CN102816213A - Method for preparing pramlintide by using solid-phase and liquid-phase combined technology - Google Patents
Method for preparing pramlintide by using solid-phase and liquid-phase combined technology Download PDFInfo
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- CN102816213A CN102816213A CN2012101713948A CN201210171394A CN102816213A CN 102816213 A CN102816213 A CN 102816213A CN 2012101713948 A CN2012101713948 A CN 2012101713948A CN 201210171394 A CN201210171394 A CN 201210171394A CN 102816213 A CN102816213 A CN 102816213A
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- 238000000034 method Methods 0.000 title claims abstract description 33
- 108010029667 pramlintide Proteins 0.000 title claims abstract description 30
- 239000007791 liquid phase Substances 0.000 title claims abstract description 11
- 239000007790 solid phase Substances 0.000 title claims abstract description 8
- 229960003611 pramlintide Drugs 0.000 title abstract description 5
- 238000005516 engineering process Methods 0.000 title abstract description 4
- NRKVKVQDUCJPIZ-MKAGXXMWSA-N pramlintide acetate Chemical compound C([C@@H](C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCCCN)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 NRKVKVQDUCJPIZ-MKAGXXMWSA-N 0.000 title abstract 4
- 239000012634 fragment Substances 0.000 claims abstract description 116
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 44
- 238000009833 condensation Methods 0.000 claims abstract description 38
- 230000005494 condensation Effects 0.000 claims abstract description 38
- 229920005989 resin Polymers 0.000 claims description 51
- 239000011347 resin Substances 0.000 claims description 51
- 230000008878 coupling Effects 0.000 claims description 27
- 238000010168 coupling process Methods 0.000 claims description 27
- 238000005859 coupling reaction Methods 0.000 claims description 27
- TZIRZGBAFTZREM-MKAGXXMWSA-N pramlintide Chemical compound C([C@@H](C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)CSSC1)[C@@H](C)O)C(C)C)C1=CC=CC=C1 TZIRZGBAFTZREM-MKAGXXMWSA-N 0.000 claims description 26
- 239000007787 solid Substances 0.000 claims description 21
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 15
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 14
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical group CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 13
- WNTGYJSOUMFZEP-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)propanoic acid Chemical compound OC(=O)C(C)OC1=CC=C(Cl)C=C1C WNTGYJSOUMFZEP-UHFFFAOYSA-N 0.000 claims description 10
- 150000001413 amino acids Chemical class 0.000 claims description 10
- 238000005336 cracking Methods 0.000 claims description 10
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 8
- TZCYLJGNWDVJRA-UHFFFAOYSA-N 6-chloro-1-hydroxybenzotriazole Chemical compound C1=C(Cl)C=C2N(O)N=NC2=C1 TZCYLJGNWDVJRA-UHFFFAOYSA-N 0.000 claims description 8
- 238000001311 chemical methods and process Methods 0.000 claims description 8
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 7
- -1 acetylmethyl Chemical group 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- 150000003053 piperidines Chemical class 0.000 claims description 7
- 229920003180 amino resin Polymers 0.000 claims description 6
- 239000007821 HATU Substances 0.000 claims description 5
- IRWJFLXBMUWAQM-UHFFFAOYSA-N 9h-xanthen-1-amine Chemical compound O1C2=CC=CC=C2CC2=C1C=CC=C2N IRWJFLXBMUWAQM-UHFFFAOYSA-N 0.000 claims description 4
- 238000005660 chlorination reaction Methods 0.000 claims description 4
- 125000006239 protecting group Chemical group 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- RDRBIXSNGAYLPT-UHFFFAOYSA-N CC1=CC=C(COC2=CC3=C(C=C2)C(NC(=O)OCC2C4=C(C=CC=C4)C4=C2C=CC=C4)C2=C(O3)C=CC=C2)C=C1 Chemical compound CC1=CC=C(COC2=CC3=C(C=C2)C(NC(=O)OCC2C4=C(C=CC=C4)C4=C2C=CC=C4)C2=C(O3)C=CC=C2)C=C1 RDRBIXSNGAYLPT-UHFFFAOYSA-N 0.000 claims description 3
- 238000006482 condensation reaction Methods 0.000 claims description 3
- 238000007363 ring formation reaction Methods 0.000 claims description 3
- 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 claims description 2
- 239000004793 Polystyrene Substances 0.000 claims description 2
- 229920002223 polystyrene Polymers 0.000 claims description 2
- 229920005990 polystyrene resin Polymers 0.000 claims description 2
- 239000006227 byproduct Substances 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 11
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 9
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 7
- 230000004913 activation Effects 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 238000010532 solid phase synthesis reaction Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108010016626 Dipeptides Proteins 0.000 description 5
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000008021 deposition Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000006340 racemization Effects 0.000 description 5
- 108700022034 Opsonin Proteins Proteins 0.000 description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 210000000496 pancreas Anatomy 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 108010041872 Islet Amyloid Polypeptide Proteins 0.000 description 3
- 102000036770 Islet Amyloid Polypeptide Human genes 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000012964 benzotriazole Substances 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 1
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 description 1
- 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 1
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 1
- URJOZSLMTIRWFW-QGZVFWFLSA-N (4r)-4-(1,3-benzodioxol-5-yl)-5,6-dimethoxy-4,9-dihydro-1h-benzo[f][2]benzofuran-3-one Chemical compound C1=C2OCOC2=CC([C@H]2C3=C(COC3=O)CC3=CC=C(C(=C32)OC)OC)=C1 URJOZSLMTIRWFW-QGZVFWFLSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 102400000320 Glicentin Human genes 0.000 description 1
- 101800002945 Glicentin Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 125000003277 amino group Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- QRUDEWIWKLJBPS-UHFFFAOYSA-N benzotriazole Chemical compound C1=CC=C2N[N][N]C2=C1 QRUDEWIWKLJBPS-UHFFFAOYSA-N 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
- 125000005519 fluorenylmethyloxycarbonyl group Chemical group 0.000 description 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 108010004034 stable plasma protein solution Proteins 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for preparing pramlintide by utilizing a solid-phase and liquid-phase combination technology, which comprises the steps of respectively synthesizing 1-12, 13-20, 21-28 and 29-37 peptide intermediate fragments by utilizing solid-phase chemistry, and then obtaining pramlintide by utilizing liquid-phase chemical condensation. The pramlintide is prepared by the route of the invention, the operation steps are short, the post-treatment is simple, the byproducts are few, the yield is high, the pollution is small, and the method is suitable for large-scale industrial production.
Description
Technical field
The present invention relates to the polypeptide drugs synthesis technical field, be specifically related to the method that a kind of solid phase and liquid phase combination technique prepare tripro-amylin.
Background technology
Tripro-amylin (Pramlitide, structure is seen formula I, 2-7; Disulfide linkage) be a kind of pancreas opsonin (Amylin, amylin) analogue of synthetic, its constitutional features is that the amino acid of the 25th (L-Ala) of pancreas opsonin, the 28th (Serine) and the 29th (Serine) substitutes with proline(Pro) respectively.It has fully kept the physiological action of endogenous pancreas opsonin, has overcome people's pancreas opsonin instability, facile hydrolysis, agglutinophilic physical property in solution.Clinical study shows that tripro-amylin can suppress the release of glicentin, delays stomach emptying and inhibition is ingested; Use with insulin combination, effectively controlling blood sugar, lose weight, reduce insulin dosage.Tripro-amylin obtains drugs approved by FDA the earliest by U.S. Amylin Pharmaceuticals company development on March 16th, 2005, is applicable to use Regular Insulin but can't reach the I type of expected effect and type ii diabetes patient's assisting therapy.
The synthetic of known tripro-amylin can pass through solid-phase synthesis (WO-A-93/10146, US-A-5424394, CN102206266A; CN101747426A), the carboxyl of C being held tyrosine on insoluble resin, is a starting point with this amino group of amino acids with the form fix of covalent linkage again; Condensation amino acid successively; Prolong peptide chain, after accomplishing peptide chain synthetic on the resin, the sulfydryl formation intramolecular disulfide bond of the 2nd and the 7th halfcystine of oxidation; Again this peptide chain is cut down and remove Side chain protective group from resin, finally prepare tripro-amylin.Obviously, for containing 37 amino acid whose tripro-amylins, this route of synthesis needs repetition tens step coupling and deprotection steps, and synthetic route is tediously long, and operating procedure is loaded down with trivial details; And along with the prolongation of peptide chain and the influence of the secondary structure that possibly exist, condensation efficiency causes impurity to become many and be difficult to and separates worse and worse, and target product yield is extremely low, and cost is very high, is not suitable for suitability for industrialized production.
Use solid phase and liquid phase bonded fragment condensation technology, be suitable for the synthetic polypeptide that contains longer aminoacid sequence, its advantage is that by product is less and is easy to purifying that product purity is high.Yet the fragment condensation strategy is hampered by the danger that (i) has increases racemization sometimes; (ii) big protection fragment poor solubility; (iii) intersegmental low condensation rate of sheet and the danger that takes place with side reaction.In order to eliminate or to reduce racemization, generally select Gly or Pro as segmental C end residue, if can not realize this point, then select Ala or Arg to hold residue, because they are difficult for racemization as C.When application fragment condensation strategy, target sequence is carried out rational segmentation can improve the result greatly.Except choose reasonable C end residue, reduce the risk of racemization as far as possible, also the segmental purity of considered synthetic on solid phase wants high, and solvability will be got well; The segmental C end and the N that participate in condensation hold the sterically hindered little of residue, to improve the intersegmental condensation rate of sheet.
Tripro-amylin is made up of 37 amino-acid residues; Thereby there are possible fragment of many kinds and a condensation order; For example CN101824086A adopts 1-8 (fragment 1), 9-19 (fragment 2), 20-28 (fragment 3); Four fragments of 29-37 (fragment 4) are with the method for the order condensation of [(4+ (3+2))+1]; CN102164608A (patent families US2010/0081788A1) reported method is similar with it; Adopt 1-8 (fragment 1), 9-19 (fragment 2), 20-29 (fragment 3); Four fragments of 30-37 (fragment 4) are held the condensation successively of N end [((4+3)+2)+1] order from C; CN101790535A (patent families WO2009/00366A1) has reported employing 1-12 (fragment 1), 13-24 (fragment 2), and three fragments of 25-37 (fragment 3) are held the method for N end order condensation from C; CN102180943A then adopts 1-16 (fragment 1), 17-28 (fragment 2), and three fragments of 29-37 (fragment 3) hold N to hold the method for condensation successively from C.In the above fragment condensation synthesis strategy; In preceding two kinds of methods in the 19th Ser (C end) and the 20th Ser (N end) and the 4th kind of method the 16th Leu (C end) and the 17th Val (N end) be not the ideal breakpoint all because the intersegmental condensation efficiency of these sheets is not high in actually operating; Though the third method Cut Selection is comparatively suitable, fragment 3 purity on solid phase synthesis is not good, will influence fragment condensation reaction efficient.
The pharmaceutical application important in view of tripro-amylin is worth, and is necessary to provide more effective tripro-amylin synthetic schemes, can overcome the defective of above method and be fit to plant-scale production.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of new tripro-amylin, and this method can overcome existing preparing method's deficiency and defective, and suitable suitability for industrialized production.
The preparation method of tripro-amylin among the present invention, employing be the combination technique of solid phase synthesis fragment and the condensation of fragment liquid phase.At first utilize the intermediate fragments peptide of solid state chemistry 4 side chain protected of synthesizing pramlintide on resin carrier: fragment a (1-12); Fragment b (13-20); Fragment c (21-28); Fragment d (29-37), then in liquid phase with the intermediate fragments peptide condensation of side chain protected, remove aminoterminal at last and Side chain protective group obtains tripro-amylin.Selected 4 suitable fragment peptides in present method, through conventional solid-phase synthesis all can high-level efficiency, high yield, highly purified acquisition each fragment peptide; Each fragment peptide solubleness in liquid phase condensation system is high, and the segmental C end and the N end spaces steric hindrance of participation condensation are little, and the condensation productive rate is high, and product is easy to purifying.
Particularly, tripro-amylin preparation method provided by the invention may further comprise the steps:
(1) utilizing the solid state chemistry method, is carrier with aminoresin, the peptide intermediate fragments a:AA of synthetic side chain protected
29-AA
37-aminoresin, the instance of fragment a:
The instance of aminoresin is: the amino xanthene of 9-Fmoc--3-oxygen base polystyrene resin [9-Fmoc-Aminoxanthen-3 – yloxy-polystyrene (Sieber Amide resin)] or the amino xanthene of 9-Fmoc--3-oxygen base acetylmethyl resin (9-Fmoc-Aminoxanthen-3-yloxy-acetamidomethyl Resin).
(2) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments b:AA of synthetic side chain protected
21-AA
28-trityl resin, the instance of fragment b:
(3) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments c:AA of synthetic side chain protected
13-AA
20-trityl resin, the instance of fragment c:
(4) utilizing the solid state chemistry method, is carrier with the trityl resin, and the peptide midbody of synthetic side chain protected forms 2,7 disulfide bridge bonds then, obtains peptide intermediate fragments d:AA
1-AA
12-trityl resin, the instance of fragment d:
P representes pseudo-proline(Pro) protection base (ψ among fragment a, c, the d
Me, MePro) or tBu protection base, preferred pseudo-proline(Pro) protection base (ψ
Me, MePro); K among the fragment d representes Fmoc protection base or Boc protection base, preferred Boc protection base.
Synthetic 2, the instance of the cyclization reagent of 7 disulfide bridge bonds is to use I
2, solvent is DMF, and the cyclisation time is 1-4h, and the cyclisation temperature is 15-25 ℃.
Trityl resin instance in step (2), (3), (4) is a 2-chloro-trityl chloride resin (CTC resin), chlorinated triphenyl methyl resin, 4-methyl chlorination trityl resin or 4-methoxyl group chlorination trityl resin.The synthetic method (SPPS) that adopts conventional solid phase synthesis of peptide of fragment.For example; Can be employed in step (1)-(4) on the resin according to peptide preface coupling protection amino acid progressively; Coupling system is DIEA and HOBt; Any one or more combination and HBTU among HOAt or the Cl-HOBt, HATU, the coupling system that any one or more combination among PyBOP or the PyAOP is formed.
(5) the peptide intermediate fragments a of side chain protected, b, c, d cracking from the resin is got off, in solution, carry out condensation reaction, obtain side chain protected peptide (d-c-b-a);
The method of liquid phase condensation can with reference to the document Chemical Synthesis of Proteins in Solution of Shumpei Sakakibara (Biopolymers (Peptide Science). 1999; 51 (4): 279-296); For example used solvent can be NMP or DMF, and coupling system is DCC and HOBt, any one or more combination among HOAt or the Cl-HOBt; Perhaps DIEA and HOBt; Any one or more combination and HBTU among HOAt or the Cl-HOBt, HATU, the condensation system that any one or more combination among PyBOP or the PyAOP is formed.
(6) aminoterminal that removes side chain protected peptide (d-c-b-a) is protected base and Side chain protective group, obtains the thick peptide of tripro-amylin;
(7) obtain tripro-amylin after purified.
Employed term among the present invention " pseudo-proline(Pro) (Pseudoprolines) " is meant C in the prolyl ring
γH
2Base is by oxygen or sulphur atom substituted proline analogs (CN101472939A).Pseudo-proline(Pro) is that Serine, Threonine and halfcystine and aldehyde or ketone cyclocondensation form.The pseudo-proline dipeptides structure of Serine of using in the embodiment of the invention and Threonine is following:
Through adopting pseudo-proline(Pro) protection strategy, not only improved the combined coefficient of difficult sequences in solid phase synthesis, and reduced the danger of C end racemization.
In above-mentioned steps (5), fragment a, b, c, d can be through different order condensations.For example, can distinguish condensation fragment b and fragment a, obtain fragment (b-a), condensation fragment d and fragment c obtain fragment (d-c), and fragment (b-a) obtains side chain protected peptide (d-c-b-a) with fragment (d-c) condensation more then; Also can first condensation fragment c and fragment b, obtain fragment (c-b), and then obtain side chain protected peptide (d-c-b-a) with fragment a and fragment d condensation respectively.Preferred scheme is according to the order condensation successively of holding the N end from C, and is specific as follows:
(5.1) fragment a cracking from the resin is got off, remove aminoterminal protection base; Fragment b cracking from the resin is got off; The two is condensation in solution; Obtain fragment (b-a), the instance of fragment (b-a):
(5.2) aminoterminal that removes fragment (b-a) is protected base, and the c fragment condensation solution with cracking from resin is got off obtains fragment (c-b-a), instance (c-b-a):
In the above structural formula, Boc (tertbutyloxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl), Trt (trityl), tBu (tertiary butyl), Pbf (2,2,4,6,7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl) are the protection bases.But the present invention is not limited to these several kinds protection bases, and those skilled in the art also can adopt other suitable protection bases.
Description of drawings
Fig. 1: the ESI-MS of tripro-amylin analyzes collection of illustrative plates.
Embodiment
The present invention comprises but is not limited to following examples.
The reagent name abbreviation
DMF:N, dinethylformamide
The NMP:N-SL 1332
DCC: NSC 57182
TFA: trifluoroacetic acid
DCM: methylene dichloride
TIS: tri isopropyl silane
DIEA:N, the N-diisopropylethylamine
The HOBt:1-hydroxybenzotriazole
HOAt:N-hydroxyl-7-azepine benzotriazole
Cl-HOBt:6-chloro-1-hydroxy benzo triazole
HBTU: benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate
HATU:2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester
PyBOP: phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus
PyAOP: (3H-1,2,3-triazolo [4,5-b] pyridine-3-oxygen base) three-1-Bi coughs up Wan Ji Phosphonium hexafluorophosphate
Embodiment 1: the preparation of fragment a
Fmoc-Tyr (tBu)-OH is coupled on the Seiber Amide insoluble resin that the 50g substitution value is 0.69mmol/g, is coupling reagent with HBTU/HOBt/DIEA, will treat that link coupled amino acid feeds intake with 2 equivalents, and first activation is then in the room temperature linked reaction.Each coupling completeness can be through Kaiser test monitoring, and after coupling was accomplished, the DMF solution of the piperidines with 20% was removed Fmoc protection base in room temperature.Divide with the TFA/DCM solution of 2-5% after the fragment coupling is accomplished and elute the synthetic polypeptide from resin for three times.Concentrate the back with ether wash white solid 12g.
The alternate fragment a ' with pseudo-proline(Pro) that can prepare 30 Thr (tBu) through similar mode, pseudo-proline(Pro) unit adopts Fmoc-Pro-Thr (ψ
Me, MePro)-importing of OH dipeptides.
Embodiment 2: the preparation of fragment b
Fmoc-Pro-OH is coupled on the CTC insoluble resin that the 50g substitution value is 0.74mmol/g, is coupling reagent with HBTU/HOBt/DIEA, will treat that link coupled amino acid feeds intake with 2 equivalents, and first activation is then in the room temperature linked reaction.Each coupling completeness can be through Kaiser test monitoring, and after coupling was accomplished, the DMF of the piperidines with 20% removed Fmoc protection base in room temperature.TFA/DCM solution with 1% after the fragment coupling is accomplished elutes the synthetic polypeptide from resin.Concentrate the back with ether wash white solid 25g.
Embodiment 3: the preparation of fragment c
Fmoc-Ser (tBu)-OH is coupled on the CTC insoluble resin that the 50g substitution value is 0.74mmol/g, is coupling reagent with HBTU/HOBt/DIEA, will treat that link coupled amino acid feeds intake with 2 equivalents, and first activation is then in the room temperature linked reaction.Each coupling completeness can be through Kaiser test monitoring, and after coupling was accomplished, the DMF solution of the piperidines with 20% was removed Fmoc protection base in room temperature.TFA/DCM solution with 1% after the fragment coupling is accomplished elutes the synthetic polypeptide from resin.Concentrate the back with ether wash white solid 11g.
The alternate fragment c ' with pseudo-proline(Pro) that can prepare 20 Ser (tBu) through similar mode, pseudo-proline(Pro) unit adopts Fmoc-Ser (tBu)-Ser (ψ
Me, MePro)-importing of OH dipeptides.
Embodiment 4: the preparation of fragment d
Fmoc-Leu-OH is coupled on the CTC insoluble resin that the 50g substitution value is 0.74mmol/g, is coupling reagent with HBTU/HOBt/DIEA, will treat that link coupled amino acid feeds intake with 2 equivalents, and first activation is then in the room temperature linked reaction.Each coupling completeness can be through Kaiser test monitoring, and after coupling was accomplished, the DMF solution of the piperidines with 20% was removed Fmoc protection base in room temperature.The fragment coupling is accomplished the back with 10 normal iodine Synthetic 2s, the disulfide bridge bond of 7 two halfcystines, and the TFA/DCM solution of following with 1% elutes the synthetic polypeptide from resin.Concentrate the back with ether wash faint yellow solid 12g.
The alternate fragment d ' with pseudo-proline(Pro) that can prepare 4 Thr (tBu) through similar mode, pseudo-proline(Pro) unit adopts Fmoc-Asn (Trt)-Thr (ψ
Me, MePro)-importing of OH dipeptides; The alternate fragment d ' ' with pseudo-proline(Pro) that perhaps prepares 6 Thr (tBu), pseudo-proline(Pro) unit adopts Fmoc-Ala-Thr (ψ
Me, MePro)-importing of OH dipeptides.
Embodiment 5: the preparation of fragment (b-a)
Earlier at HOBt, activation is 5 minutes in the DMF solution of DIEA and HBTU, adds 3.31g fragment a then, carries out coupling with 3.12g fragment b.After reacting completely mixture is poured in the cold water, had deposition to separate out, filtration washing gets white solid compound 5.83g, productive rate 90.6%.
Embodiment 6: the preparation of fragment (c-b-a)
3.22g fragment (b-a) is dissolved in the DMF solution of 20% piperidines, question response is poured mixture into after fully has deposition to separate out in the cold water, and filtration washing gets white solid.Earlier at HOBt, activation is 5 minutes in the DMF solution of DIEA and HBTU, adds white solid then, carries out coupling with 1.70g fragment c.After reacting completely mixture is poured in the cold water, had deposition to separate out, filtration washing gets white solid compound 4.42g, productive rate 94.6%.
Embodiment 7: the preparation of side chain protected peptide (d-c-b-a)
2.34g fragment (c-b-a) is dissolved in the DMF solution of 20% piperidines, question response is poured mixture into after fully has deposition to separate out in the cold water, and filtration washing gets white solid.Earlier at HOBt, activation is 5 minutes in the DMF solution of DIEA and HBTU, adds white solid then, carries out coupling with 1.35g fragment d.After reacting completely mixture is poured in the cold water, had deposition to separate out, filtration washing gets white solid compound 3.09g, productive rate 90.0%.
Embodiment 8: the preparation of tripro-amylin
The volume ratio that 2.10g side chain protected peptide (d-c-b-a) is dissolved in phenol (Phenol): TIS:TFA=2.5:2.5:95 disposes in the 20ml solution that forms.Add a large amount of cold diethyl ethers behind the reaction 2.5h, have solid to separate out, filtration washing gets the tripro-amylin bullion.The tripro-amylin bullion is carried out purifying with HPLC: chromatography column: SinoChrom ODS-BP (10um); 10mm * 200mm.Detect wavelength: 220nm.Gradient elution: B%:28%-48% (20 minutes), eluent A:0.8 ‰ TFA/ water, B:0.8 ‰ TFA/ acetonitrile.Removing freeze-drying behind the acetonitrile, to get purity be 95% the pure article 1.06g of tripro-amylin, productive rate 87.9%.
Confirm its structure through the ESI-MS collection of illustrative plates, see Fig. 1.
Claims (10)
1. method of utilizing solid phase and liquid phase combination technique to prepare tripro-amylin, tripro-amylin structural formula be suc as formula shown in the I,
Said method comprising the steps of:
(1) utilizing the solid state chemistry method, is carrier with aminoresin, the peptide intermediate fragments a:AA of synthetic side chain protected
29-AA
37-aminoresin;
(2) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments b:AA of synthetic side chain protected
21-AA
28-trityl resin;
(3) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments c:AA of synthetic side chain protected
13-AA
20-trityl resin;
(4) utilizing the solid state chemistry method, is carrier with the trityl resin, the peptide intermediate fragments d:AA of synthetic side chain protected
1-AA
12-trityl resin;
(5) the peptide intermediate fragments a of side chain protected, b, c, d cracking from the resin is got off, in solution, carry out condensation reaction, obtain side chain protected peptide (d-c-b-a);
(6) aminoterminal that removes side chain protected peptide (d-c-b-a) is protected base and Side chain protective group, obtains the thick peptide of tripro-amylin;
(7) obtain tripro-amylin behind the purifying.
2. according to the described method of claim 1; Used aminoresin is the amino xanthene of 9-Fmoc--3-oxygen base polystyrene resin [9-Fmoc-Aminoxanthen-3-yloxy-polystyrene (Sieber Amide resin)] or the amino xanthene of 9-Fmoc--3-oxygen base acetylmethyl resin (9-Fmoc-Aminoxanthen-3-yloxy-acetamidomethyl Resin) in the step (1), preferred Sieber Amide resin.
3. according to the described method of claim 1; Used trityl resin is a 2-chloro-trityl chloride resin (CTC resin) in step (2)-(4); Chlorinated triphenyl methyl resin, 4-methyl chlorination trityl resin or 4-methoxyl group chlorination trityl resin, preferred CTC resin.
4. according to the described method of claim 1; Step (1)-(4) are employed on the resin according to peptide preface coupling protection amino acid progressively; Coupling system is DIEA and HOBt; Any one or more combination and HBTU among HOAt or the Cl-HOBt, HATU, the coupling system that any one or more combination among PyBOP or the PyAOP is formed.
5. according to the described method of claim 1, step (4) Synthetic 2, the cyclization reagent of the disulfide bridge bond of 7 two halfcystines is I
2, solvent is DMF, and the cyclisation time is 1-4h, and the cyclisation temperature is 15-25 ℃.
6. according to the described method of claim 1; Used solvent is NMP or DMF in the step (5), and the condensation system is DCC and HOBt, any one or more combination among HOAt or the Cl-HOBt; Perhaps DIEA and HOBt; Any one or more combination and HBTU among HOAt or the Cl-HOBt, HATU, the condensation system that any one or more combination among PyBOP or the PyAOP is formed.
7. according to the described method of claim 1, the method that removes aminoterminal Fmoc protection base in step (5)-(6) is carried out in the solution of DMF that contains piperidines or NMP.
8. according to the described method of claim 1, step (5) comprising:
(5.1) fragment a cracking from the resin is got off, remove aminoterminal protection base; Fragment b cracking from the resin is got off, and the two is condensation in solution, obtains fragment (b-a);
(5.2) aminoterminal that removes fragment (b-a) is protected base, and the c fragment condensation solution with cracking from resin is got off obtains fragment (c-b-a);
(5.3) aminoterminal that removes fragment (c-b-a) is protected base, and the d fragment condensation solution with cracking from resin is got off obtains side chain protected peptide (d-c-b-a).
9. described according to Claim 8 method, wherein:
Fragment (b-a) is
Fragment (c-b-a) is
Fragment (d-c-b-a) is
P among fragment a, c and the d representes pseudo-proline(Pro) protection base (ψ
Me, MePro) or tBu protection base, preferred pseudo-proline(Pro) protection base (ψ
Me, MePro).
10. method according to claim 1, wherein:
Fragment a is
Fragment b is
Fragment c is
Fragment d is
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Cited By (5)
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| CN104530214A (en) * | 2014-12-23 | 2015-04-22 | 扬子江药业集团四川海蓉药业有限公司 | Preparation method of pramlintide acetate |
| CN111499719A (en) * | 2020-03-19 | 2020-08-07 | 杭州固拓生物科技有限公司 | Method for synthesizing pramlintide |
| CN114249808A (en) * | 2021-12-27 | 2022-03-29 | 杭州诺泰澳赛诺医药技术开发有限公司 | Synthesis method of Cagrilintide |
| CN115785192A (en) * | 2022-12-06 | 2023-03-14 | 生工生物工程（上海）股份有限公司 | Method for synthesizing polypeptide |
| CN118530332A (en) * | 2024-07-26 | 2024-08-23 | 南京羚诺生物医药技术研究院有限公司 | Preparation method of pramlintide |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104530214A (en) * | 2014-12-23 | 2015-04-22 | 扬子江药业集团四川海蓉药业有限公司 | Preparation method of pramlintide acetate |
| CN104530214B (en) * | 2014-12-23 | 2018-07-20 | 扬子江药业集团四川海蓉药业有限公司 | A kind of preparation method of pramlintide acetate |
| CN111499719A (en) * | 2020-03-19 | 2020-08-07 | 杭州固拓生物科技有限公司 | Method for synthesizing pramlintide |
| CN114249808A (en) * | 2021-12-27 | 2022-03-29 | 杭州诺泰澳赛诺医药技术开发有限公司 | Synthesis method of Cagrilintide |
| CN115785192A (en) * | 2022-12-06 | 2023-03-14 | 生工生物工程（上海）股份有限公司 | Method for synthesizing polypeptide |
| CN118530332A (en) * | 2024-07-26 | 2024-08-23 | 南京羚诺生物医药技术研究院有限公司 | Preparation method of pramlintide |
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Application publication date: 20121212 |