CN105713073A - Method for liquid phase preparation of atosiban - Google Patents

Method for liquid phase preparation of atosiban Download PDF

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Publication number
CN105713073A
CN105713073A CN201610195094.1A CN201610195094A CN105713073A CN 105713073 A CN105713073 A CN 105713073A CN 201610195094 A CN201610195094 A CN 201610195094A CN 105713073 A CN105713073 A CN 105713073A
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trt
asn
cys
tfa
mpa
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张颖
杨慧
王仁友
李同金
石鑫磊
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JINAN KANGHE MEDICAL TECHNOLOGY Co Ltd
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JINAN KANGHE MEDICAL TECHNOLOGY Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/16—Oxytocins; Vasopressins; Related peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention particularly relates to a method for liquid phase preparation of atosiban. The method comprises main synthesis steps as follows: (1) asparagine with an N terminal protected by a trifluoroacetyl group is prepared and then sequentially connected with four amino acids, then the trifluoroacetyl group is removed, and a pentapeptide fragment NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2 is obtained; (2) a tetrapeptide fragment Mpa(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-OH is synthesized; (3) the pentapeptide fragment and the tetrapeptide fragment are coupled, and a linear crude peptide Mpa-D-Tyr(Et)-IIe-Thr-Asn-Cys-Pro-Orn-Gly-NH2 is obtained through dissociation; (4) liquid phase oxidation is performed, and atosiban is obtained through purification and freeze-drying. The method is simple in procedure, and the linear peptide is synthesized in two stages; an amino group of asparagine is protected by the trifluoroacetyl group, three side chain protection groups including Trt, tBu and Boc can be removed by a conventional dissociation reagent containing trifluoroacetic acid, operation is convenient, and the problem that benzyl group removal conditions are harsh in a Boc strategy liquid phase synthesis method is solved; besides, resin, excessive protection amino acids, condensing agents, cleaning solvents and special production equipment are not required in liquid phase preparation of atosiban, the cost is low, and large-scale production is easy to implement.

Description

A kind of liquid phase prepares the method for atosiban
Technical field
The present invention relates to Peptides Synthesis, particularly to a kind of method that liquid phase prepares atosiban.
Technical background
Atosiban (atosiban) is the oxytocin competitive antagonist of receptor on intrauterine and decidua, fetal membrane, uses the treatment premature labor of its acetate clinically.Atosiban can suppress uterine contraction in dosage correlation ground, and makes the prostaglandin excretion that oxytocin mediates reduce, thus reaching the purpose prevented miscarriage.Atosiban acetate injection (AtosibanAcetateInjection) is developed by Huiling Co., Ltd (FerringAB), and in March, 2000 lists in Austria first.
Atosiban is a ring type polypeptide being made up of 9 aminoacid, peptide chain comprises 3 alpha-non-natural amino acid D-Tyr (Et), Mpa and Orn and a pair disulfide bond formed between Mpa and Cys, C end is the form of amide, and its structural formula is: c [Mpa-D-Tyr (Et)-IIe-Thr-Asn-Cys]-Pro-Orn-Gly-NH2。
Preparation method about atosiban, mostly adopt solid phase synthesis or solid-liquid combination: as US Patent No. 4504469 adopts Boc strategy solid phase synthesis linear peptides, cut peptide with liquefied ammonia, obtain atosiban acetate then through liquid phase oxidation, purification, complicated operation, is unfavorable for commercial production；Domestic patent CN200810043347.9, CN200910008880.6 all adopt solid-phase synthesis, aminoacid is connected on resin one by one, form disulfide bond by the method for iodine oxidation on resin again, solve the technical problem of poorly soluble response time length in air oxidation in liquid phase；CN201310411997.5 first adopts solid-phase synthesis synthesizing linear peptide; re-use 1%TFA/DCM and remove the protection base Trt on Cys (Trt); next add the solid ring of oxidising agent and obtain cyclic peptide resin; finally cracking obtains atosiban; solve existing air oxidation in liquid phase and bring a large amount of waste liquid; lagging behind in production, usefulness is not high and iodine is as the many technical problem of oxidizing by-product；CN201010604790.6 adopts solid phase synthesis linearly thick peptide, again linear atosiban is dissolved in acetonitrile solution, the synthesis of atosiban is carried out by the method for hydrogen peroxide oxidation process formation disulfide bond, this method solve linear atosiban indissoluble problem, the reducing reaction volume and decrease the response time of maximum possible.
Above method all needs to use Solid-phase synthesis peptides special equipment, and substantial amounts of resin and cleaning solvent make synthesis costly, are not suitable for large-scale production.
Patent CN201310129543.9 uses liquid phase synthesizing method synthesis pentapeptide intermediate H-IIe-Thr (Bzl)-Asn-Cys (Bzl)-Pro-OH; and then realize the liquid phase synthesis of atosiban; although solving the problem that solid-phase synthesis equipment is complicated, cost is high, be not suitable for large-scale production; but use this intermediate to need point 3 sections of synthesizing linear peptides; complex procedures; benzyl protecting group Bzl used need to process with hydrogenolysis or liquid nitrogen/sodium and remove; severe reaction conditions; and in safety, there is hidden danger, production operation is had bigger restriction.
In sum; from cost; liquid phase synthesizing method large-scale production lower than solid-phase synthesis cost, preferably; but use benzyl to limit production operation in operation easier as the liquid phase synthesizing method of Side chain protective group; and point 3 sections of synthesizing linear peptide complex procedures; thus it still remains a need find the liquid-phase synthesis process that a kind of operation is simple, operation easier is low.
Summary of the invention
The present invention provides a kind of full liquid-phase synthesis process, is first respectively synthesized fragment tetrapeptide Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH and fragment pentapeptide NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2, then two fragments are connected, crack removing Side chain protective group and obtain atosiban linearly thick peptide, then carry out liquid phase oxidation, purification, lyophilizing obtain atosiban.
For achieving the above object, the present invention provides techniques below scheme:
A kind of liquid phase prepares the method for atosiban, comprises the following steps:
(1), H-Asn-OH and trifluoroacetic acid anhydride reactant prepare Tfa-Asn-OH；
(2), under liquid-phase condition, Tfa-Asn-OH and H-Cys (Trt)-OH, H-Pro-OH, H-Orn (Boc)-OH, H-Gly-NH2Coupling successively, synthesizes fragment Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2, slough N end Tfa and protect base, obtain five fragments of peptides [5-9]: NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2；
(3), under liquid-phase condition, Mpa (Trt)-OH and H-D-Tyr (Et)-OH, H-Ile-OH, H-Thr (tBu)-OH coupling successively, synthesize four fragments of peptides [1-4]: Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH；
(4), under liquid-phase condition, four fragments of peptides [1-4] and five fragments of peptides [5-9] coupling obtain the full guard peptide chain of Side chain protective group: Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2；
(5), peptide chain warp in step (4) cracked, settle, separate to obtain atosiban linearly thick peptide: Mpa-D-Tyr (Et)-IIe-Thr-Asn-Cys-Pro-Orn-Gly-NH2；
(6), by the thick peptide in step (5) through liquid phase oxidation, preparation liquid phase purification, concentrated freeze-dried after atosiban fine peptide: c [Mpa-D-Tyr (Et)-IIe-Thr-Asn-Cys]-Pro-Orn-Gly-NH2。
In above-mentioned steps (1), the preparation method of Tfa-Asn-OH is: be dissolved in trifluoroacetic acid by H-Asn-OH, trifluoroacetic anhydride is added when-10 DEG C, it is warmed up to 10 DEG C, stirs 30 minutes, 30 DEG C of concentrating under reduced pressure, add hot toluene, place, collected by filtration, dried recrystallization, obtaining Tfa-Asn-OH, wherein the mol ratio of H-Asn-OH and trifluoroacetic anhydride is 1:1～1.2.
Fragment Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH in above-mentioned steps (2)2Preparation method be:
(2a), the preparation of Tfa-Asn-Cys (Trt)-OH
Being dissolved in organic solvent by Tfa-Asn-OH and the HOSu of preparation in step (1), add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Tfa-Asn-OSu；
Tfa-Asn-OSu is added in the alkaline salt solution of H-Cys (Trt)-OH, TLC detects reaction end, regulating pH value of solution 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Tfa-Asn-Cys (Trt)-OH；
(2b), the preparation of Tfa-Asn-Cys (Trt)-Pro-OH
Being dissolved in organic solvent by Tfa-Asn-Cys (Trt)-OH and the HOSu of preparation in step (2a), add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Tfa-Asn-Cys (Trt)-OSu；
Tfa-Asn-Cys (Trt)-OSu is added in the alkaline salt solution of H-Pro-OH, TLC detects reaction end, regulating solution ph 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Tfa-Asn-Cys (Trt)-Pro-OH；
(2c), the preparation of Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH
Being dissolved in organic solvent by Tfa-Asn-Cys (Trt)-Pro-OH and the HOSu of preparation in step (2b), add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Tfa-Asn-Cys (Trt)-Pro-OSu；
Tfa-Asn-Cys (Trt)-Pro-OSu is added in the alkaline salt solution of H-Orn (Boc)-OH, TLC detects reaction end, regulate solution ph to 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH；
(2d)、Tfa-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2Preparation
Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH and the HOSu of preparation in step (2c) is dissolved in organic solvent, add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OSu；
Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OSu is added H-Gly-NH2In the alkaline salt solution of HCl, TLC detects reaction end, regulates solution ph to 2～3, extraction into ethyl acetate, and concentrated, petroleum ether crystallize, dry, recrystallization obtain Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2；
Wherein reactions steps (2a), (2b), (2c), in (2d): Tfa-Asn-OH, Tfa-Asn-Cys (Trt)-OH, Tfa-Asn-Cys (Trt)-Pro-OH, Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH are 1:1～1.1:1～1.1 with the mol ratio of HOSu and DCC respectively；H-Cys (Trt)-OH, H-Pro-OH, H-Orn (Boc)-OH, H-Gly-NH2HCl is 1:1～2 with the mol ratio of basic salt respectively；Tfa-Asn-Osu and H-Cys (Trt)-OH, Tfa-Asn-Cys (Trt)-Osu and H-Pro-OH, Tfa-Asn-Cys (Trt)-Pro-Osu and H-Orn (Boc)-OH, Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Osu and H-Gly-NH2The mol ratio of HCl is 1:0.8～1.2.
The method sloughing N end Tfa protection base in above-mentioned steps (2) is: by fragment Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2Join in the aqueous piperidine solution of 1M dissolve, stirring reaction 20～24h, adjust solution ph to 2～3, extraction into ethyl acetate, concentration, petroleum ether crystallize, dry, recrystallization obtain five fragments of peptides [5-9] [NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2]。
In above-mentioned steps (3), the preparation method of four fragments of peptides [1-4] [Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH] is:
(3a), the preparation of Mpa (Trt)-D-Tyr (Et)-OH:
Being dissolved in organic solvent by Mpa (Trt)-OH and HOSu, add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Mpa (Trt)-OSu；
Mpa (Trt)-OSu is added in the alkaline salt solution of H-D-Tyr (Et)-OH HCl, TLC detects reaction end, regulate solution ph to 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Mpa (Trt)-D-Tyr (Et)-OH；
(3b), the preparation of Mpa (Trt)-D-Tyr (Et)-Ile-OH:
Mpa (Trt)-D-Tyr (Et)-OH and the HOSu of preparation in step (3a) is dissolved in organic solvent, add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Mpa (Trt)-D-Tyr (Et)-OSu；
Mpa (Trt)-D-Tyr (Et)-OSu is joined in the alkaline salt solution of H-Ile-OH, TLC detection reaction is eventually, regulate solution ph to 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Mpa (Trt)-D-Tyr (Et)-Ile-OH；
(3c), the preparation of Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH:
Mpa (Trt)-D-Tyr (Et)-Ile-OH and the HOSu of preparation in step (3b) is dissolved in organic solvent, add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Mpa (Trt)-D-Tyr (Et)-Ile-OSu；
Mpa (Trt)-D-Tyr (Et)-Ile-OSu is joined in the alkaline salt solution of H-Thr (tBu)-OH, TLC detects reaction end, regulate solution ph to 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain four fragments of peptides [1-4]: [Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH]；
Wherein reactions steps (3a), (3b), in (3c): in the preparation method of four fragments of peptides, Mpa (Trt)-OH, Mpa (Trt)-D-Tyr (Et)-OH, Mpa (Trt)-D-Tyr (Et)-Ile-OH are 1:1～1.1:1～1.1 with HOSu and DCC mol ratio respectively；H-D-Tyr (Et)-OH, H-Ile-OH, H-Thr (tBu)-OH are 1:1～2 with basic salt mol ratio respectively；Mpa (Trt)-Osu and H-D-Tyr (Et)-OH HCl, Mpa (Trt)-D-Tyr (Et)-Osu and H-Ile-OH, Mpa (Trt)-D-Tyr (Et)-Ile-Osu and H-Thr (tBu)-OH mol ratio be 1:0.8～1.2.
In above-mentioned steps, organic solvent is one or more in oxolane, dioxane, acetone；Basic salt is the one in sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate.
With full guard peptide chain Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (the Boc)-Gly-NH of Side chain protective group in above-mentioned steps (4)2Preparation method be:
Four fragments of peptides [1-4] Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH and the HOSu of preparation in step (3) is reacted to obtain Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OSu then five fragments of peptides [5-9] NH that prepare middle with step (2)2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2Coupling must with peptide chain Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (the Boc)-Gly-NH of Side chain protective group2；Wherein Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OSu is 1:0.8～1.2 with the ratio of five fragments of peptides.
Lytic reagent in step (5) is the trifluoroacetic acid solution adding volume ratio 1-5% scavenger；Described scavenger is one or more in tri isopropyl silane, thioanisole, 1,2-dithioglycol, phenol, water.
Preferably, a kind of liquid phase prepares the method for atosiban, comprises the following steps:
(1), preparation Tfa-Asn-OH
Dissolve H-Asn-OH with trifluoroacetic acid, at-10 DEG C, add the trifluoroacetic anhydride of 1 times of mole, react 30 minutes at 10 DEG C, through concentrating under reduced pressure at 30 DEG C, hot toluene crystallize, dry, recrystallization, obtain Tfa-Asn-OH；
(2) five fragments of peptides [5-9], are prepared
Being dissolved in oxolane by Tfa-Asn-OH and HOSu, be slowly added dropwise into the tetrahydrofuran solution containing DCC under condition of ice bath, reaction is stirred at room temperature, reaction filters insoluble matter, concentrated filtrate after terminating, add petroleum ether crystallize, dries to obtain Tfa-Asn-Osu；
H-Cys (Trt)-OH is dissolved in the sodium bicarbonate aqueous solution containing equimolar amounts, the tetrahydrofuran solution of Tfa-Asn-OSu containing equimolar amounts is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates oxolane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, and extraction into ethyl acetate 3 times merges organic facies, concentrated by rotary evaporation, saturated common salt is washed 3 times, and anhydrous sodium sulfate dries, and adds petroleum ether crystallize, collected by filtration, dried recrystallization obtains Tfa-Asn-Cys (Trt)-OH；
Coupling H-Pro-OH, H-Orn (Boc)-OH, H-Gly-NH successively2, obtain fragment Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2, slough N end Tfa with the aqueous piperidine solution of 1M and protect base, obtain five fragments of peptides [5-9]: NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2；
(3) four fragments of peptides [1-4], are prepared
Being dissolved in acetone by Mpa (Trt)-OH and HOSu, be slowly added dropwise into the acetone soln containing DCC under condition of ice bath, reaction is stirred at room temperature, reaction filters insoluble matter, concentrated filtrate after terminating, and adds petroleum ether crystallize and obtains Mpa (Trt)-OSu.
H-D-Tyr (Et)-OH HCl is dissolved in the potassium bicarbonate aqueous solution of 2 times of moles, the acetone soln of Mpa (Trt)-OSu containing equivalent molar amount is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates acetone, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, and extraction into ethyl acetate 3 times merges organic facies, concentrated by rotary evaporation, saturated common salt is washed 3 times, and anhydrous sodium sulfate dries, and adds petroleum ether crystallize, collected by filtration, dried recrystallization obtains Mpa (Trt)-D-Tyr (Et)-OH；
Then coupling H-Ile-OH, H-Thr (tBu)-OH successively, obtains four fragments of peptides [1-4]: Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH；
(4), preparation full guard peptide chain
Four fragments of peptides [1-4] Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH and HOSu is dissolved in oxolane, the tetrahydrofuran solution of DCC it is slowly added dropwise while stirring under condition of ice bath, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate, adds petroleum ether crystallize and obtains Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OSu；
By five fragments of peptides [5-9] NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2It is dissolved in aqueous sodium carbonate, the dioxane solution of Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OSu is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates organic solvent, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, solid collected by filtration, wash 3 times, dried Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2；
(5) atosiban linearly thick peptide, is prepared
The peptide chain with Side chain protective group of preparation in step (4) is added in the lysate of the TFA/ phenol/water/thioanisole/1，2-ethandithiol=82.5/5/5/5/2.5 after pre-freeze, nitrogen protection; lucifuge; stirring reaction 0.5h under ice bath, recession deicing bath, 25 DEG C of stirring reaction 3.5h；Lysate is slowly poured in the tertiary ether of cold first, stand in refrigerator-freezer, centrifugal to obtain solid, wash 6 final vacuums with the tertiary ether of first and dry to remove linear thick peptide Mpa-D-Tyr (the Et)-IIe-Thr-Asn-Cys-Pro-Orn-Gly-NH of side chain2；
(6), liquid phase oxidation, prepare the linear fine peptide of atosiban
Weigh atosiban linearly thick peptide in step (5) and be dissolved in the acetonitrile/water solution that volume ratio is 2/8 according to the ratio of 1mg/ml, carbonic acid ammonia regulates the pH value of solution to 8.0, stirring reaction under room temperature, HPLC monitors reaction process, reaction adds glacial acetic acid and regulates the pH value of solution to 4.0～5.0 after terminating, rotation is steamed, remove acetonitrile, after HPLC purification, lyophilizing, obtain atosiban acetate fine peptide c [Mpa-D-Tyr (Et)-IIe-Thr-Asn-Cys]-Pro-Orn-Gly-NH2。
The invention has the beneficial effects as follows:
(1) synthesis of atosiban linear peptides use trifluoroacetyl group as the N end protection base of five fragments of peptides; select Trt, tBu, Boc as Side chain protective group; the lytic reagent containing trifluoroacetic acid of these 3 kinds of protection bases routines can be removed; without using the liquid nitrogen/sodium under hydrogenolysis or pole cryogenic conditions to process; simple and easy to do; greatly reduce operation easier, thus it is harsh to solve side chain benzyl elimination condition in the liquid-phase synthesis process of Boc strategy, the problem of difficult operation；
(2) liquid phase process is all adopted; linear peptides divides two sections of synthesis; operation is simple; compared to solid-phase synthesis or solid-liquid combination method; production equipment is simple; do not need resin, the protected amino acid of excess and condensing agent, substantial amounts of cleaning solvent, lower in cost, it is easier to realize large-scale industrial production.
Detailed description of the invention
With specific embodiment, the present invention is described in detail below, but does not limit this patent；Rate of charge according to feed change of the present invention or reaction dissolvent or and condensing agent etc., all in protection scope of the present invention.
The abbreviation implication used in specification and claims is as follows:
The tBu tert-butyl group
Trt trityl
Tfa trifluoroacetyl group
Boc tertbutyloxycarbonyl
HOSuN-N-Hydroxysuccinimide
DCC dicyclohexylcarbodiimide
The preparation of embodiment 1:Tfa-Asn-OH
26.42g (0.20mol) H-Asn-OH is dissolved in 120ml trifluoroacetic acid, adds 28.19ml (0.20mol) trifluoroacetic anhydride when-10 DEG C, be warmed up to 10 DEG C, stir 30 minutes.30 DEG C are evaporated to 60ml, add 120ml hot toluene, place.Collected by filtration, dried recrystallization, obtain 40.18gTfa-Asn-OH, yield 88.05%.
The preparation of embodiment 2:Tfa-Asn-OH
39.64g (0.3mol) H-Asn-OH is dissolved in 150ml trifluoroacetic acid, adds 50.75ml (0.36mol) trifluoroacetic anhydride when-10 DEG C, be warmed up to 10 DEG C, stir 30 minutes.30 DEG C are evaporated to 70ml, add 150ml hot toluene, place.Collected by filtration, dried recrystallization, obtain 59.01gTfa-Asn-OH, yield 86.22%.
The preparation of embodiment 3:Tfa-Asn-Cys (Trt)-OH
99.00g (0.43mol) Tfa-Asn-OH and 49.48g (0.43mol) HOSu of preparation in embodiment 1 or 2 is dissolved in 800ml oxolane, it is slowly added dropwise into the 400ml tetrahydrofuran solution containing 88.66g (0.43mol) DCC under condition of ice bath while stirring, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate is to 500ml, add 1000ml petroleum ether crystallize, dry to obtain 123.62gTfa-Asn-Osu, yield 88.37%.
109.08g (0.30mol) H-Cys (Trt)-OH is dissolved in the 500ml aqueous solution containing 25.20g (0.30mol) sodium bicarbonate, the tetrahydrofuran solution 1000ml of Tfa-Asn-OSu containing 117.11g (0.36mol) above-mentioned preparation is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates oxolane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, the extraction into ethyl acetate of 1000ml 3 times, merge organic facies, concentrated by rotary evaporation is to 1000ml, 200ml saturated common salt is washed 3 times, anhydrous sodium sulfate dries, add 2000ml petroleum ether crystallize, collected by filtration, dried recrystallization obtains 143.64gTfa-Asn-Cys (Trt)-OH, yield 83.45%.
The preparation of embodiment 4:Tfa-Asn-Cys (Trt)-Pro-OH
143.64g (0.25mol) Tfa-Asn-Cys (Trt)-OH and 31.07g (0.27mol) HOSu of preparation in embodiment 3 is dissolved in 1000ml oxolane, it is slowly added dropwise into the 400ml tetrahydrofuran solution containing 55.67g (0.27mol) DCC under condition of ice bath while stirring, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate is to 600ml, add 1200ml petroleum ether crystallize, dry to obtain 148.74gTfa-Asn-Cys (Trt)-OSu, yield 88.69%.
25.33g (0.22mol) H-Pro-OH is dissolved in the 370ml aqueous solution containing 33.04g (0.33mol) potassium bicarbonate, the dioxane solution 1000ml of 148.74g (0.22mol) Tfa-Asn-Cys (Trt)-OSu into above-mentioned preparation it is slowly added dropwise at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates dioxane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, the extraction into ethyl acetate of 1000ml 3 times, merge organic facies, concentrated by rotary evaporation is to 1000ml, 200ml saturated common salt is washed 3 times, anhydrous sodium sulfate dries, add 2000ml petroleum ether crystallize, collected by filtration, dried recrystallization obtains 126.75gTfa-Asn-Cys (Trt)-Pro-OH, yield 85.88%.
The preparation of embodiment 5:Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH
126.75g (0.19mol) Tfa-Asn-Cys (Trt)-Pro-OH and 24.17g (0.21mol) HOSu of preparation in embodiment 4 is dissolved in 1000ml oxolane, it is slowly added dropwise into the 400ml tetrahydrofuran solution containing 43.30g (0.21mol) DCC under condition of ice bath while stirring, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate is to 700ml, add 1500ml petroleum ether crystallize, dry to obtain 128.37gTfa-Asn-Cys (Trt)-Pro-OSu, yield 87.98%.
39.49g (0.17mol) H-Orn (Boc)-OH is dissolved in the 290ml aqueous solution containing 28.56g (0.34mol) sodium bicarbonate, the tetrahydrofuran solution containing 128.37g (0.17mol) Tfa-Asn-Cys (Trt)-Pro-OSu of the 1000ml into above-mentioned preparation it is slowly added dropwise at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates oxolane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, the extraction into ethyl acetate of 1000ml 3 times, merge organic facies, concentrated by rotary evaporation is to 1000ml, 200ml saturated common salt is washed 3 times, anhydrous sodium sulfate dries, add 2000ml petroleum ether crystallize, collected by filtration, dried recrystallization obtains 124.81gTfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH, yield 82.94%.
Embodiment 6:Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2Preparation
124.81g (0.14mol) Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH and the HOSu17.26g (0.15mol) of preparation in embodiment 5 is dissolved in 1000ml oxolane, it is slowly added dropwise into the 300ml tetrahydrofuran solution containing 30.93g (0.15mol) DCC under condition of ice bath while stirring, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate is to 700ml, add 1500ml petroleum ether crystallize, dry to obtain 118.85gTfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OSu, yield 86.43%.
By 13.26g (0.12mol) H-Gly-NH2HCl is dissolved in the 200ml aqueous solution containing 20.16g (0.24mol) sodium bicarbonate, the dioxane solution 1000ml into 118.85g (0.12mol) Tfa-Asn-Cys (Trt)-Pro-Orn (the Boc)-OSu containing above-mentioned preparation it is slowly added dropwise at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates dioxane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, the extraction into ethyl acetate of 1000ml 3 times, merge organic facies, concentrated by rotary evaporation is to 1000ml, 200ml saturated common salt is washed 3 times, anhydrous sodium sulfate dries, add 2000ml petroleum ether crystallize, collected by filtration, dried recrystallization obtains 95.17gTfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2, yield 84.26%.
Embodiment 7:NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2Preparation
By 95.17gTfa-Asn-Cys (Trt)-Pro-Orn (the Boc)-Gly-NH of gained in embodiment 62Add in the aqueous piperidine solution (1000ml) of 1M and dissolve, stirring reaction 20～24h, dilute hydrochloric acid adjusts solution ph to 2～3, adds 1000ml extraction into ethyl acetate 3 times, merges organic facies, concentrated by rotary evaporation is to 1000ml, 200ml saturated common salt is washed 3 times, and anhydrous sodium sulfate dries, and adds 2000ml petroleum ether crystallize, collected by filtration, dried recrystallization obtains 76.09g five fragments of peptides [5-9] [NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2], yield 89.03%.
The preparation of embodiment 8:Mpa (Trt)-D-Tyr (Et)-OH
First 104.54g (0.30mol) Mpa (Trt)-OH and 34.52g (0.30mol) HOSu is dissolved in 1000ml oxolane, again 61.85g (0.30mol) DCC 600ml oxolane is dissolved, it is slowly added dropwise while stirring under condition of ice bath in above-mentioned solution, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate is to 800ml, add 1600ml petroleum ether crystallize and obtain 110.66gMpa (Trt)-Osu, yield 82.79%.
61.37g (0.25mol) H-D-Tyr (Et)-OH HCl is dissolved in the 450ml aqueous solution containing 26.50g (0.25mol) sodium carbonate, the 1000ml dioxane solution of 110.66g (0.25mol) Mpa (Trt)-OSu is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates dioxane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, add 1000ml extraction into ethyl acetate 3 times, merge organic facies, concentrated by rotary evaporation is to 1000ml, 200ml saturated common salt is washed 3 times, anhydrous sodium sulfate dries, add 2000ml petroleum ether crystallize, collected by filtration, dried recrystallization obtains 113.08gMpa (Trt)-D-Tyr (Et)-OH, yield 83.77%.
The preparation of embodiment 9:Mpa (Trt)-D-Tyr (Et)-Ile-OH
First 113.08g (0.21mol) Mpa (Trt)-D-Tyr (Et)-OH and 26.47g (0.23mol) HOSu of preparation in embodiment 8 is dissolved in 1000ml oxolane, 47.42g (0.23mol) DCC is dissolved again with 500ml oxolane, it is slowly added dropwise while stirring under condition of ice bath in above-mentioned solution, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate is to 700ml, add 1500ml petroleum ether crystallize and obtain 110.30gMpa (Trt)-D-Tyr (Et)-Osu, yield 82.45%.
22.30g (0.17mol) H-Ile-OH is dissolved in the 300ml aqueous solution containing 36.04g (0.34mol) sodium carbonate, the dioxane solution of 110.30g (0.17mol) Mpa (Trt)-D-Tyr (the Et)-OSu of above-mentioned preparation is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates dioxane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, add 1000ml extraction into ethyl acetate 3 times, merge organic facies, concentrated by rotary evaporation is to 1000ml, 200ml saturated common salt is washed 3 times, anhydrous sodium sulfate dries, add 2000ml petroleum ether crystallize, collected by filtration, dried recrystallization obtains 93.62gMpa (Trt)-D-Tyr (Et)-Ile-OH, yield 84.32%.
The preparation of embodiment 10:Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH
93.62g (0.14mol) Mpa (Trt)-D-Tyr (Et)-Ile-OH and 17.26g (0.15mol) HOSu of preparation in embodiment 9 is dissolved in 1000ml oxolane, dissolving with 300ml oxolane is slowly added dropwise in above-mentioned solution under 30.93g (0.15mol) DCC condition of ice bath while stirring again, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate is to 600ml, add 1200ml petroleum ether crystallize and obtain 92.68gMpa (Trt)-D-Tyr (Et)-Ile-Osu, yield 88.25%.
21.03g (0.12mol) H-Thr (tBu)-OH is dissolved in the 200ml aqueous solution containing 33.17g (0.24mol) potassium carbonate, the acetone soln 800ml of 92.68g (0.12mol) Mpa (Trt)-D-Tyr (the Et)-Ile-OSu of above-mentioned preparation is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates acetone, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, 1000ml extraction into ethyl acetate 3 times, merge organic facies, concentrated by rotary evaporation is to 1000ml, 200ml saturated common salt is washed 3 times, anhydrous sodium sulfate dries, add 2000ml petroleum ether crystallize, collected by filtration, dried recrystallization obtains 79.17g tetra-fragments of peptides [1-4] [Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH], yield 81.42%.
Embodiment 11:Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2Preparation
Weigh 36.46g (45.00mmol) fragment Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH and 5.70g (49.50mmol) HOSu of preparation in embodiment 10 and be dissolved in 400ml oxolane, the tetrahydrofuran solution 100ml into 10.21g (49.50mmol) DCC it is slowly added dropwise while stirring under condition of ice bath, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate is to 200ml, add 500ml petroleum ether crystallize and obtain 34.93gMpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Osu, yield 85.55%.
Weigh 39.05g (46.20mmol) the five fragments of peptides NH of preparation in embodiment 72-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2It is dissolved in the 100ml aqueous solution containing 7.42g (70.00mmol) sodium carbonate, the dioxane solution 400ml of 34.93g (38.50mmol) Mpa (Trt)-D-Tyr (Et)-Ile-Thr (the tBu)-OSu of above-mentioned preparation is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates dioxane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, solid collected by filtration, the washing of 400ml 3 times, obtain 48.27g peptide chain Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (the Boc)-Gly-NH with Side chain protective group after drying2, yield 76.54%.
Embodiment 12:Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2Preparation
Weigh 40.52g (50.00mmol) fragment Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH and 6.33g (55.00mmol) HOSu of preparation in embodiment 10 and be dissolved in 600ml oxolane, the tetrahydrofuran solution 50ml into 11.34g (55.00mmol) DCC it is slowly added dropwise while stirring under condition of ice bath, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate is to 300ml, add 600ml petroleum ether crystallize and obtain 39.15gMpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Osu, yield 86.28%.
Weigh 33.81g (40.00mmol) the five fragments of peptides NH of preparation in embodiment 72-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2It is dissolved in the 100ml aqueous solution containing 8.48g (80.00mmol) sodium carbonate, the tetrahydrofuran solution 400ml of 36.30g (40.00mmol) Mpa (Trt)-D-Tyr (Et)-Ile-Thr (the tBu)-OSu of above-mentioned preparation is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates oxolane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, solid collected by filtration, the washing of 400ml 3 times, obtain 53.21g peptide chain Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (the Boc)-Gly-NH with Side chain protective group after drying2, yield 81.21%.
Embodiment 13:Mpa-D-Tyr (Et)-IIe-Thr-Asn-Cys-Pro-Orn-Gly-NH2Preparation
According to TFA/ phenol/water/thioanisole/1; the proportions 200ml lysate of 2-dithioglycol=82.5/5/5/5/2.5;-20 DEG C of pre-freeze 1～2h, 48.27g full guard peptide Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (the Boc)-Gly-NH that will obtain in embodiment 112Add in the lysate after pre-freeze, nitrogen protection, lucifuge, stirring reaction 0.5h under ice bath, recession deicing bath, 25 DEG C of stirring reaction 2.5h.Lysate is slowly poured in the tertiary ether of first cold for 2L, stand 1h in refrigerator-freezer, centrifugal to obtain solid, wash 6 final vacuums with the tertiary ether of first and dry to obtain linearly thick peptide 28.07g, purity 90.32%, yield 95.62%.
Embodiment 14:Mpa-D-Tyr (Et)-IIe-Thr-Asn-Cys-Pro-Orn-Gly-NH2Preparation
Proportions 250ml lysate according to TFA/ phenol/water/tri isopropyl silane=88/5/5/2;-20 DEG C of pre-freeze 1～2h, 53.21g full guard peptide Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (the Boc)-Gly-NH that will obtain in embodiment 122Add in the lysate after pre-freeze, nitrogen protection, lucifuge, stirring reaction 0.5h under ice bath, recession deicing bath, 25 DEG C of stirring reaction 3.5h.Lysate is slowly poured in the tertiary ether of first cold for 2.5L, stand 1h in refrigerator-freezer, centrifugal to obtain solid, wash 6 final vacuums with the tertiary ether of first and dry to obtain linearly thick peptide 31.82g, purity 88.74%, yield 98.43%.
Embodiment 15: the preparation of atosiban
Weighing the 5g atosiban of preparation linearly thick peptide in embodiment 13 and be dissolved in 5L acetonitrile/water solution (volume ratio 2/8) according to the ratio of 1mg/ml, the pH value regulating solution with carbonic acid ammonia is 8.0, stirring reaction under room temperature.HPLC monitors reaction process, and reaction adds glacial acetic acid and regulates the pH value of solution to 4.0～5.0 after terminating.Rotation is steamed, and removes acetonitrile, obtains 3.12g atosiban acetate fine peptide c [Mpa-D-Tyr (Et)-IIe-Thr-Asn-Cys]-Pro-Orn-Gly-NH after HPLC purification, lyophilizing2, yield 62.43%, purity 99.72%.
Embodiment 16: the preparation of atosiban
Weighing the 10g atosiban of preparation linearly thick peptide in embodiment 14 and be dissolved in 10L acetonitrile/water solution (volume ratio 2/8) according to the ratio of 1mg/ml, the pH value regulating solution with ammonia is 8.0, stirring reaction under room temperature.HPLC monitors reaction process, and reaction adds glacial acetic acid and regulates the pH value of solution to 4.0～5.0 after terminating.Rotation is steamed, and removes acetonitrile, obtains 6.87g atosiban acetate fine peptide c [Mpa-D-Tyr (Et)-IIe-Thr-Asn-Cys]-Pro-Orn-Gly-NH after HPLC purification, lyophilizing2, yield 68.70%, purity 99.84%.

Claims (9)

1. the method that a liquid phase prepares atosiban, it is characterised in that comprise the following steps:
(1), H-Asn-OH and trifluoroacetic acid anhydride reactant prepare Tfa-Asn-OH；
(2), under liquid-phase condition, Tfa-Asn-OH and H-Cys (Trt)-OH, H-Pro-OH, H-Orn (Boc)-OH, H-Gly-NH2Coupling successively, synthesizes fragment Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2, slough N end Tfa and protect base, obtain five fragments of peptides [5-9]: NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2；
(3), under liquid-phase condition, Mpa (Trt)-OH and H-D-Tyr (Et)-OH, H-Ile-OH, H-Thr (tBu)-OH coupling successively, synthesize four fragments of peptides [1-4]: Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH；
(4), under liquid-phase condition, four fragments of peptides [1-4] and five fragments of peptides [5-9] coupling obtain the full guard peptide chain of Side chain protective group:
Mpa(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2；
(5), peptide chain warp in step (4) cracked, settle, separate to obtain atosiban linearly thick peptide:
Mpa-D-Tyr(Et)-IIe-Thr-Asn-Cys-Pro-Orn-Gly-NH2；
(6), by the thick peptide in step (5) through liquid phase oxidation, preparation liquid phase purification, concentrated freeze-dried after atosiban fine peptide:
c[Mpa-D-Tyr(Et)-IIe-Thr-Asn-Cys]-Pro-Orn-Gly-NH2。
2. the method that a kind of liquid phase according to claim 1 prepares atosiban, it is characterised in that described in step (1), the preparation method of Tfa-Asn-OH is: be dissolved in trifluoroacetic acid by H-Asn-OH, trifluoroacetic anhydride is added when-10 DEG C, it is warmed up to 10 DEG C, stirs 30 minutes, 30 DEG C of concentrating under reduced pressure, add hot toluene, place, collected by filtration, dried recrystallization, obtaining Tfa-Asn-OH, described H-Asn-OH and the mol ratio of trifluoroacetic anhydride are 1:1～1.2.
3. a kind of method that liquid phase prepares atosiban according to claim 1, it is characterised in that fragment Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH described in step (2)2Preparation method be:
(2a), the preparation of Tfa-Asn-Cys (Trt)-OH
Being dissolved in organic solvent by Tfa-Asn-OH and the HOSu of preparation in step (1), add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Tfa-Asn-OSu；
Tfa-Asn-OSu is added in the alkaline salt solution of H-Cys (Trt)-OH, TLC detects reaction end, regulating pH value of solution 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Tfa-Asn-Cys (Trt)-OH；
(2b), the preparation of Tfa-Asn-Cys (Trt)-Pro-OH
Being dissolved in organic solvent by Tfa-Asn-Cys (Trt)-OH and the HOSu of preparation in step (2a), add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Tfa-Asn-Cys (Trt)-OSu；
Tfa-Asn-Cys (Trt)-OSu is added in the alkaline salt solution of H-Pro-OH, TLC detects reaction end, regulating solution ph 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Tfa-Asn-Cys (Trt)-Pro-OH；(2c), the preparation of Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH
Being dissolved in organic solvent by Tfa-Asn-Cys (Trt)-Pro-OH and the HOSu of preparation in step (2b), add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Tfa-Asn-Cys (Trt)-Pro-OSu；
Tfa-Asn-Cys (Trt)-Pro-OSu is added in the alkaline salt solution of H-Orn (Boc)-OH, TLC detects reaction end, regulate solution ph to 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH；
(2d)、Tfa-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2Preparation
Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH and the HOSu of preparation in step (2c) is dissolved in organic solvent, add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OSu；
Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OSu is added H-Gly-NH2In the alkaline salt solution of HCl, TLC detects reaction end, regulates solution ph to 2～3, extraction into ethyl acetate, and concentrated, petroleum ether crystallize, dry, recrystallization obtain Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2；
In reactions steps (2a), (2b), (2c), (2d):
Described Tfa-Asn-OH, Tfa-Asn-Cys (Trt)-OH, Tfa-Asn-Cys (Trt)-Pro-OH, Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-OH are 1:1～1.1:1～1.1 with the mol ratio of HOSu and DCC respectively；
Described H-Cys (Trt)-OH, H-Pro-OH, H-Orn (Boc)-OH, H-Gly-NH2HCl is 1:1～2 with the mol ratio of basic salt respectively；
Described Tfa-Asn-Osu and H-Cys (Trt)-OH, Tfa-Asn-Cys (Trt)-Osu and H-Pro-OH, Tfa-Asn-Cys (Trt)-Pro-Osu and H-Orn (Boc)-OH, Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Osu and H-Gly-NH2The mol ratio of HCl is 1:0.8～1.2.
4. the method that a kind of liquid phase according to claim 1 prepares atosiban; it is characterized in that, the method sloughing N end Tfa protection base described in step (2) is: by fragment Tfa-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2Join in the aqueous piperidine solution of 1M dissolve, stirring reaction 20～24h, adjust solution ph to 2～3, extraction into ethyl acetate, concentration, petroleum ether crystallize, dry, recrystallization obtain five fragments of peptides [5-9] [NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2]。
5. the method that a kind of liquid phase according to claim 1 prepares atosiban, it is characterized in that, the preparation method of four fragments of peptides [1-4] described in step (3) [Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH] is:
(3a), the preparation of Mpa (Trt)-D-Tyr (Et)-OH:
Being dissolved in organic solvent by Mpa (Trt)-OH and HOSu, add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Mpa (Trt)-OSu；
Mpa (Trt)-OSu is added in the alkaline salt solution of H-D-Tyr (Et)-OH HCl, TLC detects reaction end, regulate solution ph to 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Mpa (Trt)-D-Tyr (Et)-OH；
(3b), the preparation of Mpa (Trt)-D-Tyr (Et)-Ile-OH:
Mpa (Trt)-D-Tyr (Et)-OH and the HOSu of preparation in step (3a) is dissolved in organic solvent, add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Mpa (Trt)-D-Tyr (Et)-OSu；
Mpa (Trt)-D-Tyr (Et)-OSu is joined in the alkaline salt solution of H-Ile-OH, TLC detection reaction is eventually, regulate solution ph to 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain Mpa (Trt)-D-Tyr (Et)-Ile-OH；
(3c), the preparation of Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH:
Mpa (Trt)-D-Tyr (Et)-Ile-OH and the HOSu of preparation in step (3b) is dissolved in organic solvent, add DCC, room temperature reaction, through filtering, concentrating, petroleum ether crystallize obtains Mpa (Trt)-D-Tyr (Et)-Ile-OSu；
Mpa (Trt)-D-Tyr (Et)-Ile-OSu is joined in the alkaline salt solution of H-Thr (tBu)-OH, TLC detects reaction end, regulate solution ph to 2～3, extraction into ethyl acetate, concentrated, petroleum ether crystallize, dry, recrystallization obtain four fragments of peptides [1-4]: [Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH]；
In reactions steps (3a), (3b), (3c):
In the preparation method of described four fragments of peptides, Mpa (Trt)-OH, Mpa (Trt)-D-Tyr (Et)-OH, Mpa (Trt)-D-Tyr (Et)-Ile-OH are 1:1～1.1:1～1.1 with HOSu and DCC mol ratio respectively；
Described H-D-Tyr (Et)-OH, H-Ile-OH, H-Thr (tBu)-OH are 1:1～2 with basic salt mol ratio respectively；
Described Mpa (Trt)-Osu and H-D-Tyr (Et)-OH HCl, Mpa (Trt)-D-Tyr (Et)-Osu and H-Ile-OH, Mpa (Trt)-D-Tyr (Et)-Ile-Osu and H-Thr (tBu)-OH mol ratio be 1:0.8～1.2.
6. the method that a kind of liquid phase according to any one of claim 3,5 prepares atosiban, it is characterised in that described organic solvent is one or more in oxolane, dioxane, acetone；Described basic salt is the one in sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate.
7. the method that a kind of liquid phase according to claim 1 prepares atosiban, it is characterised in that with the full guard peptide chain of Side chain protective group in described step (4)
Mpa(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2Preparation method be:
Four fragments of peptides [1-4] Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH and the HOSu of preparation in step (3) is reacted to obtain Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OSu then five fragments of peptides [5-9] NH that prepare middle with step (2)2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2Coupling must with peptide chain Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (the Boc)-Gly-NH of Side chain protective group2；
The ratio of described Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OSu and five fragments of peptides is 1:0.8～1.2.
8. the method that a kind of liquid phase according to claim 1 prepares atosiban, it is characterised in that the lytic reagent described in step (5) is the trifluoroacetic acid solution adding volume ratio 1-5% scavenger；Described scavenger is one or more in tri isopropyl silane, thioanisole, 1,2-dithioglycol, phenol, water.
9. a kind of method that liquid phase prepares atosiban according to claim 1, it is characterised in that comprise the following steps:
(1), preparation Tfa-Asn-OH
Dissolve H-Asn-OH with trifluoroacetic acid, at-10 DEG C, add the trifluoroacetic anhydride of 1 times of mole, react 30 minutes at 10 DEG C, through concentrating under reduced pressure at 30 DEG C, hot toluene crystallize, dry, recrystallization, obtain Tfa-Asn-OH；
(2) five fragments of peptides [5-9], are prepared
Being dissolved in oxolane by Tfa-Asn-OH and HOSu, be slowly added dropwise into the tetrahydrofuran solution containing DCC under condition of ice bath, reaction is stirred at room temperature, reaction filters insoluble matter, concentrated filtrate after terminating, add petroleum ether crystallize, dries to obtain Tfa-Asn-Osu；
H-Cys (Trt)-OH is dissolved in the sodium bicarbonate aqueous solution containing equimolar amounts, the tetrahydrofuran solution of Tfa-Asn-OSu containing equimolar amounts is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates oxolane, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, and extraction into ethyl acetate 3 times merges organic facies, concentrated by rotary evaporation, saturated common salt is washed 3 times, and anhydrous sodium sulfate dries, and adds petroleum ether crystallize, collected by filtration, dried recrystallization obtains Tfa-Asn-Cys (Trt)-OH；
Coupling H-Pro-OH, H-Orn (Boc)-OH, H-Gly-NH successively2, obtain fragment
Tfa-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2, slough N end Tfa with the aqueous piperidine solution of 1M and protect base, obtain five fragments of peptides [5-9]: NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2；
(3) four fragments of peptides [1-4], are prepared
Being dissolved in acetone by Mpa (Trt)-OH and HOSu, be slowly added dropwise into the acetone soln containing DCC under condition of ice bath, reaction is stirred at room temperature, reaction filters insoluble matter, concentrated filtrate after terminating, and adds petroleum ether crystallize and obtains Mpa (Trt)-OSu.
H-D-Tyr (Et)-OH HCl is dissolved in the potassium bicarbonate aqueous solution of 2 times of moles, the acetone soln of Mpa (Trt)-OSu containing equivalent molar amount is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates acetone, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, and extraction into ethyl acetate 3 times merges organic facies, concentrated by rotary evaporation, saturated common salt is washed 3 times, and anhydrous sodium sulfate dries, and adds petroleum ether crystallize, collected by filtration, dried recrystallization obtains Mpa (Trt)-D-Tyr (Et)-OH；Then coupling H-Ile-OH, H-Thr (tBu)-OH successively, obtains four fragments of peptides [1-4]: Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH；
(4), preparation full guard peptide chain
Four fragments of peptides [1-4] Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OH and HOSu is dissolved in oxolane, the tetrahydrofuran solution of DCC it is slowly added dropwise while stirring under condition of ice bath, reaction is stirred at room temperature, reaction filters insoluble matter after terminating, concentrated filtrate, adds petroleum ether crystallize and obtains Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OSu；
By five fragments of peptides [5-9] NH2-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly-NH2It is dissolved in aqueous sodium carbonate, the dioxane solution of Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-OSu is added at 2-8 DEG C, stirring reaction, TLC monitors reaction end, after reaction terminates, decompression evaporates organic solvent, adding 10% aqueous citric acid solution adjusts solution ph to 2～3, solid collected by filtration, wash 3 times, dried Mpa (Trt)-D-Tyr (Et)-Ile-Thr (tBu)-Asn-Cys (Trt)-Pro-Orn (Boc)-Gly-NH2；
(5) atosiban linearly thick peptide, is prepared
The peptide chain with Side chain protective group of preparation in step (4) is added in the lysate of the TFA/ phenol/water/thioanisole/1，2-ethandithiol=82.5/5/5/5/2.5 after pre-freeze, nitrogen protection; lucifuge; stirring reaction 0.5h under ice bath, recession deicing bath, 25 DEG C of stirring reaction 3.5h；Lysate is slowly poured in the tertiary ether of cold first, stand in refrigerator-freezer, centrifugal to obtain solid, wash 6 final vacuums with the tertiary ether of first and dry to remove the linear thick peptide of side chain
Mpa-D-Tyr(Et)-IIe-Thr-Asn-Cys-Pro-Orn-Gly-NH2；
(6), liquid phase oxidation, prepare the linear fine peptide of atosiban
Weigh atosiban linearly thick peptide in step (5) and be dissolved in the acetonitrile/water solution that volume ratio is 2/8 according to the ratio of 1mg/ml, carbonic acid ammonia regulates the pH value of solution to 8.0, stirring reaction under room temperature, HPLC monitors reaction process, reaction adds glacial acetic acid and regulates the pH value of solution to 4.0～5.0 after terminating, rotation is steamed, remove acetonitrile, after HPLC purification, lyophilizing, obtain atosiban acetate fine peptide c [Mpa-D-Tyr (Et)-IIe-Thr-Asn-Cys]-Pro-Orn-Gly-NH2。
CN201610195094.1A 2016-03-31 2016-03-31 Method for liquid phase preparation of atosiban Pending CN105713073A (en)

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CN109810169A (en) * 2017-11-20 2019-05-28 深圳翰宇药业股份有限公司 A kind of liquid phase preparation process of Reltecimod
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