CN107778353B - Method for synthesizing terlipressin - Google Patents
Method for synthesizing terlipressin Download PDFInfo
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- CN107778353B CN107778353B CN201610728835.8A CN201610728835A CN107778353B CN 107778353 B CN107778353 B CN 107778353B CN 201610728835 A CN201610728835 A CN 201610728835A CN 107778353 B CN107778353 B CN 107778353B
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- BENFXAYNYRLAIU-QSVFAHTRSA-N terlipressin Chemical compound NCCCC[C@@H](C(=O)NCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)CN)CSSC1 BENFXAYNYRLAIU-QSVFAHTRSA-N 0.000 title claims abstract description 64
- 108010010056 Terlipressin Proteins 0.000 title claims abstract description 63
- 229960003813 terlipressin Drugs 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 11
- 229920005989 resin Polymers 0.000 claims abstract description 103
- 239000011347 resin Substances 0.000 claims abstract description 103
- 238000003402 intramolecular cyclocondensation reaction Methods 0.000 claims abstract description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 52
- 150000001413 amino acids Chemical class 0.000 claims description 39
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical group OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 27
- 150000007530 organic bases Chemical class 0.000 claims description 20
- 239000012043 crude product Substances 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 17
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 claims description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims description 16
- 238000005859 coupling reaction Methods 0.000 claims description 16
- 230000008878 coupling Effects 0.000 claims description 15
- 238000010168 coupling process Methods 0.000 claims description 15
- -1 benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Chemical compound 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 12
- 125000003277 amino group Chemical group 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 238000009833 condensation Methods 0.000 claims description 10
- 230000005494 condensation Effects 0.000 claims description 10
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 9
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 claims description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical group C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 8
- 229910000042 hydrogen bromide Inorganic materials 0.000 claims description 8
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 claims description 6
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 claims description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical group CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 6
- 230000009471 action Effects 0.000 claims description 5
- 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 claims description 4
- ZPGDWQNBZYOZTI-UHFFFAOYSA-N 1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)C1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-UHFFFAOYSA-N 0.000 claims description 4
- 239000007825 activation reagent Substances 0.000 claims description 4
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 3
- 229920005990 polystyrene resin Polymers 0.000 claims description 3
- CSMYOORPUGPKAP-IBGZPJMESA-N (2r)-3-(acetamidomethylsulfanyl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CSCNC(=O)C)C(O)=O)C3=CC=CC=C3C2=C1 CSMYOORPUGPKAP-IBGZPJMESA-N 0.000 claims description 2
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 2
- HWBAHOVOSOAFLE-UHFFFAOYSA-N 2-[[2-[(2-methylpropan-2-yl)oxycarbonylamino]acetyl]amino]acetic acid Chemical compound CC(C)(C)OC(=O)NCC(=O)NCC(O)=O HWBAHOVOSOAFLE-UHFFFAOYSA-N 0.000 claims description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 claims description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 15
- 238000003786 synthesis reaction Methods 0.000 abstract description 15
- 150000001408 amides Chemical class 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 74
- 239000000243 solution Substances 0.000 description 71
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 27
- 238000005406 washing Methods 0.000 description 27
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- 239000012071 phase Substances 0.000 description 19
- 238000001914 filtration Methods 0.000 description 17
- 238000003756 stirring Methods 0.000 description 17
- 125000006239 protecting group Chemical group 0.000 description 16
- 238000000746 purification Methods 0.000 description 15
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000010828 elution Methods 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 208000032843 Hemorrhage Diseases 0.000 description 6
- 238000012856 packing Methods 0.000 description 6
- 229920003180 amino resin Polymers 0.000 description 5
- 230000000740 bleeding effect Effects 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 239000007821 HATU Substances 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 4
- 108010004977 Vasopressins Proteins 0.000 description 4
- 102000002852 Vasopressins Human genes 0.000 description 4
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000005086 pumping Methods 0.000 description 4
- 229960003726 vasopressin Drugs 0.000 description 4
- DEQANNDTNATYII-OULOTJBUSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-benzyl-n-[(2r,3r)-1,3-dihydroxybutan-2-yl]-7-[(1r)-1-hydroxyethyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxa Chemical compound C([C@@H](N)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)C1=CC=CC=C1 DEQANNDTNATYII-OULOTJBUSA-N 0.000 description 3
- 108010016076 Octreotide Proteins 0.000 description 3
- 102000005157 Somatostatin Human genes 0.000 description 3
- 108010056088 Somatostatin Proteins 0.000 description 3
- 239000012317 TBTU Substances 0.000 description 3
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 229960002700 octreotide Drugs 0.000 description 3
- 239000002516 radical scavenger Substances 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 3
- 229960000553 somatostatin Drugs 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- BJFIDCADFRDPIO-DZCXQCEKSA-N (2S)-N-[(2S)-6-amino-1-[(2-amino-2-oxoethyl)amino]-1-oxohexan-2-yl]-1-[[(4R,7S,10S,13S,16S,19R)-19-amino-7-(2-amino-2-oxoethyl)-10-(3-amino-3-oxopropyl)-16-[(4-hydroxyphenyl)methyl]-6,9,12,15,18-pentaoxo-13-(phenylmethyl)-1,2-dithia-5,8,11,14,17-pentazacycloeicos-4-yl]-oxomethyl]-2-pyrrolidinecarboxamide Chemical class NCCCC[C@@H](C(=O)NCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](N)CSSC1 BJFIDCADFRDPIO-DZCXQCEKSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- DSTWKJOBKSMVCV-UWVGGRQHSA-N Cys-Tyr Chemical compound SC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DSTWKJOBKSMVCV-UWVGGRQHSA-N 0.000 description 2
- 208000034189 Sclerosis Diseases 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 108010069495 cysteinyltyrosine Proteins 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 239000000902 placebo Substances 0.000 description 2
- 229940068196 placebo Drugs 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 230000009278 visceral effect Effects 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- GHONIQQBOSTHSL-UHFFFAOYSA-N 2-[[2-[[2-[(2-methylpropan-2-yl)oxycarbonylamino]acetyl]amino]acetyl]amino]acetic acid Chemical compound CC(C)(C)OC(=O)NCC(=O)NCC(=O)NCC(O)=O GHONIQQBOSTHSL-UHFFFAOYSA-N 0.000 description 1
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 108010048179 Lypressin Proteins 0.000 description 1
- 240000004674 Papaver rhoeas Species 0.000 description 1
- 235000007846 Papaver rhoeas Nutrition 0.000 description 1
- 206010046996 Varicose vein Diseases 0.000 description 1
- 206010047139 Vasoconstriction Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000001595 contractor effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960003837 lypressin Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000007243 oxidative cyclization reaction Methods 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 230000025033 vasoconstriction Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/16—Oxytocins; Vasopressins; Related peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the field of medicine synthesis, and discloses a method for synthesizing terlipressin. The invention adopts brand new MOBHA resin to synthesize terlipressin, simultaneously adjusts the time of intramolecular cyclization, has lower cost compared with the prior Rink Amide series resin, obviously improves the total yield of the obtained pure product, has simple and easy operation of the whole method and mild conditions, and is suitable for industrial production.
Description
Technical Field
The invention relates to the field of medicine synthesis, in particular to a method for synthesizing terlipressin.
Background
Terlipressin (terlipessin) is a derivative of lypressin that has the effect of increasing gastrointestinal vasoconstriction, manifested by a reduction in visceral blood flow, with only minor changes in blood pressure and heart rate. Compared with lypressin, the product has the advantages of reduced visceral blood flow, longer action time, and reduced portal vein pressure after intravenous administration, which indicates the contraction effect of the product on intestinal capillary bed.
In clinical applications, a double-blind study showed that terlipressin acetate significantly reduced the transfusion requirement, which was about 40% lower than that of placebo. The hemostasis rate was 70.7% within 12 hours, compared to 46.5% in the placebo group.
Terlipressin acetate is clearly superior to vasopressin, and in a multicenter clinical study, 70% of patients with terlipressin acetate stopped bleeding, 9% of vasopressin was present, and transfusion requirements were also lower than in the vasopressin group.
The terlipressin acetate is also obviously superior to somatostatin and octreotide, and in a multi-center clinical study, the concentrated evaluation result shows that the terlipressin acetate can obviously reduce the death rate of esophageal variceal hemorrhage, unlike the somatostatin and the octreotide.
The combined treatment of terlipressin acetate and the single sclerosis treatment are superior to each other, in a multicenter clinical study of 596 patients with cirrhosis varicose vein bleeding, 97 percent of patients using the combined treatment stop bleeding is obviously superior to the single sclerosis treatment, and the combined treatment can not only effectively control bleeding, but also reduce the possibility of rebleeding.
Because the curative effect of the terlipressin acetate is better than that of vasopressin, octreotide and somatostatin, the terlipressin acetate is taken as a first choice medicine for treating acute variceal bleeding at the second international conference on Baveno.
The prior patent CN105367627A discloses a method for preparing terlipressin, which comprises the steps of synthesizing 3-12 site peptide resin, coupling with 1-2 fragment, carrying out acidolysis, carrying out oxidative cyclization to obtain crude product, wherein the acidolysis reagent is a scavenger/TFA solution containing 15-20% of EDT and the like. The crude product is purified and converted into salt to obtain a finished product, the recorded total yield is 71 percent, and the carrier grease is conventional Rink Amide series resin. However, according to the data described in the examples of the patent, the theoretical amount of the product is 24.5g, the theoretical peptide content of the crude product is between 20 and 50 percent and cannot exceed 50 percent due to the protective agent and the scavenging agent contained in the crude product, the total amount of the crude product is about 80g calculated by the intermediate value of 30 percent, the patent only obtains 25.7g of the crude product, the maximum theoretical amount of the 25.7g of the crude product is only 12.8g, and the product cannot be obtained by 17.4g, so the total yield obtained by calculation cannot reach 70 percent. According to some terlipressin patent technologies which are disclosed at present, the total yield is basically 50-55%.
Chinese patent CN105418736A discloses a method for preparing terlipressin by solid-liquid combination, which comprises the steps of synthesizing 4-12-bit peptide resin, performing iodoxycyclization, coupling with 1-3 fragments synthesized by liquid phase, and finally performing acidolysis, wherein an acidolysis reagent is a scavenger/TFA solution containing 1-5% anisole and the like, and the carrier lipid is also conventional Rink Amide series resin. Although the final overall yield is not described, the total yield after purification is about 55% and is comparable to the current overall yield of terlipressin, which can be calculated from the batch quantities disclosed in the examples.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for synthesizing terlipressin, which has high purity and total yield.
In order to achieve the purpose, the invention provides the following technical scheme:
a method of synthesizing terlipressin, comprising the steps of:
step 1, under the action of a condensation reagent and an activation reagent, taking resin shown in formula 1 as a solid phase carrier, and sequentially coupling protected Gly, protected Lys, protected Pro, protected Cys, protected Asn, protected Gln, protected Phe, protected Tyr and protected Cys one by one according to the amino acid sequence from the C end to the N end of the terlipressin linear peptide to synthesize peptide resin 1;
step 2, under the action of a condensation reagent and an activation reagent, coupling protected Gly-Gly-Gly with peptide resin 1 to obtain peptide resin 2;
step 3, performing intramolecular cyclization on the peptide resin 2, and connecting two Cys through a disulfide bond to obtain terlipressin peptide resin;
step 4, carrying out acidolysis on the terlipressin resin by an acidolysis agent to obtain a terlipressin crude product,
step 5, purifying the crude terlipressin product and converting the purified terlipressin product into salt to obtain a pure terlipressin product;
wherein, the left round ball of formula 1 represents polystyrene resin, belonging to amino resin.
The terlipressin linear peptide main chain has 8 amino acids, and the composition is as follows:
Gly1-Gly2-Gly3-[Cys4-Tyr5-Phe6-Gln7-Asn8-Cys]9-Pro10-Lys11-Gly12-NH2
wherein, the amino group at the C terminal in the terlipressin is the amino group cracked from the amino resin by using an acidolysis agent, and the amino group does not belong to the amino group on the amino acid.
The invention provides a brand new carrier resin MOBHA resin, namely the resin shown in formula 1, which is improved on the basis of the original MBHA resin, and found by synthesizing terlipressin, the cost of the resin is lower than that of the Rink Amide resin, the total yield of the synthesized pure terlipressin can reach more than 75%, and the defect that the conventional synthesis process is generally 50-55% of the total yield is overcome.
The resin of formula 1 of the present invention can be purchased from new scientific and technological materials, Inc. of Xian lan and Xiao.
The structural formula of MBHA resin is as follows, the left round sphere representing polystyrene resin:
the protecting group is a protecting group which needs to protect the main chain of amino acid and the groups interfering the synthesis such as amino, carboxyl and the like on the side chain in the field of amino acid synthesis, and prevents the amino, carboxyl and the like from reacting to generate impurities in the process of preparing a target product, for example, the protecting group protects the side chain of Lys through Boc protecting group, and protects the side chain of Tyr through tBu protecting group; the side chain of Asn or Gln is protected by Trt protecting group, and the side chain of Cys is protected by Trt or Acm. Furthermore, in the protected amino acids involved in the process of the present invention, the N-terminus is preferably protected by Fmoc or Boc protecting group. Amino acids protected by a protecting group are collectively referred to as protected amino acids (e.g., protected D-Gly, etc.). Preferably, the protected Gly, protected Lys, protected Pro, protected Cys, protected Asn, protected Gln, protected Phe, protected Tyr, protected Gly-Gly are:
Fomc-Gly or Boc-Gly, Fmoc-Lys (Boc), Fmoc-Pro, Fmoc-Asn (Trt), Fmoc-Gln (Trt), Fmoc-Phe, Fmoc-Tyr (tBu), Fmoc-Cys (Trt) or Fmoc-Cys (Acm), Fomc-Gly-Gly-Gly or Boc-Gly-Gly.
Preferably, the molar ratio of each protected amino acid to the amino group in the resin of formula 1 when each protected amino acid is coupled to the resin of formula 1 or the synthesized peptide resin is 1-6:1, more preferably 2.5-3.5:1, i.e., the ratio of the charged amount of the protected amino acid to the molar amount of the amino group in the resin of formula 1.
Preferably, the substitution value of the resin of formula 1 is 0.2 to 1.8mmol/g amino resin, more preferably 0.5 to 1.0mmol/g amino resin.
Preferably, the condensation reagent is N, N-Diisopropylcarbodiimide (DIC), N-Dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate/organic base (PyBOP/organic base), 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate/organic base (HATU/organic base), benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate/organic base (HBTU/organic base), O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate/organic base (TBTU/organic base). The molar amount of the condensation reagent is preferably 1 to 6 times, and more preferably 2.5 to 3.5 times of the total molar number of amino groups of the resin of formula 1 or the synthesized peptide resin of formula 1.
It should be noted that the PyBOP/organic base, HATU/organic base, HBTU/organic base, TBTU/organic base are four two-system condensation reagents in the present invention, i.e. PyBOP, HATU, HBTU need to be combined with organic base to be one condensation reagent when in use, wherein the molar ratio of the organic base to PyBOP, HATU, HBTU, TBTU is preferably 1.3-3.0:1, more preferably 1.3-2: 1.
Preferably, the organic base in the condensation reagent is preferably N, N-Diisopropylethylamine (DIPEA), Triethylamine (TEA) or N-methylmorpholine (NMM), more preferably DIPEA.
Preferably, the activating reagent is 1-hydroxybenzotriazole (HOBt) or N-hydroxy-7-azabenzotriazole (HOAt). The amount of the activating agent is preferably 1 to 6 times, and more preferably 2.5 to 3.5 times, of the total number of moles of amino groups in the amino resin or the synthesized peptide resin.
Preferably, DMF is used as a reaction solvent for each reaction in the synthesis process.
The one-by-one coupling of the invention means that after the first amino acid is coupled with the resin of the formula 1, the rest amino acids are coupled with the previous coupled amino acid one by one according to the sequence from the C end to the N end of the main chain amino acid of the terlipressin linear peptide. In the coupling of the present invention, the molar ratio of the protected amino acid to the corresponding peptide resin at each coupling is preferably 1 to 6:1, more preferably 2.5 to 3.5: 1; the coupling reaction time is preferably 60 to 300 minutes, and more preferably 120 to 180 minutes. The peptide resin of the present invention refers to a peptide resin in which an arbitrary number of amino acids are linked to the resin of formula 1 in the order of amino acids of the linear peptide backbone of terlipressin, and includes not only peptide resins 1-2 but also a plurality of peptide resins obtained during the synthesis of peptide resin 1. The corresponding peptide resin is peptide resin 3 formed by coupling protected Gly and resin shown in formula 1, the peptide resin 3 is corresponding peptide resin formed by coupling protected Lys, the peptide resin coupled with the protected Lys is corresponding peptide resin formed by extension coupling of protected Pro, and by analogy, the corresponding relation between the residual protected amino acid and the peptide resin formed by coupling the last protected amino acid is formed.
In the extension coupling, since each amino acid has a protecting group at the N-terminus, it is common knowledge to those skilled in the art that the protecting group at the N-terminus needs to be removed before coupling. The invention preferably uses PIP/DMF (piperidine/N, N-dimethylformamide) mixed solution to remove the Fomc protecting group at the N end, wherein the mixed solution contains 10-30% (V) of piperidine and the balance of DMF. The time for removing the N-terminal protecting group is preferably 10 to 60 minutes, and preferably 15 to 25 minutes. The dosage of the reagent for removing the N-terminal protecting group is preferably 10mL/g of peptide resin; according to the invention, the N-terminal Boc protecting group is preferably removed by using a TFA/DCM (trifluoroacetic acid/dichloromethane) mixed solution, the trifluoroacetic acid content in the mixed solution is 20-60% (V/V), preferably 25-35% (V/V), the time for removing the N-terminal protecting group is preferably 10-50 minutes, preferably 25-35 minutes, and the dosage of the N-terminal protecting group removing reagent is preferably 10mL/g of peptide resin.
Preferably, the intramolecular cyclization of the present invention is performed by an iodine oxidation method. Can be mixed with 5% of I2the/DMF (iodine/N, N-dimethylformamide) solution was added to the peptide resin 2 for cyclization.
Preferably, the acidolysis agent is a trifluoroacetic acid solution of hydrogen bromide, and the combination of the resin of the formula 1 can improve the cracking yield and promote the improvement of the total yield. More preferably, in the trifluoroacetic acid solution of hydrogen bromide, the mass percent concentration of hydrogen bromide is preferably 5-10% wt, and more preferably 6-7% wt; the dosage of the acidolysis agent is 5-15 mL of acidolysis agent/g of peptide resin, and the dosage of the acidolysis agent is preferably 7-12 mL of acidolysis agent/g of peptide resin; the acidolysis time is 1-6 hours, preferably 3-4 hours.
Preferably, the acetylation according to the invention is carried out by Ac2The acetylation is accomplished by reaction of O with the deprotected N-terminal amino group of peptide resin 1.
Preferably, the purification trans-salt is specifically:
dissolving the crude terlipressin in 0.1% TFA/water solution, filtering the solution by using a 0.45 mu m microporous filter membrane, and purifying for later use;
purifying by high performance liquid chromatography, wherein the purification uses reversed phase C18 with chromatographic packing of 10 μm, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column of 77mm x 250mm is 90mL/min, eluting by a gradient system, purifying by circulating sample injection, sampling a crude product solution in the chromatographic column, starting the mobile phase for elution, collecting a main peak, and evaporating acetonitrile to obtain a terlipressin purified intermediate concentrated solution;
taking the intermediate concentrated solution of the terlipressin purification, and filtering the intermediate concentrated solution by using a 0.45-micrometer filter membrane for later use;
performing salt exchange by adopting a high performance liquid chromatography, wherein a mobile phase system is 1% acetic acid/water solution-acetonitrile, a reversed phase C18 with a chromatographic packing of 10 mu m for purification, a chromatographic column with a flow rate of 77mm x 250mm of 90mL/min, performing gradient elution and a circular sample loading method, loading the sample into the chromatographic column, starting the mobile phase elution, collecting a map, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, performing reduced pressure concentration to obtain a terlipressin acetic acid water solution, and performing freeze drying to obtain a terlipressin pure product.
The terlipressin synthesized by the method has a crude product purity of more than 85 percent, a product purity of more than 99.5 percent, a maximum single impurity of less than 0.12 percent and a total yield of more than 75 percent through HPLC detection.
According to the technical scheme, the invention adopts brand-new MOBHA resin to synthesize terlipressin, simultaneously adjusts the time of intramolecular cyclization, has lower cost compared with the existing Rink Amide series resin, and obviously improves the total yield of the obtained pure product.
Detailed Description
The invention discloses a method for synthesizing terlipressin, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made and the techniques of the present invention employed without departing from the spirit and scope of the invention.
In a specific embodiment of the present invention, the protected amino acids of the present invention are obtained from Yoghui bioscience, Inc., and the resins are obtained from Papaver rhoeas, Inc., and the English abbreviations used in the application documents have the Chinese meanings shown in Table 1.
TABLE 1 English abbreviation definitions
The invention is further illustrated by the following examples.
Example 1: synthesis of Fmoc-Gly-MOBHA resin
Dissolving 0.15mol of Fmoc-Gly and 0.15mol of HOBt by using a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.
Taking 0.05mol of MOBHA resin (the substitution value is about 0.6mmol/g), swelling with DMF for 25 minutes, washing and filtering, adding an activated Fmoc-Gly solution, stirring at room temperature for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3 times, wherein the washing time is 3min each time, obtaining the Fmoc-Gly-MOBHA resin, deprotecting with 20% PIP/DMF solution for 25 minutes, washing and filtering to obtain Gly-MOBHA resin.
Example 2: synthesis of Boc-Gly-MOBHA resin
Dissolving 0.15mol of Boc-Gly and 0.15mol of HOBt by using a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.
Taking 0.05mol of MOBHA resin (the substitution value is about 0.6mmol/g), swelling with DMF for 25 minutes, washing and filtering, adding an activated Boc-Gly solution, stirring at room temperature for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3 minutes, obtaining Boc-Gly-MOBHA resin, deprotecting with 30% TFA/DCM solution for 30 minutes, neutralizing with DIEA/DCM solution, washing with DMF and DCM, and filtering to obtain Gly-MOBHA resin.
Example 3: synthesis of peptide resin 1
Dissolving 0.15mol of Fmoc-Lys (Boc) and 0.15mol of HOBt with a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain the activated protected amino acid solution.
Adding the activated protected amino acid solution into the Gly-MOBHA resin prepared in example 1, stirring at room temperature for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3 times, wherein each washing time is 3min, deprotecting with 20% PIP/DMF solution for 25 min, washing and filtering to complete the access of Fmoc-Lys (Boc).
The peptide resin 1 is obtained by inoculating Fmoc-Pro, Fmoc-Cys (Trt), Fmoc-Asn (Trt), Fmoc-Gln (Trt), Fmoc-Phe, Fmoc-Tyr (tBu) and Fmoc-Cys (Trt) by the same method, then using 20% PIP/DMF solution for deprotection, washing and filtering, and finally obtaining the peptide resin 1, Cys (Trt) -Tyr (tBu) -Phe-Gln (Trt) -Asn (Trt) -Cys (Trt) -Pro-Lys (Boc) -Gly-MOBHA resin.
Example 4: synthesis of peptide resin 1
Dissolving 0.15mol of Fmoc-Lys (Boc) and 0.15mol of HOBt with a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain the activated protected amino acid solution.
Adding the activated protected amino acid solution into the Gly-MOBHA resin prepared in example 2, stirring at room temperature for 3 hours, pumping out the reaction solution, washing with DMF for 3 times, washing with DCM for 3 times, wherein each washing time is 3min, deprotecting with 20% PIP/DMF solution for 25 min, washing and filtering to complete the access of Fmoc-Lys (Boc).
The same method is used for inoculating Fmoc-Pro, Fmoc-Cys (Trt), Fmoc-Asn (Trt), Fmoc-Gln (Trt), Fmoc-Phe, Fmoc-Tyr (tBu) and Fmoc-Cys (Trt), then using 20% PIP/DMF solution for deprotection, washing and filtering to obtain peptide resin 1, Cys (Trt) -Tyr (tBu) -Phe-Gln (Trt) -Asn (Trt) -Cys (Trt) -Pro-Lys (Boc) -Gly-MOBHA resin.
Example 5: synthesis of peptide resin 2
Dissolving 0.15mol of Fmoc-Gly-Gly-Gly and 0.15mol of HOBt by using a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain the activated protected amino acid solution.
The activated protected amino acid solution was added to the peptide resin 1 obtained in example 3, and the mixture was stirred at room temperature for 3 hours, the reaction solution was aspirated, after 3 washes with DMF, 3 washes with DCM for 3min each, and then deprotected with 20% PIP/DMF solution for 25 minutes, followed by washing and filtration to give peptide resin 2, Gly-Gly-Gly-Cys (Trt) -Tyr (tBu) -Phe-Gln (Trt) -Asn (Trt) -Cys (Trt) -Pro-Lys (Boc) -Gly-MOBHA resin.
Example 6: synthesis of peptide resin 2
Dissolving 0.15mol of Boc-Gly-Gly-Gly and 0.15mol of HOBt by using a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain the activated protected amino acid solution.
The activated protected amino acid solution was added to the peptide resin 1 obtained in example 4, and the mixture was stirred at room temperature for 3 hours, and the reaction solution was withdrawn, after 3 times of DMF washing, 3 times of DCM washing was carried out for 3min each time, to obtain Boc-protected peptide resin 2, Boc-Gly-Gly-Gly-Cys (Trt) -Tyr (tBu) -Phe-Gln (Trt) -Asn (Trt) -Cys (Trt) -Pro-Lys (Boc) -Gly-MOBHA resin.
Example 7: synthesis of terlipressin peptide resin
Taking 5% of I2DMF solution (10 mL/g resin) was added to the peptide resin 2 obtained in example 5, the reaction was stirred at 40 ℃ for 4 hours, the reaction solution was aspirated off, after washing 6 times with DMF, DCM was washed 3 times for 3min each time, and the resulting peptide resin, Gly-Gly-Gly- [ Cys-Tyr (tBu) -Phe-Gln (Trt) -Asn (Trt) -Cys, was obtained]-Pro-Lys (Boc) -Gly-MOBHA resin.
Example 8: synthesis of terlipressin peptide resin
Taking 5% of I2DMF solution (10 mL/g resin) was added to Boc protected peptide resin 2 obtained in example 6, and the reaction was stirred at 40 ℃ for 4 hours, the reaction solution was aspirated off, after DMF washing was performed 6 times, DCM washing was performed 3 times for 3min each time to obtain Boc protected terlipressin peptide resin, Gly-Gly-Gly- [ Cys-Tyr (tBu) -Phe-Gln (Trt) -Asn (Trt) -Cys]-Pro-Lys (Boc) -Gly-MOB HA resin.
Example 9: preparation of crude terlipressin
Taking the terlipressin peptide resin prepared in the example 7, adding 8% HBr/TFA solution (acidolysis solution 10 mL/gram of terlipressin resin), stirring and reacting for 6 hours, filtering and collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and draining to obtain off-white powder, namely a crude product of the terlipressin, wherein the purity of the crude product is 86.3%.
Example 10: preparation of crude terlipressin
The Boc protected terlipressin peptide resin prepared in the example 8 is taken, 8% HBr/TFA solution (acidolysis solution 10 mL/crilipressin resin) is added, the mixture is stirred and reacted for 6 hours, filtrate is collected by filtration, the resin is washed by a small amount of TFA for 3 times, the filtrate is combined and concentrated under reduced pressure, anhydrous ether is added for precipitation, the precipitate is washed by the anhydrous ether for 3 times, and after being dried by suction, white-like powder which is a terlipressin crude product is obtained, wherein the purity of the crude product is 87.2%.
Example 11: purification of crude terlipressin
Dissolving the crude terlipressin obtained in the example 9 by using 20 percent acetic acid solution, filtering the solution by using a 0.45 mu m microporous membrane, and purifying for later use;
purifying by high performance liquid chromatography, wherein the purification uses reversed phase C18 with chromatographic packing of 10 μm, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column of 77mm x 250mm is 90mL/min, eluting by a gradient system, purifying by circulating sample injection, sampling a crude product solution in the chromatographic column, starting the mobile phase for elution, collecting a main peak, and evaporating acetonitrile to obtain a terlipressin purified intermediate concentrated solution;
taking the intermediate concentrated solution of the terlipressin purification, and filtering the intermediate concentrated solution by using a 0.45-micrometer filter membrane for later use;
performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the purification is performed by reversed phase C18 with chromatographic packing of 10 μm, the flow rate of a chromatographic column of 77mm × 250mm is 90mL/min, gradient elution and cyclic sample loading method are adopted, the sample is loaded in the chromatographic column, the mobile phase elution is started, the chromatogram is collected, the change of the absorbance is observed, the main peak of salt exchange is collected and the purity is detected by analyzing the liquid phase, the main peak solutions of salt exchange are combined, the reduced pressure concentration is performed to obtain the acetic acid water solution of terlipressin, and the freeze drying is performed to obtain 46.3g pure terlipressin product
The total yield was 75.3%, molecular weight: 1228.6, purity: 99.6 percent and 0.11 percent of maximum single impurity.
Example 12: purification of crude terlipressin
Dissolving the crude terlipressin obtained in the example 10 by using a purification mobile phase A, and filtering the solution by using a 0.45 mu m microporous membrane for purification for later use;
purifying by high performance liquid chromatography, wherein the purification uses reversed phase C18 with chromatographic packing of 10 μm, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column of 77mm x 250mm is 90mL/min, eluting by a gradient system, purifying by circulating sample injection, sampling a crude product solution in the chromatographic column, starting the mobile phase for elution, collecting a main peak, and evaporating acetonitrile to obtain a terlipressin purified intermediate concentrated solution;
taking the intermediate concentrated solution of the terlipressin purification, and filtering the intermediate concentrated solution by using a 0.45-micrometer filter membrane for later use;
performing salt exchange by adopting a high performance liquid chromatography, wherein a mobile phase system is 1% acetic acid/water solution-acetonitrile, a reversed phase C18 with a chromatographic packing of 10 mu m for purification, a chromatographic column with a flow rate of 77mm × 250mm is 90mL/min, performing gradient elution and a circular sample loading method, loading the sample into the chromatographic column, starting mobile phase elution, collecting a map, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, performing reduced pressure concentration to obtain a terlipressin acetic acid water solution, and performing freeze drying to obtain 47.1g of a terlipressin pure product.
The total yield was 76.6%, molecular weight: 1228.4, purity: 99.5 percent and 0.10 percent of maximum single impurity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A method of synthesizing terlipressin, comprising the steps of:
step 1, under the action of a condensation reagent and an activation reagent, taking resin shown in formula 1 as a solid phase carrier, and sequentially coupling protected Gly, protected Lys, protected Pro, protected Cys, protected Asn, protected Gln, protected Phe, protected Tyr and protected Cys one by one according to the amino acid sequence from the C end to the N end of the terlipressin linear peptide to synthesize peptide resin 1;
step 2, under the action of a condensation reagent and an activation reagent, coupling protected Gly-Gly-Gly with peptide resin 1 to obtain peptide resin 2;
step 3, performing intramolecular cyclization on the peptide resin 2, and connecting two Cys through a disulfide bond to obtain terlipressin peptide resin;
step 4, carrying out acidolysis on the terlipressin resin by using an acidolysis agent to obtain a terlipressin crude product, wherein the acidolysis agent is a trifluoroacetic acid solution of hydrogen bromide, and the mass percentage concentration of the hydrogen bromide in the trifluoroacetic acid solution of the hydrogen bromide is 5-10%;
step 5, purifying the crude terlipressin product and converting the purified terlipressin product into salt to obtain a pure terlipressin product;
wherein, the left round ball of formula 1 represents polystyrene resin.
2. The method of claim 1, wherein the protected Gly, protected Lys, protected Pro, protected Cys, protected Asn, protected Gln, protected Phe, protected Tyr, protected Gly-Gly are:
Fomc-Gly or Boc-Gly, Fmoc-Lys (Boc), Fmoc-Pro, Fmoc-Asn (Trt), Fmoc-Gln (Trt), Fmoc-Phe, Fmoc-Tyr (tBu), Fmoc-Cys (Trt) or Fmoc-Cys (Acm), Fomc-Gly-Gly-Gly or Boc-Gly-Gly.
3. The method of claim 1 or 2, wherein the molar ratio of the charge amount of each protected amino acid to the amino group of the resin represented by formula 1 is 1-6: 1.
4. The method of claim 1, wherein the condensation reagent is one of N, N-diisopropylcarbodiimide, N, N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate/organic base, 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate/organic base, benzotriazol-N, N, N ', N' -tetramethyluronium hexafluorophosphate/organic base, O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate/organic base.
5. The method of claim 4, wherein the organic base is N, N-diisopropylethylamine, triethylamine, or N-methylmorpholine.
6. The method of claim 1, wherein the activating reagent is 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole.
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