CN102702325B

CN102702325B - A kind of preparation method of anticoagulant peptides

Info

Publication number: CN102702325B
Application number: CN201210203147.1A
Authority: CN
Inventors: 宓鹏程; 覃亮政; 马亚平; 袁建成
Original assignee: Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Co Ltd
Priority date: 2012-06-19
Filing date: 2012-06-19
Publication date: 2015-09-23
Anticipated expiration: 2032-06-19
Also published as: CN102702325A

Abstract

The invention belongs to field of pharmaceutical chemistry technology, relate to a kind of preparation method of anticoagulant peptides, disclose a kind of preparation method of Bivalirudin.The method adopts anti-phase/thick peptide of weak cation exchange mixed mode HPLC method purifying Bivalirudin, then adopts Reversed phase HPLC method to turn salt, collects solution freeze-drying and namely obtains Bivalirudin.Preparation method of the present invention is simple to operate, cost is low, Bivalirudin gram yield is high, be suitable for Bivalirudin large-scale industrialized production, hydrophobicity can be disposablely utilized well to be separated by the impurity in thick peptide with charging property difference and to remove, obtained Bivalirudin purity is high, foreign matter content is low, has considerable economical and practical value and application prospect widely.

Description

A kind of preparation method of anticoagulant peptides

Technical field

The invention belongs to field of pharmaceutical chemistry technology, relate to a kind of preparation method of anticoagulant peptides, particularly relate to a kind of preparation method of Bivalirudin.

Background technology

Bivalirudin (Bivalirudin, Angiomax) be synthetic anticoagulant, its anti-freezing composition is the peptide that hirudin derivative C holds 20 amino-acid residues, and structure sequence is D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Gl u-Ile-Pro-Glu-Glu-Tyr-Leu-OH.Bivalirudin belongs to strong direct thrombin inhibitor, and the negatively charged ion by being incorporated into catalyst site and circulation and zymoplasm clot exports site and direct Trombin inhibiting.Early stage clinical study shows Bivalirudin anticoagulant therapy definite effect, and the incidence of bleeding episode is lower, compares to use safer with traditional heparin anticoagulant therapy, is the wider thrombin inhibitors of clinical application in recent years.

At present about the preparation method of Bivalirudin, existing a large amount of report both at home and abroad.Chinese patent 200910028793.7 reports and adopts full liquid phase process synthesis Bivalirudin; first progressively synthesizing three full guard fragment: N-holds full guard 6 peptide, middle full guard 6 peptide, C-to hold full guard 8 peptide; then by the coupling of three fragments; Bivalirudin crude product is obtained after deprotection base; again through high-efficient liquid phase chromatogram purification, obtain Bivalirudin sterling.Chinese patent 200910051311 discloses a kind of Fmoc solid phase order synthetic method of Bivalirudin, and adopt king's resin to be initial resin, access protected amino acid successively, gained peptide resin adopts trifluoroacetic acid acidolysis to obtain Bivalirudin crude product.US20070093423 reports a kind of preparation method of Bivalirudin, what it adopted is that fragment condensation approach obtains crude product, and the purity obtaining Bivalirudin essence peptide through HPLC purifying is greater than 98.5%, and total mixing is less than 1.5%, single mixing is less than 1.0%, and [Asp9-Bivalirudin] is less than 0.5%.

But above-mentioned patent be all difficult to solve intrinsic in Bivalirudin building-up process, be difficult to the contaminant problem removed.Containing-Gly-Gly-Gly-Gly a fragment in Bivalirudin structure, in coupling-Gly process successively, due to Gly self-characteristic, before and after main peak, very easily produce the impurity of Bivalirudin-Gly and Bivalirudin+Gly, these two impurity are difficult to when later separation remove.Meanwhile, due to the existence of Asn-Gly structure, Asn (removes Fmoc group) in the basic conditions, and mutability is configured to Asp.

Chinese patent 201110170669 and Chinese patent 201110144317 all have employed Fmoc-Gly-Gly-Gly-Gly-OH solves above-mentioned Gly impurity problem as reaction raw materials.But above-mentioned patented method can not solve the allosteric problem of Asn, and reaction raw materials needs self-control, and complex steps, is unfavorable for suitability for industrialized production.

It is reported, which kind of synthetic technology no matter present stage adopt, synthesize the impurity such as Bivalirudin-Gly, Bivalirudin+Gly, Asp9-Bivalirudin, D-Phe 12-Bivalirudin and D-Tyr19-Bivalirudin more or less containing removing in distress in the crude product obtained, all cannot reach the purity of requirements for pharmaceuticals, thus need to carry out polishing purification.Current report or disclosed purification process less.Chinese patent 201110144317.9 discloses a two-step purifying and adds the HPLC purification process that a step turns salt, but the method purification step is many, and cost is higher; The purity that the preparation method of the Bivalirudin of US20070093423 report obtains Bivalirudin essence peptide through HPLC purifying is only greater than 98.5%, Asp9-Bivalirudin and is also only less than 0.5%, and result is all undesirable.Therefore the purifying of Bivalirudin is the difficult point in preparation technology, and especially more than feather weight preparing purifying has on a large scale become one of bottleneck of restriction Bivalirudin industrialization.

Summary of the invention

In view of this, the object of the invention is the defect for prior art, there is provided that a kind of with low cost, technological process is simple, sample impurity is few and stablize the preparation method of controlled Bivalirudin, the yield of the Bivalirudin essence peptide that described preparation method obtains is high, purity is high, and the industrialization being suitable for Bivalirudin is produced.

For realizing object of the present invention, the present invention adopts following technical scheme:

A preparation method for Bivalirudin, adopts anti-phase/thick peptide of weak cation exchange mixed mode HPLC method purifying Bivalirudin, then adopts Reversed phase HPLC method to turn salt, collects solution freeze-drying and namely obtains Bivalirudin.

Bivalirudin is the amphiprotic substance of iso-electric point about 3.7, containing the difficult separating impurity such as Bivalirudin-Gly, Bivalirudin+Gly, Asp9-Bivalirudin, D-Phe12-Bivalirudin and D-Tyr19-Bivalirudin in thick peptide.The present invention, by adopting anti-phase/weak cation exchange mixed mode HPLC method purifying, can disposablely utilize hydrophobicity be well separated by the impurity in thick peptide with charging property difference and remove.Then utilize Reversed phase HPLC method to change into trifluoroacetate API, improve yield and the purity of product, simultaneously easy and simple to handle, be conducive to the preparation realizing mass-producing.

As preferably, of the present invention anti-phase/stationary phase of weak cation exchange mixed mode HPLC method is the chromatograph packing material that surface is bonded with octadecyl and weak cation exchange groups simultaneously.

As preferably, of the present invention anti-phase/mobile phase A of weak cation exchange mixed mode HPLC method is 10-50mmol/L phosphate buffer soln containing 5-10v/v% acetonitrile.

As preferably, of the present invention anti-phase/Mobile phase B of weak cation exchange mixed mode HPLC method is the buffered soln of 10-50mmol/L phosphoric acid salt containing 30-50v/v% acetonitrile and 200-500mmol/L an alkali metal salt.

Wherein, as preferably, described phosphoric acid salt is one or more in sodium phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassiumphosphate, dipotassium hydrogen phosphate, potassium primary phosphate, ammonium phosphate or primary ammonium phosphate.

Further, as preferably, described an alkali metal salt is sodium salt or sylvite.Preferred, be one or more in sodium-chlor, Repone K, sodium-acetate, Potassium ethanoate, sodium phosphate or potassiumphosphate.

As preferably, of the present invention anti-phase/the buffered soln pH of the mobile phase A of weak cation exchange mixed mode HPLC method is 1.5-3.5.。

As preferably, of the present invention anti-phase/the buffered soln pH of the Mobile phase B of weak cation exchange mixed mode HPLC method is 6.0-8.0.

As preferably, the stationary phase that Reversed phase HPLC method of the present invention turns salt is octadecylsilane or eight alkyl silane bonded silica gels.

As preferably, the mobile phase A that Reversed phase HPLC method of the present invention turns salt is 0.05-0.20v/v% trifluoroacetic acid solution.

As preferably, the Mobile phase B that Reversed phase HPLC method of the present invention turns salt is trifluoroacetic acid aqueous solution.

In certain embodiments, the preparation method of Bivalirudin of the present invention, the preparation method of the thick peptide of described Bivalirudin comprises the following steps:

1) solid phase synthesis process is adopted to prepare peptide resin Segment A: Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-resin;

2) solid phase synthesis process is adopted to prepare peptide resin fragment B:NH2-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-resin;

3) peptide resin Segment A, after obtaining full guard peptide fragment, adopts solid phase method and fragment B coupling, obtains Bivalirudin peptide resin fragment;

4) cutting reagent cracking Bivalirudin peptide resin fragment, obtains the thick peptide of Bivalirudin.

The present invention, by the selection in suitable fragment site, makes the amino acid Asn easily producing impurity in the basic conditions be in the end of amino group, decreases the number of times contacted with basic solution DBLK, effectively reduce the generation of Asn allosteric.

As preferably, in step 1) of the present invention, solid phase synthesis process prepares the solid phase carrier of Segment A is CTC resin.

As preferably, the synthesis material that in step 1) of the present invention, in Segment A ,-Gly-Gly-Gly-Gly-structure adopts is Fmoc-Gly-Gly-OH.Namely after the coupling of Fmoc-Gly-Gly-OH and CTC resin, and then carry out coupling with Fmoc-Gly-Gly-OH, thus obtain-Gly-Gly-Gly-Gly-structure.

As preferably, in step 1) of the present invention after the coupling of Fmoc-Gly-Gly-OH and CTC resin, the substitution degree scope of gained is 0.6 ~ 1.0mmol/g.Be more preferably 0.8mmol/g;

As preferably, step 2 of the present invention) in the solid phase synthesis process solid phase carrier of preparing fragment B be king's resin;

As preferably, step 2 of the present invention) in the substitution degree prepared in fragment B after first amino acid Leu and the coupling of Wang resin of solid phase synthesis process be 0.6 ~ 0.8mmol/g.Be more preferably 0.7mmol/g

As preferably, the mixing solutions of the reagent of crack fragment A to be TFE and DCM volume ratio be 1:4 in step 3) of the present invention;

As preferably, the reagent of Segment A and fragment B coupling in step 3) of the present invention is HATU, HOAt and DIPEA, the wherein mol ratio 3-5:3.15-6:6-10 of HATU, HOAt and DIPEA.

As preferably, cutting reagent in step 4) of the present invention is the mix reagent of trifluoroacetic acid, thioanisole, tri isopropyl silane and water, in wherein said mix reagent, trifluoroacetic acid accounts for 88-92v/v%, and thioanisole accounts for 2-4v/v%, tri isopropyl silane accounts for 3-6v/v%, and surplus is water.The volume ratio being more preferably trifluoroacetic acid, thioanisole, tri isopropyl silane and water is 90:4:3:3.

Compared with prior art, preparation method of the present invention is simple to operate, cost is low, Bivalirudin gram yield is high, be suitable for Bivalirudin large-scale industrialized production, and obtained Bivalirudin purity is high, foreign matter content is low, there is considerable economical and practical value and application prospect widely.

Accompanying drawing explanation

Fig. 1 shows the process flow sheet that the present invention is total;

Fig. 2 shows that embodiment 17 Bivalirudin solid essence peptide detects color atlas.

Embodiment

The embodiment of the invention discloses a kind of preparation method of anticoagulant peptides.Those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope method as herein described is changed or suitably change with combination, realize and apply the technology of the present invention.

In order to understand the present invention further, below in conjunction with embodiment, the present invention is described in detail.Wherein, Wang resin and 2-CTC resin purchased from Tianjin Nankai with become company limited; anti-phase/weak cation exchange mixed mode stationary phase is purchased from the Dalian Chemistry and Physics Institute of the Chinese Academy of Sciences, and various protected amino acid is purchased from the biochemical company limited of gill, and other solvent and reagent are common commercially available product.In specification sheets and claims use the implication of english abbreviation to be listed in the following table:

Fmoc	9-fluorenylmethyloxycarbonyl
		2-CTC resin	2-chlorine trityl resin
Boc	Tertbutyloxycarbonyl
		tBu	The tertiary butyl
OtBu	Tert.-butoxy
		Pbf	2,2,4,6,7-pentamethyl-cumarone-5-alkylsulfonyl
Trt	Trityl
		DCM	Methylene dichloride
DBLK	20% hexahydropyridine/DMF solution
		DIPEA	DIPEA
TFE	Trifluoroethanol
		TIS	Tri isopropyl silane
DMF	DMF
		DMAP	DMAP
HPLC	High performance liquid chromatography
		TFA	Trifluoroacetic acid

Embodiment 1: substitution degree is the preparation of the Fmoc-Gly-Gly-CTC Resin of 0.6mmol/g

Take the 2-CTC resin 300g that substitution degree is 1.0mmol/g, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 79.65g Fmoc-Gly-Gly-OH DMF to dissolve, add after 97.8mL DIPEA activates under ice-water bath, add and be above-mentionedly equipped with in the reaction column of resin, react after 2 hours, add 243mL anhydrous methanol and close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol shrinks to be drained, and obtain Fmoc-Gly-Gly-CTC resin, detection substitution degree is 0.628mmol/g.

Embodiment 2: substitution degree is the preparation of the Fmoc-Gly-Gly-CTC Resin of 1.0mmol/g

Take the 2-CTC resin 300g that substitution degree is 1.2mmol/g, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 159.3g Fmoc-Gly-Gly-OH DMF to dissolve, add after 195.6mL DIPEA activates under ice-water bath, add and be above-mentionedly equipped with in the reaction column of resin, react after 2 hours, add 243mL anhydrous methanol and close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol shrinks to be drained, and obtain Fmoc-Gly-Gly-CTC resin, detection substitution degree is 0.988mmol/g.

Embodiment 3: substitution degree is the preparation of the Fmoc-Gly-Gly-CTC Resin of 0.8mmol/g

Take the 2-CTC resin 300g that substitution degree is 1.0mmol/g, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 106.2g Fmoc-Gly-Gly-OH DMF to dissolve, add after 130.4mL DIPEA activates under ice-water bath, add and be above-mentionedly equipped with in the reaction column of resin, react after 2 hours, add 243mL anhydrous methanol and close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol shrinks to be drained, and obtain Fmoc-Gly-Gly-CTC resin, detection substitution degree is 0.806mmol/g.

The preparation of embodiment 4:Fmoc-Gly-Gly-Gly-Gly-CTC Resin

Take the Fmoc-Gly-Gly-CTC resin 372g(300mmol that substitution degree is 0.806mmol/g), add in solid state reaction post, wash 2 times with DMF; with the swelling Fmoc-Gly-Gly-CTC resin of DMF after 30 minutes; remove Fmoc protection with DBLK, then wash 4 times with DMF, DCM washes 2 times.By 318.6gFmoc-Gly-Gly-OH(900mmol), 145.9g HOBt(1080mmol), 136.1gDIC(1080mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add in solid state reaction post, room temperature reaction 2h(reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then react completely, resin develops the color, and represents that reaction not exclusively, needs linked reaction 1h again).Obtain Fmoc-Gly-Gly-Gly-Gly-CTC Resin

The preparation of embodiment 5:Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-CTC resin

Fmoc-Gly-Gly-Gly-Gly-CTC Resin DMF is washed 4 times, and DCM washes 2 times, and remove Fmoc protection with DBLK, then wash 4 times with DMF, DCM washes 2 times.Take 303.7gFmoc-Pro-OH(900mmol), 145.9g HOBt(1080mmol), 136.1gDIC(1080mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add in solid state reaction post, room temperature reaction 2h(reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then react completely, resin develops the color, and represents that reaction not exclusively, needs linked reaction 1h again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to the order of FragmentA; complete Fmoc-Arg (Pbf)-OH successively, Fmoc-Pro-OH, Boc-D--Phe-OH reaction terminates rear methyl alcohol and shrinks; resin vacuum dried overnight, weighs and obtains Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-CTC resin 670.3g.

Embodiment 6: substitution degree is the preparation of the Fmoc-Leu-Wang Resin of 0.6mmol/g

Take the Wang resin 150g(150mmol that substitution degree is 1.0mmol/g), add in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 106.1g Fmoc-Leu-OH, 48.6g HOBt, 45.4g DIC, 3.7g DMAP be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add in solid state reaction post, room temperature reaction 2h.Reaction terminates rear DMF and washs 4 times, and DCM washes 2 times.Then 237.3g pyridine is added and resin 6h closed by 306.3g diacetyl oxide mixed solution.Wash 4 times with DMF, after DCM washs 2 times, methyl alcohol shrinks to be drained, and obtain Fmoc-Leu-Wang resin, detection substitution degree is 0.608mmol/g.

Embodiment 7: substitution degree is the preparation of the Fmoc-Leu-Wang Resin of 0.8mmol/g

Take the Wang resin 150g(150mmol that substitution degree is 1.0mmol/g), add in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 159.1g Fmoc-Leu-OH, 73.0g HOBt, 68.0g DIC, 5.5g DMAP be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add in solid state reaction post, room temperature reaction 2h.Reaction terminates rear DMF and washs 4 times, and DCM washes 2 times.Then 237.3g pyridine is added and resin 6h closed by 306.3g diacetyl oxide mixed solution.Wash 4 times with DMF, after DCM washs 2 times, methyl alcohol shrinks to be drained, and obtain Fmoc-Leu-Wang resin, detection substitution degree is 0.794mmol/g.

Embodiment 8: substitution degree is the preparation of the Fmoc-Leu-Wang Resin of 0.7mmol/g

Take the Wang resin 150g(150mmol that substitution degree is 1.0mmol/g), add in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 132.6g Fmoc-Leu-OH, 60.8g HOBt, 56.7g DIC, 4.6g DMAP be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add in solid state reaction post, room temperature reaction 2h.Reaction terminates rear DMF and washs 4 times, and DCM washes 2 times.Then 237.3g pyridine is added and resin 6h closed by 306.3g diacetyl oxide mixed solution.Wash 4 times with DMF, after DCM washs 2 times, methyl alcohol shrinks to be drained, and obtain Fmoc-Leu-Wang resin, detection substitution degree is 0.682mmol/g.

Embodiment 9:NH ₂the preparation of-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin

Take the Fmoc-Leu-wang resin 183.3g(125mmol that substitution degree is 0.682mmol/g), add in solid state reaction post, wash 2 times with DMF, with DMF swellable resins after 30 minutes, remove Fmoc protection with DBLK, then wash 4 times with DMF, DCM washes 2 times.By 172.3g Fmoc-Tyr (tBu)-OH(375mmol), 60.8g HOBt(450mmol), 56.7g DIC(450mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add in solid state reaction post, room temperature reaction 2h(reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then react completely, resin develops the color, and represents that reaction not exclusively, needs linked reaction 1h again).Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to the order of fragment, complete Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH successively.Reaction terminates rear methyl alcohol and shrinks, and resin vacuum dried overnight, weigh NH ₂-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-wang resin 378.6g.

The preparation of embodiment 10:Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-OH

By 670.3g Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-resin scission reaction wherein, 6703ml lysate (volume ratio, TFE:DCM=1:4) is added, room temperature reaction 2h.Reaction terminates, and filters resin, collects filtrate.Filtrate volume is revolved and steams to < 25vol%, drop to (volume ratio in 20L precipitation reagent, normal hexane: ether=1:4), centrifugal, anhydrous diethyl ether washs, and vacuum-drying, obtains Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-OH fragment 302.8g.Yield is 92.0%, and purity is 95.6%.

The preparation of embodiment 11:Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin

Take 378.6g H ₂-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-wang resin (125mmol); add in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; Fmoc protection is removed with DBLK; then wash 4 times with DMF, DCM washes 2 times.By 274.2g Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-OH(250mmol), 95.1g HATU(250mmol), 40.8g HOAt(300mmol), 64.5g DIPEA(500mmol) be dissolved in DMF mixing solutions, activate 15min under 0 DEG C of condition of ice bath after, add solid state reaction post room temperature reaction 5h(reaction end to be as the criterion with ninhydrin method detection, if resin water white transparency, then react completely, resin develops the color, represent that reaction not exclusively, needs linked reaction 1h again).Reaction terminates rear methyl alcohol and shrinks, resin vacuum dried overnight, weighs and obtains Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin 695.4g.

The preparation of embodiment 12:Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin

Take 379.1g H ₂-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-wang resin (125mmol); add in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; Fmoc protection is removed with DBLK; then wash 4 times with DMF, DCM washes 2 times.By 273.9g Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-OH(250mmol), 80.3g TBTU(250mmol), 40.5g HOBt(300mmol), 64.5g DIPEA(500mmol) be dissolved in DMF mixing solutions, activate 15min under 0 DEG C of condition of ice bath after, add solid state reaction post room temperature reaction 5h(reaction end to be as the criterion with ninhydrin method detection, if resin water white transparency, then react completely, resin develops the color, represent that reaction not exclusively, needs linked reaction 1h again).Reaction terminates rear methyl alcohol and shrinks, resin vacuum dried overnight, weighs and obtains Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin 679.8g.

The preparation of embodiment 13:Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin

Take 377.9g H ₂-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-wang resin (125mmol); add in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; Fmoc protection is removed with DBLK; then wash 4 times with DMF, DCM washes 2 times.By 274.6g Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-OH(250mmol), 94.8g HBTU(250mmol), 40.5g HOBt(300mmol), 64.5g DIPEA(500mmol) be dissolved in DMF mixing solutions, activate 15min under 0 DEG C of condition of ice bath after, add solid state reaction post room temperature reaction 5h(reaction end to be as the criterion with ninhydrin method detection, if resin water white transparency, then react completely, resin develops the color, represent that reaction not exclusively, needs linked reaction 1h again).Reaction terminates rear methyl alcohol and shrinks, resin vacuum dried overnight, weighs and obtains Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin 682..8g.

Embodiment 14: the preparation of Bivalirudin crude product

695.4g Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin is placed in scission reaction wherein, lytic reagent (TFA: thioanisole: TIS: water=90:4:3:3(V/V) is added with the ratio of 10ml/g resin), stirring at room temperature 2.5h.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide 293.6g of Bivalirudin.Weight yield is 107.7%, HPLC purity is 87.9%.Wherein impurity bivalirudin-Gly is 0.12%, bivalirudin+Gly be 0.09%, Asp9-Bivalirudin is 0.03%.

Embodiment 15: the preparation of Bivalirudin crude product

695.4g Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin is placed in scission reaction wherein, lytic reagent (TFA: thioanisole: TIS: water=88:3:2:7(V/V) is added with the ratio of 10ml/g resin), stirring at room temperature 2.5h.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide 270.5g of Bivalirudin.Weight yield is 99.3%, HPLC purity is 84.3%.Wherein impurity bivalirudin-Gly is 0.15%, bivalirudin+Gly be 0.12%, Asp9-Bivalirudin is 0.13%.

Embodiment 16: the preparation of Bivalirudin crude product

695.4g Boc-D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Gly-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-Wang resin is placed in scission reaction wherein, lytic reagent (TFA: thioanisole: TIS: water=92:5:2:1(V/V) is added with the ratio of 10ml/g resin), stirring at room temperature 2.5h.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide 285.6g of Bivalirudin.Weight yield is 104.8%, HPLC purity is 85.8%.Wherein impurity bivalirudin-Gly is 0.12%, bivalirudin+Gly be 0.14%, Asp9-Bivalirudin is 0.08%.

Embodiment 17: the thick peptide purification of Bivalirudin

Dissolved by thick for Bivalirudin peptide 20.0g purified water 1000ml, filter, collection filtrate is for subsequent use.

Purifying chromatographic condition:

Chromatographic column: 100 × 250mm, in-built anti-phase/weak cation exchange mixed mode fixed phase stuffing.

Flow velocity: 250ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: the 20mmol/L sodium dihydrogen phosphate buffer being added with 5% acetonitrile (V/V), adjusts pH to be 2.5 with phosphoric acid.

Mobile phase B phase: the 20mmol/L SODIUM PHOSPHATE, MONOBASIC+300mmol/L sodium chloride buffer solution being added with 40% acetonitrile (V/V), adjusts pH to be 7.5 with sodium hydroxide.

Gradient:

Applied sample amount: 10.0g (500ml).

Purge process: chromatographic column is balanced loading after 5min, run gradient-purified, monitoring and before swarming, behind summit, peak three sections collect object peak cuts.Before peak, peak after cut reclaims purifying after removing most of acetonitrile; Summit cut turns round salt after removing most of acetonitrile.

Divide three sample introductions, repeat above operation.

Turn salt chromatographic condition:

Chromatographic column: 100 × 250mm, in-built anti-phase C18 chromatograph packing material.

Flow velocity: 250ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: 0.1%TFA(V/V) solution.

Mobile phase B phase: trifluoroacetic acid aqueous solution.

Gradient:

Loading volume: 500ml.

Purge process: chromatographic column is balanced loading after 5min, run gradient-purified, monitor and collect object peak cut.Object peak cut vacuum rotary steam is concentrated into freeze-drying after 100ml.

White powdery solids essence peptide 9.85g is obtained after freeze-drying.Chromatogram detects obtained Bivalirudin solid essence peptide, and the results are shown in Figure 2, statistics is in table 1.

The chromatogram detected result of table 1 Bivalirudin solid essence peptide

Peak is numbered	Retention time (min)	Area	Peak height	Area percentage (%)
					1	1.801	7797	856	0.01
2	2.135	8312	1055	0.02
					3	2.693	1972	579	0.00
4	3.154	12265	1267	0.02
					5	3.666	8145	656	0.02
6	4.178	7276	1842	0.01
					7	8.646	4771	547	0.01
8	11.346	16246	1017	0.03
					9	12.492	23998	1558	0.05
10	17.787	8215	402	0.02
					11	19.188	31115	2194	0.06
12	19.981	52089591	1829719	99.63
					13	21.609	21019	771	0.04
14	22.901	19960	661	0.04
					15	25.388	21768	610	0.04

From table 1 result, obtained Bivalirudin solid essence peptide purity 99.63%, Bivalirudin-Gly is 0.04%, Bivalirudin+Gly is 0.06%, and other impurity is all less than 0.05%.Purification yield 78.2%(is with Bivalirudin cubage in crude product), total recovery 49.2%.

Embodiment 18: the thick peptide purification of Bivalirudin

Dissolved by thick for Bivalirudin peptide 6.0g purified water 300ml, filter, collection filtrate is for subsequent use.

Purifying chromatographic condition:

Chromatographic column: 50 × 250mm, in-built anti-phase/weak cation exchange mixed mode fixed phase stuffing.

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: the 20mmol/L sodium dihydrogen phosphate buffer being added with 10% acetonitrile (V/V), adjusts pH to be 2.0 with phosphoric acid.

Mobile phase B phase: the 20mmol/L SODIUM PHOSPHATE, MONOBASIC+200mmol/L sodium chloride buffer solution being added with 40% acetonitrile (V/V), adjusts pH to be 7.5 with sodium hydroxide.

Gradient:

Applied sample amount: 2.0g (100ml).

Divide three sample introductions, repeat above operation.

Turn salt chromatographic condition:

Chromatographic column: 50 × 250mm, in-built anti-phase C8 chromatograph packing material.

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: 0.1%TFA(V/V) solution.

Mobile phase B phase: trifluoroacetic acid aqueous solution.

Gradient:

Loading volume: 300ml.

Purge process: chromatographic column is balanced loading after 5min, run gradient-purified, monitor and collect object peak cut.Object peak cut vacuum rotary steam is concentrated into freeze-drying after 30ml.

Obtain white powdery solids essence peptide 2.92g after freeze-drying, purity 99.62%, Bivalirudin-Gly is 0.04%, Bivalirudin+Gly is 0.05%, and other impurity is all less than 0.05%.Purification yield 77.3%(is with Bivalirudin cubage in crude product), total recovery 48.7%.

Embodiment 19: the thick peptide purification of Bivalirudin

Purifying chromatographic condition:

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: the 30mmol/L potassium dihydrogen phosphate buffer solution being added with 5% acetonitrile (V/V), adjusts pH to be 3.5 with phosphoric acid.

Mobile phase B phase: the 30mmol/L potassium primary phosphate+400mmol/L sodium chloride buffer solution being added with 50% acetonitrile (V/V), adjusts pH to be 6.0 with sodium hydroxide.

Gradient:

Applied sample amount: 2.0g (100ml).

Divide three sample introductions, repeat above operation.

Turn salt chromatographic condition:

Chromatographic column: 50 × 250mm, in-built anti-phase C18 chromatograph packing material.

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: 0.15%TFA(V/V) solution.

Mobile phase B phase: trifluoroacetic acid aqueous solution.

Gradient:

Loading volume: 250ml.

Obtain white powdery solids essence peptide 3.01g after freeze-drying, purity 99.48%, Bivalirudin-Gly is 0.07%, Bivalirudin+Gly is 0.04%, and other impurity is all less than 0.05%.Purification yield 79.7%(is with Bivalirudin cubage in crude product), total recovery 50.2%.

Embodiment 20: the thick peptide purification of Bivalirudin

Dissolved by thick for Bivalirudin peptide 4.0g purified water 300ml, filter, collection filtrate is for subsequent use.

Purifying chromatographic condition:

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: the 50mmol/L buffer solution of sodium phosphate being added with 5% acetonitrile (V/V), adjusts pH to be 1.5 with phosphoric acid.

Mobile phase B phase: the 50mmol/L sodium phosphate+400mmol/L sodium chloride buffer solution being added with 30% acetonitrile (V/V), adjusts pH to be 8.0 with sodium hydroxide.

Gradient:

Applied sample amount: 2.0g (100ml).

Sample introduction at twice, repeats above operation.

Turn salt chromatographic condition:

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: 0.2%TFA(V/V) solution.

Mobile phase B phase: trifluoroacetic acid aqueous solution.

Gradient:

Loading volume: 300ml.

After freeze-drying white powdery solids essence peptide 1.98g, purity is 99.51%, Bivalirudin-Gly be 0.04%, Bivalirudin+Gly is 0.04%, and other impurity is all less than 0.05%.Purification yield 78.6%(is with Bivalirudin cubage in crude product), total recovery 49.5%.

Embodiment 21: the thick peptide purification of Bivalirudin

Dissolved by thick for Bivalirudin peptide 4.0g purified water 200ml, filter, collection filtrate is for subsequent use.

Purifying chromatographic condition:

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: the 50mmol/L primary ammonium phosphate buffered soln being added with 5% acetonitrile (V/V), adjusts pH to be 3.0 with phosphoric acid.

Mobile phase B phase: the 50mmol/L primary ammonium phosphate+100mmol/L sodium chloride buffer solution being added with 50% acetonitrile (V/V), adjusts pH to be 7.0 with sodium hydroxide.

Gradient:

Applied sample amount: 2.0g (100ml).

Sample introduction at twice, repeats above operation.

Turn salt chromatographic condition:

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: 0.1%TFA(V/V) solution.

Mobile phase B phase: trifluoroacetic acid aqueous solution.

Gradient:

Loading volume: 250ml.

Purge process: chromatographic column is balanced loading after 5min, run gradient-purified, monitor and collect object peak cut.Object peak cut vacuum rotary steam is concentrated into freeze-drying after 25ml.

Obtain white powdery solids essence peptide 1.87g after freeze-drying, purity 99.57%, Bivalirudin-Gly is 0.06%, Bivalirudin+Gly is 0.05%, and other impurity is all less than 0.05%.Purification yield 74.3%(is with Bivalirudin cubage in crude product), total recovery 46.8%.

Embodiment 22: the thick peptide purification of Bivalirudin

Purifying chromatographic condition:

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: the 40mmol/L primary ammonium phosphate buffered soln being added with 10% acetonitrile (V/V), adjusts pH to be 3.0 with phosphoric acid.

Mobile phase B phase: the 50mmol/L primary ammonium phosphate+500mmol/L potassium chloride buffer solution being added with 40% acetonitrile (V/V), adjusts pH to be 7.5 with sodium hydroxide.

Gradient:

Applied sample amount: 2.0g (100ml).

Sample introduction at twice, repeats above operation.

Turn salt chromatographic condition:

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: 0.1%TFA(V/V) solution.

Mobile phase B phase: trifluoroacetic acid aqueous solution.

Gradient:

Loading volume: 250ml.

Obtain white powdery solids essence peptide 2.03g after freeze-drying, purity 99.55%, Bivalirudin-Gly is 0.08%, Bivalirudin+Gly is 0.06%, and other impurity is all less than 0.05%.Purification yield 80.6%(is with Bivalirudin cubage in crude product), total recovery 50.8%.

Embodiment 23: the thick peptide purification of Bivalirudin

Purifying chromatographic condition:

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase B phase: the 20mmol/L SODIUM PHOSPHATE, MONOBASIC+300mmol/L sodium chloride buffer solution being added with 50% acetonitrile (V/V), adjusts pH to be 7.0 with sodium hydroxide.

Gradient:

Applied sample amount: 2.0g (100ml).

Divide three sample introductions, repeat above operation.

Turn salt chromatographic condition:

Flow velocity: 80ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: 0.05%TFA(V/V) solution.

Mobile phase B phase: trifluoroacetic acid aqueous solution.

Gradient:

Loading volume: 300ml.

Obtain white powdery solids essence peptide 2.97g after freeze-drying, purity 99.42%, Bivalirudin-Gly is 0.06%, Bivalirudin+Gly is 0.04%, and other impurity is all less than 0.05%.Purification yield 78.5%(is with Bivalirudin cubage in crude product), total recovery 49.5%.

Embodiment 24: the thick peptide purification of Bivalirudin

Dissolved by thick for Bivalirudin peptide 210g purified water 10000ml, filter, collection filtrate is for subsequent use.

Purifying chromatographic condition:

Chromatographic column: 150 × 250mm, in-built anti-phase/weak cation exchange mixed mode fixed phase stuffing.

Flow velocity: 500ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: the 30mmol/L sodium dihydrogen phosphate buffer being added with 10% acetonitrile (V/V), adjusts pH to be 2.5 with phosphoric acid.

Mobile phase B phase: the 30mmol/L SODIUM PHOSPHATE, MONOBASIC+400mmol/L potassium chloride buffer solution being added with 35% acetonitrile (V/V), adjusts pH to be 8.0 with sodium hydroxide.

Gradient:

Applied sample amount: 30.0g (1500ml).

Divide seven sample introductions, repeat above operation.

Turn salt chromatographic condition:

Chromatographic column: 150 × 250mm, in-built anti-phase C18 chromatograph packing material.

Flow velocity: 500ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: 0.07%TFA(V/V) solution.

Mobile phase B phase: trifluoroacetic acid aqueous solution.

Gradient:

Loading volume: 2000ml.

Purge process: chromatographic column is balanced loading after 5min, run gradient-purified, monitor and collect object peak cut.Object peak cut vacuum rotary steam is concentrated into freeze-drying after 750ml.

Obtain white powdery solids essence peptide 104.4g after freeze-drying, purity 99.58%, Bivalirudin-Gly is 0.05%, Bivalirudin+Gly is 0.06%, and other impurity is all less than 0.05%.Purification yield 79.0%(is with Bivalirudin cubage in crude product), total recovery 49.7%.

Embodiment 25: the thick peptide purification of Bivalirudin

Dissolved by thick for Bivalirudin peptide 2200g purified water 75L, filter, collection filtrate is for subsequent use.

Purifying chromatographic condition:

Chromatographic column: 450 × 250mm, in-built anti-phase/weak cation exchange mixed mode fixed phase stuffing.

Flow velocity: 4500ml/min.

Monitoring wavelength: 280nm.

Mobile phase B phase: the 30mmol/L SODIUM PHOSPHATE, MONOBASIC+400mmol/L potassium chloride buffer solution being added with 40% acetonitrile (V/V), adjusts pH to be 8.5 with sodium hydroxide.

Gradient:

Applied sample amount: 250g (8.5L).

Divide seven sample introductions, repeat above operation.

Turn salt chromatographic condition:

Chromatographic column: 450 × 250mm, in-built anti-phase C18 chromatograph packing material.

Flow velocity: 4500ml/min.

Monitoring wavelength: 280nm.

Mobile phase A phase: 0.05%TFA(V/V) solution.

Mobile phase B phase: trifluoroacetic acid aqueous solution.

Gradient:

Loading volume: 15L.

Purge process: chromatographic column is balanced loading after 5min, run gradient-purified, monitor and collect object peak cut.Object peak cut vacuum rotary steam is concentrated into freeze-drying after 8L.

Obtain white powdery solids essence peptide 1102.2g after freeze-drying, purity 99.58%, Bivalirudin-Gly is 0.05%, Bivalirudin+Gly is 0.07%, and other impurity is all less than 0.05%.Purification yield 79.6%(is with Bivalirudin cubage in crude product), total recovery 50.1%.

The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.

Claims

1. the preparation method of a Bivalirudin, it is characterized in that, adopt anti-phase/thick peptide of weak cation exchange mixed mode HPLC method purifying Bivalirudin, then Reversed phase HPLC method is adopted to turn salt, collect solution freeze-drying and namely obtain Bivalirudin, wherein said anti-phase/stationary phase of weak cation exchange mixed mode HPLC method is the chromatograph packing material that surface is bonded with octadecyl and weak cation exchange groups simultaneously; The mobile phase A of anti-phase/weak cation exchange mixed mode HPLC method is the 10-50mmol/L phosphate buffer soln containing 5-10v/v% acetonitrile, and pH is 1.5-3.5; Described anti-phase/Mobile phase B of weak cation exchange mixed mode HPLC method is the buffered soln of 10-50mmol/L phosphoric acid salt containing 30-50v/v% acetonitrile and 200-500mmol/L an alkali metal salt, pH is 6.0-8.0; The stationary phase that described Reversed phase HPLC method turns salt is octadecylsilane or eight alkyl silane bonded silica gels, the mobile phase A that described Reversed phase HPLC method turns salt is 0.05-0.20v/v% trifluoroacetic acid solution, and the Mobile phase B that described Reversed phase HPLC method turns salt is trifluoroacetic acid aqueous solution.

2. preparation method according to claim 1, it is characterized in that, the preparation method of the thick peptide of described Bivalirudin comprises the following steps:

3. preparation method according to claim 2, is characterized in that, described step 1) in the solid phase synthesis process solid phase carrier of preparing Segment A be CTC resin.

4. preparation method according to claim 2, is characterized in that, described step 1) in-Gly-Gly-Gly-Gly-structure adopts in Segment A synthesis material be Fmoc-Gly-Gly-OH.

5. preparation method according to claim 2, is characterized in that, described step 1) in after the coupling of Fmoc-Gly-Gly-OH and CTC resin, the substitution degree of gained is 0.6 ~ 1.0mmol/g.

6. preparation method according to claim 2, is characterized in that, described step 2) in the solid phase synthesis process solid phase carrier of preparing fragment B be king's resin;

7. preparation method according to claim 2, is characterized in that, described step 2) in the substitution degree prepared in fragment B after first amino acid Leu and the coupling of Wang resin of solid phase synthesis process be 0.6 ~ 0.8mmol/g.

8. preparation method according to claim 2, is characterized in that, described step 3) in the reagent of crack fragment A be TFE and DCM volume ratio be the mixing solutions of 1:4.

9. preparation method according to claim 2, is characterized in that, described step 3) in the reagent of Segment A and fragment B coupling be HATU, HOAt and DIPEA, the wherein mol ratio 3-5:3.15-6:6-10 of HATU, HOAt and DIPEA.

10. preparation method according to claim 2, it is characterized in that, described step 4) in cutting reagent be the mix reagent of trifluoroacetic acid, thioanisole, tri isopropyl silane and water, in described mix reagent, gifblaar poison accounts for 88-92v/v%, thioanisole accounts for 2-4v/v%, tri isopropyl silane accounts for 3-6v/v%, and surplus is water.