 华北制药集团有限责任公司 FK506 oral medicinal composition

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
H. Ok,等.Thermal rearrangement of the immunosuppressant FK-506.《Tetrahedron Letters》.1990,第31卷(第45期),第6477-6480页. *

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