CN104774229A - Preparation method for doxorubicin and derivative thereof - Google Patents

Preparation method for doxorubicin and derivative thereof Download PDF

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Publication number
CN104774229A
CN104774229A CN201410012304.XA CN201410012304A CN104774229A CN 104774229 A CN104774229 A CN 104774229A CN 201410012304 A CN201410012304 A CN 201410012304A CN 104774229 A CN104774229 A CN 104774229A
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China
Prior art keywords
derivative
zorubicin
preparation
organic solvent
filler
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CN201410012304.XA
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吴晏旻
周永正
张慧
张晗
于连丹
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ZHEJIANG ACCHROM TECHNOLOGIES Co Ltd
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ZHEJIANG ACCHROM TECHNOLOGIES Co Ltd
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Priority to CN201410012304.XA priority Critical patent/CN104774229A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/252—Naphthacene radicals, e.g. daunomycins, adriamycins

Abstract

A purpose of the present invention is to provide a preparation method for doxorubicin and a derivative thereof. The preparation method is characterized in that the separation mechanism is a reverse phase pattern, and an organic solvent and a buffer salt water solvent system are used to carry out separation preparation, wherein the used filler is a reversed phase silica gel filler adopting SiO2 or a derivative thereof as a matrix and adopting a C8-C18 aliphatic hydrocarbon chain as a bonded phase, the particle size is 5-50 [mu]m, the pore size is 50-300 angstrom, the mobile phase is an organic solvent and buffer salt water solution mixed solvent, the organic solvent is a low substituent, a derivative thereof or a mixture of -OH, -CN, halogen atom, oxo, carbonyl or carboxyl of a lower aliphatic hydrocarbon containing 1-3 carbon atoms, the viscosity is less than or equal to 5 mPa.S, the buffer salt water solution adopts one or a plurality of ions selected from Na<+>, K<+>, H<+> and NH4<+> as cation and adopts one or a plurality of ions selected from Cl<->, COOH<->, F<->, PO4<3->, HClO4<-> and HCO3<-> as anion, and the organic solvent accounts for 5-70% of the total volume of the mobile phase.

Description

The preparation method of a kind of Zorubicin and derivative thereof
Technical field
The invention belongs to chemical pharmacy field, relate to a kind of method for preparing purified of organic compound, specifically, the present invention relates to a kind of purification process preparing high purity antitumor antibiotics Zorubicin and derivative and derivative thereof.
Background technology
Zorubicin and derivative thereof are a kind of Anthraquinones microbiotic, can suppress the synthesis of RNA and DNA, the strongest to the restraining effect of RNA, antitumor spectra is wider, all there is effect to kinds of tumors, belong to cell cycle nonspecific agent (CCNSA), have killing action to the tumour cell of various growth cycle.Mainly be applicable to acute leukemia, all effective to acute lymphoblastic leukemia and granulocyte leukemia, generally as Second line Drug, namely can consider to apply this medicine when choice drug resistance.Malignant lymphoma, can be used as the first medicine be used alternatingly.Certain curative effect is had, many and other anticarcinogen conbined usage to other various cancers such as mammary cancer, sarcoma, lung cancer, bladder cancer.Its chemical structural formula is as follows:
Summary of the invention
A kind of half preparation based on dynamic axial compression column (Dynamic AxialCompression, DAC) or preparative high performance liquid phase system (Pre-HPLC) is the object of the present invention is to provide to carry out the new preparation method of Zorubicin and derivative purifying thereof.
In order to realize foregoing invention object, present invention employs following technical scheme: the preparation method of a kind of Zorubicin and derivative thereof, is characterized in that: separating mechanism is rp mode, being with an organic solvent separated with buffering saline solvent system.
The preparation method of Zorubicin of the present invention and derivative thereof, is characterized in that: filler used is with SiO 2or derivatives thereof is matrix, the silica gel being Bonded Phase with C8 ~ C18 fat hydrocarbon chain, and its particle diameter is 5 μm ~ 50 μm, and aperture is reverse phase silica gel filler, filler shape is irregular shape or spherical.
The preparation method of Zorubicin of the present invention and derivative thereof, it is characterized in that: moving phase used is organic solvent and buffered saline solution mixed solvent, wherein organic solvent is containing the low substituent of-the OH ,-CN of lower aliphatic hydrocarbon of 1 ~ 3 carbon atom, halogen atom, oxo, carbonyl or carboxylic group, derivative or its mixture, its viscosity≤5mPaS; Buffered saline solution adopts Na +, K +, H +, NH 4 +one or more be positively charged ion, Cl -, COOH -, F -, PO 4 3-, HClO 4 -, HCO 3 -one or more be negatively charged ion (preferred NaH 2pO 4, KH 2pO 4, NaHClO 4, KHClO 4, NH 4cl, NH 4hClO 4one or more); Wherein organic solvent 5% ~ 70%(preferably 10% ~ 50% of accounting for moving phase cumulative volume).
The preparation method of Zorubicin of the present invention and derivative thereof, is characterized in that: the volume containing the sample of upper prop absorption is weight percentage 0.2% ~ 2.0% for reverse phase silica gel filler.
The preparation method of Zorubicin of the present invention and derivative thereof, it is characterized in that: when Fractional Collections according to UV detector monitoring peak-shaped curve Fractional Collections desorbed solution, collect point 3 large sections to complete (as shown in Figure 1), being respectively the ultraviolet curve 60% tack place, peak height place to peak that starts significantly to rise is first section, curve flat head section is second largest section, curve tack place is the third-largest section to the 60% peak height place of curve decline stage, each large section further segmentation segment collect, collect duration 5 seconds/sample ~ 120 second/sample.
The preparation method of Zorubicin of the present invention and derivative thereof, is characterized in that: Zorubicin and derivative crude product solution thereof adopt the absorption of single needle upper prop, resolve and carry out Fractional Collections; Or adopt the continuous sample introduction way of purification of spininess continuous mode upper prop, parsing in batches, and carry out Fractional Collections to often criticizing desorbed solution.
Zorubicin of the present invention and derivative thereof be Zorubicin, pirarubicin, epirubicin one or more.
The concrete steps of Zorubicin of the present invention and derivative preparation method thereof are as follows:
(1), using reverse phase silica gel as DAC preparative column material, with organic solvent and the slurrying of filler mixing and stirring, described filler is with SiO 2or derivatives thereof is matrix, the silica gel being Bonded Phase with C4 ~ C18 fat hydrocarbon chain, and its particle diameter is 5 μm ~ 50 μm (preferably 20 μm ~ 40 μm), and aperture is (preferably ) reverse phase silica gel filler, filler shape is irregular shape or spherical (preferably spherical);
Described filler is preferably with the silica gel that octyl group C8 or octadecyl C18 are Bonded Phase; Organic solvent for slurrying is the low molecule water-miscible organic solvent of 2mPas ~ 5mPas with the year of filler used and moving phase solution chemistry compatibility, and preferably the homologue of preparation mobile phase solvent is as slurrying solvent, as acetonitrile etc.Obtained its concentration of filler slurries is generally 50% ~ 80%(volume/volume, and namely containing packing volume in every 100mL slurries is 50mL ~ 80mL).
(2), by even good filler slurries load in DAC preparative column column casing, open packing column machine and carry out axial compression;
(3), with preparing pump moving phase to be pumped in DAC preparative column organic solvent in coupled columns and carry out displacement washing, balance preparative column, post effect theory of testing stage number is not less than 10000N/m simultaneously;
(4), by Zorubicin and derivative crude product solution thereof be injected to upper prop absorption in DAC preparative column by quantitative loop, the volume containing the sample of upper prop absorption is weight percentage 0.2% ~ 2.0% for reverse phase silica gel filler;
Zorubicin of the present invention and derivative thereof and derivative crude product solution thereof can directly adopt commercially available Zorubicin and derivative crude product thereof obtained after 33% acetonitrile-aqueous solution dissolved dilution, Zorubicin and derivative sample sample concentration thereof are 0.01% ~ 1.5%(mass/volume, represent in every 100mL solution containing sample 0.01g ~ 0.15g), preferably 0.05% ~ 0.1%.
(5), upper prop absorption terminates rear moving phase and resolves, and to desorbed solution Fractional Collections, and DAC preparative column is the preferred 0.10B.V./min ~ 0.20B.V./min of 0.02B.V./min ~ 0.50B.V./min(at the solution flow rate of balance, loading and parsing);
(6), by purity qualified samples undertaken merging, concentrated and dry, obtained Zorubicin and derivative finished product thereof;
Described moving phase is organic solvent and buffered saline solution mixed solvent, organic solvent is containing the low substituent of-the OH ,-CN of lower aliphatic hydrocarbon of 1 ~ 3 carbon atom, halogen atom, oxo, carbonyl or carboxylic group, derivative or its mixture, its viscosity≤5mPaS; Buffered saline solution adopts NaH 2pO 4, KH 2pO 4, NaHClO 4, KHClO 4, NH 4cl, NH 4hClO 4one or more; Wherein organic solvent account for moving phase total 10% ~ 50%.
Select suitably to fill column parameter according to wire feeding and specification, column pressure is generally 0.5MPa ~ 20MPa, post bed height is generally 20cm ~ 35cm to enter silica filler conventional dress.
As another kind of embodiment, described Zorubicin and derivative crude product solution thereof adopt the continuous sample introduction way of purification of continuous mode upper prop, parsing in batches, carry out Fractional Collections for often criticizing desorbed solution.
Accompanying drawing explanation
Fig. 1 Fractional Collections schematic diagram.
Fig. 2 product integration spectrogram.
Fig. 3 product integration spectrogram (enlarged view).
Embodiment
Embodiment 1
About 300g is in harmonious proportion particle diameter is 30 μm, aperture is anti-phase C8 spherical silica gel filler import in about 1000mL methyl alcohol, stir and make filler slurries, after removing filler fragment and fine powder, rapid dumps to internal diameter is in the DAC preparative column column casing of 500mm, setting dress column pressure is 20MPa, open dress base for post pneumatic piston rod and carry out axial compression, open DAC preparative column lower end valve simultaneously and methyl alcohol is discharged, reclaims.When Piston Compression is to setting pressure 20MPa, complete dress post, post bed height 250mm, post bed specification 500 × 250mm.The DAC preparative column installed is through the test of post effect, and theoretical plate number reaches 25338N/m.
Zorubicin (content 97.43%) 33% acetonitrile solution is dissolved, stand-by after dissolved dilution.
Open UV detector, setting determined wavelength is 245nm, with 2B.V. acetonitrile solution and 10% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate, the methyl alcohol in DAC preparative column is replaced, washs, balanced respectively successively, baseline zeroing after stable to UV detector monitoring curve level.B.V. in the present invention refers to Bed Volume, i.e. column volume.
Get the above-mentioned solution containing 1g Zorubicin crude product, by upper prop absorption in quantitative loop loading to DAC preparative column.Immediately with 10% ~ 35% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate linear gradient washing after end of the sample, Fractional Collections is carried out according to peak-shaped curve immediately by 30/ sample ~ 60/ sample per second after going out peak, flow velocity is 0.32B.V./min, and each collection liquid samples respectively and carries out HPLC detection.
Zorubicin main peak purity > 99.0%, single assorted < 0.10% will be detected and the collection liquid meeting medicine quality standard-required of total assorted < 1.0% merges, concentrated, desalination and drying, desalination and dryly adopt prior art through HPLC.The obtained Zorubicin 0.75g meeting current medicinal standard.Through inspection, finished product purity is 99.79%, and maximum list assorted 0.09%, always mix 0.21%, purification yield 73.12%, obtain product integration spectrogram and see Fig. 2,3, integration data is in table 1.
Table 1 product table of integral
Retention time Area % area
1 4.326 8746 0.04
2 9.483 3941 0.02
3 10.883 9428 0.05
4 12.224 20873195 99.79
5 14.837 10811 0.05
6 15.256 10997 0.05
Purity of the present invention is HPLC method detected value, and its concrete meaning and method of calculation all have explanation at " Chinese Pharmacopoeia " annex " high performance liquid chromatography " related Sections.Purity of the present invention specifically obtains according to " area normalization method " detection computations under pharmacopeia annex " high performance liquid chromatography " item, and content obtains according to external standard method detection computations.
Embodiment 2
About 330g is in harmonious proportion particle diameter is 40 μm, aperture is anti-phase C18 spherical silica gel filler import in about 1000mL methyl alcohol, stir and make filler slurries, after removing filler fragment and fine powder, rapid dumps to internal diameter is in the DAC preparative column column casing of 500mm, setting dress column pressure is 20MPa, open dress base for post pneumatic piston rod and carry out axial compression, open DAC simultaneously and account for preparative column lower end valve by methyl alcohol discharge, recovery.When Piston Compression is to setting pressure 20MPa, complete dress post, post bed height 250mm, post bed specification 500 × 250mm.The DAC preparative column installed is through the test of post effect, and theoretical plate number reaches 21220N/m.
Zorubicin (content 97.43%) 33% acetonitrile solution is dissolved, stand-by after dissolved dilution.
Open UV detector, setting determined wavelength is 245nm, with 2B.V. aqueous acetone solution and 20% acetone-ammoniumper chlorate/potassium dihydrogen phosphate, the methyl alcohol in DAC preparative column is replaced, washs, balanced respectively successively, baseline zeroing after stable to UV detector monitoring curve level.
Get the above-mentioned solution containing 1g Zorubicin crude product, by upper prop absorption in quantitative loop loading to DAC preparative column.Immediately with 10% ~ 35% acetone-ammoniumper chlorate/potassium dihydrogen phosphate linear gradient washing after end of the sample, Fractional Collections is carried out according to peak-shaped curve immediately by 30/ sample ~ 60/ sample per second after going out peak, flow velocity is 0.32B.V./min, and each collection liquid samples respectively and carries out HPLC detection.
Zorubicin main peak purity > 99.0%, single assorted < 0.10% will be detected and the collection liquid meeting medicine quality standard-required of total assorted < 1.0% merges, concentrated, desalination and drying, desalination and dryly adopt prior art through HPLC.The obtained Zorubicin 0.70g meeting current medicinal standard.Through inspection, finished product purity is 95.21%, and maximum list assorted 0.18%, always mixes 4.79%.
Embodiment 3
About 280g is in harmonious proportion particle diameter is 50 μm, aperture is anti-phase C4 spherical silica gel filler import in about 1000mL methyl alcohol, stir and make filler slurries, after removing filler fragment and fine powder, rapid dumps to internal diameter is in the DAC preparative column column casing of 500mm, setting dress column pressure is 20MPa, open dress base for post pneumatic piston rod and carry out axial compression, open DAC simultaneously and account for preparative column lower end valve by methyl alcohol discharge, recovery.When Piston Compression is to setting pressure 20MPa, complete dress post, post bed height 250mm, post bed specification 500 × 250mm.The DAC preparative column installed is through the test of post effect, and theoretical plate number reaches 28764N/m.
Zorubicin (content 97.43%) 33% acetonitrile solution is dissolved, stand-by after dissolved dilution.
Open UV detector, setting determined wavelength is 245nm, with 2B.V. aqueous acetone solution and 70% acetone-potassium primary phosphate/sodium dihydrogen phosphate, the methyl alcohol in DAC preparative column is replaced, washs, balanced respectively successively, baseline zeroing after stable to UV detector monitoring curve level.
Get the above-mentioned solution containing 1g Zorubicin, by upper prop absorption in quantitative loop loading to DAC preparative column.Immediately with 10% ~ 35% acetone-potassium primary phosphate/sodium dihydrogen phosphate linear gradient washing after end of the sample, Fractional Collections is carried out according to peak-shaped curve immediately by 30/ sample ~ 60/ sample per second after going out peak, flow velocity is 0.32B.V./min, and each collection liquid samples respectively and carries out HPLC detection.
Obtaining finished product purity is 90.75%, and maximum list assorted 0.18%, always mixes 0.79%.
Embodiment 4
The present embodiment is based on embodiment 1.After completing the preparation works such as dress post, Ultraviolet Detector balance and stability and zeroing, get the Zorubicin crude product solution containing being equivalent to silica filler weight 0.35% ~ 0.70% in DAC preparative column at every turn, adsorbed by quantitative loop loading, after end of the sample, use 50% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate with the linear gradient wash of the flow velocity of 0.32B.V./min, parsing immediately.After parsing is flowed out to Zorubicin peak, appropriate time carries out the second pin purifying upper prop absorption, and upper prop terminates to resolve immediately.So carry out sample " upper prop absorption-10% ~ 35% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate washing " the circulation continuous purification preparation of many batches.In continuous circulation sample introduction purge process, before and after sample introduction density, the parsing eluting peak of adjacent twice sample introduction completely separately and between neighboring samples, impurity peaks and main peak are not overlapping is advisable, after parsing sample goes out peak, the every 30 seconds/sample of amine ~ every 60 seconds/sample carries out Fractional Collections immediately, and each collection liquid samples respectively and carries out HPLC detection.Zorubicin main peak purity > 99.0%, single assorted < 0.10% will be detected through HPLC and the collection liquid meeting clinical application quality criteria requirements of total assorted < 1.0% merges, concentrated, desalination, drying, the Zorubicin of obtained medicinal standard.
This example adopts the test conditions identical with embodiment 1 based on embodiment 1(), continuous sample introduction three pin, every pin sample size is all about 1g containing Zorubicin, merges three pin purifying and resolves qualified sample, concentrates and dry, obtains Zorubicin finished product 2.1g.Through inspection, finished product purity is 99.64%, and maximum list assorted 0.07%, always mixes 0.36%, purification yield 70.03%.
Embodiment 5
About 300g is in harmonious proportion particle diameter is 30 μm, aperture is anti-phase C8 spherical silica gel filler import in about 1000mL methyl alcohol, stir and make filler slurries, after removing filler fragment and fine powder, rapid dumps to internal diameter is in the DAC preparative column column casing of 500mm, setting dress column pressure is 20MPa, open dress base for post pneumatic piston rod and carry out axial compression, open DAC simultaneously and account for preparative column lower end valve by methyl alcohol discharge, recovery.When Piston Compression is to setting pressure 20MPa, complete dress post, post bed height 250mm, post bed specification 500 × 250mm.The DAC preparative column installed is through the test of post effect, and theoretical plate number reaches 28997N/m.
Pirarubicin (content 95.08%) 33% acetonitrile solution is dissolved, stand-by after dissolved dilution.
Open UV detector, setting determined wavelength is 245nm, with 2B.V. acetonitrile solution and 10% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate, the methyl alcohol in DAC preparative column is replaced, washs, balanced respectively successively, baseline zeroing after stable to UV detector monitoring curve level.
Get the above-mentioned solution containing 0.7g pirarubicin crude product, by upper prop absorption in quantitative loop loading to DAC preparative column.Immediately with 10% ~ 35% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate linear gradient washing after end of the sample, Fractional Collections is carried out according to peak-shaped curve immediately by 30/ sample ~ 60/ sample per second after going out peak, flow velocity is 0.32B.V./min, and each collection liquid samples respectively and carries out HPLC detection.
Pirarubicin main peak purity > 99.0%, single assorted < 0.10% will be detected and the collection liquid meeting medicine quality standard-required of total assorted < 1.0% merges, concentrated, desalination and drying, desalination and dryly adopt prior art through HPLC.The obtained pirarubicin 0.48g meeting current medicinal standard.Through inspection, finished product purity is 98.93%, and maximum list assorted 0.07%, always mixes 1.09%, purification yield 69.75%.
Purity of the present invention is HPLC method detected value, and its concrete meaning and method of calculation all have explanation at " Chinese Pharmacopoeia " annex " high performance liquid chromatography " related Sections.Purity of the present invention specifically obtains according to " area normalization method " detection computations under pharmacopeia annex " high performance liquid chromatography " item, and content obtains according to external standard method detection computations.
Embodiment 6
About 330g is in harmonious proportion particle diameter is 40 μm, aperture is anti-phase C18 spherical silica gel filler import in about 1000mL methyl alcohol, stir and make filler slurries, after removing filler fragment and fine powder, rapid dumps to internal diameter is in the DAC preparative column column casing of 500mm, setting dress column pressure is 20MPa, open dress base for post pneumatic piston rod and carry out axial compression, open DAC simultaneously and account for preparative column lower end valve by methyl alcohol discharge, recovery.When Piston Compression is to setting pressure 20MPa, complete dress post, post bed height 250mm, post bed specification 500 × 250mm.The DAC preparative column installed is through the test of post effect, and theoretical plate number reaches 28768N/m.
Pirarubicin (content 95.08%) 33% acetonitrile solution is dissolved, stand-by after dissolved dilution.
Open UV detector, setting determined wavelength is 245nm, with 2B.V. aqueous acetone solution and 10% acetone-ammoniumper chlorate/potassium dihydrogen phosphate, the methyl alcohol in DAC preparative column is replaced, washs, balanced respectively successively, baseline zeroing after stable to UV detector monitoring curve level.
Get the above-mentioned solution containing 0.7g pirarubicin crude product, by upper prop absorption in quantitative loop loading to DAC preparative column.Immediately with 70% acetone-ammoniumper chlorate/potassium dihydrogen phosphate linear gradient washing after end of the sample, Fractional Collections is carried out according to peak-shaped curve immediately by 30/ sample ~ 60/ sample per second after going out peak, flow velocity is 0.32B.V./min, and each collection liquid samples respectively and carries out HPLC detection.
Pirarubicin main peak purity > 99.0%, single assorted < 0.10% will be detected and the collection liquid meeting medicine quality standard-required of total assorted < 1.0% merges, concentrated, desalination and drying, desalination and dryly adopt prior art through HPLC.The obtained pirarubicin 0.43g meeting current medicinal standard.Through inspection, finished product purity is 98.04%, and maximum list assorted 0.25%, always mixes 1.96%, purification yield 60.37%.
Embodiment 7
About 280g is in harmonious proportion particle diameter is 50 μm, aperture is anti-phase C4 spherical silica gel filler import in about 1000mL methyl alcohol, stir and make filler slurries, after removing filler fragment and fine powder, rapid dumps to internal diameter is in the DAC preparative column column casing of 500mm, setting dress column pressure is 20MPa, open dress base for post pneumatic piston rod and carry out axial compression, open DAC simultaneously and account for preparative column lower end valve by methyl alcohol discharge, recovery.When Piston Compression is to setting pressure 20MPa, complete dress post, post bed height 250mm, post bed specification 500 × 250mm.The DAC preparative column installed is through the test of post effect, and theoretical plate number reaches 21324N/m.
Pirarubicin (content 95.08%%) 33% acetonitrile solution is dissolved, stand-by after dissolved dilution.
Open UV detector, setting determined wavelength is 245nm, with 2B.V. aqueous acetone solution and 10% acetone-potassium primary phosphate/sodium dihydrogen phosphate, the methyl alcohol in DAC preparative column is replaced, washs, balanced respectively successively, baseline zeroing after stable to UV detector monitoring curve level.
Get the above-mentioned solution containing 1g pirarubicin, by upper prop absorption in quantitative loop loading to DAC preparative column.Immediately with 5% acetone-potassium primary phosphate/sodium dihydrogen phosphate linear gradient washing after end of the sample, Fractional Collections is carried out according to peak-shaped curve immediately by 30/ sample ~ 60/ sample per second after going out peak, flow velocity is 0.32B.V./min, and each collection liquid samples respectively and carries out HPLC detection.
Obtaining finished product purity is 90.75%, and maximum list assorted 6.37%, always mixes 9.25%.
Embodiment 8
The present embodiment is based on embodiment 5.After completing the preparation works such as dress post, Ultraviolet Detector balance and stability and zeroing, get the pirarubicin crude product solution containing being equivalent to silica filler weight 0.35% ~ 0.70% in DAC preparative column at every turn, adsorbed by quantitative loop loading, after end of the sample, use 10% ~ 35% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate with the linear gradient wash of the flow velocity of 0.32B.V./min, parsing immediately.After parsing is flowed out to pirarubicin peak, appropriate time carries out the second pin purifying upper prop absorption, and upper prop terminates to resolve immediately.So carry out sample " upper prop absorption-10% ~ 35% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate washing " the circulation continuous purification preparation of many batches.In continuous circulation sample introduction purge process, before and after sample introduction density, the parsing eluting peak of adjacent twice sample introduction completely separately and between neighboring samples, impurity peaks and main peak are not overlapping is advisable, after parsing sample goes out peak, the every 30 seconds/sample of amine ~ every 60 seconds/sample carries out Fractional Collections immediately, and each collection liquid samples respectively and carries out HPLC detection.Pirarubicin main peak purity > 99.0%, single assorted < 0.10% will be detected through HPLC and the collection liquid meeting clinical application quality criteria requirements of total assorted < 1.0% merges, concentrated, desalination, drying, the pirarubicin of obtained medicinal standard.
This example adopts the test conditions identical with embodiment 5 based on embodiment 5(), continuous sample introduction three pin, every pin sample size is all about 0.7g containing pirarubicin, merges three pin purifying and resolves qualified sample, concentrates and dry, obtains pirarubicin finished product 1.3g.Through inspection, finished product purity is 99.33%, and maximum list assorted 0.09%, always mixes 0.67%, purification yield 72.35%.
Embodiment 9
About 300g is in harmonious proportion particle diameter is 30 μm, aperture is anti-phase C8 spherical silica gel filler import in about 1000mL methyl alcohol, stir and make filler slurries, after removing filler fragment and fine powder, rapid dumps to internal diameter is in the DAC preparative column column casing of 500mm, setting dress column pressure is 20MPa, open dress base for post pneumatic piston rod and carry out axial compression, open DAC simultaneously and account for preparative column lower end valve by methyl alcohol discharge, recovery.When Piston Compression is to setting pressure 20MPa, complete dress post, post bed height 250mm, post bed specification 500 × 250mm.The DAC preparative column installed is through the test of post effect, and theoretical plate number reaches 15338N/m.
Epirubicin (content 90.37%) 33% acetonitrile solution is dissolved, stand-by after dissolved dilution.
Open UV detector, setting determined wavelength is 245nm, with 2B.V. acetonitrile solution and 10% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate, the methyl alcohol in DAC preparative column is replaced, washs, balanced respectively successively, baseline zeroing after stable to UV detector monitoring curve level.
Get containing the soft above-mentioned solution than star crude product of 1g epirubicin, by upper prop absorption in quantitative loop loading to DAC preparative column.Immediately with 10% ~ 35% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate linear gradient washing after end of the sample, Fractional Collections is carried out according to peak-shaped curve immediately by 30/ sample ~ 60/ sample per second after going out peak, flow velocity is 0.32B.V./min, and each collection liquid samples respectively and carries out HPLC detection.
Epirubicin main peak purity > 99.0%, single assorted < 0.10% will be detected and the collection liquid meeting medicine quality standard-required of total assorted < 1.0% merges, concentrated, desalination and drying, desalination and dryly adopt prior art through HPLC.The obtained epirubicin 0.72g meeting current medicinal standard.Through inspection, finished product purity is 99.93%, and maximum list assorted 0.03%, always mixes 0.07%, purification yield 73.57%, and this method is applicable to the separation of this sample very much.
Purity of the present invention is HPLC method detected value, and its concrete meaning and method of calculation all have explanation at " Chinese Pharmacopoeia " annex " high performance liquid chromatography " related Sections.Purity of the present invention specifically obtains according to " area normalization method " detection computations under pharmacopeia annex " high performance liquid chromatography " item, and content obtains according to external standard method detection computations.
Embodiment 10
About 330g is in harmonious proportion particle diameter is 40 μm, aperture is anti-phase C18 spherical silica gel filler import in about 1000mL methyl alcohol, stir and make filler slurries, after removing filler fragment and fine powder, rapid dumps to internal diameter is in the DAC preparative column column casing of 500mm, setting dress column pressure is 20MPa, open dress base for post pneumatic piston rod and carry out axial compression, open DAC simultaneously and account for preparative column lower end valve by methyl alcohol discharge, recovery.When Piston Compression is to setting pressure 20MPa, complete dress post, post bed height 250mm, post bed specification 500 × 250mm.The DAC preparative column installed is through the test of post effect, and theoretical plate number reaches 11220N/m.
Epirubicin (content 90.37%) 33% acetonitrile solution is dissolved, stand-by after dissolved dilution.
Open UV detector, setting determined wavelength is 245nm, with 2B.V. aqueous acetone solution and 10% acetone-ammoniumper chlorate/potassium dihydrogen phosphate, the methyl alcohol in DAC preparative column is replaced, washs, balanced respectively successively, baseline zeroing after stable to UV detector monitoring curve level.
Get the above-mentioned solution containing 1g epirubicin crude product, by upper prop absorption in quantitative loop loading to DAC preparative column.Immediately with 10% ~ 35% acetone-ammoniumper chlorate/potassium dihydrogen phosphate linear gradient washing after end of the sample, Fractional Collections is carried out according to peak-shaped curve immediately by 30/ sample ~ 60/ sample per second after going out peak, flow velocity is 0.32B.V./min, and each collection liquid samples respectively and carries out HPLC detection.
Epirubicin main peak purity > 99.0%, single assorted < 0.10% will be detected and the collection liquid meeting medicine quality standard-required of total assorted < 1.0% merges, concentrated, desalination and drying, desalination and dryly adopt prior art through HPLC.The obtained epirubicin 0.43g meeting current medicinal standard.Through inspection, finished product purity is 98.97%, and maximum list assorted 0.13%, always mixes 1.03%, purification yield 65.24%.
Embodiment 11
About 280g is in harmonious proportion particle diameter is 50 μm, aperture is anti-phase C4 spherical silica gel filler import in about 1000mL methyl alcohol, stir and make filler slurries, after removing filler fragment and fine powder, rapid dumps to internal diameter is in the DAC preparative column column casing of 500mm, setting dress column pressure is 20MPa, open dress base for post pneumatic piston rod and carry out axial compression, open DAC simultaneously and account for preparative column lower end valve by methyl alcohol discharge, recovery.When Piston Compression is to setting pressure 20MPa, complete dress post, post bed height 250mm, post bed specification 500 × 250mm.The DAC preparative column installed is through the test of post effect, and theoretical plate number reaches 18764N/m.
Epirubicin (content 90.37%) 33% acetonitrile solution is dissolved, stand-by after dissolved dilution.
Open UV detector, setting determined wavelength is 245nm, with 2B.V. aqueous acetone solution and 10% acetone-potassium primary phosphate/sodium dihydrogen phosphate, the methyl alcohol in DAC preparative column is replaced, washs, balanced respectively successively, baseline zeroing after stable to UV detector monitoring curve level.
Get the above-mentioned solution containing 1g epirubicin, by upper prop absorption in quantitative loop loading to DAC preparative column.Immediately with 65% acetone-potassium primary phosphate/sodium dihydrogen phosphate linear gradient washing after end of the sample, Fractional Collections is carried out according to peak-shaped curve immediately by 30/ sample ~ 60/ sample per second after going out peak, flow velocity is 0.32B.V./min, and each collection liquid samples respectively and carries out HPLC detection.
Obtaining finished product purity is 97.64%, and maximum list assorted 1.08%, always mixes 2.36%, purification yield 60.45%
Embodiment 12
The present embodiment is based on embodiment 9.After completing the preparation works such as dress post, Ultraviolet Detector balance and stability and zeroing, get the epirubicin crude product solution containing being equivalent to silica filler weight 0.35% ~ 0.70% in DAC preparative column at every turn, adsorbed by quantitative loop loading, after end of the sample, use 10% ~ 35% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate with the linear gradient wash of the flow velocity of 0.32B.V./min, parsing immediately.After parsing is flowed out to epirubicin peak, appropriate time carries out the second pin purifying upper prop absorption, and upper prop terminates to resolve immediately.So carry out sample " upper prop absorption-10% ~ 35% acetonitrile-sodium perchlorate/sodium dihydrogen phosphate washing " the circulation continuous purification preparation of many batches.In continuous circulation sample introduction purge process, before and after sample introduction density, the parsing eluting peak of adjacent twice sample introduction completely separately and between neighboring samples, impurity peaks and main peak are not overlapping is advisable, after parsing sample goes out peak, the every 30 seconds/sample of amine ~ every 60 seconds/sample carries out Fractional Collections immediately, and each collection liquid samples respectively and carries out HPLC detection.Pirarubicin main peak purity > 99.0%, single assorted < 0.10% will be detected through HPLC and the collection liquid meeting clinical application quality criteria requirements of total assorted < 1.0% merges, concentrated, desalination, drying, the pirarubicin of obtained medicinal standard.
This example adopts the test conditions identical with embodiment 9 based on embodiment 9(), continuous sample introduction three pin, every pin sample size is all about 1g containing epirubicin, merges three pin purifying and resolves qualified sample, concentrates and dry, obtains epirubicin finished product 2.2g.Through inspection, finished product purity is 99.72%, and maximum list assorted 0.07%, always mixes 0.28%, purification yield 71.56%.
This example adopts continuous sample introduction purification Zorubicin and derivative thereof, and under ensureing that purification yield is in high-caliber situation, the production cycle foreshortens on average often criticizes about 60 minutes, and solvent consumption amount reduces, and has considerable application prospect.
Above-described embodiment, only for technical conceive of the present invention and feature are described, its object is to person skilled in the art can be understood content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalences done according to spirit of the present invention change or modify, and all should be encompassed within protection scope of the present invention.

Claims (10)

1. a preparation method for Zorubicin and derivative thereof, is characterized in that: separating mechanism is rp mode, is with an organic solvent separated with buffering saline solvent system.
2., according to the preparation method of Zorubicin described in claim 1 and derivative thereof, it is characterized in that: filler used is with SiO 2or derivatives thereof is matrix, the silica gel being Bonded Phase with C8 ~ C18 fat hydrocarbon chain, and its particle diameter is 5 μm ~ 50 μm, and aperture is reverse phase silica gel filler, filler shape is irregular shape or spherical.
3. according to the preparation method of Zorubicin described in claim 1 and derivative thereof, it is characterized in that: moving phase used is organic solvent and buffered saline solution mixed solvent, wherein organic solvent is containing the low substituent of the groups such as-the OH ,-CN of lower aliphatic hydrocarbon of 1 ~ 3 carbon atom, halogen atom, oxo, carbonyl or carboxyl, derivative or its mixture, its viscosity≤5mPaS; Buffered saline solution adopts Na +, K +, H +, NH 4 +one or more be positively charged ion, Cl -, COOH -, F -, PO 4 3-, HClO 4 -, HCO 3 -one or more be negatively charged ion; Wherein organic solvent accounts for 5% ~ 70% of moving phase cumulative volume.
4. according to the preparation method of Zorubicin described in claim 2 and derivative thereof, it is characterized in that: the volume containing the sample of upper prop absorption is weight percentage 0.2% ~ 2.0% for reverse phase silica gel filler.
5., according to the preparation method of Zorubicin described in claim 3 and derivative thereof, it is characterized in that: in moving phase, buffered saline solution is NaH 2pO 4, KH 2pO 4, NaHClO 4, KHClO 4, NH 4cl, NH 4hClO 4one or more.
6., according to the preparation method of Zorubicin described in claim 3 and derivative thereof, it is characterized in that: organic solvent accounts for 10% ~ 50% of moving phase cumulative volume.
7. according to the preparation method of Zorubicin described in claim 1 and derivative thereof, it is characterized in that: when Fractional Collections according to UV detector monitoring peak-shaped curve Fractional Collections desorbed solution, collect point 3 large sections to complete, being respectively the ultraviolet curve 60% tack place, peak height place to peak that starts significantly to rise is first section, curve flat head section is second largest section, curve tack place is the third-largest section to the 60% peak height place of curve decline stage, each large section further segmentation segment collect, collect duration 5 seconds/sample ~ 120 second/sample.
8. according to the preparation method of Zorubicin described in claim 1 and derivative thereof, it is characterized in that: Zorubicin and derivative crude product solution thereof adopt the absorption of single needle upper prop, resolve and carry out Fractional Collections; Or adopt the continuous sample introduction way of purification of spininess continuous mode upper prop, parsing in batches, and carry out Fractional Collections to often criticizing desorbed solution.
9., according to the preparation method of Zorubicin described in claim 1 and derivative thereof, it is characterized in that: described Zorubicin and derivative thereof be Zorubicin, pirarubicin, epirubicin one or more.
10., according to the preparation method of Zorubicin described in claim 1 and derivative thereof, it is characterized in that, concrete steps are as follows:
(1), using reverse phase silica gel as DAC preparative column material, with organic solvent and the slurrying of filler mixing and stirring, described filler is with SiO 2or derivatives thereof is matrix, the silica gel being Bonded Phase with C4 ~ C18 fat hydrocarbon chain, and its particle diameter is 5 μm ~ 50 μm, and aperture is reverse phase silica gel filler, filler shape is irregular shape or spherical;
(2), by even good filler slurries load in DAC preparative column column casing, open packing column machine and carry out axial compression;
(3), with preparing pump moving phase to be pumped in DAC preparative column organic solvent in coupled columns and carry out displacement washing, balance preparative column, post effect theory of testing stage number is not less than 10000N/m simultaneously;
(4), by Zorubicin and derivative crude product solution thereof be injected to upper prop absorption in DAC preparative column by quantitative loop, the volume containing the sample of upper prop absorption is weight percentage 0.2% ~ 2.0% for reverse phase silica gel filler;
(5), upper prop absorption terminates rear moving phase and resolves, and to desorbed solution Fractional Collections, and DAC preparative column is 0.02B.V./min ~ 0.50B.V./min at the solution flow rate of balance, loading and parsing;
(6), by purity qualified samples undertaken merging, concentrated and dry, obtained Zorubicin and derivative finished product thereof;
Described moving phase is organic solvent and buffered saline solution mixed solvent, organic solvent is containing the low substituent of-the OH ,-CN of lower aliphatic hydrocarbon of 1 ~ 3 carbon atom, halogen atom, oxo, carbonyl or carboxylic group, derivative or its mixture, its viscosity≤5mPaS; Buffered saline solution adopts NaH 2pO 4, KH 2pO 4, NaHClO 4, KHClO 4, NH 4cl, NH 4hClO 4one or more; Wherein organic solvent account for moving phase total 10% ~ 50%.
CN201410012304.XA 2014-01-10 2014-01-10 Preparation method for doxorubicin and derivative thereof Pending CN104774229A (en)

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