CN103467575B - A kind of preparation method of Pa Xirui peptide - Google Patents
A kind of preparation method of Pa Xirui peptide Download PDFInfo
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- CN103467575B CN103467575B CN201310370228.5A CN201310370228A CN103467575B CN 103467575 B CN103467575 B CN 103467575B CN 201310370228 A CN201310370228 A CN 201310370228A CN 103467575 B CN103467575 B CN 103467575B
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- 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
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Abstract
The invention belongs to medicinal chemistry art, particularly relate to a kind of preparation method of Pa Xirui peptide.Preparation method of the present invention obtains H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C by changing fragment coupling order
2h
4-NH-CO-O) Pro-OH, then carry out liquid phase cyclisation, cracking obtains target product Pa Xirui peptide crude product, purifying obtains Pa Xirui peptide sterling.Compared with prior art, during preparation method's liquid phase cyclisation of the present invention, the activation of carboxyl is Pro, and the racemization comparing carboxyl terminal Phe reduces greatly, and HPLC does not detect racemization, thus improves Pa Xirui peptide sterling yield, is suitable for large-scale industrialized production.
Description
Technical field
The invention belongs to medicinal chemistry art, particularly relate to a kind of preparation method of Pa Xirui peptide.
Background technology
Cushing's syndrome (Cushingsyndromes), also known as hypercortisolism or hypercortisolism, too much causes primarily of glucocorticoid secretion.The dissimilar cushing's syndrome cause of disease is different, and the suprarenal gland type cushing's syndrome cause of disease is that adrenal cortex has the adenoma or tumour that generate hydrocortisone or other glucocorticosteroid; The hypophysis type cushing's syndrome cause of disease is that adenohypophysis tumour is with thyroliberin supersecretion or ectopic adrenocorticotropic hormone generative nature tumour; The essential cushing's syndrome cause of disease is that adrenal cortical cell improves the susceptibility of thyroliberin.Usually this few type can be distinguished by measuring thyroliberin level at present.The substance metabolism state of diabetes sample that cushing's syndrome cardinal symptom has hyperglycemia, glycosuria and occurs together, is called steroid diabetes; Body fat is redistributed, and causes central obesity and moon shaped face; Breaks down proteins is strengthened causing the structural protein of peripheral organs to reduce, shows as muscle and consumes, unable, skin malnutrition and a kind of osteoporosis; Lymphopenia and eosinopenia disease; The male sex hormone of reticular zone secretion increases, and can cause amenorrhoea, manlike and hirsutism women.
Pa Xirui peptide (Pasireotide), also referred to as ring [(4-NH
2-C
2h
4-NH-CO-O) Pro-Phg-DTrp-Lys-Tyr (4-Bzl)-Phe], wherein Phg represents-HN-CH (C
6h
5)-CO-, Bzl represent benzyl.Pa Xirui peptide structural formula is as follows:
Pa Xirui peptide, commodity are called SIGNIFOR, and 2012 granted, are a kind of injectable ring six peptide somatostatin analogue, are applicable to the treatment not selecting hypophysis operation or the Cushing syndrome adult patients of not curing of performing the operation.Pa Xirui peptide plays its pharmacological activity by combining with somatostatin receptor (ssts).Known 5 people's somatostatin receptor subtypes: hsst1, hsst2, hsst3, hsst4 and hsst5.Under normal physiological conditions, these receptor subtypes are expressed in different tissues.And the frequent process LAN hsst5 of Cushing syndrome patient thyroliberin tumour cell and usually do not express other receptor subtypes or in low expression level.Pa Xirui peptide combines and activates the suppression that hsst acceptor causes thyroliberin (adreno-cortico-tropic-hormone, ACTH) to be secreted, and causes cortisol secretion to lower.
At present about the preparation method of Pa Xirui peptide, have been reported both at home and abroad.Wherein US2009137462, US6225284, US2005014686 and patent families CN01813686, CN96195120 are the basic patent of this compound, the Pa Xirui peptide symthesis method that they are mentioned is first by the Fmoc/tBu strategy of standard, be initial resin with SASRINResin, solid phase condensation obtains { (4-Boc-NH-C
2h
4-NH-CO-O) Pro}-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe, last liquid phase cyclisation, TFA cracking obtain the thick peptide of target product Pa Xirui peptide, then carry out HPLC purifying obtain qualified Pa Xirui peptide essence peptide.But researchist finds, although SASRINResin is also acid sensitive resin, it is expensive that it compares conventional 2-ClTrtResin.And the fragment to obtain due to above-mentioned patent is H-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-OH, carries out liquid phase cyclisation and can produce certain racemization, thus affect final product yield when carboxyl terminal is Phe.
Summary of the invention
In view of this, the object of the invention is to the defect for prior art, the preparation method of the Pa Xirui peptide providing a kind of yield high, the generation of racemization when described method can avoid liquid phase cyclisation, thus improve Pa Xirui peptide yield.
For realizing object of the present invention, the present invention adopts following technical scheme:
A preparation method for Pa Xirui peptide, comprising:
Step 1: by Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH and solid phase carrier coupling, obtain Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin;
Step 2:Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin coupling Fmoc-Phe-OH, Fmoc-Tyr (4-Bzl)-OH, Fmoc-Lys (Boc)-OH, Fmoc-DTrp (Boc)-OH, Fmoc-Phe-OH successively, obtain H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-{(4-Boc-NH-C
2h
4-NH-CO-O) Pro}-Resin;
Step 3:H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-{(4-Boc-NH-C
2h
4-NH-CO-O) Pro}-Resin cracking obtains full guard fragment H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH;
Step 4: by full guard fragment H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH organic solvent dissolution, add activator and carry out liquid phase cyclisation and obtain ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro];
Step 5: ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro] cracking obtains Pa Xirui peptide crude product;
Step 6: the thick peptide purification of Pa Xirui peptide obtains Pa Xirui peptide sterling.
Preferably, solid phase carrier described in step 1) is TrtResin or 2-ClTrtResin, is more preferably 2-ClTrtResin.
Preferably, the initial substitution degree of solid phase carrier described in step 1) is 0.4mmol/g ~ 1.0mmol/g, is more preferably 0.5 ~ 0.6mmol/g.
Preferably, the coupling agent of coupling described in step 1) is diisopropylethylamine.
Preferably, step 2) coupling agent of described coupling is DIC/HOBt, PyBOP/HOBt/DIPEA, TBTU/HOBt/DIPEA or HBTU/HOBt/DIPEA, preferred DIC/HOBt.
Preferably, the cracking agent of cracking described in step 3) is the mixture of TFA and DCM or the mixture of TFE and DCM.
Preferably, organic solvent described in step 4) is DCM, DMF or NMP.
Preferably, activator described in step 4) is PyBop/HOBT/DIPEA, PyBop/DIPEA, DIC/HOAT or DIC/HOBT.More preferably PyBop/HOBT/DIPEA.
Preferably, the mixing solutions of the cracking agent of cracking described in step 5) to be TFA, thioanisole, EDT, TIS and water volume ratio be 86:5:5:3:1.
Preferably, step 6) described purifying is RPLC purifying.
Preparation method of the present invention obtains H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C2H4-NH-CO-O) Pro-OH by changing fragment coupling order, carry out liquid phase cyclisation again, cracking obtains target product Pa Xirui peptide crude product, purifying obtains Pa Xirui peptide sterling.Compared with prior art, during preparation method's liquid phase cyclisation of the present invention, the activation of carboxyl is Pro, and the racemization comparing carboxyl terminal Phe reduces greatly, and HPLC does not detect racemization, thus improves Pa Xirui peptide sterling yield, is suitable for large-scale industrialized production.
Accompanying drawing explanation
Fig. 1 shows that embodiment 17 Pa Xirui peptide crude product detects color atlas.
Embodiment
The embodiment of the invention discloses a kind of preparation method of Pa Xirui peptide.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.
For realizing object of the present invention, the present invention adopts following technical scheme:
A preparation method for Pa Xirui peptide, comprising:
Step 1: by Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH and solid phase carrier coupling, obtain Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin;
Step 2:Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin coupling Fmoc-Phe-OH, Fmoc-Tyr (4-Bzl)-OH, Fmoc-Lys (Boc)-OH, Fmoc-DTrp (Boc)-OH, Fmoc-Phe-OH successively, obtain H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-{(4-Boc-NH-C
2h
4-NH-CO-O) Pro}-Resin;
Step 3:H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-{(4-Boc-NH-C
2h
4-NH-CO-O) Pro}-Resin cracking obtains full guard fragment H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH;
Step 4: by full guard fragment H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH organic solvent dissolution, add activator and carry out liquid phase cyclisation and obtain ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro];
Step 5: ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro] cracking obtains Pa Xirui peptide crude product;
Step 6: the thick peptide purification of Pa Xirui peptide obtains Pa Xirui peptide sterling.
The preparation method of Pa Xirui peptide of the present invention adopts Fmoc/tBu synthesis strategy; first the solid phase carrier coupling of Fmoc-(4-Boc-NH-C2H4-NH-CO-O) Pro-OH and suitable substitution degree is obtained Fmoc-(4-Boc-NH-C2H4-NH-CO-O) Pro-Resin; the amino acid of coupling Fmoc blocking group one by one afterwards, cracking obtains H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH, then liquid phase cyclisation, then carry out cracking and obtain the thick peptide of target product Pa Xirui peptide, purifying obtains Pa Xirui peptide sterling.
Wherein Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH can by commercial channel buy obtain or prepare voluntarily according to existing document.
Preferably, solid phase carrier described in step 1 is TrtResin or 2-ClTrtResin, can reduce resin cost.Be more preferably 2-ClTrtResin.
Further, the initial substitution degree of solid phase carrier described in step 1 is preferably 0.4mmol/g ~ 1.0mmol/g.Be more preferably 0.5 ~ 0.6mmol/g.
Usually coupling agent is needed, with activated amino acid in the amino acid of Fmoc blocking group and the process of solid phase carrier coupling.Preferably, the coupling agent of coupling described in step 1 is diisopropylethylamine (DIPEA).
Preparation method's step 2 of the present invention adopts the mode of coupling successively that Fmoc-Phe-OH, Fmoc-Tyr (4-Bzl)-OH, Fmoc-Lys (Boc)-OH, Fmoc-DTrp (Boc)-OH, Fmoc-Phe-OH are coupled to Fmoc-(4-Boc-NH-C by polypeptide solid-state reaction method
2h
4-NH-CO-O) on Pro-Resin.Wherein, the coupling agent that described coupling mode successively uses is preferably DIC/HOBt, PyBOP/HOBt/DIPEA, TBTU/HOBt/DIPEA or HBTU/HOBt/DIPEA.More preferably DIC/HOBt.
Preparation method's step 3 of the present invention by cracking agent to H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-{(4-Boc-NH-C
2h
4-NH-CO-O) Pro}-Resin carries out cracking, obtains full guard fragment H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH.Wherein, described cracking agent is preferably the mixture of TFA and DCM or the mixture of TFE and DCM.
Further, in the mixture of TFA and DCM described in above-mentioned cracking agent, the volume ratio of TFA and DCM is preferably 0.5:99.5 ~ 5:95, and preferred volume ratio is 1:99.
Further, in the mixture of TFE and DCM described in above-mentioned cracking agent, the volume ratio of TFE and DCM is 20:80.
Preparation method's step 4 of the present invention, by full guard fragment organic solvent dissolution, adds activator and carries out liquid phase cyclisation and obtain ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro].Wherein said organic solvent is preferably DCM, DMF or NMP.Be more preferably DCM.
Further, above-mentioned activator is preferably PyBop/HOBT/DIPEA, PyBop/DIPEA, DIC/HOAT or DIC/HOBT, is more preferably PyBop/HOBT/DIPEA.
Preparation method's step 5 of the present invention by cracking agent to ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro] carry out cracking, obtain Pa Xirui peptide crude product.Wherein, the cracking agent of described cracking is preferably the mixing solutions that TFA, thioanisole, EDT, TIS and water volume ratio are 86:5:5:3:1.
The Pa Xirui peptide crude product that cracking obtains by preparation method's step 6 of the present invention is further purified and obtains Pa Xirui peptide sterling, and wherein said purifying is preferably RPLC purifying.
RPLC, English name reversedphasehighperformanceliquidchromatography, is called for short, RP-HPLC, the liquid chromatographic system be made up of non-polar stationary phase and polarity moving phase.It is just in time contrary with the liquid chromatographic system (normal-phase chromatography) be made up of polar stationary phase and low-pole moving phase.RP-HPLC is the topmost clastotype of current liquid chromatography, almost can be used for all organic separation and purification that can be dissolved in polarity or weak polar solvent.The Pa Xirui peptide crude product that step 5 cracking obtains by preparation method of the present invention adopts reversed-phased high performace liquid chromatographic purifying to obtain Pa Xirui peptide sterling.
Preferably, described reversed-phased high performace liquid chromatographic is specially: with anti-phase octadecylsilane or eight alkyl silane bonded silica gels for stationary phase, after the Pa Xirui peptide crude product solution loading that cracking obtains, purify with 0.2%TFA/ acetonitrile mobile phase, collects object peak cut; With anti-phase octadecylsilane or eight alkyl silane bonded silica gels for stationary phase, turn salt with 0.1% aspartic acid solution/acetonitrile mobile phase, freeze-drying and get final product.
In order to understand the present invention further, below in conjunction with embodiment, the present invention is described in detail.
The implication of the abbreviation used in specification sheets and claims is listed in the following table:
| Abbreviation and English | Implication |
| TRT Resin | Trityl chloride resin |
| 2-Cl TRT Resin | 2-chlorine trityl chloride resin |
| HOAt | 1-hydroxyl-7-azo benzotriazole |
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| DIPCDI | DIC |
| HOBt | I-hydroxybenzotriazole |
| TBTU | O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid |
| PyBOP | Phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl |
| HBTU | Benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate |
| DIPEA | DIPEA |
| DMF | DMF |
| DCM | Methylene dichloride |
| NMP | N-Methyl pyrrolidone |
| TFA | Trifluoroacetic acid |
| PhSMe | Thioanisole |
| DIC | N, N'-DIC |
| TFE | Trifluoroethanol |
| TIS | Tri isopropyl silane |
| EDT | Dithioglycol |
Embodiment 1:Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-2-ClTrtResin
Take the 2-ClTrtResin250g (100mmol) that substitution degree is 0.4mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 107.8gFmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH200mmol is dissolved in DMF solution, adds 69.5mLDIPEA(400mmol under ice-water bath) activate 3min after add in solid state reaction post, again add 34.8mLDIPEA(200mmol after room temperature reaction 5min).Room temperature reaction 60min.Wash 3 times with DMF, add 81mL confining liquid (methyl alcohol: 2000mmol) and close 8 hours (if resin does not spread completely add DMF as solvent).Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-2-ClTrtResin.Detection substitution degree is 0.316mmol/g.
Embodiment 2:Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-2-ClTrtResin
Take the 2-ClTrtResin100g (100mmol) that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 107.8gFmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH200mmol is dissolved in DMF solution, adds 69.5mLDIPEA(400mmol under ice-water bath) activate 3min after add in solid state reaction post, again add 34.8mLDIPEA(200mmol after room temperature reaction 5min).Room temperature reaction 60min.Wash 3 times with DMF, add 81mL confining liquid (methyl alcohol: 2000mmol) and close 8 hours (if resin does not spread completely add DMF as solvent).Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-2-ClTrtResin.Detection substitution degree is 0.903mmol/g.
Embodiment 3:Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-2-ClTrtResin
Take the 2-ClTrtResin200g (100mmol) that substitution degree is 0.5mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 107.8gFmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH200mmol is dissolved in DMF solution, adds 69.5mLDIPEA(400mmol under ice-water bath) activate 3min after add in solid state reaction post, again add 34.8mLDIPEA(200mmol after room temperature reaction 5min).Room temperature reaction 60min.Wash 3 times with DMF, add 81mL confining liquid (methyl alcohol: 2000mmol) and close 8 hours (if resin does not spread completely add DMF as solvent).Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-2-ClTrtResin.Detection substitution degree is 0.413mmol/g.
Embodiment 4:Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-TrtResin
Take the TrtResin250g (100mmol) that substitution degree is 0.4mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 107.8gFmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH200mmol is dissolved in DMF solution, adds 69.5mLDIPEA(400mmol under ice-water bath) activate 3min after add in solid state reaction post, again add 34.8mLDIPEA(200mmol after room temperature reaction 5min).Room temperature reaction 60min.Wash 3 times with DMF, add 81mL confining liquid (methyl alcohol: 2000mmol) and close 8 hours (if resin does not spread completely add DMF as solvent).Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-2TrtResin.Detection substitution degree is 0.302mmol/g.
Embodiment 5:Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-TrtResin
Take the 2-ClTrtResin100g (100mmol) that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 107.8gFmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH200mmol is dissolved in DMF solution, adds 69.5mLDIPEA(400mmol under ice-water bath) activate 3min after add in solid state reaction post, again add 34.8mLDIPEA(200mmol after room temperature reaction 5min).Room temperature reaction 60min.Wash 3 times with DMF, add 81mL confining liquid (methyl alcohol: 2000mmol) and close 8 hours (if resin does not spread completely add DMF as solvent).Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-TrtResin.Detection substitution degree is 0.835mmol/g.
Embodiment 6:Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-TrtResin
Take the 2-ClTrtResin200g (100mmol) that substitution degree is 0.5mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 107.8gFmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH200mmol is dissolved in DMF solution, adds 69.5mLDIPEA(400mmol under ice-water bath) activate 3min after add in solid state reaction post, again add 34.8mLDIPEA(200mmol after room temperature reaction 5min).Room temperature reaction 60min.Wash 3 times with DMF, add 81mL confining liquid (methyl alcohol: 2000mmol) and close 8 hours (if resin does not spread completely add DMF as solvent).Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin.Detection substitution degree is 0.402mmol/g.
Embodiment 7:H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-Resin
Take the Fmoc-(4-Boc-NH-C that embodiment 3 is synthesized
2h
4-NH-CO-O) Pro-2-ClTrtResin24.213g(10mmol), join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Add 20% piperidines/DMF, remove Fmoc by the mode of 10min+10min.Drain reaction solution, and wash resin 6 times with DMF.Take 11.622gFmoc-Phe-OH(30mmol), 4.053gHOBt(30mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 5.19mLDIC(33mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; coupling Fmoc-Tyr (4-Bzl)-OH, Fmoc-Lys (Boc)-OH, Fmoc-DTrp (Boc)-OH, Fmoc-Phg-OH, finally remove Fmoc successively.Methyl alcohol shrinks, and resin vacuum dried overnight, weighs and obtain H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin32.65g.
Embodiment 8:H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-Resin
Take the Fmoc-(4-Boc-NH-C that embodiment 3 is synthesized
2h
4-NH-CO-O) Pro-2-ClTrtResin24.213g(10mmol), join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Add 20% piperidines/DMF, remove Fmoc by the mode of 10min+10min.Drain reaction solution, and wash resin 6 times with DMF.Take 11.622gFmoc-Phe-OH(30mmol), 4.053gHOBt(30mmol), 15.612gPyBop (30mmol) is dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 9.93mLDIPEA(60mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; coupling Fmoc-Tyr (4-Bzl)-OH, Fmoc-Lys (Boc)-OH, Fmoc-DTrp (Boc)-OH, Fmoc-Phg-OH, finally remove Fmoc successively.Methyl alcohol shrinks, and resin vacuum dried overnight, weighs and obtain H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin32.33g.
Embodiment 9:H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-Resin
Take the Fmoc-(4-Boc-NH-C that embodiment 3 is synthesized
2h
4-NH-CO-O) Pro-2-ClTrtResin24.213g(10mmol), join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Add 20% piperidines/DMF, remove Fmoc by the mode of 10min+10min.Drain reaction solution, and wash resin 6 times with DMF.Take 11.622gFmoc-Phe-OH(30mmol), 4.053gHOBt(30mmol), 9.151gTBTU (28.5mmol) is dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 9.93mLDIPEA(60mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; coupling Fmoc-Tyr (4-Bzl)-OH, Fmoc-Lys (Boc)-OH, Fmoc-DTrp (Boc)-OH, Fmoc-Phg-OH, finally remove Fmoc successively.Methyl alcohol shrinks, and resin vacuum dried overnight, weighs and obtain H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin32.39g.
Embodiment 10:H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) synthesis of Pro-Resin
Take the Fmoc-(4-Boc-NH-C that embodiment 3 is synthesized
2h
4-NH-CO-O) Pro-2-ClTrtResin24.213g(10mmol), join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Add 20% piperidines/DMF, remove Fmoc by the mode of 10min+10min.Drain reaction solution, and wash resin 6 times with DMF.Take 11.622gFmoc-Phe-OH(30mmol), 4.053gHOBt(30mmol), 10.810gHBTU (28.5mmol) is dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 9.93mLDIPEA(60mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; coupling Fmoc-Tyr (4-Bzl)-OH, Fmoc-Lys (Boc)-OH, Fmoc-DTrp (Boc)-OH, Fmoc-Phg-OH, finally remove Fmoc successively.Methyl alcohol shrinks, and resin vacuum dried overnight, weighs and obtain H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin32.13g.
Embodiment 11:H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) preparation of Pro-OH
H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C prepared by embodiment 7
2h
4-NH-CO-O) Pro-Resin32.65g is placed in scission reaction bottle, adds lytic reagent (TFA:DCM=1:99(V/V) 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 DCM again, is evaporated to oily after merging filtrate.Adding minimum ethyl acetate dissolves completely by precipitate, lysate is added 10 times of freezing anhydrous diethyl ether precipitations of volume, and wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH.Weight 13.521g, yield 99.0%, HPLC purity is 91.2%.
Embodiment 12:H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) preparation of Pro-OH
H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C prepared by embodiment 7
2h
4-NH-CO-O) Pro-Resin32.65g is placed in scission reaction bottle, adds lytic reagent (TFE:DCM=20:80(V/V) 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 DCM again, is evaporated to oily after merging filtrate.Adding minimum ethyl acetate dissolves completely by precipitate, lysate is added 10 times of freezing anhydrous diethyl ether precipitations of volume, and wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH.Weight 13.384g, yield 98.0%, HPLC purity is 91.0%.
Embodiment 13: ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro] preparation
H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C prepared by embodiment 11
2h
4-NH-CO-O) Pro-OH11.231g is placed in 500mL round-bottomed flask, and it is complete by dissolution of solid to add the dry DCM of 250mL, stir under ice-water bath and add 1.73mLDIC (11mmol), HOAT1.497g(11mmol), maintain ice bath 2h and return to room temperature and continue stirring reaction 2h.Evaporated under reduced pressure solvent is thick to precipitate yellowly; Precipitate is complete with appropriate acetic acid ethyl dissolution, use saturated NaHCO
3, water, washing, dry final vacuum revolves steaming and obtains white solid, is ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro], weight 13.413g, purity 86.8%.
Embodiment 14: ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro] preparation
H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C prepared by embodiment 11
2h
4-NH-CO-O) Pro-OH11.231g is placed in 500mL round-bottomed flask, and it is complete by dissolution of solid to add the dry DCM of 250mL, stir under ice-water bath and add 1.73mLDIC (11mmol), HOBT1.486g(11mmol), maintain ice bath 2h and return to room temperature and continue stirring reaction 2h.Evaporated under reduced pressure solvent is thick to precipitate yellowly; Precipitate is complete with appropriate acetic acid ethyl dissolution, use saturated NaHCO
3, water, washing, dry final vacuum revolves steaming and obtains white solid, is ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro], weight 13.401g, purity 83.2%.
Embodiment 15: ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro] preparation
H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C prepared by embodiment 11
2h
4-NH-CO-O) Pro-OH11.231g is placed in 500mL round-bottomed flask, and it is complete by dissolution of solid to add the dry DCM of 250mL, stir under ice-water bath and add PyBoP5.724g (11mmol), HOBT1.486g(11mmol), DIPEA3.31ml (20mmol), maintain ice bath 2h and return to room temperature and continue stirring reaction 2h.Evaporated under reduced pressure solvent is thick to precipitate yellowly; Precipitate is complete with appropriate acetic acid ethyl dissolution, use saturated NaHCO
3, water, washing, dry final vacuum revolves steaming and obtains white solid, is ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro], weight 13.413g, purity 87.5%.
Embodiment 16: ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro] preparation
H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C prepared by embodiment 11
2h
4-NH-CO-O) Pro-OH11.231g is placed in 500mL round-bottomed flask, and it is complete by dissolution of solid to add the dry DCM of 250mL, stir under ice-water bath and add PyBoP5.724g (11mmol), DIPEA3.31ml (20mmol), maintain ice bath 2h and return to room temperature and continue stirring reaction 2h.Evaporated under reduced pressure solvent is thick to precipitate yellowly; Precipitate is complete with appropriate acetic acid ethyl dissolution, use saturated NaHCO
3, water, washing, dry final vacuum revolves steaming and obtains white solid, is ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro], weight 13.403g, purity 86.1%.
The preparation of the thick peptide of embodiment 17: Pa Xirui peptide
Ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C prepared by embodiment 15
2h
4-NH-CO-O) Pro] 13.413g is placed in cracking reactor, adds lytic reagent 80mL(TFA: thioanisole: EDT:TIS: water=86:5:5:3:1(V/V)), stirring at room temperature 2h.Then this product is added the anhydrous diethyl ether precipitation that 800mL is freezing, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide 9.68g of Pa Xirui peptide, and thick peptide weight yield 92.5%, HPLC purity is 85.2%.HPLC detects and sees Fig. 1.
The preparation of embodiment 18: Pa Xirui peptide essence peptide aspartate
After taking embodiment 17 Pa Xirui peptide thick peptide 9.68g 3000mL water dissolution, adopt Waters2545RP-HPLC system, wavelength 230nm, chromatographic column is the anti-phase C18 post of 50 × 250mm, conventional 0.2%TFA/ acetonitrile mobile phase purifying, collects object peak cut, obtains purity and be greater than 98.5% smart peptide.Smart peptide solution is adopted Waters2545RP-HPLC system, chromatographic column is the anti-phase C18 post of 50 × 250mm, 0.1% aspartic acid solution/acetonitrile mobile phase turns salt, collect object peak cut, rotary evaporation concentrates, freeze-drying obtains Pa Xirui peptide aspartate essence peptide 5.133g, RP-HPLC purity >=98.5%.
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 (9)
1. the preparation method of Yi Zhong Pa Xirui peptide, is characterized in that, comprising:
Step 1: by Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH and solid phase carrier coupling, obtain Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin;
Step 2:Fmoc-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-Resin coupling Fmoc-Phe-OH, Fmoc-Tyr (4-Bzl)-OH, Fmoc-Lys (Boc)-OH, Fmoc-DTrp (Boc)-OH, Fmoc-Phe-OH successively, obtain H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-{ (4-Boc-NH-C
2h
4-NH-CO-O) Pro}-Resin;
Step 3:H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-{ (4-Boc-NH-C
2h
4-NH-CO-O) Pro}-Resin cracking obtains full guard fragment H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH;
Step 4: by full guard fragment H-Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro-OH organic solvent dissolution, add activator and carry out liquid phase cyclisation and obtain ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro];
Step 5: ring [Phg-DTrp (Boc)-Lys (Boc)-Tyr (4-Bzl)-Phe-(4-Boc-NH-C
2h
4-NH-CO-O) Pro] cracking obtains Pa Xirui peptide crude product;
Step 6: the thick peptide purification of Pa Xirui peptide obtains Pa Xirui peptide sterling;
Step 2) coupling agent of described coupling is DIC/HOBt, PyBOP/HOBt/DIPEA, TBTU/HOBt/DIPEA or HBTU/HOBt/DIPEA.
2. preparation method according to claim 1, is characterized in that, step 1) described solid phase carrier is TrtResin or 2-ClTrtResin.
3. preparation method according to claim 2, is characterized in that, step 1) the initial substitution degree of described solid phase carrier is 0.4mmol/g ~ 1.0mmol/g.
4. preparation method according to claim 1, is characterized in that, step 1) coupling agent of described coupling is diisopropylethylamine.
5. preparation method according to claim 1, is characterized in that, step 3) cracking agent of described cracking is the mixture of TFA and DCM or the mixture of TFE and DCM.
6. preparation method according to claim 1, is characterized in that, step 4) described organic solvent is DCM, DMF or NMP.
7. preparation method according to claim 1, is characterized in that, step 4) described activator is PyBop/HOBT/DIPEA, PyBop/DIPEA, DIC/HOAT or DIC/HOBT.
8. preparation method according to claim 1, is characterized in that, step 5) mixing solutions of the cracking agent of described cracking to be TFA, thioanisole, EDT, TIS and water volume ratio be 86:5:5:3:1.
9. preparation method according to claim 1, is characterized in that, step 6) described purifying is RPLC purifying.
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| CN106188231B (en) * | 2015-05-25 | 2021-10-22 | 中国医学科学院药物研究所 | Synthesis and application of pasireotide pentapeptide intermediate |
| WO2016207912A1 (en) * | 2015-06-22 | 2016-12-29 | Biophore India Pharmaceuticals Pvt. Ltd. | Novel process for the preparation of pasireotide |
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| CN101883785A (en) * | 2007-12-03 | 2010-11-10 | 意大利法尔马科有限公司 | New non-selective somatostatin analogues |
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| WO2005014624A2 (en) * | 2003-08-08 | 2005-02-17 | Novartis Ag | Preparation of somatostatin peptides |
| CN101883785A (en) * | 2007-12-03 | 2010-11-10 | 意大利法尔马科有限公司 | New non-selective somatostatin analogues |
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