CN117088966A - Synthesis method of bivalirudin impurity - Google Patents
Synthesis method of bivalirudin impurity Download PDFInfo
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- CN117088966A CN117088966A CN202211711002.2A CN202211711002A CN117088966A CN 117088966 A CN117088966 A CN 117088966A CN 202211711002 A CN202211711002 A CN 202211711002A CN 117088966 A CN117088966 A CN 117088966A
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- bivalirudin
- gly
- fmoc
- impurity
- glu
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- OIRCOABEOLEUMC-GEJPAHFPSA-N bivalirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)CNC(=O)CNC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 OIRCOABEOLEUMC-GEJPAHFPSA-N 0.000 title claims abstract description 52
- 108010055460 bivalirudin Proteins 0.000 title claims abstract description 50
- 229960001500 bivalirudin Drugs 0.000 title claims abstract description 49
- 239000012535 impurity Substances 0.000 title claims abstract description 44
- 238000001308 synthesis method Methods 0.000 title description 9
- 150000001413 amino acids Chemical class 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 10
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 10
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 8
- FBNFRRNBFASDKS-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-prop-2-enoxybutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OCC=C)C(=O)O)C3=CC=CC=C3C2=C1 FBNFRRNBFASDKS-IBGZPJMESA-N 0.000 claims abstract description 7
- 238000001556 precipitation Methods 0.000 claims abstract description 7
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 3
- 238000010511 deprotection reaction Methods 0.000 claims abstract description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 3
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 16
- 238000005859 coupling reaction Methods 0.000 claims description 11
- 230000008878 coupling Effects 0.000 claims description 7
- 238000010168 coupling process Methods 0.000 claims description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 5
- 238000005336 cracking Methods 0.000 claims description 5
- 239000007810 chemical reaction solvent Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000000197 pyrolysis Methods 0.000 claims description 2
- ZYJPUMXJBDHSIF-LLVKDONJSA-N (2r)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylpropanoic acid Chemical compound CC(C)(C)OC(=O)N[C@@H](C(O)=O)CC1=CC=CC=C1 ZYJPUMXJBDHSIF-LLVKDONJSA-N 0.000 claims 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 32
- 238000007363 ring formation reaction Methods 0.000 abstract description 6
- 238000003776 cleavage reaction Methods 0.000 abstract description 5
- 230000007017 scission Effects 0.000 abstract description 5
- 229920001184 polypeptide Polymers 0.000 abstract description 3
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract 1
- YBYIRNPNPLQARY-UHFFFAOYSA-N 1H-indene Chemical compound C1=CC=C2CC=CC2=C1 YBYIRNPNPLQARY-UHFFFAOYSA-N 0.000 description 18
- 239000011347 resin Substances 0.000 description 17
- 229920005989 resin Polymers 0.000 description 17
- 239000000243 solution Substances 0.000 description 16
- 239000002904 solvent Substances 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 13
- 239000007790 solid phase Substances 0.000 description 9
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 7
- 229940024606 amino acid Drugs 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- -1 aspartyl imine Chemical class 0.000 description 7
- 238000001035 drying Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 6
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 description 6
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 6
- NTFTULBKHJJQAW-HNNXBMFYSA-N 9h-fluoren-9-ylmethyl n-[(2s)-4-methyl-1-oxopentan-2-yl]carbamate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C=O)C3=CC=CC=C3C2=C1 NTFTULBKHJJQAW-HNNXBMFYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000008961 swelling Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 4
- 108090000190 Thrombin Proteins 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 229960004072 thrombin Drugs 0.000 description 4
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 3
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 3
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 108010007267 Hirudins Proteins 0.000 description 2
- 102000007625 Hirudins Human genes 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940006607 hirudin Drugs 0.000 description 2
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000013146 percutaneous coronary intervention Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 229940003354 angiomax Drugs 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for synthesizing bivalirudin impurities, and belongs to the technical field of polypeptide synthesis. According to the invention, 2% DBU/DMF is selected as a deprotection reagent by a solid phase synthesis method, and bivalirudin impurity amino acid is sequentially coupled from a carbon end to a nitrogen end, wherein the bivalirudin impurity has the following sequence: H-D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH; fmoc-Asp (OAll) -OH is selected at position 12, 2% of DBU/DMF solution is added after synthesis is finished, cyclization operation is carried out, and bivalirudin impurity is obtained through cleavage and precipitation. The method improves the purity of the crude peptide and greatly reduces the material cost and the purification cost.
Description
Technical Field
The invention relates to the field of polypeptide drug synthesis, in particular to an artificially synthesized anticoagulation drug, which is an impurity synthesis method of hirudin 20 peptide analogues.
Background
Bivalirudin is an artificially synthesized anticoagulant drug, a 20-peptide analog of hirudin, approved for sale in the united states in 2000. Bivalirudin is capable of specifically binding to thrombin catalytic site and anionic exosite, directly inhibiting thrombin activity, thereby inhibiting thrombin catalyzed and induced reaction, and its effect is reversible. Unlike common heparin, bivalirudin does not require cofactors to assist its anticoagulant properties, and bivalirudin targets inhibition of thrombin (platelet agonists), thus effectively reducing platelet reactivity, a biological property that is very beneficial to patients receiving interventional therapy in the cutaneous coronary arteries. Bivalirudin is mainly used as an anticoagulant for Percutaneous Coronary Intervention (PCI) in adulthood. The bivalirudin is available in the form of powder for injection, with the trade name of Angiomax and the specification of 250mg.
Bivalirudin produces a variety of impurities during its production and storage, and formation of aspartyl imine is believed to be a serious side reaction in peptide synthesis, which can be both acid and base catalyzed, affecting both solid and liquid phase reactions. In order to ensure the safety and effectiveness of bivalirudin medicines, the content of impurities in the product must be controlled. The discovery and synthesis of impurities have important practical significance for quality control of bivalirudin bulk drugs.
Disclosure of Invention
The invention aims to solve the problems of more impurities, low purity and yield, high cost, complex operation steps and the like in the existing synthesis process, and provides an optimized synthesis method of bivalirudin impurities.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the synthesis method of the bivalirudin impurity is characterized in that the bivalirudin impurity sequence is as follows:
H-D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH; 2% DBU/DMF is selected as a deprotection reagent through a solid phase synthesis method, bivalirudin impurity amino acid is sequentially coupled from a carbon end to a nitrogen end, fmoc-Asp (OAll) -OH is selected at 12 positions, 2% DBU/DMF solution is added after synthesis is finished for ring forming operation, and finally bivalirudin impurity is obtained through pyrolysis precipitation.
The invention relates to a synthesis method for optimizing bivalirudin impurities, which has the further preferable technical scheme that: when coupling bivalirudin impurity amino acid, boc-D-Phe-OH is selected at position 20.
The invention relates to a synthesis method for optimizing bivalirudin impurities, which has the further preferable technical scheme that: condensing agents used for synthesizing the respective amino acids are one or more of DIC/HOBt, HBTU/HOBt/DIEA, pyBop/HOBt/DIEA.
The invention relates to a synthesis method for optimizing bivalirudin impurities, which has the further preferable technical scheme that: the reaction solvent used is one or more of DCM, DMF, NMP, DMSO.
The invention relates to a synthesis method for optimizing bivalirudin impurities, which has the further preferable technical scheme that: the cracking reagent used is: TFA: TIS: H 2 O=90:5:5。
The applicant found that during the development of bivalirudin, an asparagine impurity is produced during the production and storage of bivalirudin, i.e. nucleophilic substitution reaction between Gly at position 11 and Asn at position 12 in the bivalirudin residue occurs to form a five-membered ring. The invention replaces the amino acid Fmoc-Asn (Trt) -OH at position 12 of bivalirudin sequence with Fmoc-Asp (OAll) -OH; and then sequentially and solid-phase synthesizing amino acids at other sites of the bivalirudin impurity sequence to obtain bivalirudin impurities.
Compared with the prior art, the invention has the following beneficial effects:
the method for synthesizing the bivalirudin impurity is simple to operate, improves the purity of crude peptide of the bivalirudin impurity, greatly reduces the difficulty of purification, and correspondingly improves the yield.
Drawings
FIG. 1 is a chromatogram of a crude peptide obtained in example 1 of the present invention;
FIG. 2 is a chromatogram of a crude peptide obtained in example 2 of the present invention;
FIG. 3 is a chromatogram of a crude peptide obtained in example 3 of the present invention.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1
Preparation of bivalirudin impurity peptide resin:
using Wang Resin as a carrier (sub=0.50 mmol/g), 60.00. 60.00 g was weighed into a solid phase reactor and 400mL of DMF swelling Resin 0.5. 0.5h was added. The solvent was drained, fmoc-Leu-OH 31.80. 31.80 g, HOBt 14.61. 14.61 g, DIC 16.8. 16.8 ml, DMAP 1.11 g,400 ml DMF solvent were added, coupling reaction 5h was performed, the solvent was drained, and after washing 3 times with DMF, the degree of substitution was measured to obtain Fmoc-Leu-Wang Resin (sub=0.44 mmol/g).
Fmoc-Leu-Wang Resin (sub=0.44 mmol/g) 22.73. 22.73 g was weighed into a solid phase reactor and 200mL DMF swelling Resin was added for 0.5h. The solvent was drained and deprotected by addition of 200mL v/v 20% piperidine/DMF solution and reacted for 10+20min. The mixture was drained and washed 6 times with 250mL of DMF. The indene test result is positive. Fmoc-Tyr (tBu) -OH 13.79 g,HOBt 4.82g,DIC 5.5mL,200mLDMF solution was weighed, activated in an ice bath for 10 minutes at a temperature of no more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each.
The steps are repeated, and after Fmoc-Tyr (tBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Pro-OH, fmoc-Ile-OH, fmoc-Glu (OtBu) -OH, fmoc-Phe-OH, fmoc-Asp (OtBu) -OH, fmoc-Gly-OH and Fmoc-Asp (OAll) -OH are sequentially coupled to the 12 th amino acid, the ring forming reaction is carried out, and then DIC 10.00 ml and HOBt 4.82g are added for cutting test to judge the end point of the reaction. If the ring formation has been completed, the coupling is continued sequentially according to the amino acid sequences Fmoc-Gly-OH, fmoc-Pro-OH, fmoc-Arg (Pbf) -OH, fmoc-Pro-OH, boc-D-Phe-OH. After the coupling, 250mL of DMF was added for 3 times and the mixture was drained. The DCM was washed alternately 3 times with 400mL each. And (5) drying in vacuum to obtain the bivalirudin impurity peptide resin.
Cracking:
preparation of 300mL of cleavage reagent as TFA: TIS: H 2 O=90:5:5, the full-protection peptide is added under ice bath condition, the reaction is continued for 2 hours at room temperature, and anhydrous isopropyl ether is added for precipitation after the reaction is finished. The precipitate was centrifuged 4 times, and washed with 500mL of isopropyl ether each time. The product obtained after drying is bivalirudin impurity crude peptide, the crude peptide is 17.88 g, and the crude peptide is quantified by a reference substance to be 10.72 g, total yield is 38.5%, purity is 85.52%. The chromatogram of the crude peptide obtained is shown in FIG. 1.
Example 2:
fragment preparation:
2-chloro-trityl chloride resin was used as a carrier (sub=1.10 mmol/g), 45.46 g was weighed and added to a polypeptide synthesizer, and the resin was washed with 300mL DCM and the solvent was drained. Fmoc-Gly-OH 44.62 g,DIEA 38 mL,300 mL DMF solution was added, after 3h of coupling reaction, methanol 80 ml was added to block the active part on the resin for 30 min. The solvent was drained, washed 3 times with 300mL DMF, washed with 300mL methanol, and dried under vacuum to constant weight to give Fmoc-Gly-2-chloro-trityl resin. The degree of substitution was found to be 0.89 mmol/g.
Fmoc-Gly-2-chloro-trityl resin (sub=0.89 mmol/g) 45.00. 45.00 g was weighed into a solid phase reactor and 300mL DMF was added to swell the resin for 0.5h. The solvent was drained and deprotected by addition of 200mL v/v 20% piperidine/DMF solution and reacted for 10+20min. The mixture was drained and washed 6 times with DMF 300 mL. The indene test result is positive. Fmoc-Gly-OH 35.70 g,HOBt 19.28 g,DIC 22 mL,300 mLDMF solution is weighed, and the solution is activated for 10 minutes in an ice bath, wherein the activation temperature is not more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each. The steps are repeated, and Fmoc-Gly-OH, fmoc-Pro-OH, fmoc-Arg (pbf) -OH, fmoc-Pro-OH, fmoc-Phe-OH and Boc-D-Phe are coupled in sequence according to the amino acid sequences. After the coupling, 300mL of DMF was added for 3 times and the mixture was drained. The DCM was washed alternately 3 times with 400mL each. Vacuum drying to obtain Boc-D-Phe-Pro-Arg (pbf) -Pro-Gly-Gly-Gly-Gly-CTC fragment peptide resin.
300mL of cleavage reagent was prepared as TFE: DCM=1:3, the fragment peptide resin was added under ice bath conditions, the reaction was continued for 2 hours after 0.5 hour and after the completion of the reaction, anhydrous isopropyl ether was added for precipitation. The precipitate was centrifuged 4 times, and washed with 500mL of isopropyl ether each time. The product obtained after drying is Boc-D-Phe-Pro-Arg (pbf) -Pro-Gly-Gly-Gly-Gly fragment, and the purity reaches 98%.
Preparation of bivalirudin impurity peptide resin:
using Wang Resin as a carrier (sub=0.50 mmol/g), 60.00. 60.00 g was weighed into a solid phase reactor and 400mL of DMF swelling Resin was added for 0.5h. The solvent was drained, fmoc-Leu-OH 31.80. 31.80 g, HOBt 14.61. 14.61 g, DIC 16.8. 16.8 ml, DMAP 1.11 g,400 ml DMF solvent were added, coupling reaction 5h was performed, the solvent was drained, and after washing 3 times with DMF, the degree of substitution was measured to obtain Fmoc-Leu-Wang Resin (sub=0.44 mmol/g).
Fmoc-Leu-Wang Resin (sub=0.44 mmol/g) 22.75. 22.75 g was weighed into a solid phase reactor and 200mL DMF swelling Resin was added for 0.5h. The solvent was drained and deprotected by addition of 200mL v/v 20% piperidine/DMF solution and reacted for 10+20min. The mixture was drained and washed 6 times with 250mL of DMF. The indene test result is positive. Fmoc-Tyr (tBu) -OH 13.79 g,HOBt 4.82g,DIC 5.5mL,200mL DMF solution was weighed, activated in an ice bath for 10 minutes at a temperature of no more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each.
The steps are repeated, and after Fmoc-Tyr (tBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Pro-OH, fmoc-Ile-OH, fmoc-Glu (OtBu) -OH, fmoc-Phe-OH, fmoc-Asp (OtBu) -OH, fmoc-Gly-OH and Fmoc-Asp (OAll) -OH are sequentially coupled to the 12 th amino acid, the ring forming reaction is carried out, and then DIC 10.00 ml and HOBt 4.82g are added for cutting test to judge the end point of the reaction. If the ring formation is carried out, adding Boc-D-Phe-Pro-Arg (pbf) -Pro-Gly-Gly-Gly fragment 32.70 g,HOBt 4.82g for fragment coupling, and draining after the indene detection result is negative. DMF was added to 250mL and washed 3 times and dried. The DCM was washed alternately 3 times with 400mL each. And (5) drying in vacuum to obtain the bivalirudin impurity peptide resin.
Cracking:
preparation of 300mL of cleavage reagent as TFA: TIS: H 2 O=90:5:5, the full-protection peptide is added under ice bath condition, the reaction is continued for 2 hours after 0.5 hour and the reaction is completed, and anhydrous isopropyl ether is added for precipitation. The precipitate was centrifuged 4 times, and washed with 500mL of isopropyl ether each time. The product obtained after drying is bivalirudin impurity crude peptide, the crude peptide is obtained by 18.90 g, the crude peptide is quantified by 12.90 g by a reference substance, the total yield is 42.5%, and the purity is 87.15%. Is prepared intoReference is made to figure 2 for a chromatogram of the crude peptide.
Example 3
Preparation of bivalirudin impurity peptide resin:
using Wang Resin as a carrier (sub=0.50 mmol/g), 60.00. 60.00 g was weighed into a solid phase reactor and 400mL of DMF swelling Resin 0.5. 0.5h was added. The solvent was drained, fmoc-Leu-OH 31.80. 31.80 g, HOBt 14.61. 14.61 g, DIC 16.8. 16.8 ml, DMAP 1.11 g,400 ml DMF solvent were added, coupling reaction 5h was performed, the solvent was drained, and after washing 3 times with DMF, the degree of substitution was measured to obtain Fmoc-Leu-Wang Resin (sub=0.44 mmol/g).
Fmoc-Leu-Wang Resin (sub=0.44 mmol/g) 22.80. 22.80 g was weighed into a solid phase reactor and 200mL DMF swelling Resin was added for 0.5h. The solvent was drained and deprotected by addition of 200mL v/v 2% DBU/DMF solution and reacted for 10+20min. The mixture was drained and washed 6 times with 250mL of DMF. The indene test result is positive. Fmoc-Tyr (tBu) -OH 13.79 g,HOBt 4.82g,DIC 5.5mL,200mLDMF solution was weighed, activated in an ice bath for 10 minutes at a temperature of no more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each.
The above steps were repeated and the amino acid sequences Fmoc-Tyr (tBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Pro-OH, fmoc-Ile-OH, fmoc-Glu (OtBu) -OH, fmoc-Phe-OH, fmoc-Asp (OtBu) -OH, fmoc-Gly-OH, fmoc-Asp (OAll) -OH, fmoc-Gly-OH, fmoc-Pro-OH, fmoc-Arg (pbf) -OH, fmoc-Pro-OH, fmoc-Phe-OH, boc-D-Phe were sequentially coupled. After the coupling, 250mL of DMF was added for 3 times and the mixture was drained. The cyclization reaction was carried out by adding 200mL v/v 2% DBU/DMF solution, and the end point of the reaction was judged by cutting small pieces. If the ring had been formed, 250mL of DMF was added and washed 3 times and dried. The DCM was washed alternately 3 times with 400mL each. And (5) drying in vacuum to obtain the bivalirudin impurity peptide resin.
Cracking:
preparation of 300mL of cleavage reagent as TFA: TIS: H 2 O=90:5:5, the full-protection peptide is added under ice bath condition, the reaction is continued for 2 hours after 0.5 hour and the reaction is completed, and anhydrous isopropyl ether is added for precipitation. Centrifuging and precipitating for 4 times, adding each timeIsopropyl ether 500 mL. The product obtained after drying is bivalirudin impurity crude peptide, the crude peptide is obtained by 21.50 and g, the crude peptide is quantified by 18.15 and g by a reference substance, the total yield is 65.5%, and the purity is 93.06%. The chromatogram of the crude peptide obtained is shown in FIG. 3.
Claims (5)
1. A method for synthesizing bivalirudin impurity is characterized in that: the sequences of bivalirudin impurities are:
H-D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH; 2% DBU/DMF is selected as a deprotection reagent through a solid phase synthesis method, bivalirudin impurity amino acid is sequentially coupled from a carbon end to a nitrogen end, fmoc-Asp (OAll) -OH is selected at 12 positions, 2% DBU/DMF solution is added as an Fmoc removal reagent after synthesis is finished, ring forming operation is carried out, and finally bivalirudin impurity is obtained through pyrolysis precipitation.
2. The method for synthesizing an optimized bivalirudin impurity according to claim 1, wherein the 20 th position is selected from Boc-D-Phe-OH when coupling the bivalirudin impurity amino acid.
3. The method for synthesizing an optimized bivalirudin impurity according to claim 1, wherein the condensing agent used for synthesizing each amino acid is one or more of DIC/HOBt, HBTU/HOBt/DIEA, pyBop/HOBt/DIEA.
4. A method of synthesizing an optimized bivalirudin impurity as claimed in claim 1, wherein the reaction solvent used is one or a combination of DCM, DMF, NMP, DMSO.
5. The method for synthesizing the optimized bivalirudin impurity according to claim 1, wherein the adopted cracking reagent is as follows: TFA: TIS: H 2 O=90:5:5。
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| WO1990003391A1 (en) * | 1988-09-29 | 1990-04-05 | Biogen, Inc. | Hirudin peptides |
| US20070093423A1 (en) * | 2005-09-14 | 2007-04-26 | Avi Tovi | Process for production of Bivalirudin |
| US7598343B1 (en) * | 2008-07-27 | 2009-10-06 | The Medicines Company | Pharmaceutical formulations of bivalirudin and processes of making the same |
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| WO2011071799A2 (en) * | 2009-12-11 | 2011-06-16 | Dr. Reddy's Laboratories Ltd. | Purification of bivalirudin |
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