CN103265624B - Method for preparing copaxone - Google Patents
Method for preparing copaxone Download PDFInfo
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- CN103265624B CN103265624B CN201310200501.XA CN201310200501A CN103265624B CN 103265624 B CN103265624 B CN 103265624B CN 201310200501 A CN201310200501 A CN 201310200501A CN 103265624 B CN103265624 B CN 103265624B
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Abstract
The invention belongs to the technical field of polypeptide medicament preparation, and in particular relates to a method for preparing copaxone, aiming to solve the technical problems that the operation is complex, the productivity is low, and the production cost is high in an existing method. According to the method, the technical scheme for solving the technical problems is that the method for preparing copaxone comprises the following steps of: carrying out copolymerization on L-alanine N-carboxy anhydride, protected L-glutamyl Gamma-benzyl ester N-carboxy anhydride, protected L-lysine N-carboxy anhydride and protected tyrosine N-carboxy anhydride under base catalysis, thereby obtaining a protected copolymer; carrying out acidolysis or hydrogenolysis on the protected copolymer, thereby obtaining a copaxone crude product; and purifying the copaxone crude product, thereby obtaining a copaxone product. According to the method provided by the invention, is simple to the operation is simple, the yield is high, an application prospect and economical value are wide. The invention provides a novel high-yield method for preparing copaxone.
Description
Technical field
The invention belongs to polypeptide drugs preparation method technical field, particularly lattice draw the preparation method for thunder.
Background technology
Ge La has following structure for thunder:
(Glu,Ala,Lys,Tyr)x·xCH
3COOH
Ge La is a kind of peptides of synthesis for thunder (Glatiramer), by Israel's pharmaceutical factory manufacture that TEVA researches and develops, and trade(brand)name
, obtained U.S. FDA approval what in 1996 and treat multiple sclerosis, medicine freezes crystalline flour end for white to linen sterilizing, packs another attached water for injection to be modulated into the use of injection solution.
In the western countries with more multiple sclerosis patients,
curative effect and tolerance all obtain pure affirmative.Except much research and clinical experience confirm that it can lower the recurrence rate of patient significantly, improve the patient's condition of anergy, reduce outside brain injury, the incidence of side effects of Copaxone is few, helps the tolerance that what promotes patient.In addition, correlative study also shows
more can reduce recurrence rate significantly than Interferon, rabbit, prevent neural further damage and worsen, and there is better tolerance.In the effectiveness study of other life-time service (6 years to the longest 12 years not etc.),
also confirm the deterioration that can delay the course of disease in its neuroscience of patient constantly or the patient's condition allowing patient remain stable, reduce in brain and damage and prevent neurone from wrecking in a large number eventually to the deterioration situation of loss of function.
the main immunization schedule by regulating and controlling in multiple sclerosis patients body is to reach the object for the treatment of.Enter after in body,
regulatory factor in immunity system is combined and enters nervus centralis, reverses its autoimmunity process of multiple sclerosis patients through a series of immunization; That is stimulate " being harmful to " or front inflammatory T cell to be converted to the T cell of " useful " or anti-inflammation.
also directly can reduce inflammation situation in direct effect what neural cell injury position.These benefits all help what and reduce multiple sclerosis patients its recurrence rate, and effectively reduce it and further damage.
Clinical existing much research confirms
effectively can reduce its recurrence frequency of relapsive sclerosis patients.These researchs comprise the recurrence situation after monitored patient dispensing nearly, the assessment that attendant symptoms improves, and the damage location shown by MRI (magnetic resonance imaging) improves situation.Also have research to safeguard that the impact of patient's physiological function is assessed for Copaxone to what in addition, result shows
taking in sail property lesion development is permanent nerve damage, recovers axon function and neuronic integrity, that is
prevent the deterioration of further infringement and the patient's condition in patient's nervus centralis.
Acetic acid copaxone (
) be made up of 4 seed amino acids: L-glutamic acid (Pidolidone), L-alanine(L-L-Ala), L-lysine(L-Methionin) and L-tyrosine(L-TYR), various amino acid whose ratio is in table 1, and molecular-weight average is about 4000 ~ 7000daltons.
Both at home and abroad draw the report for thunder preparation report a lot of about lattice, as United States Patent (USP) 3849550,5800808 and No. 2006/0172942nd, U.S. Patent Publication case, No. 2006/0154862, No. 2007/0141663 etc.Preparation method involved by these patents prepares with the copolymerization of protection 1B N-carboxylic acid the multipolymer protected based on the L-paddy ammonia Γ-benzyl fat N-carboxylic acid of tyrosine N-carboxylic acid, ALANINE N-carboxylic acid, protection.Wherein have employed two steps and go protection: the benzyl protecting group first being removed the L-paddy ammonia Γ-benzyl fat N-carboxylic acid γ position carboxyl of protection by acidolysis or hydrogenolysis; Again by the trifluoroacetyl group protecting group of the N-carboxylic acid of the 1B as cracking removal protections such as piperidines alkali.
In the preparation patent reported, the phenolic hydroxyl group in the N-carboxylic acid of tyrosine is not protected, and phenolic hydroxyl group is easily oxidized in the course of the polymerization process; thus increase the generation of by product, and have employed the operation that two steps go protecting group simultaneously, make complex operation; productive rate is low, and production cost is high.
Summary of the invention
The technical problem to be solved in the present invention is existing method complex operation, and productive rate is low, and production cost is high.
The technical scheme that the present invention solves the problems of the technologies described above is to provide a kind of lattice and draws preparation method for thunder, comprises the following steps:
A, L-paddy ammonia γ-benzyl fat N-carboxylic acid with ALANINE N-carboxylic acid, protection, the 1B N-carboxylic acid of protection and tyrosine N-carboxylic acid copolymerization under base catalysis of protection, prepare the multipolymer of protection;
The multipolymer of b, above-mentioned protection, again through acidolysis or hydrogenolysis, obtains lattice and draws for thunder crude product;
C, lattice draw for thunder crude product the more purified lattice that obtain draw for thunder product.
Above-mentioned lattice draw in the preparation method for thunder, and the reaction consumption mol ratio of the L-paddy ammonia γ-benzyl fat N-carboxylic acid of the ALANINE N-carboxylic acid described in step a, protection, the 1B N-carboxylic acid of protection and the tyrosine N-carboxylic acid of protection is respectively: 0.90 ~ 1.10 ︰ 0.30 ~ 0.36 ︰ 0.75 ~ 0.85 ︰ 0.20 ~ 0.24.
Above-mentioned lattice draw in the preparation method for thunder, and the time of the polyreaction described in step a is 10 ~ 30 hours.
Above-mentioned lattice draw in the preparation method for thunder, and the structure of the tyrosine N-carboxylic acid of described protection is:
Wherein R
1for benzyl or be 2-bromo-benzyloxy carbonyl acyl group (2-Br-Z).
Above-mentioned lattice draw in the preparation method for thunder, and the structure of described ALANINE N-carboxylic acid is:
Above-mentioned lattice draw in the preparation method for thunder, and the structure of the L-paddy ammonia γ-benzyl fat N-carboxylic acid of described protection is:
Wherein R
2for benzyl.
Above-mentioned lattice draw in the preparation method for thunder, and the structure of the 1B N-carboxylic acid of described protection is:
Wherein R
3for benzyloxy carbonyl acyl group (Z), 2-chlorine benzyloxy carbonyl acyl group (2-Cl-Z) or 2-bromo-benzyloxy carbonyl acyl group (2-Br-Z).
Above-mentioned lattice draw in the preparation method for thunder, and the alkali described in step a is diethylamine or triethylamine.
Above-mentioned lattice draw in the preparation method for thunder, and the consumption of alkali described in step a is 0.5 ~ 2.0% of protected amino acid gross weight used.
Above-mentioned lattice draw in the preparation method for thunder, and the multipolymer of the protection described in step b obtains lattice through acidolysis and draws for thunder crude product.
Further, above-mentioned lattice draw in the preparation method for thunder, the acidolysis described in step b, and its acidolysis agent is hydrogen bromide trifluoracetic acid (TFA) solution; Wherein, the concentration of hydrogen bromide is 5 ~ 10%wt, and preferred concentration is 6 ~ 7%wt.
Further, the consumption of described acidolysis agent is multipolymer needs 5 ~ 15mL acidolysis agent of every gram of protection.Preferably, the consumption of acidolysis agent is multipolymer needs 8 ~ 11mL acidolysis agent of every gram of protection.
Further, the time of described acidolysis is 1 ~ 6 hour, preferably 3 ~ 4 hours.
Preferably, above-mentioned lattice draw in the preparation method for thunder, and the multipolymer hydrogenolysis of the protection described in step b obtains lattice and draws for thunder crude product.
Further, above-mentioned lattice draw in the preparation method for thunder, the hydrogenolysis described in step b, adopt normal pressure hydrogenolysis or pressurization hydrogenolysis; Wherein, hydrogenolysis catalyst is the Pd/C(palladium/carbon of 5% ~ 15%wt), be preferably the Pd/C of 10%wt.
Further, above-mentioned lattice draw in the preparation method for thunder, the hydrogenolysis described in step b, and the lattice of every gram of protection draw and need hydrogenolysis catalyst 0.05 ~ 0.2g for thunder multipolymer.
Above-mentioned lattice draw in the preparation method for thunder, and the purifying described in step c is ultrafiltration purification.
Lattice provided by the invention draw the preparation method for thunder, adopt the amino acid N-carboxylic acid of side chain full guard to prepare lattice and draw for thunder, only through one-step polymerization reaction and a step protective reaction, can prepare lattice and draw for thunder.Method technological operation provided by the invention is simple, and yield is high, has a wide range of applications future and economic worth.
Embodiment
Ge La, for the preparation method of thunder, comprises the following steps:
A, the tyrosine N-carboxylic acid using protection, ALANINE N-carboxylic acid, the L-paddy ammonia γ-benzyl fat N-carboxylic acid of protection and 1B N-carboxylic acid copolymerization under base catalysis of protection, prepare the multipolymer of protection.Wherein, the reaction consumption mol ratio of the L-paddy ammonia γ-benzyl fat N-carboxylic acid of ALANINE N-carboxylic acid, protection, the 1B N-carboxylic acid of protection and the tyrosine N-carboxylic acid of protection is respectively: 0.90 ~ 1.10 ︰ 0.30 ~ 0.36 ︰ 0.75 ~ 0.85 ︰ 0.20 ~ 0.24, the time of described polyreaction is 10 ~ 30 hours.
The multipolymer of b, above-mentioned protection, again through acidolysis or hydrogenolysis, obtains lattice and draws for thunder crude product;
C, lattice draw and obtain lattice through ultrafiltration purification again for thunder crude product and draw for thunder product.
Above-mentioned lattice draw in the preparation method for thunder, and the alkali described in step a is diethylamine or triethylamine, and the consumption of alkali is 0.5 ~ 2.0% of protected amino acid gross weight used.
Above-mentioned lattice draw in the preparation method for thunder, when the lattice of the protection described in step b draw for thunder multipolymer adopt acid hydrolysis method obtain lattice draw for thunder crude product time, its acidolysis agent is hydrogen bromide trifluoracetic acid (TFA) solution; Wherein, the concentration of hydrogen bromide is 5 ~ 10%wt, and preferred concentration is 6 ~ 7%wt.The consumption of described acidolysis agent is that every gram of lattice protected draw for thunder multipolymer needs 5 ~ 15mL acidolysis agent.Preferably, the consumption of acidolysis agent is that every gram of lattice protected draw for thunder multipolymer needs 8 ~ 11mL acidolysis agent.The time of described acidolysis is 1 ~ 6 hour, preferably 3 ~ 4 hours.
Above-mentioned lattice draw in the preparation method for thunder, when the lattice of the protection described in step b draw for thunder multipolymer adopt hydrogenolysis mode obtain lattice draw for thunder crude product time, be by normal pressure hydrogenolysis or pressurization hydrogenolysis; Wherein, hydrogenolysis catalyst is the Pd/C(palladium/carbon of 5% ~ 15%wt), be preferably the Pd/C of 10%wt.The amount ranges of hydrogenolysis catalyst is that every gram of lattice protected draw for thunder multipolymer needs 0.05 ~ 0.2g hydrogenolysis catalyst.
The preparation of the multipolymer that embodiment 1 is protected
By the N-carboxylic acid (11.3g of ALANINE; 98.18mmol), the N-carboxylic acid (8.7g of Pidolidone γ-benzyl fat; 33.05mmol), the N-carboxylic acid (23.5g of N-benzyloxy carbonyl acyl-lysine; 76.72mmol) be placed in single neck flask with magnetic stirrer with the N-carboxylic acid (6.5g, 21.86mmol) of TYR.This mixture is dissolved by adding anhydrous DMF (DMF) (400mL).Add distillation diethylamine (0.94mL).At room temperature gained mixture is stirred 24 hours.Pour in the water of 1000mL by above-mentioned reaction solution, stir 30 minutes, solid collected by filtration, drying under reduced pressure, obtain the multipolymer of 43.3g protection, yield is 86.6%.
Embodiment 2 acidolysis is prepared lattice and is drawn for thunder crude product
The copolymer 1 0g that Example 1 is obtained, be dissolved in the HBr/TFA of 100mL8%, stirring reaction 3 hours, underpressure distillation, residue ether sedimentation, solid collected by filtration, get Ge La is for thunder crude product.
Embodiment 3 hydrogenolysis is prepared lattice and is drawn for thunder crude product
In autoclave, first add 100mL tetrahydrofuran (THF), add multipolymer, 1mL Glacial acetic acid that Pd/C, 20mL water of 1g10%, 10g embodiment 1 are obtained under stirring, pass into H
2, hydrogenolysis pressure is 0.8 ~ 0.95Mpa, and hydrogenolysis temperature is 40 ~ 50 DEG C, reaction 20h.React complete, pressurization is concentrated, residue ether sedimentation, solid collected by filtration, and get Ge La is for thunder crude product.
Embodiment 4 lattice draw the purifying for thunder crude product
The obtained lattice of Example 2 draw for thunder crude product, dissolve with the sodium carbonate solution of 1%wt, and with the sodium carbonate solution adjust pH 8 of 1%wt, and are diluted to 10mg/mL lattice and draw solution for thunder.Use 3000 dalton's films to carry out ultrafiltration, remove low-molecular-weight impurity, when ultrafiltration is to original volume 50%, add to original volume with water, add 4 times altogether, gained solution is carried out concentrating under reduced pressure and lyophilize, obtain 7.2g white sponge solid, yield is 72%.
Above-described embodiment shows, lattice provided by the invention draw the preparation method for thunder, and technological operation is simple, and yield is high, has a wide range of applications future and economic worth.
Claims (1)
1. lattice draw the preparation method for thunder, it is characterized in that: the method comprises the following steps:
The preparation of the multipolymer of a, protection: the N-carboxylic acid 23.5g of N-carboxylic acid 8.7g, N-benzyloxy carbonyl acyl-lysine of the N-carboxylic acid 11.3g of ALANINE, Pidolidone γ-benzyl fat and the N-carboxylic acid 6.5g of TYR is placed in single neck flask with magnetic stirrer, this mixture is dissolved by adding anhydrous DMF 400mL; Add distillation diethylamine 0.94mL, at room temperature gained mixture is stirred 24 hours; Above-mentioned reaction solution is poured in the water of 1000mL, stirs 30 minutes, solid collected by filtration, drying under reduced pressure, obtain the multipolymer of 43.3g protection;
B, acidolysis are prepared lattice and are drawn for thunder crude product: get the copolymer 1 0g that step a is obtained, be dissolved in the HBr/TFA of 100mL 8%, stirring reaction 3 hours, underpressure distillation, residue ether sedimentation, solid collected by filtration, and get Ge La is for thunder crude product;
Or hydrogenolysis is prepared lattice and drawn for thunder crude product: first add 100mL tetrahydrofuran (THF) in autoclave, add multipolymer, 1mL Glacial acetic acid that Pd/C, 20mL water of 1g 10%, 10g step a are obtained under stirring, pass into H
2, hydrogenolysis pressure is 0.8 ~ 0.95Mpa, and hydrogenolysis temperature is 40 ~ 50 DEG C, reaction 20h; React complete, pressurization is concentrated, residue ether sedimentation, solid collected by filtration, and get Ge La is for thunder crude product;
C, lattice draw the purifying for thunder crude product: the lattice getting step b obtained draw for thunder crude product, dissolve with the sodium carbonate solution of 1%wt, and with the sodium carbonate solution adjust pH 8 of 1%wt, and are diluted to 10mg/mL lattice and draw solution for thunder; Use 3000 dalton's films to carry out ultrafiltration, remove low-molecular-weight impurity, when ultrafiltration is to original volume 50%, add to original volume with water, add 4 times altogether, gained solution is carried out concentrating under reduced pressure and lyophilize, obtain 7.2g lattice and draw for thunder.
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| CN201310200501.XA CN103265624B (en) | 2013-05-27 | 2013-05-27 | Method for preparing copaxone |
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| CN201310200501.XA CN103265624B (en) | 2013-05-27 | 2013-05-27 | Method for preparing copaxone |
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| CN103265624B true CN103265624B (en) | 2015-04-22 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105884866B (en) * | 2015-01-26 | 2021-04-20 | 漳州未名博欣生物医药有限公司 | Chemical synthesis method of glatiramer |
| CN112649538B (en) * | 2015-04-28 | 2024-03-29 | 深圳翰宇药业股份有限公司 | High performance liquid chromatography analysis method for polypeptide mixture |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3849550A (en) * | 1971-04-21 | 1974-11-19 | Yeda Res & Dev | Therapeutic copolymer |
| US5800808A (en) * | 1994-05-24 | 1998-09-01 | Veda Research And Development Co., Ltd. | Copolymer-1 improvements in compositions of copolymers |
| CN101111252A (en) * | 2005-02-02 | 2008-01-23 | 泰华制药工业有限公司 | Process for producing polypeptide mixtures using hydrogenolysis |
-
2013
- 2013-05-27 CN CN201310200501.XA patent/CN103265624B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3849550A (en) * | 1971-04-21 | 1974-11-19 | Yeda Res & Dev | Therapeutic copolymer |
| US5800808A (en) * | 1994-05-24 | 1998-09-01 | Veda Research And Development Co., Ltd. | Copolymer-1 improvements in compositions of copolymers |
| CN101111252A (en) * | 2005-02-02 | 2008-01-23 | 泰华制药工业有限公司 | Process for producing polypeptide mixtures using hydrogenolysis |
Non-Patent Citations (1)
| Title |
|---|
| 陈家念 等.4-(对羟基苄基)- 噁唑- 2- 硫酮的合成研究.《湖北大学学报》.2005, * |
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