CN102952174A - Method for synthesizing degarelix - Google Patents
Method for synthesizing degarelix Download PDFInfo
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- CN102952174A CN102952174A CN2012104601959A CN201210460195A CN102952174A CN 102952174 A CN102952174 A CN 102952174A CN 2012104601959 A CN2012104601959 A CN 2012104601959A CN 201210460195 A CN201210460195 A CN 201210460195A CN 102952174 A CN102952174 A CN 102952174A
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- 238000000034 method Methods 0.000 title claims abstract description 24
- 108010052004 acetyl-2-naphthylalanyl-3-chlorophenylalanyl-1-oxohexadecyl-seryl-4-aminophenylalanyl(hydroorotyl)-4-aminophenylalanyl(carbamoyl)-leucyl-ILys-prolyl-alaninamide Proteins 0.000 title abstract description 11
- 229960002272 degarelix Drugs 0.000 title abstract description 11
- MEUCPCLKGZSHTA-XYAYPHGZSA-N degarelix Chemical compound C([C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCNC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@H](C)C(N)=O)NC(=O)[C@H](CC=1C=CC(NC(=O)[C@H]2NC(=O)NC(=O)C2)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](CC=1C=NC=CC=1)NC(=O)[C@@H](CC=1C=CC(Cl)=CC=1)NC(=O)[C@@H](CC=1C=C2C=CC=CC2=CC=1)NC(C)=O)C1=CC=C(NC(N)=O)C=C1 MEUCPCLKGZSHTA-XYAYPHGZSA-N 0.000 title abstract description 11
- 230000002194 synthesizing effect Effects 0.000 title abstract 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 25
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 13
- 229920003180 amino resin Polymers 0.000 claims abstract description 11
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007822 coupling agent Substances 0.000 claims abstract description 3
- 239000002994 raw material Substances 0.000 claims abstract description 3
- 229920005989 resin Polymers 0.000 claims description 59
- 239000011347 resin Substances 0.000 claims description 59
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 35
- 238000005406 washing Methods 0.000 claims description 28
- 238000002360 preparation method Methods 0.000 claims description 20
- 150000001408 amides Chemical class 0.000 claims description 17
- 125000006239 protecting group Chemical group 0.000 claims description 16
- 150000001413 amino acids Chemical class 0.000 claims description 14
- 238000003746 solid phase reaction Methods 0.000 claims description 12
- 238000010671 solid-state reaction Methods 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 11
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 10
- 235000001014 amino acid Nutrition 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 230000004913 activation Effects 0.000 claims description 9
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 9
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 8
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000010511 deprotection reaction Methods 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 8
- 229920001184 polypeptide Polymers 0.000 claims description 7
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008961 swelling Effects 0.000 claims description 6
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 claims description 6
- 239000006166 lysate Substances 0.000 claims description 5
- QWVGKYWNOKOFNN-UHFFFAOYSA-N o-cresol Chemical compound CC1=CC=CC=C1O QWVGKYWNOKOFNN-UHFFFAOYSA-N 0.000 claims description 5
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 5
- ZHGNHOOVYPHPNJ-UHFFFAOYSA-N Amigdalin Chemical compound FC(F)(F)C(=O)OCC1OC(OCC2OC(OC(C#N)C3=CC=CC=C3)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C2OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C1OC(=O)C(F)(F)F ZHGNHOOVYPHPNJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 238000005336 cracking Methods 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- YDJXDYKQMRNUSA-UHFFFAOYSA-N tri(propan-2-yl)silane Chemical compound CC(C)[SiH](C(C)C)C(C)C YDJXDYKQMRNUSA-UHFFFAOYSA-N 0.000 claims description 4
- JYUTZJVERLGMQZ-AREMUKBSSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-naphthalen-2-ylpropanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@@H](C(=O)O)CC1=CC=C(C=CC=C2)C2=C1 JYUTZJVERLGMQZ-AREMUKBSSA-N 0.000 claims description 3
- JQLPMTXRCLXOJO-OAQYLSRUSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-pyridin-3-ylpropanoic acid Chemical compound C([C@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CN=C1 JQLPMTXRCLXOJO-OAQYLSRUSA-N 0.000 claims description 3
- CQPNKLNINBUUOM-JOCHJYFZSA-N (2r)-3-(4-chlorophenyl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C([C@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=C(Cl)C=C1 CQPNKLNINBUUOM-JOCHJYFZSA-N 0.000 claims description 3
- 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 claims description 3
- 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 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000012982 microporous membrane Substances 0.000 claims description 3
- 239000012071 phase Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- QWXZOFZKSQXPDC-LLVKDONJSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-LLVKDONJSA-N 0.000 claims description 2
- 239000012043 crude product Substances 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 10
- 238000003786 synthesis reaction Methods 0.000 abstract description 10
- 238000007086 side reaction Methods 0.000 abstract description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 abstract description 4
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 abstract description 3
- 230000008707 rearrangement Effects 0.000 abstract description 3
- UFIVEPVSAGBUSI-UHFFFAOYSA-N dihydroorotic acid Chemical group OC(=O)C1CC(=O)NC(=O)N1 UFIVEPVSAGBUSI-UHFFFAOYSA-N 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 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 abstract description 2
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 abstract 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 abstract 1
- 108090000279 Peptidyltransferases Proteins 0.000 abstract 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 22
- 239000000243 solution Substances 0.000 description 20
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 12
- 239000000047 product Substances 0.000 description 8
- 239000006227 byproduct Substances 0.000 description 6
- 229960003604 testosterone Drugs 0.000 description 6
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 5
- 206010060862 Prostate cancer Diseases 0.000 description 5
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 5
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 5
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 5
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- UFIVEPVSAGBUSI-REOHCLBHSA-N (S)-dihydroorotic acid Chemical compound OC(=O)[C@@H]1CC(=O)NC(=O)N1 UFIVEPVSAGBUSI-REOHCLBHSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 235000004400 serine Nutrition 0.000 description 3
- 108010000817 Leuprolide Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- GFIJNRVAKGFPGQ-LIJARHBVSA-N leuprolide Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 GFIJNRVAKGFPGQ-LIJARHBVSA-N 0.000 description 2
- 229960004338 leuprorelin Drugs 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- PXQPEWDEAKTCGB-UHFFFAOYSA-N orotic acid Chemical compound OC(=O)C1=CC(=O)NC(=O)N1 PXQPEWDEAKTCGB-UHFFFAOYSA-N 0.000 description 2
- 150000003053 piperidines Chemical class 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- IONOZYFSQPGBAT-NRFANRHFSA-N (2s)-3-[tert-butyl(dimethyl)silyl]oxy-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CO[Si](C)(C)C(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 IONOZYFSQPGBAT-NRFANRHFSA-N 0.000 description 1
- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 description 1
- UFIVEPVSAGBUSI-REOHCLBHSA-M (S)-dihydroorotate Chemical compound [O-]C(=O)[C@@H]1CC(=O)NC(=O)N1 UFIVEPVSAGBUSI-REOHCLBHSA-M 0.000 description 1
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 1
- BLCLNMBMMGCOAS-URPVMXJPSA-N Goserelin Chemical compound C([C@@H](C(=O)N[C@H](COC(C)(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(=O)NNC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 BLCLNMBMMGCOAS-URPVMXJPSA-N 0.000 description 1
- 108010069236 Goserelin Proteins 0.000 description 1
- 208000012766 Growth delay Diseases 0.000 description 1
- 206010062767 Hypophysitis Diseases 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
- 102000008238 LHRH Receptors Human genes 0.000 description 1
- 108010021290 LHRH Receptors Proteins 0.000 description 1
- 102000007066 Prostate-Specific Antigen Human genes 0.000 description 1
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 229960000846 camphor Drugs 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- WQXNXVUDBPYKBA-YFKPBYRVSA-N ectoine Chemical compound CC1=[NH+][C@H](C([O-])=O)CCN1 WQXNXVUDBPYKBA-YFKPBYRVSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229960002913 goserelin Drugs 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- RYDVBNBVYVMEDT-UHFFFAOYSA-N n-carbamoyl-2-hydroxyacetamide Chemical group NC(=O)NC(=O)CO RYDVBNBVYVMEDT-UHFFFAOYSA-N 0.000 description 1
- YZNDBOCHGHZKCG-UHFFFAOYSA-N n-carbamoyl-2-hydroxyacetamide;imidazolidine-2,4-dione Chemical group O=C1CNC(=O)N1.NC(=O)NC(=O)CO YZNDBOCHGHZKCG-UHFFFAOYSA-N 0.000 description 1
- 229960005010 orotic acid Drugs 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 230000006965 reversible inhibition Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 150000003355 serines Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-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 discloses a method for synthesizing degarelix, wherein an amino resin protected with Fmoc is used as a raw material; according to a solid-phase synthesis method, DIC/HOBt (N,N'-diisopropylcarbodiimide/1-hydroxybenzotrizole) is used as a coupling agent for transpeptidase reaction; 4-serine uses tert-butyl dimethyl to replace tertiary butyl for protection protecting hydroxyl; a hydroorotic acid fragment connected with 4-amino of 5-phenylalanine benzene ring is protected with triphenylmethyl at first, and then introduced, so that the rearrangement side reaction is prevented; and since D-4Aph (Cbm) is used for replacing D-4Aph (Cbm-tBu), a t-Bu removal difficulty is prevented and the occurrence of the side reaction is reduced. A synthesis technology of the method for synthesizing degarelix is simple in steps, easy to control, small in environment pollution and high in yield, thereby being applicable for industrial production.
Description
Technical field
The present invention relates to a kind of synthetic method with polypeptide of physiologically active, specifically the artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Background technology
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, English popular name Degarelix, it is a kind of gonadotropin releasing hormone (GnRH) acceptor inhibitor class medicine, reversible inhibition hypophysis GnRH acceptor reduces the release that gonadotropin releasing hormone suppresses testosterone then, by suppressing that prostate cancer is continued growth and the deterioration that the vital testosterone of growth delays prostate cancer.It is present unique clinical GnRH receptor antagonist for advanced prostate cancer.Its aminoacid sequence is Ac-D-2Nal-D-4 Cpa-D-3Pal-Ser-4Aph (L-Hor)-D-4Aph (Cbm)-Leu-Ilys
-Pro-D-Ala-NH
2 ,Structural formula is as follows:
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 begins just can reduce testosterone concentration from medication, can not produce such as GnRH agonist (such as goserelin, Leuprolide etc.) and bring " falling after rising " phenomenon.The effect that Degarelix reduces testosterone concentration is suitable with Leuprolide depot controlled release injection, and it is significantly fast statistically to reduce testosterone concentration, suppresses all the time prostate specific antigen in whole 1 year for the treatment of.
Quick, the lasting control testosterone levels of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 energy suppresses the development of prostate cancer, and tolerance is with marketed drug is suitable at present.Along with Chinese society's aging is accelerated, the morbidity of prostate cancer improves year by year, and the market potential of Degarelix is huge.
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is the linear decapeptide that contains 7 alpha-non-natural amino acids, and wherein containing 5 amino acid is D configuration amino acid.
Patent documentation is at first by the Semple Graeme of Ferring company and Jiang Guangcheng R ﹠ D Cooperation
,The Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solid phase synthesis process of patent documentation WO9846634 report adopts the Boc strategy, on resin, then removes the Boc protecting group with TFA with the N-Boc-D-Ala coupling, then connects reactive polypeptide according to amino acid whose order, deprotection.Wherein 5 and 6 s' 4-amino-benzene L-Ala (
Aph) need to adopt the Fmoc protecting group to protect, and then remove with piperidines/DMF solution, react with tertiary butyl isocyanic ester or L-hydroorotic acid again, and then next peptide bond in succession in turn.After the acetic anhydride acidylate, remove 4 Ser-OBn with HF, 6 Aph-Cbm-tBu and 8 s' Lys-NCbz.WO9846634 has also mentioned another method, and just the vitamin B13 on the 5 amino acids side chains is placed on whole peptide chain and finishes afterwards again introducing.
Patent documentation US6214798B1 discloses a kind of liquid-phase synthesis process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and the liquid phase synthesis mode of proposition is more suitable for a large amount of preparing products.The purity of the finished product is greatly about 98%, yet point out at existing document (J.Med.Chem., 2005,48,4851-4860) to measure purity be that 98%, HPLC measures only is 96% by kapillary zone gel electrophoresis according to the product of the same manner preparation.And use at last the hazardous gases such as HF, be unfavorable for industrial production.
Patent documentation WO2010121835 discloses the technical scheme that a kind of Fmoc of employing method prepares Degarelix, by document (J.Chem.Soc.Perkin, Trans.2,1984, pg1077-1081; Org.Biomol. Chem., 2004, pg1098-1103) as can be known, the L-hydroorotic acid structure on the 5 amino acids side chains is unstable under alkaline condition, easily resets the by product that generates glycolylurea (Hydantoin) structure.And found through experiments at dense NaOH and regulate the by product that can generate 4.5% weight ratio when the pH value is processed, 2% DBU/DMF (1,8-diazabicylo [5.4.0] 11 carbon-7-alkene/yl) process degarelix and produce 1.8% glycolylurea structure product, if contain 5% water, by product then increases to 7%.But the piperidines 20%/DMF(dimethyl sulfoxide (DMSO)) in the solution degarelix without any degraded.Adopt at last this method can prepare degarelix less than the related substances of 0.3% weight percent, and the rearrangement product that has reduced the L-hydroorotic acid generates, avoided in addition above-mentioned Boc method to use in a large number TFA, reduced environmental hazard, reduced the TFA(trifluoroacetic acid to the high poison of human body) harm.
Patent documentation WO2011066386 points out to use in the above-mentioned Fmoc strategy hydroxyl of t-Bu protection Serine; the deprotection test shows; general condition can not remove the t-Bu protection fully; need 100% TFA(trifluoroacetic acid) process and just can finish deprotection in 25 hours; under this strong reaction condition, increasing of side reaction and by product can affect the quality of the finished product; if adopt 45 ℃ of reactions to remove fully, but still can produce the quality that degraded product affects the finished product.Adopt the hydroxyl of Trt protection Serine, can be at the 95%TFA(trifluoroacetic acid), the 2.5%TIS(tri isopropyl silane), 2.5EDT(1,2-dithioglycol) in the solution 45 ℃ removed in lower 1 ~ 2 hour.Adopt the Degarelix HPLC purity of this strategy preparation to reach more than 98.5%; the Trt protection of adopting acid easily to dissociate; because the space bit of Trt group is rented large and hydrophobicity; cause peptide bond to form difficulty, need the long reaction times and then produce more by product and sequence peptide disappearance and racemization.
Therefore, be badly in need of a kind of technique simple, be easy to the artificial chemistry synthesis technique controlling quality and be suitable for large industrial (S)-2-methyl-1,4,5,6-tetra-hydro pyrimidine-4-carboxylic acid.
Summary of the invention
For above-mentioned prior art, the invention provides Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 artificial synthesis.
The present invention is achieved by the following technical solutions:
A kind of solid phase synthesis process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 may further comprise the steps:
Take the aminoresin of Fmoc protection as starting raw material, according to the method for solid phase synthesis, use DIC/ HOBt
For coupling agent connects reactive polypeptide; the amino acid that connects successively the Fmoc protection; obtain the decapeptide resin of protection; the Fmoc group in the deaminize acid successively during this time; after obtaining the decapeptide resin of protection, remove amino acid whose Side chain protective group, cut peptide and obtain the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product; then the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of purifying may further comprise the steps:
1) resin swelling and deprotection
The aminoresin of Fmoc protection is packed in the solid state reaction post, with DMF washing 1 ~ 4 time, then with
DCM swelling 20 ~ 40 minutes, suction filtration is removed DCM, adds the Fmoc protecting group that 20% DBLK solution removes aminoresin, then with DMF washing 4 ~ 6 times;
2) preparation Fmoc-D-Ala-resin:
Under the ice bath Fmoc-D-Ala-OH and HOBt are dissolved among the DMF, then add DIC activation 5 ~ 10
Minute, the solution weight that activation is good is added with in the resin of step 1) gained reacts, stopped reaction when detecting resin and be negative to the triketohydrindene hydrate detection method, and resin gets Fmoc-D-Ala-resin with DMF washing 4 ~ 6 times;
3) preparation Fmoc-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-
Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin:
With step 2) the Fmoc-D-Ala-resin of gained is with DMF washing 2 ~ 6 times, adds 20% DBLK
Solution removes the Fmoc protecting group, with DMF washing 4 ~ 6 times, then according to step 2) operation connect reactive polypeptide, successively with Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH, Fmoc-L-4Aph (Trt)-OH, Fmoc-Ser (x)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH, Fmoc-D-2Nal-OH connects according to the aminoacid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2;
4) preparation Ac-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-
D-3Pal-D-Phe(4Cl)-the D-2Nal-resin:
After the step 3) reaction is complete resin is washed 2 ~ 6 times with DMF, add 20% DBLK solution and remove
Then the Fmoc protecting group adds diacetyl oxide and pyridine, detects with the triketohydrindene hydrate detection method, drains after reaction is finished, with DMF washing 3 ~ 8 times;
5) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (x)-D-3Pal-
The D-4-Cpa-D-2Nal-resin:
Ac-D-Ala-Pro-ILys(Boc is being housed)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin the solid state reaction post in to add volume ratio be 10% TFA/DCM solution, after the stirring at room reaction, drain, with DMF washing 3 ~ 8 times;
6) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (x)-
D-3Pal-D-4-Cpa-D-2Nal-resin:
With L-4, the 5-dihydroorotate is dissolved among the DMF, adds HOBt, adds the DIC activation again, then will
The solution that activation is good add Ac-Ala-Pro-ILys(Boc is housed)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin the solid state reaction post in, behind the stirring reaction 2 hours, triketohydrindene hydrate detects and is negative, drain DMF washing 3 ~ 8 times;
7) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser-
D-3Pal-D-4-Cpa-D-2Nal-resin:
Ac-Ala-Pro-ILys(Boc is being housed)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (x)-
In the solid state reaction post of D-3Pal-D-4-Cpa-D-2Nal-resin, add the DMF solution of TBAF, stirring reaction washed 3 ~ 8 times with DMF after 2 hours;
8) the standby thick peptide of cracking:
Freezing lysate is joined in the resin of oven dry, stirring reaction 2 hours adds ether with reaction solution
In, freezing 20 ~ 60 minutes, centrifugal, with DMF washing 6 times, be neutrality to pH till, separate, after the drying thick peptide;
9) purifying gets Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide:
The thick peptide of step 8) gained is got Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide through separation and purification;
Described aminoresin is selected from a kind of among Rink amide AM, the Rink amide MBHA; Step 2
The protecting group x of described Fmoc-Ser (x)-OH is selected from a kind of among TBDMS, the OBn; The described lysate of step 8) is comprised of TFA, TIS, phenol, methyl-phenoxide, and the volume ratio of TFA, TIS, phenol, methyl-phenoxide is 95:2:2:1.
The substitution degree of described aminoresin is 0.4 ~ 0.8mmol/g.
The volume ratio of the described diacetyl oxide of step 4) and pyridine is 1:1.
The described separation and purification of step 9) may further comprise the steps: thick peptide is dissolved in 5% aqueous acetic acid, behind 0.45 μ m filtering with microporous membrane, directly be loaded to C18 bonded silica gel filling chromatographic column, adopt gradient 6 ~ 9% methanol-water moving phases to carry out gradient elution separation, collect the purpose component.
The artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the present invention, with the Fmoc solid phase synthesis, the problem of having avoided the Boc method repeatedly to come deprotection to bring with acid: such as the joint at peptide and resin, when the each 50%TFA(of using trifluoroacetic acid) when taking off the Boc base, 1.4% the peptide of having an appointment comes off from resin, synthetic peptide chain is longer, loses more serious; In addition, the acid treatment meeting causes some side reactions of side chain.Avoid HF gas to use, alleviated environmental protection pressure; Greatly reduce the TFA(trifluoroacetic acid) use, avoided in the building-up process by product to the pollution of environment; The artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the present invention, 4 Serines are with TBDMS(tertiary butyl dimethyl) replace the tertiary butyl to protect hydroxyl.Can use the TBAF(tetrabutyl ammonium fluoride at last) remove under the mild conditions, can not bring other side reactions, reduced the impurity generation; The artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the present invention, 5 continuous hydroorotic acid fragments of phenylalanine phenyl ring 4 bit aminos are easily reset under alkaline condition, first with trityl as protecting group, introduce at last again, have avoided the rearrangement side reaction to occur; The artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the present invention replaces D-4Aph (Cbm-tBu) with D-4Aph (Cbm), has avoided t-Bu to remove difficulty, has reduced the side reaction generation.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, should be understood that, following examples only are used for explaining the present invention, rather than restriction protection scope of the present invention.
The solid phase synthesis of embodiment 1 Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
1) Rink amide AM resin swelling and the deprotection of Fmoc protection
Rink amide AM(substitution degree 0.49mmol/g with the Fmoc protection; 0.82g; 0.4mmol) in the solid state reaction of packing into the post; with DMF washing 1 ~ 4 time; then with DCM swelling 20 ~ 40 minutes, suction filtration was removed DCM, adds 20% DBLK solution deprotection 5 minutes; 10 minutes respectively once, then with DMF washing 4 ~ 6 times.
2) preparation Fmoc-D-Ala-Rink amide AM resin
Under the ice bath with F-D-Ala-OH(249mg, 0.8mmol), HOBt-Cl(164mg, 0.88mmol)
Be dissolved among the 2ml DMF, then adding 124 μ L DIC activation will activate in the resin that the solution weight of getting well is added with the step 1) gained and react in 5 ~ 10 minutes, stopped reaction when being negative to triketohydrindene hydrate detection method detection resin, resin gets Fmoc-D-Ala-Rink amide AM resin with DMF washing 4 ~ 6 times;
3) preparation Fmoc-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (TBDMS)-D-3Pal-D-Phe(4Cl)-D-2Nal-Rink amide AM resin
With step 2) the Fmoc-D-Ala-Rink amide AM of gained is with DMF washing 2 ~ 6 times, adds
20% DBLK solution removes the Fmoc protecting group, with DMF washing 4 ~ 6 times, then according to step 2) operation connect reactive polypeptide, successively with Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH, Fmoc-L-4Aph (Trt)-OH, Fmoc-Ser (TBDMS)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH, Fmoc-D-2Nal-OH connects according to the aminoacid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2;
4) preparation Ac-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (TBDMS)-D-
3Pal-D-Phe(4Cl)-D-2Nal-Rink amide AM resin:
After the step 3) reaction is complete resin is washed 2 ~ 6 times with DMF, add 20% DBLK solution and remove
Then the Fmoc protecting group adds 2ml diacetyl oxide and 2ml pyridine, detects with the triketohydrindene hydrate detection method, drains after reaction is finished, with DMF washing 3 ~ 8 times;
5) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (TBDMS)-D-3Pal-D-
4-Cpa-D-2Nal-resin:
Ac-D-Ala-Pro-ILys(Boc is being housed)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-
Ser (TBDMS)-D-3Pal-D-Phe(4Cl)-D-2Nal-Rink amide AM resin the solid state reaction post in to add volume ratio be 10% TFA/DCM solution, after the stirring at room reaction, drain, with DMF washing 3 ~ 8 times;
6) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (TBDMS)-D
-3Pal-D-4-Cpa-D-2Nal-Rink amide AM resin:
With L-4,5-dihydroorotate (190mg, 1.2mmol) is dissolved among the 1mlDMF, adding HOBt (183mg,
1.2mmol), add again DIC(124 μ L) activation, then will activate good solution add Ac-Ala-Pro-ILys(Boc is housed)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (TBDMS)-D-3Pal-D-4-Cpa-D-2Nal-resin the solid state reaction post in, behind the stirring reaction 2 hours, triketohydrindene hydrate detects and is negative, drain DMF washing 3 ~ 8 times;
7) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser-D-3Pal-D-4
-Cpa-D-2Nal-Rink amide AM resin:
Ac-Ala-Pro-ILys(Boc is being housed)-the solid state reaction post of Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (TBDMS)-D-3Pal-D-4-Cpa-D-2Nal-Rink amide AM resin in, add TBAF(314mg, 1.2mmol) DMF solution, stirring reaction after 2 hours with DMF washing 3 ~ 8 times;
8) the standby thick peptide of cracking:
Lysate (TFA:TIS: phenol: methyl-phenoxide=95:2:2:1,1ml) join with freezing (5 ℃)
In the resin of oven dry, stirring reaction 2h.Reaction solution is poured in the ether of 6ml freezing 20-60 minute.Centrifugal (4000rpm, 5min),, with DMF washing 6 times, be neutrality to pH till.Separate, get thick peptide 0.85g after the drying.
9 purifying get Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide:
The thick peptide of step 8) gained is dissolved in 5% aqueous acetic acid, behind 0.45 μ m filtering with microporous membrane, directly be loaded to C18 bonded silica gel filling chromatographic column, adopt gradient 6 ~ 9% methanol-water moving phases to carry out gradient elution separation, collect the purpose component, get Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide 0.357g, yield 42%, purity 99.8%;
The solid phase synthesis of embodiment 2 Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2s
The Rink amide AM resin of Fmoc protection among the embodiment 1 is changed into the Rink amide MBHA(substitution degree=0.73mmol/g of Fmoc protection, 0.55g, 0.4mmol).All the other operations get peptide resin 1.17g with embodiment 1 after the contraction, get thick peptide 0.52g, smart peptide 0.21g, yield 40% after the cracking.
The solid phase synthesis of embodiment 3 Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2s
The Fmoc-Ser (TBDMS) of step 3) among the embodiment 1-OH is changed to Fmoc-Ser (OBn)-OH, and all the other operate with embodiment 1, and the reaction conditions that removes the Obn protecting group is identical in embodiment, gets smart peptide, and yield is 47%, purity 99.6%
The abbreviation that embodiment and aforementioned process adopt and the material of representative thereof are as follows:
Claims (4)
1. the solid phase synthesis process of an Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is characterized in that, may further comprise the steps:
Take the aminoresin of Fmoc protection as starting raw material; method according to solid phase synthesis; be that coupling agent connects reactive polypeptide with DIC/ HOBt, connect successively the amino acid of Fmoc protection, obtain the decapeptide resin of protection; the Fmoc group in the deaminize acid successively during this time; after obtaining the decapeptide resin of protection, remove amino acid whose Side chain protective group, cut peptide and obtain the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product; then the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of purifying may further comprise the steps:
1) resin swelling and deprotection
The aminoresin of Fmoc protection is packed in the solid state reaction post, and with DMF washing 1 ~ 4 time, then with DCM swelling 20 ~ 40 minutes, suction filtration was removed DCM, adds the Fmoc protecting group that 20% DBLK solution removes aminoresin, then with DMF washing 4 ~ 6 times;
2) preparation Fmoc-D-Ala-resin:
Under the ice bath Fmoc-D-Ala-OH and HOBt are dissolved among the DMF, then add DIC activation 5 ~ 10 minutes, the solution weight that activation is good is added with in the resin of step 1) gained reacts, stopped reaction when being negative to triketohydrindene hydrate detection method detection resin, resin gets Fmoc-D-Ala-resin with DMF washing 4 ~ 6 times;
3) preparation Fmoc-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin:
With step 2) the Fmoc-D-Ala-resin of gained is with DMF washing 2 ~ 6 times, add 20% DBLK solution and remove the Fmoc protecting group, with DMF washing 4 ~ 6 times, then according to step 2) operation connect reactive polypeptide, successively with Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH, Fmoc-L-4Aph (Trt)-OH, Fmoc-Ser (x)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH, Fmoc-D-2Nal-OH connects according to the aminoacid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2;
4) preparation Ac-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin:
After the step 3) reaction is complete resin is washed 2 ~ 6 times with DMF, add 20% DBLK solution and remove the Fmoc protecting group, then add diacetyl oxide and pyridine, detect with the triketohydrindene hydrate detection method, drain after reaction is finished, with DMF washing 3 ~ 8 times;
5) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin:
Ac-D-Ala-Pro-ILys(Boc is being housed)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin the solid state reaction post in to add volume ratio be 10% TFA/DCM solution, after the stirring at room reaction, drain, with DMF washing 3 ~ 8 times;
6) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin:
With L-4, the 5-dihydroorotate is dissolved among the DMF, add HOBt, add again the DIC activation, then will activate good solution add Ac-Ala-Pro-ILys(Boc is housed)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin the solid state reaction post in, stirring reaction is after 2 hours, triketohydrindene hydrate detects and is negative, drain DMF washing 3 ~ 8 times;
7) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser-D-3Pal-D-4-Cpa-D-2Nal-resin:
Ac-Ala-Pro-ILys(Boc is being housed)-the solid state reaction post of Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin in, the DMF solution that adds TBAF, stirring reaction washed 3 ~ 8 times with DMF after 2 hours;
8) the standby thick peptide of cracking:
Freezing lysate is joined in the resin of oven dry, stirring reaction 2 hours adds reaction solution in the ether, freezing 20 ~ 60 minutes, centrifugal, with DMF washing 6 times, be neutrality to pH till, separate, after the drying thick peptide;
9) purifying gets Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide:
The thick peptide of step 8) gained is got Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide through separation and purification;
Described aminoresin is selected from a kind of among Rink amide AM, the Rink amide MBHA; The protecting group x of the described Fmoc-Ser of step 2 (x)-OH is selected from a kind of among TBDMS, the OBn; The described lysate of step 8) is comprised of TFA, TIS, phenol, methyl-phenoxide, and the volume ratio of TFA, TIS, phenol, methyl-phenoxide is 95:2:2:1.
2. solid phase synthesis process according to claim 1 is characterized in that, the substitution degree of described aminoresin is 0.4 ~ 0.8mmol/g.
3. solid phase synthesis process according to claim 1 is characterized in that, the volume ratio of the described diacetyl oxide of step 4) and pyridine is 1:1.
4. solid phase synthesis process according to claim 1, it is characterized in that, the described separation and purification of step 9) may further comprise the steps: thick peptide is dissolved in 5% aqueous acetic acid, behind 0.45 μ m filtering with microporous membrane, directly be loaded to C18 bonded silica gel filling chromatographic column, adopt gradient 6 ~ 9% methanol-water moving phases to carry out gradient elution separation, collect the purpose component.
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| CN103351428A (en) * | 2013-08-05 | 2013-10-16 | 海南双成药业股份有限公司 | Synthesis of degarelix by solid phase segment method |
| CN103992378A (en) * | 2013-11-01 | 2014-08-20 | 杭州诺泰制药技术有限公司 | Method for preparing Degarelix acetate |
| CN103992392A (en) * | 2014-05-19 | 2014-08-20 | 泰州施美康多肽药物技术有限公司 | Solid-phase synthesis method of degarelix |
| CN104177478A (en) * | 2014-08-27 | 2014-12-03 | 成都圣诺生物科技股份有限公司 | Method for synthesizing degarelix |
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| CN107417771A (en) * | 2017-05-04 | 2017-12-01 | 苏州强耀生物科技有限公司 | A kind of preparation method of the cysteine polypeptide of farnesyl modification |
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| CN112125956A (en) * | 2019-06-25 | 2020-12-25 | 深圳市健元医药科技有限公司 | Preparation method of degarelix |
| US11168114B2 (en) | 2015-12-17 | 2021-11-09 | Fresenius Kabi iPSUM S.r.l | Process for the manufacture of degarelix and its intermediates |
| US11332495B2 (en) | 2019-09-21 | 2022-05-17 | RK Pharma Solutions LLC | Process for the preparation of Degarelix acetate and Degarelix acetate-mannitol premix |
| CN115141257A (en) * | 2021-03-31 | 2022-10-04 | 浙江苏泊尔制药有限公司 | Purification method of degarelix |
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| CN103351428A (en) * | 2013-08-05 | 2013-10-16 | 海南双成药业股份有限公司 | Synthesis of degarelix by solid phase segment method |
| CN103992378A (en) * | 2013-11-01 | 2014-08-20 | 杭州诺泰制药技术有限公司 | Method for preparing Degarelix acetate |
| CN103992392A (en) * | 2014-05-19 | 2014-08-20 | 泰州施美康多肽药物技术有限公司 | Solid-phase synthesis method of degarelix |
| CN103992392B (en) * | 2014-05-19 | 2017-05-31 | 泰州启瑞医药科技有限公司 | A kind of solid phase synthesis process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
| CN104177478B (en) * | 2014-08-27 | 2018-04-03 | 成都圣诺生物制药有限公司 | A kind of method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
| CN104177478A (en) * | 2014-08-27 | 2014-12-03 | 成都圣诺生物科技股份有限公司 | Method for synthesizing degarelix |
| CN105085634A (en) * | 2015-08-29 | 2015-11-25 | 中肽生化有限公司 | Preparation method for degarelix |
| US11168114B2 (en) | 2015-12-17 | 2021-11-09 | Fresenius Kabi iPSUM S.r.l | Process for the manufacture of degarelix and its intermediates |
| EP3981781A1 (en) | 2015-12-17 | 2022-04-13 | Fresenius Kabi iPSUM S.r.l. | Process for the manufacture of degarelix and its intermediates |
| CN107417771B (en) * | 2017-05-04 | 2021-06-29 | 苏州强耀生物科技有限公司 | Preparation method of farnesyl-modified cysteine polypeptide |
| CN107417771A (en) * | 2017-05-04 | 2017-12-01 | 苏州强耀生物科技有限公司 | A kind of preparation method of the cysteine polypeptide of farnesyl modification |
| CN109929007A (en) * | 2017-12-15 | 2019-06-25 | 连云港恒运药业有限公司 | The preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 key dipeptides intermediate |
| CN109929007B (en) * | 2017-12-15 | 2023-07-28 | 连云港恒运药业有限公司 | Preparation method of key dipeptide intermediate of degarelix |
| CN112125956A (en) * | 2019-06-25 | 2020-12-25 | 深圳市健元医药科技有限公司 | Preparation method of degarelix |
| WO2020259714A1 (en) * | 2019-06-25 | 2020-12-30 | 深圳市健元医药科技有限公司 | Method for preparing degarelix |
| US11332495B2 (en) | 2019-09-21 | 2022-05-17 | RK Pharma Solutions LLC | Process for the preparation of Degarelix acetate and Degarelix acetate-mannitol premix |
| CN115141257A (en) * | 2021-03-31 | 2022-10-04 | 浙江苏泊尔制药有限公司 | Purification method of degarelix |
| CN115141257B (en) * | 2021-03-31 | 2025-01-03 | 浙江苏泊尔制药有限公司 | A method for purifying degarelix |
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