CN102875650B - Preparation method of barusiban - Google Patents

Abstract

The invention relates to a method for preparing barusiban, which comprises steps of solid-phase synthesis and solid-phase cyclization.

Description

The preparation method of Ba Luxiban

Technical field

The present invention relates to the synthetic field of a peptide species, in particular to the solid phase synthesis field of polypeptide drugs.

Background technology

As the representative medicine of Oxytocin receptor antagonists, Atosiban has obtained clinical verification, and it can be directly and oxytocin competition oxytocin receptor, suppresses oxytocin and oxytocin receptor combination, acts on uterus thereby directly suppress oxytocin, suppresses uterine contraction; Its also can inhibition of phosphatidylinositol3 hydrolytic action, blocking-up second messenger's generation and Ca ²⁺activity, thereby indirectly suppress the reaction of uterus to pitocin, uterine contraction is inhibited, but still has certain limitation, as bigger than normal in dosage, medication is inconvenient etc.

Ba Luxiban is a kind of Atosiban analogue of Ferring company exploitation, and it has higher susceptibility and select specificity as Oxytocin receptor antagonists, but current preparation method is limited and have some shortcomings.U.S. Pat 6143722 has been introduced the method for synthetic Ba Luxiban a kind of, it adopts 2-CTC resin (2-chlorine trityl chloride resin) solid phase synthesis linear peptides resin, then make peptide chain and resin isolation with cracking agent, and make the esterification of peptide chain end, in liquid phase, make again the cyclisation of linear peptides chain, finally make the group reduction of above-mentioned esterification.In this method, because cyclization carries out in liquid phase, so reactions steps is many and last handling process is complicated, cause purity low low with yield, be unfavorable for scale operation.

Kazimierz Wisniewski proposes a kind of method of preparing Ba Luxiban with different material in 29 European polypeptide academic conferences, but this method still adopts the method for solid phase synthesis and liquid phase cyclisation combination, so the shortcoming of liquid phase cyclisation still exists.

Summary of the invention

The object of this invention is to provide a kind of combination solid phase synthesis and solid phase cyclization and prepare the method for Ba Luxiban, comprising:

1) Fmoc-N-Me-Orn (Boc)-ol is reacted under the existence of organic bases in solvent with carboxy resin, obtain Fmoc-N-Me-Orn (Boc)-resin;

2) make Fmoc-N-Me-Orn (Boc)-resin remove Fmoc under the existence of organic bases, then under the existence of coupling agent system in solvent with Fmoc-HomoCys (mmt)-OH coupling, repeat de-Fmoc step and coupling step, coupling Fmoc-Asn (Trt)-OH, Fmoc-AlloIle-OH, Fmoc-Ile-OH, Fmoc-D-Trp (Boc)-OH and 3-halopropanoic acid, obtain linear peptides resin successively

XCH ₂cH ₂cO-NH-D-Trp (Boc)-Ile-AlloIle-Asn (Trt)-HomoCys (mmt)-N-Me-Orn (Boc)-resin,

Wherein X=halogen;

3) make described linear peptides resin remove mmt side chain with side chain deprotection liquid;

4) make the cyclisation of described linear peptides resin with organic bases solution, obtain Ba Luxiban peptide resin;

5) Ba Luxiban peptide resin reacts and obtains Ba Luxiban under the existence of lysate.

Method of the present invention combines solid phase synthesis and solid phase cyclization, and not only reactions steps is few, and aftertreatment is simple, and then has improved purity and yield.

Embodiment

Herein, " substitution degree " refers to the quantity of the resin-carried material (such as phenylformic acid, amino acid etc.) of unit vol, and unit is " mmol/g ".

Herein, " in right amount " represents that the consumption of the material of modifying is not crucial for reaction, as long as can reach required object, without being limited to a concrete scope, but also can once add or add several times, those skilled in the art can rule of thumb select in conjunction with practical situation, for example, control consumption by detection reaction terminal.

Herein, " appropriate time " represents that the time of modifying is not crucial for reaction, as long as can reach required object, without being limited to a concrete scope, those skilled in the art can rule of thumb select in conjunction with practical situation, for example, carry out the period by detection reaction terminal.

The represented Chinese implication of abbreviation occurring is herein set forth in table 1.

The implication of abridging in table 1. literary composition

Fmoc	9-fluorenylmethyloxycarbonyl
		Boc	Tertbutyloxycarbonyl
tBu	The tertiary butyl
		Trt	Trityl
mmt	4-methoxyl group trityl
		TMP	2，4，6-trimethylpyridine
DIPEA	DIPEA

DMAP	4-dimethylamino pyridine
		TEA	Triethylamine
NMP	N-Methyl pyrrolidone
		DMSO	Dimethyl sulfoxide (DMSO)
DMF	DMF
		THF	Tetrahydrofuran (THF)
DCM	Methylene dichloride
		DIC	N, N-DIC
PyBOP	Phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl
		TBTU	O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid
HOAT	1-hydroxyl-7-azo benzotriazole
		HOBT	I-hydroxybenzotriazole
HBTU	2-(7-azo benzotriazole)-tetramethyl-urea phosphofluoric acid ester
		HATU	2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester
TFA	Trifluoroacetic acid
		EDT	1，2-ethandithiol
PhOH	Phenol
		TIS	Tri isopropyl silane
TES	Triethyl silicane
		HPLC	High performance liquid chromatography
Fmoc-N-Me-Orn(Boc)-ol	N-α-fluorenylmethyloxycarbonyl-N-methyl-ornithine (tertbutyloxycarbonyl)-ol
		Fmoc-HomoCys(mmt)-OH	N-α-fluorenylmethyloxycarbonyl-S-4-methoxyl group trityl-L-homocysteine
Fmoc-D-Trp(Boc)-OH	N-α-fluorenylmethyloxycarbonyl-N-in-tertbutyloxycarbonyl-D-trp
		Fmoc-Asn(Trt)-OH	N-α-fluorenylmethyloxycarbonyl-N '-trityl-altheine
Fmoc-Ile-OH	N-α-fluorenylmethyloxycarbonyl-ILE
		Fmoc-AlloIle-OH	N-α-fluorenylmethyloxycarbonyl-L-alloisoleucine

A kind of method that the invention provides Ba Luxiban of preparation, comprising:

1) Fmoc-N-Me-Orn (Boc)-ol is reacted under the existence of organic bases in solvent with carboxy resin, obtain Fmoc-N-Me-Orn (Boc)-resin;

2) make Fmoc-N-Me-Orn (Boc)-resin remove Fmoc under the existence of organic bases, then under the existence of coupling agent system in solvent with Fmoc-HomoCys (mmt)-OH coupling, repeat de-Fmoc step and coupling step, coupling Fmoc-Asn (Trt)-OH, Fmoc-AlloIle-OH, Fmoc-Ile-OH, Fmoc-D-Trp (Boc)-OH and 3-halopropanoic acid, obtain linear peptides resin successively

XCH ₂cH ₂cO-NH-D-Trp (Boc)-Ile-AlloIle-Asn (Trt)-HomoCys (mmt)-N-Me-Orn (Boc)-resin,

Wherein X=halogen;

3) make described linear peptides resin remove mmt side chain with side chain deprotection liquid;

4) make the cyclisation of described linear peptides resin with organic bases solution, obtain Ba Luxiban peptide resin;

5) Ba Luxiban peptide resin reacts and obtains Ba Luxiban under the existence of lysate.

Step 1):

In step 1) in, described carboxy resin includes, but are not limited to phenylformic acid resin, carboxyl vinyl chloride-vinyl acetate resin, carboxyl butyronitrile resin etc., preferably phenylformic acid resin; Before reacting with amino acid, the substitution degree of described carboxy resin is 0.05-3.0mmol/g, preferably 0.1-2.5mmolg, more preferably 0.2-2.0mmol/g; After reacting with amino acid, the substitution degree of resin is 0.05-1.5mmol/g, preferably 0.1-1.2mmol/g, more preferably 0.2-1.0mmol/g; Can be by those skilled in the art's experience, select the consumption of Fmoc-N-Me-Orn (Boc)-ol according to the substitution degree before the consumption of resin, reaction and required reacted substitution degree; Step 1) in organic bases used be the organic bases that is usually used in this object, for example, but be not limited to, TEA, TMP, DMAP, DIPEA etc. and composition thereof, preferably DIPEA or DMAP, its consumption is generally the 0.03-5 equivalent of amino acid mole number, preferably 0.05-3 equivalent, more preferably 0.08-1 equivalent; Solvent used is the solvent that is usually used in this object, such as, but not limited to, DMF, DCM, DMSO, NMP, THF, ethyl acetate, methyl alcohol, ether, etc. and any mixture, preferably DMF, the consumption of solvent is unrestricted, in right amount.

In some embodiments, in step 1) in, preferably under nitrogen bubble, make resin swelling appropriate time (such as, but not limited to, 5-60 minute, preferably 10-50 minute, more preferably 20-40 minute) with appropriate solvent, and Fmoc-N-Me-Orn (Boc)-ol is dissolved in to solvent, then be preferably cooled to-20 ℃ to 20 ℃, preferably-10 ℃ to 10 ℃, more preferably-5 ℃ to 5 ℃, then make its under the existence of alkali in room temperature with through swelling resin reaction appropriate time (such as, but not limited to, 10 minutes to 10 hours, preferably 30 minutes to 5 hours, more preferably 1 hour to 3 hours), carry out afterwards conventional purification process, then preferably under the existence of appropriate above-mentioned organic bases with appropriate alcohol (for example, but be not limited to methyl alcohol, ethanol, propyl alcohol, Virahol, butanols etc., particular methanol) process resin, make alcohol and the carboxyl reaction appropriate time not reacting with amino acid in resin (for example, but be not limited to, 10 minutes to 5 hours, preferably 20 minutes to 3 hours, more preferably 30 minutes to 2 hours), again carry out purifying aftertreatment, then (for example preferably use appropriate above-mentioned alcohol shrinkage resin, but be not limited to, shrink 1-5 time, preferably 2-4 time, more preferably 3 times), finally by product drying (preferably vacuum-drying).Afterwards preferably by metric measurement substitution degree.

Step 2):

In some embodiments, in step 2) in, making the reagent that Fmoc removes is step 1) described in the solution of organic bases, preferred volume concentration is 5-90%, preferably 10-70%, more preferably the DMF solution of the piperidines of 15-50%, its consumption is unrestricted, in right amount, based on the amino acid whose molar weight meter of institute's load on resin, the total free aminoacids of participation coupling consumption is separately 1.5-10 equivalent, preferably 2-5 equivalent, more preferably 2.5-4 equivalent, described coupling agent system is the coupling agent system that is usually used in this object, for example, but be not limited to, DIC/HOBT, DIC/HOAT, HOBT/PyBOP/DIPEA, HOBT/HBTU/DIPEA, HOBT/HBTU/TMP, HOBT/HATU/DIPEA, HOBT/HATU/TMP, HOBT/TBTU/DIPEA, HOBT/TBTU/TMP, HOAT/HBTU/DIPEA, HOAT/HBTU/TMP, HOAT/HATU/DIPEA, HOAT/HATU/TMP, HOAT/TBTU/DIPEA, HOAT/TBTU/TMP, preferably DIC/HOBT, HOBT/PyBOP/DIPEA, the wherein molar weight meter of the total free aminoacids based on participation coupling, DIC, HOBT, HOAT, PyBOP, HBTU, the consumption of HATU and TBTU is respectively 0.1-10 equivalent, preferably 0.5-5 equivalent, more preferably 1-1.5 equivalent, and the consumption of DIPEA and TMP is respectively 0.1-10 equivalent, preferably 0.5-5 equivalent, more preferably 1-3 equivalent, step 2) solvent used is the solvent that is usually used in this object, for example above-mentioned steps 1) described in solvent, the wherein halogen X in linear peptides resin, is fluorine, chlorine, bromine, iodine, preferably chlorine, bromine, iodine.

In some embodiments, in step 2) in, preferably under nitrogen bubble, make Fmoc-N-Me-Orn (Boc)-resin swelling for some time with solvent, then with organic bases solution, Fmoc protecting group is removed, after reaction reasonable time, (preferably carry out above removing twice, every secondary response 5-60 minute, preferably 8-50 minute, more preferably 10-40 minute), with after solvent wash, after triketohydrindene hydrate detection reaction is complete, the amino acid of Fmoc protection will be had, coupling agent system and solvent are in-20 ℃ to 20 ℃, preferably-10 ℃ to 10 ℃, at the temperature of more preferably-5 ℃ to 5 ℃, mix, then make it in room temperature and the swelling resin reaction appropriate time of above-mentioned process (for example, but be not limited to 0.5-10 hour, preferably 1-5 hour, more preferably 1.5-3 hour), then after triketohydrindene hydrate detection reaction is complete, alternately repeat above de-Fmoc and coupling step, successively coupling Fmoc-Asn (Trt)-OH, Fmoc-AlloIle-OH, Fmoc-Ile-OH, Fmoc-D-Trp (Bo c)-OH and 3-halopropanoic acid.Obtain Ba Luxiban linear peptides resin.

Step 3):

In some embodiments, in step 3) in, the reagent that makes HomoCys (mmt) in Ba Luxiban linear peptides resin remove side chain mmt is that volume ratio is the TFA:TES:EDT:DCM side chain deprotection liquid of 1-3:0-5:0-3:89-99; Remove and preferably carry out several, at every turn with appropriate side chain deprotection liquid reaction appropriate time, the color of then observing side chain deprotection liquid, until the color of side chain deprotection liquid while no longer showing yellow, represents that mmt side chain removes completely; Preferably use afterwards step 1) described in solvent wash for several times and dry.

Step 4):

In some embodiments, step 4) described in organic bases such as, but not limited to, DIPEA, TEA, TMP, tetramethyl guanidine, N-methylmorpholine, preferably TEA, DIPEA and tetramethyl guanidine, and the solvent of organic bases solution is step 1) described in solvent; Step 4) organic bases solution in, the volumetric concentration of organic bases is 0.05-10%, preferably 0.1-8%, more preferably 0.5-5%; The consumption of organic bases solution is 0.5-50mL/g resin, preferably 1-30mL/g resin, more preferably 2-20mL/g resin; Reaction times is 1-24 hour, preferably 2-18 hour, more preferably 3-16 hour; Temperature of reaction is room temperature; Reaction end can be tested by spectrophotometry; Preferably purify with conventional purification process afterwards, and preferably use alcohol (such as, but not limited to methyl alcohol, ethanol, propyl alcohol, Virahol, butanols etc., particular methanol) shrink process 1-5 time, preferably 2-4 time, more preferably 3 times, finally by product drying, preferably vacuum-drying.

Step 5):

In some embodiments, in step 5) in, described lysate is that volume ratio is the TFA:TIS:EDT:PhOH:H of 85-95:2-5:0-3:0-2:1-5 ₂o mixture, it can not only make peptide and resin isolation, can also remove Boc and Trt side chain; The consumption of lysate is 2-30mL/g resin, preferably 3-20mL/g resin, and more preferably 5-15mL/g resin, the reaction times is 0.5-10 hour, preferably 1-8 hour, more preferably 1.5-5 hour; Temperature of reaction is room temperature; Reaction mixture can adopt conventional purification process to carry out aftertreatment, such as, but not limited to, adopting the poor solvent precipitator method, HPLC method, gel chromatography or ion chromatography etc., preferably obtain, after thick peptide, further obtaining smart peptide by HPLC by the poor solvent precipitator method; In some preferred embodiments, first filter the resin in reaction mixture, with appropriate TFA washing, merging filtrate, then filtrate being added to appropriate poor solvent (refers to that the solubleness to Ba Luxiban is less than 10 % by weight, is preferably less than 5% under room temperature and normal pressure, is more preferably less than 1% solvent.Such as, but not limited to, anhydrous diethyl ether, methyl tertiary butyl ether etc.) in, by the solid collection of separating out dry, obtain thick peptide, be further purified the thick peptide of gained the dry Ba Luxiban essence peptide that obtains by HPLC afterwards.

Herein, as illustrated without contrary, described reaction is carried out at normal temperatures and pressures.

Wide in range, preferred, preferred and most preferred definition and scope in the present invention can combine mutually.

Following examples are used for explaining the present invention, rather than for limiting the present invention.

Embodiment

Fmoc-N-Me-Orn (Boc)-ol, according to people such as Sun Binyuan, Peking University's journal (natural science edition), the 35th volume, the 4th phase, the method preparation (molecular weight is 454.6g/mol) described in 441-445 page; Carboxy resin purchased from Tianjin Nankai with become (degree of crosslinking 1%; Order number: 100-200 order); Amino acid is all purchased from gill biochemistry.

Reaction column is purchased from dawn glassware factory; Whizzer DR600 is purchased from the vertical whizzer of system in Beijing Jing company limited; Waters 2545RP-HPLC is purchased from Waters company.

Embodiment 1.Fmoc-N-Me-Orn (Boc)-resin is standby

The carboxy resin that is 0.3mmol/g by 100g substitution degree and appropriate DMF add solid state reaction post, swelling 30 minutes of nitrogen bubble.By 36.4g(80mmol) Fmoc-N-Me-Orn (Boc)-ol is dissolved in 200mL DMF, 0 ℃ of ice-water bath 5 minutes, this mixture is added to solid state reaction post, after 2 minutes, add wherein 0.96g DMAP(8mmol), room temperature reaction 2 hours, take out reaction solution, with appropriate DMF washing.Take out after washings, in reaction column, add 200mLDMF, 4.5mL DIPEA(27mmol) and 32mL methyl alcohol in room temperature reaction 1.5 hours, with the carboxyl not reacting with amino acid on sealing resin, take out afterwards confining liquid, with appropriate DMF washing, then wash with appropriate DCM, then shrink appropriate time with appropriate methyl alcohol respectively, vacuum-drying, obtains amino-acid resin 106.5g, and recording substitution degree is 0.25mmol/g.

Embodiment 2.Fmoc-N-Me-Orn (Boc)-resin is standby

The carboxy resin that is 0.8mmol/g by 100g substitution degree and appropriate DMF add solid state reaction post, swelling 30 minutes of nitrogen bubble.By 73.8g(162mmol) Fmoc-N-Me-Orn (Boc)-ol is dissolved in 200mL DMF, 0 ℃ of ice-water bath 5 minutes, this mixture is added to solid state reaction post, after 2 minutes, add wherein 2.1g DMAP(17mmol), room temperature reaction 2 hours, take out reaction solution, with appropriate DMF washing.Take out after washings, in reaction column, add 200mLDMF, 10mL DIPEA(61mmol) and 65mL methyl alcohol in room temperature reaction 1.5 hours, with the carboxyl not reacting with amino acid on sealing resin, take out afterwards confining liquid, with appropriate DMF washing, then wash with appropriate DCM, then shrink appropriate time with appropriate methyl alcohol respectively, vacuum-drying, obtains amino-acid resin 107.7g, and recording substitution degree is 0.58mmol/g.

Embodiment 3.Fmoc-N-Me-Orn (Boc)-resin is standby

The carboxy resin that is 1.2mmol/g by 100g substitution degree and appropriate DMF add solid state reaction post, swelling 30 minutes of nitrogen bubble.By 109.3g(240mmol) Fmoc-N-Me-Orn (Boc)-ol is dissolved in 200mL DMF, 0 ℃ of ice-water bath 5 minutes, this mixture is added to solid state reaction post, after 2 minutes, add wherein 3.2g DMAP(26mmol), room temperature reaction 2 hours, take out reaction solution, with appropriate DMF washing.Take out after washings, in reaction column, add 200mL DMF, 16.3mL DIPEA(99mmol) and 100mL methyl alcohol in room temperature reaction 1.5 hours, with the carboxyl not reacting with amino acid on sealing resin, take out afterwards confining liquid, with appropriate DMF washing, then wash with appropriate DCM, then shrink appropriate time with appropriate methyl alcohol respectively, vacuum-drying, obtains amino-acid resin 108.5g, and recording substitution degree is 0.98mmol/g.

Embodiment 4.

ClCH ₂cH ₂the preparation of CONH-D-Trp (Boc)-Ile-AlloIle-Asn (Trt)-HomoCys (mmt)-N-Me-Orn (Boc)-resin

By 40g(10mmol) substitution degree Fmoc-N-Me-Orn (the Boc)-resin that is 0.25mmol/g and appropriate DMF add reaction column, swelling 30 minutes of nitrogen bubble, with appropriate 20% piperidines/DMF solution reaction appropriate time, to remove Fmoc group, then with appropriate DMF washing, after triketohydrindene hydrate detection reaction is complete, by 18.9g(30mmol) Fmoc-HomoCys (mmt)-OH and 4.45g(33mmol) HOBT is dissolved in 610mL DMF, under 0 ℃ of ice-water bath, add 5.2mL(33mmol) DIC, activate 5 minutes, mixture is added to reaction column, react 2 hours, with (if the colour developing of triketohydrindene hydrate detection reaction terminal, continue reaction 1 hour, if water white transparency, termination reaction).Repeat above step, successively coupling Fmoc-Asn (Trt)-OH, Fmoc-AlloIle-OH, Fmoc-Ile-OH, Fmoc-D-Trp (Boc)-OH and 3-halopropanoic acid.Obtain Ba Luxiban linear peptides resin, drain for subsequent use.

Embodiment 5.

BrCH ₂cH ₂the preparation of CONH-D-Trp (Boc)-Ile-AlloIle-Asn (Trt)-HomoCys (mmt)-N-Me-Orn (Boc)-resin

The Fmoc-N-Me-Orn that is 0.25mmol/g by 40g substitution degree (Boc)-resin and appropriate DMF add reaction column, swelling 30 minutes of nitrogen bubble, with appropriate 20% piperidines/DMF solution reaction appropriate time, to remove Fmoc group, then with appropriate DMF washing, after triketohydrindene hydrate detection reaction is complete, by 18.9g(30mmol) Fmoc-HomoCys (mmt)-OH, 4.45g(33mmol) HOBT and 15.6g(30mmol) PyBOP is dissolved in 610mL DMF, under 0 ℃ of ice-water bath, add 10.5mL DIPEA(60mmol), activate 5 minutes, mixture is added to reaction column, react 2 hours, with (if the colour developing of triketohydrindene hydrate detection reaction terminal, continue reaction 1 hour, if water white transparency, termination reaction).Repeat above step, successively coupling Fmoc-Asn (Trt)-OH, Fmoc-AlloIle-OH, Fmoc-Ile-OH, Fmoc-D-Trp (Boc)-OH and 3-halopropanoic acid.Obtain Ba Luxiban linear peptides resin, drain for subsequent use.

Embodiment 6.

ICH ₂cH ₂the preparation of CONH-D-Trp (Boc)-Ile-AlloIle-Asn (Trt)-HomoCys (mmt)-N-Me-Orn (Boc)-resin

The Fmoc-N-Me-Orn that is 0.25mmol/g by 40g substitution degree (Boc)-resin and appropriate DMF add reaction column, swelling 30 minutes of nitrogen bubble, with appropriate 20% piperidines/DMF solution reaction appropriate time, to remove Fmoc group, then with appropriate DMF washing, after triketohydrindene hydrate detection reaction is complete, by 18.9g(30mmol) Fmoc-HomoCys (mmt)-OH and 4.45g(33mmol) HOBT is dissolved in 610mL DMF, under 0 ℃ of ice-water bath, add 5.2mL(33mmol) DIC, activate 5 minutes, mixture is added to reaction column, react 2 hours, with (if the colour developing of triketohydrindene hydrate detection reaction terminal, continue reaction 1 hour, if water white transparency, termination reaction).Repeat above step, successively coupling Fmoc-Asn (Trt)-OH, Fmoc-AlloIle-OH, Fmoc-Ile-OH, Fmoc-D-Trp (Boc)-OH and 3-halopropanoic acid.Obtain Ba Luxiban linear peptides resin, drain for subsequent use.

Embodiment 7.

ClCH ₂cH ₂the preparation of CONH-D-Trp (Boc)-Ile-AlloIle-Asn (Trt)-HomoCys (mmt)-N-Me-Orn (Boc)-resin

17.24g is got to the Fmoc-N-Me-Orn that substitution degree is 0.58mmol/g (Boc)-resin and add reaction column with appropriate DMF, swelling 30 minutes of nitrogen bubble, with appropriate 20% piperidines/DMF solution reaction appropriate time, to remove Fmoc group, then with appropriate DMF washing, after triketohydrindene hydrate detection reaction is complete, by 18.9g(30mmol) Fmoc-HomoCys (mmt)-OH and 4.45g(33mmol) HOBT is dissolved in 610mL DMF, under 0 ℃ of ice-water bath, add 5.2mL(33mmol) DIC, activate 5 minutes, mixture is added to reaction column, react 2 hours, with (if the colour developing of triketohydrindene hydrate detection reaction terminal, continue reaction 1 hour, if water white transparency, termination reaction).Repeat above step, successively coupling Fmoc-Asn (Trt)-OH, Fmoc-AlloIle-OH, Fmoc-Ile-OH, Fmoc-D-Trp (Boc)-OH and 3-halopropanoic acid.Obtain Ba Luxiban linear peptides resin, drain for subsequent use.

The preparation of embodiment 8. Ba Luxiban resins

The linear peptides resin that embodiment 7 is obtained; with appropriate DCM washing, add wherein afterwards 80mL side chain deprotection liquid TFA:TES:EDT:DCM=3:5:1:91(volume ratio) react 10 minutes, altogether add 3 times; after end, successively with appropriate DCM, appropriate DMF and appropriate DCM washing, drain for subsequent use.

Triethylamine/DMF the solution that is 3% by 100mL volumetric concentration, adds above-mentioned reaction column, room temperature reaction 6 hours, take out reaction solution, with appropriate DMF and appropriate DCM washing, then shrink appropriate time with appropriate methyl alcohol respectively, vacuum-drying, obtains Ba Luxiban resin 25.19g, and yield is 85.06%.

The preparation of embodiment 9. Ba Luxiban resins

The linear peptides resin that embodiment 7 is obtained; with appropriate DCM washing, add wherein afterwards 80mL side chain deprotection liquid TFA:EDT:DCM=2:5:93(volume ratio) react 10 minutes, altogether add 6 times; after end, successively with appropriate DCM, appropriate DMF and appropriate DCM washing, drain for subsequent use.

The DIPEA/DMF solution that is 3% by 100mL volumetric concentration, adds above-mentioned reaction column, room temperature reaction 10 hours, take out reaction solution, with appropriate DMF and appropriate DCM washing, then shrink appropriate time with appropriate methyl alcohol respectively, vacuum-drying, obtains Ba Luxiban resin 26.2g, and yield is 88.33%.

The preparation of embodiment 10. Ba Luxiban resins

The linear peptides resin that embodiment 7 is obtained; with appropriate DCM washing, add wherein afterwards 80mL side chain deprotection liquid TFA:TES:DCM=1:3:96(volume ratio) react 10 minutes, altogether add 12 times; after end, successively with appropriate DCM, appropriate DMF and appropriate DCM washing, drain for subsequent use.

The DIPEA/DMF solution that is 2% by 100mL volumetric concentration, adds above-mentioned reaction column, room temperature reaction 6 hours, take out reaction solution, with appropriate DMF and appropriate DCM washing, then shrink appropriate time with appropriate methyl alcohol respectively, vacuum-drying, obtains Ba Luxiban resin 27.2g, and yield is 91.71%.

The preparation of embodiment 11. Ba Luxiban resins

The linear peptides resin that embodiment 7 is obtained; with appropriate DCM washing, add wherein afterwards 80mL side chain deprotection liquid TFA:EDT:DCM=2:3:95(volume ratio) react 10 minutes, altogether add 5 times; after end, successively with appropriate DCM, appropriate DMF and appropriate DCM washing, drain for subsequent use.

Tetramethyl guanidine/DMF the solution that is 1% by 100mL volumetric concentration, adds above-mentioned reaction column, room temperature reaction 3.5 hours, take out reaction solution, with appropriate DMF and appropriate DCM washing, then shrink appropriate time with appropriate methyl alcohol respectively, vacuum-drying, obtains Ba Luxiban resin 27.9g, and yield is 94.07%.

The preparation of embodiment 12. Ba Luxiban resins

The linear peptides resin that embodiment 7 is obtained; with appropriate DCM washing, add wherein afterwards 80mL side chain deprotection liquid TFA:EDT:DCM=2:3:95(volume ratio) react 10 minutes, altogether add 5 times; after end, successively with appropriate DCM, appropriate DMF and appropriate DCM washing, drain for subsequent use.

Tetramethyl guanidine/DMF the solution that is 1% by 100mL volumetric concentration, adds above-mentioned reaction column, room temperature reaction 8 hours, take out reaction solution, with appropriate DMF and appropriate DCM washing, then shrink appropriate time with appropriate methyl alcohol respectively, vacuum-drying, obtains Ba Luxiban resin 28.3g, and yield is 95.41%.

The preparation of embodiment 13. Ba Luxiban resins

The linear full guard peptide resin that embodiment 7 is obtained; with appropriate DCM washing, add wherein afterwards 80mL side chain deprotection liquid TFA:EDT:DCM=2:3:95(volume ratio) react 10 minutes, altogether add 5 times; after end, successively with appropriate DCM, appropriate DMF and appropriate DCM washing, drain for subsequent use.

Tetramethyl guanidine/DMF the solution that is 1% by 100mL volumetric concentration, adds above-mentioned reaction column, room temperature reaction 16 hours, take out reaction solution, with appropriate DMF and appropriate DCM washing, then shrink appropriate time with appropriate methyl alcohol respectively, vacuum-drying, obtains Ba Luxiban resin 26.5g, and yield is 89.35%.

The preparation of the thick peptide of embodiment 14. Ba Luxiban

The 28.3g peptide resin that embodiment 12 is obtained joins in 500mL single port flask, by 300mL lysate (TFA:TIS:EDT:PhOH:H ₂o=90:3:2:1:4(volume ratio)) join in flask, room temperature reaction 2 hours, filter resin, 20mL TFA washing for resin, merging filtrate, then joins filtrate in 3000mL anhydrous diethyl ether and separates out white solid, centrifugal, wash solid with anhydrous diethyl ether, vacuum-drying obtains white solid 7.7g, yield 92.77%.HPLC purity 82.34%.

The preparation of the thick peptide of embodiment 15. Ba Luxiban

The 28.3g peptide resin that embodiment 12 is obtained joins in 500mL single port flask, by 300mL lysate (TFA:TIS:EDT:H ₂o=91:3:2:4(volume ratio)) join in flask, room temperature reaction 2.5 hours, filter resin, 20mL TFA washing for resin, merging filtrate, then joins filtrate in 3000mL anhydrous diethyl ether and separates out white solid, centrifugal, wash solid with anhydrous diethyl ether, vacuum-drying obtains white solid 7.5g, yield 90.36%.HPLC purity 72.14%.

The preparation of embodiment 16. Ba Luxiban essence peptides

The thick peptide of Ba Luxiban that 3.8g embodiment 14 is obtained is dissolved in 40mL water, and employing Waters2545RP-HPLC system (wavelength 220nm, chromatographic column is the anti-phase C18 post of 50 × 250mm, moving phase: A phase: 0.3%TFA/ acetonitrile solution (v/v); B phase: acetonitrile, gradient: B%:10%-40%, flow velocity: 6 ml/min) collect object peak cut, obtain the smart peptide that purity is greater than 98.5%.Essence peptide is Waters 2545RP-HPLC system (chromatographic column is the anti-phase C18 post of 50 × 250mm, and 0.2% acetum/acetonitrile moving phase turns salt) collection target peak cut for the aqueous solution, and rotary evaporation is concentrated, freeze-drying, obtains Ba Luxiban acetate essence peptide 1.22g, ESI, m/z, C ₄₀h ₆₃n ₉o ₈the calculated value of SNa+ ([M+Na]+) is 851.45, and measured value is 851.6, HPLC purity 99.38%, yield 32.1%.

Claims (31)

1. a method of preparing Ba Luxiban, comprising:

1) Fmoc-N-Me-Orn (Boc)-ol is reacted under the existence of organic bases in solvent with carboxy resin, obtain Fmoc-N-Me-Orn (Boc)-resin;

2) make Fmoc-N-Me-Orn (Boc)-resin remove Fmoc under the existence of organic bases, then under the existence of coupling agent system in solvent with Fmoc-HomoCys (mmt)-OH coupling, repeat de-Fmoc step and coupling step, coupling Fmoc-Asn (Trt)-OH, Fmoc-AlloIle-OH, Fmoc-Ile-OH, Fmoc-D-Trp (Boc)-OH and 3-halopropanoic acid, obtain linear peptides resin XCH successively ₂cH ₂cO-NH-D-Trp (Boc)-Ile-AlloIle-Asn (Trt)-HomoCys (mm t)-N-Me-Orn (Boc)-resin,

Wherein X=halogen;

3) make described linear peptides resin remove mmt side chain with side chain deprotection liquid;

4) make the cyclisation of described linear peptides resin with organic bases solution, obtain Ba Luxiban peptide resin;

5) Ba Luxiban peptide resin reacts and obtains Ba Luxiban under the existence of lysate.

2. the process of claim 1 wherein step 4) described in organic bases be DIPEA, TEA, TMP, tetramethyl guanidine, N-methylmorpholine, its volumetric concentration is 0.05-10%; Organic bases liquor capacity consumption is 0.5-50mL/g resin; Wherein step 4) temperature of reaction be room temperature.

3. the method for claim 2, wherein step 4) described in organic bases be TEA, DIPEA and tetramethyl guanidine.

4. the method for claim 2, wherein step 4) described in organic bases volumetric concentration be 0.1-8%.

5. the method for claim 4, wherein step 4) described in organic bases volumetric concentration be 0.5-5%.

6. the method for claim 2, wherein step 4) described in organic bases liquor capacity consumption be 1-30mL/g resin.

7. the method for claim 6, wherein step 4) described in organic bases liquor capacity consumption be 2-20mL/g resin.

8. the method for claim 1-7 any one, the X in described linear peptides resin, is fluorine, chlorine, bromine, iodine.

9. the method for claim 8, the X in described linear peptides resin, is chlorine, bromine, iodine.

10. the method for claim 1-7 any one, step 5) in the Ba Luxiban that obtains adopt the poor solvent precipitator method, HPLC, gel chromatography or chromatography of ions to carry out purifying.

The method of 11. claim 1-7 any one, step 5) in the Ba Luxiban that obtains adopt the poor solvent precipitator method to carry out purifying, relend and help HPLC to be further purified.

The method of 12. claim 1-7 any one, wherein in step 1) in, described carboxy resin is phenylformic acid resin, carboxyl vinyl chloride-vinyl acetate resin, carboxyl butyronitrile resin; Before reacting with amino acid, the substitution degree of described carboxy resin is 0.05-3.0mmol/g.

The method of 13. claims 12, wherein in step 1) in, described carboxy resin is phenylformic acid resin.

The method of 14. claims 12, wherein in step 1) in, before described carboxy resin reacts with amino acid, the substitution degree of described carboxy resin is 0.1-2.5mmol/g.

The method of 15. claims 14, wherein in step 1) in, before described carboxy resin reacts with amino acid, the substitution degree of described carboxy resin is 0.2-2.0mmol/g.

The method of 16. claim 1-7 any one, wherein in step 1) in, after reacting with amino acid, the substitution degree of resin is 0.05-1.5mmol/g.

The method of 17. claims 16, wherein in step 1) in, after reacting with amino acid, the substitution degree of resin is 0.1-1.2mmol/g.

The method of 18. claims 17, wherein in step 1) in, after reacting with amino acid, the substitution degree of resin is 0.2-1.0mmol/g.

The method of 19. claim 1-7 any one, wherein in step 1) in, after Fmoc-N-Me-Orn (Boc)-ol reacts with carboxy resin, under the existence of appropriate organic bases by appropriate alcohol process resin.

The method of 20. claims 19, wherein said alcohol is methyl alcohol, ethanol, propyl alcohol, Virahol, butanols.

The method of 21. claims 20, wherein said alcohol is methyl alcohol.

The method of 22. claim 1-7 any one, wherein in step 2) in, making the reagent that Fmoc removes is step 1) described in the solution of organic bases, its consumption is unrestricted, in right amount.

The method of 23. claims 22, the solution of wherein said organic bases is that volumetric concentration is the DMF solution of the piperidines of 5-90%.

The method of 24. claims 23, the solution of wherein said organic bases is that volumetric concentration is the DMF solution of the piperidines of 10-70%.

The method of 25. claims 24, the solution of wherein said organic bases is that volumetric concentration is the DMF solution of the piperidines of 15-50%.

The method of 26. claim 1-7 any one; wherein in step 3) in, the reagent that makes HomoCys (mmt) in Ba Luxiban linear peptides resin remove side chain mmt is that volume ratio TFA:TES:EDT:DCM is the side chain deprotection liquid of 1-3:0-5:0-3:89-99.

The method of 27. claim 1-7 any one, wherein in step 5) in, described lysate is that volume ratio TFA:TIS:EDT:PhOH:H2O is the mixture of 85-95:2-5:0-3:0-2:1-5; The consumption of lysate is 2-30mL/g resin, and the reaction times is 0.5-10 hour.

The method of 28. claims 27, the consumption of described lysate is 3-20mL/g resin.

The method of 29. claims 28, the consumption of described lysate is 5-15mL/g resin.

The method of 30. claims 27, wherein in step 5) in, the described lysate reaction times is 1-8 hour.

The method of 31. claims 30, wherein in step 5) in, the described lysate reaction times is 1.5-5 hour.