CN106279375A - A kind of preparation method of acetic acid copaxone - Google Patents

A kind of preparation method of acetic acid copaxone Download PDF

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CN106279375A
CN106279375A CN201610953974.0A CN201610953974A CN106279375A CN 106279375 A CN106279375 A CN 106279375A CN 201610953974 A CN201610953974 A CN 201610953974A CN 106279375 A CN106279375 A CN 106279375A
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王波
万家强
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Shanghai Bai Yu Pharmaceutical Technology Co Ltd
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    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/34—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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Abstract

The invention discloses the preparation method of a kind of acetic acid copaxone, the steps include: first to prepare four amino acid N CA monomers, by its preparation white paper-like solid Polymer 1 and white solid Polymer 2；Finally preparing acetic acid copaxone, specifically add the Polymer 2 of 80.0g, the water of 4L and the piperidines of 250mL, argon shield in 10L reaction bulb, 23 25 DEG C of lucifuge stirrings are reacted 24 hours, obtain light yellow reactant liquor；Filtering, select 3KDa film bag, alkali liquor, deionized water clean film bag successively；By ultrafiltration apparatus, reactant liquor being concentrated into 700mL, adds deionized water 700mL dilution, then ultrafiltration to reactant liquor volume is 700mL, the pH value of loop ultrafiltration to system is 10～11；Being transferred to by reaction concentrated solution in 1L reaction bulb, it is 3.0 that stirring adds the pH value of acetic acid regulation system, reaction concentrated solution is passed through ultrafiltration apparatus again, is washed with deionized water filter, until the pH value of reactant liquor is to 5.0 6.0；Filtrate subpackage, lyophilizing, obtain acetic acid copaxone, the method is easy and simple to handle, and qualification rate is high.

Description

A kind of preparation method of acetic acid copaxone
Technical field
The present invention relates to polypeptide drugs synthesis technical field, be specifically related to the preparation method of a kind of acetic acid copaxone.
Background technology
Be referred to as acetic acid copaxone (Glatiramer acetate, GA), be not that all there is same acid sequence Mixtures of polypeptides carry out commercially available under trade (brand) name Copaxone, it includes the acetate of such polypeptide, and described polypeptide is respectively Comprising Pidolidone, ALANINE, L-TYR with the molar average fraction of 0.141,0.427,0.095 and 0.338, L-relies ammonia Acid.The mean molecule quantity of Copaxone is 4700-11000 dalton.In chemistry, acetic acid copaxone is referred to as containing L-third Propylhomoserin, 1B, L-TYR, the polymer acetate of Pidolidone.Its structural formula is:
(Glu, Ala, Lys, Tyr)x.XCH3COOH
(C5H9NO4.C3H7NO2.C6H14N2O2.C9H11NO3)x.XC2H4O2
CAS:147245-92-9。
Acetic acid copaxone is approved for reducing the frequency of recurrence in the patient suffering from interval relapsive hardening Rate.Multiple sclerosis is classified as autoimmune disease.Have also been disclosed and be used for acetic acid copaxone treating other certainly Body immunological diseases (the publication number US2002/0055466 A1 of R.Aharoni etc.), inflammatory non-autoimmune disease (V.Wee The publication number US2005/0014694 A1 of Yong etc.；With the U.S. Patent Application No. 2002/ being disclosed on June 20th, 2002 0077278 A1, (Young etc.)), and be used for promoting neuranagenesis and/or prevent or suppress secondary degeneration, described secondary becomes Property can then primary nervous system injury occur (the publication number US2003/0004099 of M.Eisenbach-Schwartz etc. A1；With U.S. Patent Application No. 2002/0037848(Eisenbach-Schwartz being disclosed on March 28th, 2002)).This Outward, disclose and acetic acid copaxone is used for treats immunologically mediated disease (such as, is issued to the United States Patent (USP) on February 4th, 2003 Number 6514938 B1(Gad etc.)；It is published in PCT International Publication WO 00/27417 on 23 days Augusts calendar year 2001, (Aharoni Deng)) and the disease relevant to demyelination (it is published in calendar year 2001 December PCT International Publication WO-1/97846 of 27 days, (Moses etc.)).
The preparation method described in detail in above-mentioned patent includes making protected polypeptide and 33% hydrobromic acid in acetic acid Reaction (JIUYUE was issued to the U.S. Patent number 5800808 of Konfino etc. on the 1st in 1998).This protective reaction is by Gama benzyl Protection group is removed from the 5-carboxylate of glutaminic acid residue, and is that less polypeptide is to form trifluoro by described polymer cracking Acetyl group polypeptide (JIUYUE was issued to the U.S. Patent number 5800808 of Konfino etc. on the 1st in 1998).Obtain between 7000 ± 2000 Time required for the GA of daltonian correct mean molecule quantity depends on reaction temperature and protected acetic acid copaxone Molecular weight modes (JIUYUE was issued to the U.S. Patent number 5800808 of Konfino etc. on the 1st in 1998).Test reaction is when different Between the stage carry out in each batch, with determine on fixed temperature obtain correct molecular weight pattern trifluoroacetyl group polypeptide institute The response time needed.The scope of the amount of the time required for reaction, such as between 10 and 50 hours.Additionally, the U.S. is special Profit number 5981589,6048898,6054430,6342476,6362161 and 6620847 further relates to the mixing for preparing polypeptide Thing, including compositions and the method for GA.
Report about copaxone preparation report is a lot, such as United States Patent (USP) 3849550,5800808 and the U.S. both at home and abroad Patent publication the 2006/0172942nd, No. 2006/0154862, No. 2007/0141663 etc..Involved by these patents To preparation method be by ALANINE, TYR, Pidolidone-γ-benzyl ester, the N-of L-ε-trifluoroacetyl lysine Formic anhydride (NCA), in anhydrous Isosorbide-5-Nitrae-dioxane, uses diethylamine to cause and is polymerized at random, shielded many to produce Peptide.The deprotection of γ-benzyl group realizes by stirring shielded polypeptide under room temperature in hydrogen bromide/acetic acid.With Time, this condition can also cut copolymer.Next step is processed by piperidines can remove ε-trifluoroacetyl group.Finally by dialysis Purification copolymer, obtains acetic acid copaxone.In this method, the amino acid monomer that synthetic polymer uses is amino acid N CA, The quality of amino acid N CA can directly affect the quality of polymer and (include the mean molecule quantity of polymer, molecular weight distribution, impurity Kind and content etc.), thus cause the quality problems of finished product acetic acid copaxone, such as mean molecule quantity bigger than normal or less than normal, Molecular weight distribution is broadening；The stability problem of product quality is relatively big on industrialized production impact, is unfavorable for that wide scale security produces.
When being prepared mixtures of polypeptides (e.g., acetic acid copaxone) by N-carboxy acid anhydride (NCA), the quality of N-carboxy acid anhydride, Especially content of dissociative chlorine ion, the qualitative effects for polyreaction and product is the biggest.Especially, if N-carboxy acid anhydride not Can reach there is specification, the mean molecule quantity of acetic acid copaxone and aminoacid composition all can not reach due scope: acetic acid The amino acid molar ratio of copaxone is about 0.392-0.462:0.129-0.153:0.300-0.374:0.086-0.100, Mean molecule quantity about 5000-9000 dalton.
In prior art, amino acid N CA that synthetic polymer uses only has simple preparation method, does not finds clear and definite matter Amount standard and synthesize the method detailed of qualified monomer.
Summary of the invention
For problem above, the invention provides the preparation method of a kind of acetic acid copaxone, the method is easy and simple to handle, closes Lattice rate is high, can stablize the size of mean molecule quantity, molecular weight distribution by controlling amino acid monomer quality, be suitable to industrialization, can Effectively to solve the problem in background technology.
To achieve these goals, the technical solution used in the present invention is as follows: the preparation method of a kind of acetic acid copaxone, Comprise the steps:
S1, preparation four amino acid N CA monomers, respectively alanine monomer, lysine monomer, tyrosine monomer and paddy ammonia Acid monomers；Its chemical formula is as follows:
S2, with four amino acid N CA monomers as raw material, preparation white paper-like solid Polymer-1；Its chemical formula is:
S3, the preparation white paper-like solid Polymer-1 obtained with step S2, prepare white solid Polymer-2, and it is changed Formula is:
S4, preparation acetic acid copaxone, add the water of Polymer-2,4L of 80.0g and 250mL in 10L reaction bulb Piperidines, argon shield, 23-25 DEG C of lucifuge stirring is reacted 24 hours, obtains light yellow reactant liquor；Reacting liquid filtering, selects 3KDa Film bag, alkali liquor, deionized water clean film bag successively；Reactant liquor is concentrated into 700mL by ultrafiltration apparatus, adds deionized water 700mL dilutes, then ultrafiltration to reactant liquor volume is 700mL, and the pH value of loop ultrafiltration to system is 10～11；Concentrated solution will be reacted Transferring in 1L reaction bulb, under stirring, the pH value adding acetic acid regulation system is 3.0, stirs one hour；Concentrated solution will be reacted again By ultrafiltration apparatus, it is washed with deionized water filter, until the pH value of reactant liquor is to 5.0-6.0；Filtrate subpackage, lyophilizing, obtain white Fluffy solid i.e. acetic acid copaxone；Its chemical formula is:
Preferably, the preparation method of described glutamic acid monomer is as follows:
S2.1, in 2L reaction bulb, add the Pidolidone-γ-benzyl ester of 63.6g, the anhydrous THF of 640mL, and evacuation Logical argon is replaced three times, is heated with stirring to 55 DEG C under argon shield；
S2.2,32.0g triphosgene solid is dissolved in the anhydrous THF of 100mL, transfers in the constant pressure funnel of 250mL, Evacuation leads to argon replaces three times, within 10-15 minute, is added drop-wise in reactant liquor, continues stirring reaction 4 hours；
S2.3, reaction end are cooled to room temperature, and sucking filtration removes impurity, and under 20 DEG C of environment, filtrate are instilled dry under stirring In dry 2000mL normal hexane, dripped off in 1 hour, separate out a large amount of white precipitate, at 0～-5 DEG C, then stand crystallize 3 little Time；
S2.4, the filter cake after filter cake is dried normal hexane washing with a small amount of, then 25 DEG C be vacuum dried white powder i.e. Its NCA monomer.
S2.5, this monomer 55.0g is dissolved in the THF of dry 150mL, the lower dry normal hexane dripping 450mL of stirring, Dripping off and be cooled to 0-5 DEG C, stir three hours, filter, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried white Powder, obtains high-purity glutamic acid monomer.
Preferably, the preparation method of described tyrosine monomer is as follows:
S3.1, adding the O-Benzyl-L-tyrosine of 48.0g in 1L reaction bulb, the anhydrous THF of 480mL, evacuation leads to argon Gas is replaced three times, is heated with stirring to 55 DEG C under argon shield；
S3.2, the triphosgene solid of 24.0g is dissolved in the anhydrous THF of 70mL, transfers to the constant pressure funnel of 250mL In, evacuation leads to argon replaces three times, within 10-15 minute, is added drop-wise in reactant liquor, continues stirring reaction 4 hours；
S3.3, reaction end are cooled to room temperature, and sucking filtration removes impurity；And under 20 DEG C of environment, under stirring, filtrate is instilled dry In dry 1500mL normal hexane, dripped off in 1 hour, separate out a large amount of yellow-white precipitation, 0～-5 DEG C at stand crystallize 3 hours；
S3.4, carrying out sucking filtration, and with a small amount of, filter cake is dried normal hexane washing, then 25 DEG C are vacuum dried to obtain greyish white toner End i.e. its NCA monomer；
S3.5, this monomer of 40.0g is dissolved in dry 120mLTHF, the lower dry normal hexane dripping 400mL of stirring, Dripping off and be cooled to 0-5 DEG C, stir three hours, filter, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried white Powder, obtains high-purity tyrosine monomer.
Preferably, the preparation method of described lysine monomer is as follows:
S4.1, in 2L reaction bulb, add the N-6-trifluoroacetyl-1B of 150.0g and the anhydrous of 1500mL THF, evacuation leads to argon replaces three times, is heated with stirring to 55 DEG C under argon shield；
S4.2, the triphosgene solid of 78.0g is dissolved in the anhydrous THF of 140mL, transfers to the constant pressure addition leakage of 250mL In bucket, evacuation leads to argon replaces three times, is added drop-wise in reactant liquor, continues stirring reaction 4 hours；
S4.3, reaction end are cooled to room temperature, and sucking filtration removes impurity, and filtrate is concentrated into 1L volume below 40 DEG C；And 20 Under DEG C environment, under stirring, filtrate is instilled in the dry normal hexane of 4000mL, dripped off in 1 hour, separate out a large amount of white precipitate, 0～-5 DEG C at stand crystallize 3 hours；
S4.4, through sucking filtration, filter cake is dried normal hexane washing with a small amount of, then 25 DEG C be vacuum dried white powder i.e. Its NCA monomer；
S4.5, this monomer of 150.0g is dissolved in the THF that 500mL is dried, the lower dry normal hexane dripping 1500mL of stirring, Dripping off and be cooled to 0-5 DEG C, stir three hours, filter, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried white Powder, i.e. high-purity lysine monomer.
Preferably, the preparation method of described alanine monomer is as follows:
The anhydrous THF of S5.1, the ALANINE adding 70.0g in 5L reaction bulb and 3500mL, evacuation leads to argon Replace three times, under argon shield, be heated with stirring to 55 DEG C；
S5.2, the triphosgene solid of 113.0g is dissolved in the anhydrous THF of 300mL, transfers to the constant pressure addition leakage of 500mL In bucket, evacuation leads to argon replaces three times, is added drop-wise in reactant liquor, continues stirring reaction 4 hours；
S5.3, reaction end are cooled to room temperature, and sucking filtration removes impurity, and filtrate is concentrated into 500mL volume below 40 DEG C；And Under 20 DEG C of environment, filtrate is instilled in the normal hexane that 2000mL is dried under stirring, within 1 hour, drips off, separate out a large amount of pale pink precipitation, 0～-5 DEG C at stand crystallize 3 hours；
S5.4, through sucking filtration, and filter cake is dried normal hexane washing with a small amount of, then 25 DEG C are vacuum dried to obtain white powder Its NCA monomer i.e.；
S5.5, this monomer of 60.0g is dissolved in the THF that 200mL is dried, stirring lower dropping 1000mL be dried just oneself Alkane, drips off and is cooled to 0-5 DEG C, stirs three hours, filters, and filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried White powder, i.e. high purity N CA monomer.
Preferably, the preparation method of described white paper-like solid Polymer-1 is as follows:
S6.1, four amino acid N CA monomers of accurate weighing, respectively alanine monomer 40.0g；Lysine monomer 64.0g； Tyrosine monomer 20.6g；Glutamic acid monomer 28.6g；Insert in the four-hole bottle of 3L；Evacuation argon is replaced three times；
S6.2, under an argon, adds the dioxane that 1600mL is dried, and vacuum argon is replaced three times；
S6.3, stir at 22～24 DEG C to solid and all dissolve；The diethylamide that disposable addition drying is heavily steamed Dioxane solution, 25 DEG C are stirred 24 hours；
S6.4, the most slowly dropping to reactant liquor, in the deionized water of 12L, within 3 hours, drip off, sucking filtration, filter cake is used Pure water；
S6.5, filter cake is smashed to pieces after proceed in flask, add 12L pure water, be stirred vigorously 1 hour, sucking filtration, repeat making beating Once；Sucking filtration obtains white paper-like solid, and 60 DEG C of vacuum is dried 6～8 hours, and to weightlessness, < 0.1g obtains white paper-like solid Polymer-1。
Preferably, the preparation method of described white paper-like solid Polymer-2 is as follows:
S7.1, in 2L reaction bulb, add the POLYMER-1 of the phenol of 5.0g, 110.0g, add the HBr-acetic acid of 900mL Solution, argon shield, 23-25 DEG C of lucifuge stirring is reacted 16 hours, obtains peony reactant liquor；
S7.2, reactant liquor 3 times amount n-hexane extraction three times, is under agitation added drop-wise to the deionized water of 8L by reactant liquor In, within three hours, drip off, continue stirring one hour；
S7.3, through sucking filtration, by filter cake pure water；Obtaining shallow white solid, 40 DEG C of vacuum is dried, and obtains white solid Body Polymer-2.
Beneficial effects of the present invention:
The solution of the present invention is easy and simple to handle, and qualification rate is high, can stablize mean molecule quantity by controlling amino acid monomer quality Size, molecular weight distribution, be suitable to industrialization.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, to the present invention It is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not used to Limit the present invention.
Example one:
Operating procedure:
Pidolidone-γ-benzyl ester (63.6g, 0.27mol), anhydrous THF(640mL is added in 2L reaction bulb), evacuation Logical argon is replaced three times, is heated with stirring to 55 DEG C under argon shield；Triphosgene (32.0g, 0.11mol) solid is dissolved in anhydrous THF(100mL) in, transferring in the constant pressure funnel of 250mL, evacuation leads to argon replaces three times, within 10-15 minute, is added drop-wise to In reactant liquor, continue stirring reaction 4 hours；Reaction end is cooled to room temperature, and sucking filtration removes impurity；At 20 DEG C, by filtrate under stirring Instill in the normal hexane (2000mL) being dried, within 1 hour, drip off, separate out a large amount of white precipitate, 0～-5 DEG C at stand crystallize 3 hours； Sucking filtration, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried to obtain white powder i.e. its NCA monomer about (57.0g). This monomer (55.0g) is dissolved in dry THF(150mL) in, the lower dropping of stirring is dried normal hexane (450mL), drips off and is cooled to 0-5 DEG C, stirring three hours, filter, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried to obtain white powder, obtain high-purity Degree glutamic acid monomer (50.0g).
Example two:
Operating procedure:
O-Benzyl-L-tyrosine (48.0g, 0.18mol), anhydrous THF(480mL is added in 1L reaction bulb), evacuation Logical argon is replaced three times, is heated with stirring to 55 DEG C under argon shield；Triphosgene (24.0g, 0.08mol) solid is dissolved in anhydrous THF(70mL) in, transferring in the constant pressure funnel of 250mL, evacuation leads to argon replaces three times, within 10-15 minute, is added drop-wise to In reactant liquor, continue stirring reaction 4 hours；Reaction end is cooled to room temperature, and sucking filtration removes impurity；At 20 DEG C, by filtrate under stirring Instill be dried normal hexane (1500mL) in, within 1 hour, drip off, separate out a large amount of yellow-white precipitation, 0～-5 DEG C at stand crystallize 3 little Time；Sucking filtration, filter cake is dried normal hexane washing with a small amount of, then 25 DEG C be vacuum dried pale powder i.e. its NCA monomer about (42.0g).This monomer (40.0g) is dissolved in dry THF(120mL) in, the lower dropping of stirring is dried normal hexane (400mL), drips Complete being cooled to 0-5 DEG C, stir three hours, filter, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried to obtain white powder End, obtains high-purity tyrosine monomer (37.0g).
Example three:
Operating procedure:
N-6-trifluoroacetyl-1B (150.0g, 0.62mol), anhydrous THF is added in 2L reaction bulb (1500mL), evacuation leads to argon replaces three times, is heated with stirring to 55 DEG C under argon shield；By triphosgene (78.0g, 0.27mol) solid is dissolved in anhydrous THF(140mL) in, to transfer in the constant pressure funnel of 250mL, evacuation leads to argon displacement Three times, it is added drop-wise in reactant liquor, continues stirring reaction 4 hours；Reaction end is cooled to room temperature, and sucking filtration removes impurity, and filtrate is 40 1L volume it is concentrated into below DEG C；At 20 DEG C, under stirring, filtrate is instilled in the normal hexane (4000mL) being dried, within 1 hour, drip off, analysis Go out a large amount of white precipitate, 0～-5 DEG C at stand crystallize 3 hours；Sucking filtration, filter cake washs with a small amount of normal hexane that is dried, then 25 DEG C It is vacuum dried to obtain white powder i.e. its NCA monomer about (152.0g).This monomer (150.0g) is dissolved in dry THF(500mL) In, the lower dropping of stirring is dried normal hexane (1500mL), drips off and is cooled to 0-5 DEG C, stir three hours, filters, and filter cake is dried with a small amount of Normal hexane washs, and then 25 DEG C are vacuum dried to obtain white powder, i.e. high-purity lysine monomer (130.0g).
Example four:
Operating procedure:
ALANINE (70.0g, 0.80mol), anhydrous THF(3500mL is added in 5L reaction bulb), evacuation leads to argon Replace three times, under argon shield, be heated with stirring to 55 DEG C；Triphosgene (113.0g, 0.38mol) solid is dissolved in anhydrous THF (300mL) in, transferring in the constant pressure funnel of 500mL, evacuation leads to argon replaces three times, is added drop-wise in reactant liquor, continues Continuous stirring reaction 4 hours；Reaction end is cooled to room temperature, and sucking filtration removes impurity, and filtrate is concentrated into 500mL volume below 40 DEG C； At 20 DEG C, under stirring, filtrate is instilled in the normal hexane (2000mL) being dried, within 1 hour, drip off, separate out a large amount of pale pink and precipitate, 0 ～at-5 DEG C, stand crystallize 3 hours；Sucking filtration, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried to obtain white powder End i.e. its NCA monomer about (66.0g).This monomer (60.0g) is dissolved in dry THF(200mL) in, the lower dropping of stirring is just being dried Hexane (1000mL), drips off and is cooled to 0-5 DEG C, stirs three hours, filters, and filter cake washs with a small amount of normal hexane that is dried, then 25 DEG C It is vacuum dried to obtain white powder, i.e. high-purity alanine monomer (52.0g).
Example five:
Operating procedure:
Accurate weighing four amino acid N CA monomers (alanine monomer 40.0g, 0.35mol；Lysine monomer 64.0g, 0.48mol；Tyrosine monomer 20.6g, 0.07mol；Glutamic acid monomer 28.6g, 0.11mol；), insert in the four-hole bottle of 3L；Take out Vacuum argon is replaced three times；Under an argon, adding the dioxane (1600mL) being dried, vacuum argon is replaced three times.22～ Stir at 24 DEG C to solid and all dissolve；Disposable dioxane solution (the 1mL diethyl adding the diethylamide that drying is heavily steamed Amine+20mL dioxane), 25 DEG C are stirred 24 hours；The most slowly being dropped to by reactant liquor in deionized water (12L), 3 is little Time drip off, sucking filtration, filter cake pure water；Filter cake proceeds in flask after smashing to pieces, adds 12L pure water, is stirred vigorously 1 hour, takes out Filter, repeats making beating once；Sucking filtration obtains white paper-like solid, and 60 DEG C of vacuum is dried 6～8 hours, and to weightlessness, < 0.1g obtains white Paper-like solid Polymer-1(119.0g).
Example six:
Operating procedure:
Phenol (5.0g), POLYMER-1(110.0g is added in 2L reaction bulb), add HBr-acetic acid solution (900mL), Argon shield, 23-25 DEG C of lucifuge stirring is reacted 16 hours, obtains peony reactant liquor；Reactant liquor is with 3 times amount n-hexane extractioies three Secondary, reactant liquor is under agitation added drop-wise in deionized water (8L), within three hours, drips off, continue stirring one hour, sucking filtration, filter cake is used Pure water；Obtaining shallow white solid, 40 DEG C of vacuum is dried, and obtains white solid Polymer-2(87.0g).
Example seven:
Operating procedure:
In 10L reaction bulb, add Polymer-2(80.0g), water (4L) and piperidines (250mL), argon shield, 23-25 DEG C lucifuge stirring reaction 24 hours, obtains light yellow reactant liquor；Reacting liquid filtering, selects 3KDa film bag, successively alkali liquor, deionization Water cleans film bag；Reactant liquor is concentrated into 700mL by ultrafiltration apparatus, adds deionized water 700mL dilution, then ultrafiltration is to reaction Liquid amasss as 700mL, and the pH value of loop ultrafiltration to system is 10～11；Reaction concentrated solution is transferred in 1L reaction bulb, stirring Under, the pH value adding acetic acid regulation system is 3.0, stirs one hour；Reaction concentrated solution is passed through ultrafiltration apparatus again, uses deionization Washing filter, until the pH value of reactant liquor is to 5.0-6.0；Filtrate subpackage, lyophilizing, obtain white fluffy solid i.e. acetic acid copaxone (51.0g).
The solution of the present invention is easy and simple to handle, and qualification rate is high, can stablize mean molecule quantity by controlling amino acid monomer quality Size, molecular weight distribution, be suitable to industrialization.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention Any amendment, equivalent and the improvement etc. made within god and principle, should be included within the scope of the present invention.

Claims (7)

1. the preparation method of an acetic acid copaxone, it is characterised in that comprise the steps:
S1, preparation four amino acid N CA monomers, respectively alanine monomer, lysine monomer, tyrosine monomer and glutamic acid list Body；Its chemical formula is as follows:
S2, with four amino acid N CA monomers as raw material, preparation white paper-like solid Polymer-1；Its chemical formula is:
S3, the preparation white paper-like solid Polymer-1 obtained with step S2, prepare white solid Polymer-2, its chemical formula For:
S4, preparation acetic acid copaxone, add the water of Polymer-2,4L of 80.0g and the piperazine of 250mL in 10L reaction bulb Pyridine, argon shield, 23-25 DEG C of lucifuge stirring is reacted 24 hours, obtains light yellow reactant liquor；Reacting liquid filtering, selects 3KDa film Bag, alkali liquor, deionized water clean film bag successively；Reactant liquor is concentrated into 700mL by ultrafiltration apparatus, adds deionized water 700mL dilutes, then ultrafiltration to reactant liquor volume is 700mL, and the pH value of loop ultrafiltration to system is 10～11；Concentrated solution will be reacted Transferring in 1L reaction bulb, under stirring, the pH value adding acetic acid regulation system is 3.0, stirs one hour；Concentrated solution will be reacted again By ultrafiltration apparatus, it is washed with deionized water filter, until the pH value of reactant liquor is to 5.0-6.0；Filtrate subpackage, lyophilizing, obtain white Fluffy solid i.e. acetic acid copaxone；Its chemical formula is:
The preparation method of a kind of acetic acid copaxone the most according to claim 1, it is characterised in that described glutamic acid monomer Preparation method as follows:
S2.1, in 2L reaction bulb, add the Pidolidone-γ-benzyl ester of 63.6g, the anhydrous THF of 640mL, and evacuation leads to argon Gas is replaced three times, is heated with stirring to 55 DEG C under argon shield；
S2.2,32.0g triphosgene solid is dissolved in the anhydrous THF of 100mL, transfers in the constant pressure funnel of 250mL, take out true Empty logical argon is replaced three times, within 10-15 minute, is added drop-wise in reactant liquor, continues stirring reaction 4 hours；
S2.3, reaction end are cooled to room temperature, and sucking filtration removes impurity, and under 20 DEG C of environment, filtrate instillation are dried under stirring In 2000mL normal hexane, dripped off in 1 hour, separate out a large amount of white precipitate, at 0～-5 DEG C, then stand crystallize 3 hours；
S2.4, with a small amount of, filter cake after filter cake being dried normal hexane washing, then 25 DEG C are vacuum dried to obtain white powder i.e. its NCA Monomer.
S2.5, this monomer 55.0g is dissolved in the THF of dry 150mL, the lower dry normal hexane dripping 450mL of stirring, drips off Being cooled to 0-5 DEG C, stir three hours, filter, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried to obtain white powder End, obtains high-purity glutamic acid monomer.
The preparation method of a kind of acetic acid copaxone the most according to claim 1, it is characterised in that described tyrosine monomer Preparation method as follows:
S3.1, adding the O-Benzyl-L-tyrosine of 48.0g in 1L reaction bulb, the anhydrous THF of 480mL, evacuation leads to argon to be put Change and under three times, argon shield, be heated with stirring to 55 DEG C；
S3.2, the triphosgene solid of 24.0g is dissolved in the anhydrous THF of 70mL, transfers in the constant pressure funnel of 250mL, Evacuation leads to argon replaces three times, within 10-15 minute, is added drop-wise in reactant liquor, continues stirring reaction 4 hours；
S3.3, reaction end are cooled to room temperature, and sucking filtration removes impurity；And under 20 DEG C of environment, under stirring, filtrate instillation is dried In 1500mL normal hexane, dripped off in 1 hour, separate out a large amount of yellow-white precipitation, 0～-5 DEG C at stand crystallize 3 hours；
S3.4, carry out sucking filtration, and filter cake be dried normal hexane washing with a small amount of, then 25 DEG C be vacuum dried pale powder i.e. Its NCA monomer；
S3.5, this monomer of 40.0g is dissolved in dry 120mLTHF, the lower dry normal hexane dripping 400mL of stirring, drips off Being cooled to 0-5 DEG C, stir three hours, filter, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried to obtain white powder End, obtains high-purity tyrosine monomer.
The preparation method of a kind of acetic acid copaxone the most according to claim 1, it is characterised in that described lysine monomer Preparation method as follows:
S4.1, the N-6-trifluoroacetyl-1B adding 150.0g in 2L reaction bulb and the anhydrous THF of 1500mL, take out Vacuum leads to argon displacement three times, is heated with stirring to 55 DEG C under argon shield；
S4.2, the triphosgene solid of 78.0g is dissolved in the anhydrous THF of 140mL, transfers in the constant pressure funnel of 250mL, Evacuation leads to argon replaces three times, is added drop-wise in reactant liquor, continues stirring reaction 4 hours；
S4.3, reaction end are cooled to room temperature, and sucking filtration removes impurity, and filtrate is concentrated into 1L volume below 40 DEG C；And at 20 DEG C of rings Under border, under stirring, filtrate is instilled in the dry normal hexane of 4000mL, dripped off in 1 hour, separate out a large amount of white precipitate, 0～- Crystallize 3 hours are stood at 5 DEG C；
S4.4, through sucking filtration, filter cake is dried normal hexane washing with a small amount of, then 25 DEG C be vacuum dried white powder i.e. its NCA monomer；
S4.5, this monomer of 150.0g is dissolved in the THF that 500mL is dried, the lower dry normal hexane dripping 1500mL of stirring, drips off Being cooled to 0-5 DEG C, stir three hours, filter, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried to obtain white powder End, i.e. high-purity lysine monomer.
The preparation method of a kind of acetic acid copaxone the most according to claim 1, it is characterised in that described alanine monomer Preparation method as follows:
The anhydrous THF of S5.1, the ALANINE adding 70.0g in 5L reaction bulb and 3500mL, evacuation leads to argon displacement Three times, under argon shield, it is heated with stirring to 55 DEG C；
S5.2, the triphosgene solid of 113.0g is dissolved in the anhydrous THF of 300mL, transfers to the constant pressure funnel of 500mL In, evacuation leads to argon replaces three times, is added drop-wise in reactant liquor, continues stirring reaction 4 hours；
S5.3, reaction end are cooled to room temperature, and sucking filtration removes impurity, and filtrate is concentrated into 500mL volume below 40 DEG C；And at 20 DEG C Under environment, filtrate is instilled in the normal hexane that 2000mL is dried under stirring, within 1 hour, drips off, separate out a large amount of pale pink precipitation, 0～- Crystallize 3 hours are stood at 5 DEG C；
S5.4, through sucking filtration, and filter cake is dried normal hexane washing with a small amount of, then 25 DEG C be vacuum dried white powder i.e. its NCA monomer；
S5.5, this monomer of 60.0g is dissolved in the THF that 200mL is dried, the normal hexane that stirring lower dropping 1000mL is dried, drips Complete being cooled to 0-5 DEG C, stir three hours, filter, filter cake is dried normal hexane washing with a small amount of, and then 25 DEG C are vacuum dried to obtain white powder End, i.e. high purity N CA monomer.
The preparation method of a kind of acetic acid copaxone the most according to claim 1, it is characterised in that described white paper-like is solid The preparation method of body Polymer-1 is as follows:
S6.1, four amino acid N CA monomers of accurate weighing, respectively alanine monomer 40.0g；Lysine monomer 64.0g；Cheese ammonia Acid monomers 20.6g；Glutamic acid monomer 28.6g；Insert in the four-hole bottle of 3L；Evacuation argon is replaced three times；
S6.2, under an argon, adds the dioxane that 1600mL is dried, and vacuum argon is replaced three times；
S6.3, stir at 22～24 DEG C to solid and all dissolve；The disposable dioxy adding the diethylamide that drying is heavily steamed Six ring solution, 25 DEG C are stirred 24 hours；
S6.4, the most slowly drop to reactant liquor, in the deionized water of 12L, within 3 hours, drip off, sucking filtration, filter cake pure water Washing；
S6.5, filter cake is smashed to pieces after proceed in flask, add 12L pure water, be stirred vigorously 1 hour, sucking filtration, repeat making beating one Secondary；Sucking filtration obtains white paper-like solid, and 60 DEG C of vacuum is dried 6～8 hours, and to weightlessness, < 0.1g obtains white paper-like solid Polymer-1。
The preparation method of a kind of acetic acid copaxone the most according to claim 1, it is characterised in that described white paper-like is solid The preparation method of body Polymer-2 is as follows:
S7.1, adding the POLYMER-1 of the phenol of 5.0g, 110.0g in 2L reaction bulb, the HBr-acetic acid adding 900mL is molten Liquid, argon shield, 23-25 DEG C of lucifuge stirring is reacted 16 hours, obtains peony reactant liquor；
S7.2, reactant liquor 3 times amount n-hexane extraction three times, is under agitation added drop-wise to reactant liquor in the deionized water of 8L, three Hour drip off, continue stirring one hour；
S7.3, through sucking filtration, by filter cake pure water；Obtaining shallow white solid, 40 DEG C of vacuum is dried, and obtains white solid Polymer-2。
CN201610953974.0A 2016-11-03 2016-11-03 A kind of preparation method of acetic acid copaxone Pending CN106279375A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103910784A (en) * 2014-04-10 2014-07-09 山东大学 Simple and convenient preparation method of copaxone
CN104610436A (en) * 2015-02-03 2015-05-13 郑州大明药物科技有限公司 Preparation method of glatiramer acetate
CN104844697A (en) * 2014-09-26 2015-08-19 深圳翰宇药业股份有限公司 Glatirameracetate preparation method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103910784A (en) * 2014-04-10 2014-07-09 山东大学 Simple and convenient preparation method of copaxone
CN104844697A (en) * 2014-09-26 2015-08-19 深圳翰宇药业股份有限公司 Glatirameracetate preparation method
CN104610436A (en) * 2015-02-03 2015-05-13 郑州大明药物科技有限公司 Preparation method of glatiramer acetate

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Application publication date: 20170104