WO2023030278A1 - Full-liquid-phase synthesis method for reelin drug - Google Patents
Full-liquid-phase synthesis method for reelin drug Download PDFInfo
- Publication number
- WO2023030278A1 WO2023030278A1 PCT/CN2022/115637 CN2022115637W WO2023030278A1 WO 2023030278 A1 WO2023030278 A1 WO 2023030278A1 CN 2022115637 W CN2022115637 W CN 2022115637W WO 2023030278 A1 WO2023030278 A1 WO 2023030278A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- compound
- tbu
- reaction
- boc
- liquid phase
- Prior art date
Links
- 239000007791 liquid phase Substances 0.000 title claims abstract description 80
- 239000003814 drug Substances 0.000 title claims abstract description 31
- 229940079593 drug Drugs 0.000 title claims abstract description 28
- 238000001308 synthesis method Methods 0.000 title abstract description 4
- 102000043322 Reelin Human genes 0.000 title abstract 3
- 108700038365 Reelin Proteins 0.000 title abstract 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 143
- 238000000034 method Methods 0.000 claims abstract description 57
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 55
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 55
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 13
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical group C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 claims abstract description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 72
- 150000001875 compounds Chemical class 0.000 claims description 65
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 59
- 239000007787 solid Substances 0.000 claims description 56
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 54
- 239000003795 chemical substances by application Substances 0.000 claims description 51
- 150000007530 organic bases Chemical class 0.000 claims description 38
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims description 28
- 239000002904 solvent Substances 0.000 claims description 27
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 26
- 239000002244 precipitate Substances 0.000 claims description 21
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 20
- 238000001914 filtration Methods 0.000 claims description 19
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 19
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 229920001184 polypeptide Polymers 0.000 claims description 18
- 230000002194 synthesizing effect Effects 0.000 claims description 18
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 17
- 239000012141 concentrate Substances 0.000 claims description 15
- 238000006482 condensation reaction Methods 0.000 claims description 15
- 239000012190 activator Substances 0.000 claims description 14
- 229940125904 compound 1 Drugs 0.000 claims description 14
- 239000012043 crude product Substances 0.000 claims description 14
- 239000007821 HATU Substances 0.000 claims description 13
- 239000012317 TBTU Substances 0.000 claims description 13
- 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 claims description 13
- 238000010511 deprotection reaction Methods 0.000 claims description 13
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 12
- -1 benzyl ester Chemical class 0.000 claims description 12
- SNZIFNXFAFKRKT-NSHDSACASA-N (2s)-2-azaniumyl-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoate Chemical compound CC(C)(C)OC1=CC=C(C[C@H]([NH3+])C([O-])=O)C=C1 SNZIFNXFAFKRKT-NSHDSACASA-N 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 10
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 10
- 229940126214 compound 3 Drugs 0.000 claims description 10
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 8
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 8
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 8
- 229940125773 compound 10 Drugs 0.000 claims description 8
- 229940125898 compound 5 Drugs 0.000 claims description 8
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 7
- 239000006166 lysate Substances 0.000 claims description 7
- UVNPEUJXKZFWSJ-LMTQTHQJSA-N (R)-N-[(4S)-8-[6-amino-5-[(3,3-difluoro-2-oxo-1H-pyrrolo[2,3-b]pyridin-4-yl)sulfanyl]pyrazin-2-yl]-2-oxa-8-azaspiro[4.5]decan-4-yl]-2-methylpropane-2-sulfinamide Chemical compound CC(C)(C)[S@@](=O)N[C@@H]1COCC11CCN(CC1)c1cnc(Sc2ccnc3NC(=O)C(F)(F)c23)c(N)n1 UVNPEUJXKZFWSJ-LMTQTHQJSA-N 0.000 claims description 6
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 6
- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 claims description 6
- NUGPIZCTELGDOS-QHCPKHFHSA-N N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclopentanecarboxamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CC[C@@H](C=1C=NC=CC=1)NC(=O)C1CCCC1)C NUGPIZCTELGDOS-QHCPKHFHSA-N 0.000 claims description 6
- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 claims description 6
- 229940125782 compound 2 Drugs 0.000 claims description 6
- 125000006239 protecting group Chemical group 0.000 claims description 6
- WDBQJSCPCGTAFG-QHCPKHFHSA-N 4,4-difluoro-N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclohexane-1-carboxamide Chemical compound FC1(CCC(CC1)C(=O)N[C@@H](CCN1CCC(CC1)N1C(=NN=C1C)C(C)C)C=1C=NC=CC=1)F WDBQJSCPCGTAFG-QHCPKHFHSA-N 0.000 claims description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 125000004494 ethyl ester group Chemical group 0.000 claims description 2
- 150000004702 methyl esters Chemical class 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 238000009833 condensation Methods 0.000 abstract description 7
- 230000005494 condensation Effects 0.000 abstract description 7
- 239000012634 fragment Substances 0.000 abstract description 6
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 3
- ABRICLFKFRFDKS-IHPCNDPISA-N Trp-Ser-Tyr Chemical group C([C@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)N)C(O)=O)C1=CC=C(O)C=C1 ABRICLFKFRFDKS-IHPCNDPISA-N 0.000 abstract description 2
- XFWCSGJOVUQCME-YUMQZZPRSA-N pEH Chemical group C([C@@H](C(=O)O)NC(=O)[C@H]1NC(=O)CC1)C1=CNC=N1 XFWCSGJOVUQCME-YUMQZZPRSA-N 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 143
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 86
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 72
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 67
- 238000004128 high performance liquid chromatography Methods 0.000 description 60
- 238000001514 detection method Methods 0.000 description 58
- 239000000243 solution Substances 0.000 description 51
- 239000000463 material Substances 0.000 description 49
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 49
- 239000012071 phase Substances 0.000 description 44
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 37
- 230000005526 G1 to G0 transition Effects 0.000 description 22
- 239000008213 purified water Substances 0.000 description 22
- 239000003208 petroleum Substances 0.000 description 20
- 230000007935 neutral effect Effects 0.000 description 19
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 17
- 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 12
- 102400000932 Gonadoliberin-1 Human genes 0.000 description 9
- 101500026183 Homo sapiens Gonadoliberin-1 Proteins 0.000 description 9
- 108010050144 Triptorelin Pamoate Proteins 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 229960001442 gonadorelin Drugs 0.000 description 9
- VXKHXGOKWPXYNA-PGBVPBMZSA-N triptorelin Chemical compound C([C@@H](C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=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 VXKHXGOKWPXYNA-PGBVPBMZSA-N 0.000 description 9
- 229960004824 triptorelin Drugs 0.000 description 9
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 6
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 6
- 108010021717 Nafarelin Proteins 0.000 description 6
- AKRYBBWYDSDZHG-UHFFFAOYSA-N nitrosobis(2-oxopropyl)amine Chemical compound CC(=O)CN(N=O)CC(C)=O AKRYBBWYDSDZHG-UHFFFAOYSA-N 0.000 description 6
- 229960002333 nafarelin Drugs 0.000 description 5
- RWHUEXWOYVBUCI-ITQXDASVSA-N nafarelin Chemical compound C([C@@H](C(=O)N[C@H](CC=1C=C2C=CC=CC2=CC=1)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)NCC(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 RWHUEXWOYVBUCI-ITQXDASVSA-N 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 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 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- XXMYDXUIZKNHDT-QNGWXLTQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1-tritylimidazol-4-yl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(N=C1)=CN1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XXMYDXUIZKNHDT-QNGWXLTQSA-N 0.000 description 3
- JPZXHKDZASGCLU-GFCCVEGCSA-N 3-(2-Naphthyl)-D-Alanine Chemical compound C1=CC=CC2=CC(C[C@@H](N)C(O)=O)=CC=C21 JPZXHKDZASGCLU-GFCCVEGCSA-N 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- DODCBMODXGJOKD-RGMNGODLSA-N methyl (2s)-2-amino-4-methylpentanoate;hydrochloride Chemical compound Cl.COC(=O)[C@@H](N)CC(C)C DODCBMODXGJOKD-RGMNGODLSA-N 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- MGHMWKZOLAAOTD-XMMPIXPASA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1h-indol-3-yl)propanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@@H](C(=O)O)CC1=CNC2=CC=CC=C12 MGHMWKZOLAAOTD-XMMPIXPASA-N 0.000 description 2
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 2
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 2
- 206010062767 Hypophysitis Diseases 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- QIGLJVBIRIXQRN-ZETCQYMHSA-N ethyl (2s)-2-amino-4-methylpentanoate Chemical compound CCOC(=O)[C@@H](N)CC(C)C QIGLJVBIRIXQRN-ZETCQYMHSA-N 0.000 description 2
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 2
- 210000003635 pituitary gland Anatomy 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 229960003604 testosterone Drugs 0.000 description 2
- 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 description 1
- OIXLLKLZKCBCPS-RZVRUWJTSA-N (2s)-2-azanyl-5-[bis(azanyl)methylideneamino]pentanoic acid Chemical compound OC(=O)[C@@H](N)CCCNC(N)=N.OC(=O)[C@@H](N)CCCNC(N)=N OIXLLKLZKCBCPS-RZVRUWJTSA-N 0.000 description 1
- DVBUCBXGDWWXNY-SFHVURJKSA-N (2s)-5-(diaminomethylideneamino)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C3=CC=CC=C3C2=C1 DVBUCBXGDWWXNY-SFHVURJKSA-N 0.000 description 1
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-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
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 description 1
- VRPJIFMKZZEXLR-UHFFFAOYSA-N 2-[(2-methylpropan-2-yl)oxycarbonylamino]acetic acid Chemical compound CC(C)(C)OC(=O)NCC(O)=O VRPJIFMKZZEXLR-UHFFFAOYSA-N 0.000 description 1
- HJBLUNHMOKFZQX-UHFFFAOYSA-N 3-hydroxy-1,2,3-benzotriazin-4-one Chemical compound C1=CC=C2C(=O)N(O)N=NC2=C1 HJBLUNHMOKFZQX-UHFFFAOYSA-N 0.000 description 1
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 description 1
- LQJAALCCPOTJGB-YUMQZZPRSA-N Arg-Pro Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O LQJAALCCPOTJGB-YUMQZZPRSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000015474 Central precocious puberty Diseases 0.000 description 1
- 125000003941 D-tryptophan group Chemical group [H]C1=C([H])C([H])=C2C(C([C@@](N([H])[H])(C(=O)[*])[H])([H])[H])=C([H])N([H])C2=C1[H] 0.000 description 1
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 108700042658 GAP-43 Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010071119 Hormone-dependent prostate cancer Diseases 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- ODHCTXKNWHHXJC-GSVOUGTGSA-N Pyroglutamic acid Natural products OC(=O)[C@H]1CCC(=O)N1 ODHCTXKNWHHXJC-GSVOUGTGSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 206010046798 Uterine leiomyoma Diseases 0.000 description 1
- NOUDPBCEONUCOV-FJXQXJEOSA-N [(2s)-1-ethoxy-4-methyl-1-oxopentan-2-yl]azanium;chloride Chemical compound Cl.CCOC(=O)[C@@H](N)CC(C)C NOUDPBCEONUCOV-FJXQXJEOSA-N 0.000 description 1
- ODHCTXKNWHHXJC-UHFFFAOYSA-N acide pyroglutamique Natural products OC(=O)C1CCC(=O)N1 ODHCTXKNWHHXJC-UHFFFAOYSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- OROGSEYTTFOCAN-DNJOTXNNSA-N codeine Chemical compound C([C@H]1[C@H](N(CC[C@@]112)C)C3)=C[C@H](O)[C@@H]1OC1=C2C3=CC=C1OC OROGSEYTTFOCAN-DNJOTXNNSA-N 0.000 description 1
- 229960004126 codeine Drugs 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 description 1
- MGHPNCMVUAKAIE-UHFFFAOYSA-N diphenylmethanamine Chemical compound C=1C=CC=CC=1C(N)C1=CC=CC=C1 MGHPNCMVUAKAIE-UHFFFAOYSA-N 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 201000010260 leiomyoma Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- WOOWBQQQJXZGIE-UHFFFAOYSA-N n-ethyl-n-propan-2-ylpropan-2-amine Chemical compound CCN(C(C)C)C(C)C.CCN(C(C)C)C(C)C WOOWBQQQJXZGIE-UHFFFAOYSA-N 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- VWBWQOUWDOULQN-UHFFFAOYSA-N nmp n-methylpyrrolidone Chemical compound CN1CCCC1=O.CN1CCCC1=O VWBWQOUWDOULQN-UHFFFAOYSA-N 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 208000006155 precocious puberty Diseases 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000003746 solid phase reaction Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229960002317 succinimide Drugs 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
- WHRNULOCNSKMGB-UHFFFAOYSA-N tetrahydrofuran thf Chemical compound C1CCOC1.C1CCOC1 WHRNULOCNSKMGB-UHFFFAOYSA-N 0.000 description 1
- 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 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
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
- C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- 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
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
Definitions
- the invention relates to the technical field of medicine synthesis, in particular to the technical field of synthesis of ralin drugs, in particular to a method for synthesizing ralin drugs in full liquid phase.
- Relin drugs refer to a class of artificially synthesized polypeptide drugs based on the structure of gonadotropin-releasing hormone (GnRH).
- GnRH gonadotropin-releasing hormone
- the mechanism of action of ralin drugs is to first act on the pituitary gland, leading to the synthesis and release of luteinizing hormone and follicle-stimulating hormone, causing male testosterone to stimulate the production of testosterone, and female ovaries to induce estrogen synthesis, which is called the ignition effect.
- the active ingredients of ralin drugs are generally composed of 10 amino acids, and their biological activity can be improved by substituting the 6th position.
- the preparation method of ralin drugs is mainly based on one-by-one condensation.
- U.S. Patent US4010125 uses Benzhydryl amine resin as a starting material, and uses Boc-protected amino acids as monomers.
- Chinese patents CN200710044419.7, CN201310013712.2, and CN201310014882.2 all use Rink Amide MBHA resin or RinkAmide AM resin as a starting material. , taking Fmoc-protected amino acids as monomers, connecting amino acids one by one in turn, cleavage to obtain crude triptorelin, and finally separating and purifying by HPLC to obtain the target product.
- Chinese patent CN201510524010.X is a method for synthesizing the pharmaceutical polypeptide nafarelin by microwave solid-phase synthesis. This method requires high production conditions, high cost, and low yield, and is not suitable for large-scale production.
- the above method has the following problems: a) Arg-Pro exists in the amino acid sequence, and under normal conditions, the solid-phase method condensation requires excessive amino acids, and the condensation efficiency is low, and defective peptides are prone to occur. This is because the steric hindrance caused by the structure of the amino acid itself makes the condensation reaction extremely difficult. b) The carboxy-terminal amino acid substitution value of Rink resin should not be too high, and the synthesis is uneconomical; and it is expensive compared to 2-chloro-trityl chloride resin. c) It is difficult to have both yield and purity. At present, there are few reports on the synthesis of ralin drugs by the total solution method.
- the technical problem to be solved by the present invention is to overcome the disadvantages of the current mainstream solid-phase reaction, such as high cost, many solvents, precursor reagents, high pressure on environmental protection, and low purity of the crude product, thereby providing an all-liquid phase synthesis
- a method for synthesizing ralin drugs in full liquid phase comprises the following steps:
- R 1 is an amino protecting group, including any one of Fmoc, Z, Boc
- R 2 is a carboxyl protecting group, including methyl ester Me, ethyl ester Et, benzyl ester Bzl, trityl ester Tr Any one
- R 3 includes any one of Boc or Trt
- R 4 is an amino protecting group, including any one of Fmoc, Z, Boc
- the R 5 is D-Trp, Gly, D-2 Any of -Nal.
- R is D-Trp
- the obtained ralin drug is triptorelin
- R is Gly
- the obtained ralin drug is gonadorelin
- R is D-2-Nal
- the class drug is nafarelin.
- step S1 specifically includes the following steps:
- step S2 specifically includes the following steps:
- Condensation reaction is carried out with R 1 -R 5 -OH, H-Leu-OR 2 as the reaction unit, the molar ratio of R 1 -R 5 -OH to H-Leu-OR 2 is 1:1.05-2, adding activator, Organic base, condensing agent, the ratio of H-Leu-OR 2 to activator, condensing agent, and organic base is 1:1:1:1, after the reaction is complete, filter, precipitate, wash, dry, and collect the solid to obtain compound 2;
- the activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt;
- the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU;
- the organic base includes any one of DIEA, TEA, NMM;
- the solvent includes any one of THF, DCM, DMF, NMP, dioxane.
- step S3 specifically includes the following steps:
- step S2 Using compound 2 prepared in step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, separating out, filtering, and drying in vacuo to obtain compound 3;
- the deprotection reagent includes any one of trifluoroacetic acid, diethylamine, piperazine, and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM, and THF.
- step S4 specifically includes the following steps:
- Condensation reaction is carried out with compound 1 synthesized in step S1 and compound 3 synthesized in step S3 as reaction units, wherein the molar ratio of compound 1 and compound 3 is 1: 1.05-2, adding organic base and condensing agent, wherein compound 3 and organic base 1.
- the molar ratio of the condensing agent is 1:1:1.
- the condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU;
- the organic base includes any one of DIEA, TEA, and NMM species;
- the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
- step S5 specifically includes the following steps:
- the deprotection reagent includes any one of diethylamine, piperazine, and piperidine solutions .
- step S6 specifically includes the following steps:
- Condensation reaction is carried out with Fmoc-His(R 3 )-OH and compound 5 synthesized in step S5 as the reaction unit, wherein the molar ratio of compound 5 to Fmoc-His(R 3 )-OH is 1:1.05-2, adding activating agent, organic base, condensing agent, wherein the ratio of Fmoc-His(R 3 )-OH to activator, condensing agent, and organic base is 1:1:1:1, the reaction is complete in the solvent, concentrated, filtered, washed, Drying, deprotection to obtain compound 6;
- the activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt;
- the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU;
- the organic base includes any one of DIEA, TEA, NMM;
- the solvent includes any one of THF, DCM, DMF, NMP, dioxane.
- step S7 specifically includes the following steps:
- step S6 Perform condensation reaction with R 4 -Pyr-OH and compound 6 synthesized in step S6, wherein the molar ratio of compound 6 to R 4 -Pyr-OH is 1:1.05-2; add organic base and condensing agent, wherein R4-Pyr- The molar ratio of OH to condensing agent and organic base is 1:1:1. After the reaction is complete, filter, wash, and dry to obtain compound 7;
- the condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU;
- the organic base includes any one of DIEA, TEA, and NMM species;
- the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
- step S8 specifically includes the following steps:
- the molar ratio of NaOH and compound 7 is 1.5:1-20:1;
- Step S9 specifically includes the following steps:
- the condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU;
- the organic base includes any one of DIEA, TEA, and NMM species;
- the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
- step S10 specifically includes the following steps:
- the condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU;
- the organic base includes any one of DIEA, TEA, and NMM A kind;
- Described solvent comprises any one in THF, DCM, DMF, NMP, dioxane;
- Step S11 specifically includes the following steps:
- reagents used in the above-mentioned technical schemes are common commercially available medicaments; in the above-mentioned technical schemes, ether reagents are usually used for precipitation or solid precipitation operations, including any one of petroleum ether, isopropyl ether, diethyl ether or Any combination, preferably, petroleum ether.
- the invention creatively invents a green and mild production process through the full liquid phase synthesis method, without using any highly toxic and precursor reagents, greatly reducing the cost, and is very suitable for large-scale production.
- the yield of the crude product of gonadorelin produced by the synthetic method of the present invention is more than 82%, and the purity can reach more than 95%; the yield of the crude product of triptorelin is about 90%, and the purity can reach more than 90%; The yield of the crude product is above 86%, and the purity can reach above 81%.
- Fig. 1 is the HPLC spectrogram of the crude product of gonadorelin prepared in Example 1 of the present invention
- Fig. 2 is the HPLC spectrogram of the triptorelin crude product that the embodiment of the present invention 3 prepares;
- Fig. 3 is the HPLC spectrogram of the nafarelin crude product prepared in Example 4 of the present invention.
- a method for synthesizing gonadorelin in full liquid phase the steps are as follows:
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- HPLC detection condition of compound 2 is:
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- HPLC detection condition of compound 5 is:
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- HPLC detection condition of compound 7 is:
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- a method for synthesizing gonadorelin in full liquid phase the steps are as follows:
- a method for synthesizing triptorelin in full liquid phase the steps are as follows:
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- HPLC detection and analysis conditions are as follows:
- A 5% acetonitrile/H 2 O solution, 0.1% TFA
- B acetonitrile/H 2 O, 0.1% TFA (HPLC);
- Mobile phase A 0.1% TFA/water
- mobile phase B 0.1% TFA/acetonitrile
- Detection wavelength 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5 ⁇ ,
- a method for synthesizing nafarelin in full liquid phase the steps are as follows.
- reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids and put them in containers and weigh them;
- Boc-Pyr-OH (76.9mmol) and BOP (76.9mmol) in a reaction flask, dissolve them completely in DMF, and then add TEA (76.9mmol) in a cold bath for 10min, then dissolve compound 6 (69.9mmol) in DMF After adding to the reaction to start the reaction.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Provided is a full-liquid-phase synthesis method for a reelin drug, belonging to the technical field of medicine synthesis. A liquid-phase method is used to synthesize "Trp-Ser-Tyr" fragments, "R5-Leu" fragments, "PYR-His" fragments, and "Arg-Pro-Gly-NH2" respectively, wherein R=D-Trp, Gly or D-2-NAL; and a reelin drug is synthesized in a "3+2+2+3" fragment condensation manner. The method has the characteristics of low reaction costs, non use of toxic reagents, a high product yield, and high purity, and is environmentally friendly, and is thus suitable for large-scale production.
Description
本发明涉及医药合成技术领域，具体涉及瑞林类药物合成技术领域，特别涉及一种全液相合成瑞林类药物的方法。The invention relates to the technical field of medicine synthesis, in particular to the technical field of synthesis of ralin drugs, in particular to a method for synthesizing ralin drugs in full liquid phase.
瑞林类药物是指以促性腺激素释放激素(gonadotropin-releasing hormone，GnRH)结构为基础的一类人工合成的多肽类药物。FDA批准其用于治疗中枢性性早熟，激素依赖性前列腺癌、卵巢癌、乳腺癌、子宫内膜异位症、子宫肌瘤以及辅助生殖等。瑞林类药物的作用机制是首先作用于垂体，导致促黄体生成激素和促卵泡激素的合成和释放，引起雄性睾丸激素刺激睾酮的产生，雌性卵巢诱导雌激素合成，称为点火效应。然后通过负反馈作用于垂体，达到垂体脱敏，通过降调节使性激素水平下降。瑞林类药物的活性成分一般由10个氨基酸组成，通过第6位取代提高其生物活性，氨基酸序列为：H-Pyr-His-Trp-Ser-Tyr-R-Leu-Arg-Pro-Gly-NH
2，R＝D-Trp(曲普瑞林)、Gly(戈那瑞林)或D-2-Nal(那法瑞林)等。
Relin drugs refer to a class of artificially synthesized polypeptide drugs based on the structure of gonadotropin-releasing hormone (GnRH). FDA approved it for the treatment of central precocious puberty, hormone-dependent prostate cancer, ovarian cancer, breast cancer, endometriosis, uterine fibroids and assisted reproduction. The mechanism of action of ralin drugs is to first act on the pituitary gland, leading to the synthesis and release of luteinizing hormone and follicle-stimulating hormone, causing male testosterone to stimulate the production of testosterone, and female ovaries to induce estrogen synthesis, which is called the ignition effect. Then it acts on the pituitary gland through negative feedback to achieve pituitary desensitization, and the level of sex hormones is reduced by down-regulation. The active ingredients of ralin drugs are generally composed of 10 amino acids, and their biological activity can be improved by substituting the 6th position. The amino acid sequence is: H-Pyr-His-Trp-Ser-Tyr-R-Leu-Arg-Pro-Gly- NH 2 , R=D-Trp (triptorelin), Gly (gonadorelin) or D-2-Nal (nafarelin), etc.
目前瑞林类药物的制备方法主要以逐个缩合为主。例如美国专利US4010125使用Benzhydryl amine树脂为起始原料，以Boc保护的氨基酸为单体，中国专利CN200710044419.7和CN201310013712.2以及CN201310014882.2，均以Rink Amide MBHA树脂或者RinkAmide AM树脂为起始原料，以Fmoc保护的氨基酸为单体，依次逐个接上氨基酸，裂解获得曲普瑞林粗品，最后HPLC进行分离纯化，获得目标产物。捷克专利C22014976A3采用了类似的氨基酸单体和Rink树脂为载体的合成方法。中国专利CN201410743707.1采用片段缩合的方式，所得曲普瑞林收率＞40％，纯度为98.5％。美国专利US4024248提及了戈那瑞林类似物可以采用片段缩合的方法：分片段采用叠氮法进行缩合，缩合片段未经保护，副反应较多，成本较高。中国发明CN202011087410.6一种多肽固液组合合成戈那瑞林的方法，该法在固相法的基础上进行了改进，但还是存在杂质难以纯化的问题。中国专利CN201510524010.X一种利用微波固相合成法合成药物多肽那法瑞林的方法，该法生产条件要求高，成本高，收率低，不适合规模化生产。At present, the preparation method of ralin drugs is mainly based on one-by-one condensation. For example, U.S. Patent US4010125 uses Benzhydryl amine resin as a starting material, and uses Boc-protected amino acids as monomers. Chinese patents CN200710044419.7, CN201310013712.2, and CN201310014882.2 all use Rink Amide MBHA resin or RinkAmide AM resin as a starting material. , taking Fmoc-protected amino acids as monomers, connecting amino acids one by one in turn, cleavage to obtain crude triptorelin, and finally separating and purifying by HPLC to obtain the target product. Czech patent C22014976A3 adopts a similar synthesis method of amino acid monomer and Rink resin as a carrier. Chinese patent CN201410743707.1 adopts the method of fragment condensation, and the yield of triptorelin obtained is >40%, and the purity is 98.5%. US Patent No. 4,024,248 mentions that gonadorelin analogues can be condensed by fragments: fragments are condensed by the azide method, and the condensed fragments are unprotected, with many side reactions and high costs. Chinese invention CN202011087410.6 is a method for synthesizing gonadorelin by solid-liquid combination of polypeptides. This method is improved on the basis of the solid-phase method, but there is still the problem that impurities are difficult to purify. Chinese patent CN201510524010.X is a method for synthesizing the pharmaceutical polypeptide nafarelin by microwave solid-phase synthesis. This method requires high production conditions, high cost, and low yield, and is not suitable for large-scale production.
以上方法有如下一些问题：a)氨基酸序列中存在Arg-Pro，在常规条件下用 固相方法缩合需要过量氨基酸，且缩合效率低，容易出现缺陷肽。这是由于氨基酸自身结构导致的立体位阻使得缩合反应极为困难。b)使用Rink树脂羧基端氨基酸取代值不能太高，合成不经济；并且相对于2-氯-三苯甲基氯树脂价格不菲。c)收率和纯度难以兼得。目前，关于全液相法合成瑞林类药物的报道较少。The above method has the following problems: a) Arg-Pro exists in the amino acid sequence, and under normal conditions, the solid-phase method condensation requires excessive amino acids, and the condensation efficiency is low, and defective peptides are prone to occur. This is because the steric hindrance caused by the structure of the amino acid itself makes the condensation reaction extremely difficult. b) The carboxy-terminal amino acid substitution value of Rink resin should not be too high, and the synthesis is uneconomical; and it is expensive compared to 2-chloro-trityl chloride resin. c) It is difficult to have both yield and purity. At present, there are few reports on the synthesis of ralin drugs by the total solution method.
发明内容Contents of the invention
因此，本发明要解决的技术问题在于克服了目前主流的固相反应成本高，使用溶剂多，使用易制毒试剂、环保压力大，产品粗品纯度低的缺点，从而提供一种全液相合成瑞林类药物的方法。采用液相法分别合成“Trp-Ser-Tyr”片段、“R
5-Leu”片段、“Pyr-His”片段、“Arg-Pro-Gly-NH
2”，其中R＝D-Trp、Gly或D-2-Nal；通过“3+2+2+3”的片段缩合方式合成瑞林类药物。
Therefore, the technical problem to be solved by the present invention is to overcome the disadvantages of the current mainstream solid-phase reaction, such as high cost, many solvents, precursor reagents, high pressure on environmental protection, and low purity of the crude product, thereby providing an all-liquid phase synthesis The ralin-like approach. The "Trp-Ser-Tyr" fragment, the "R 5 -Leu" fragment, the "Pyr-His" fragment, and the "Arg-Pro-Gly-NH 2 " were synthesized by liquid phase method, wherein R=D-Trp, Gly or D-2-Nal; synthesizing ralin drugs through the "3+2+2+3" fragment condensation method.
本发明提供的一种全液相合成瑞林类药物的方法，包括如下步骤：A method for synthesizing ralin drugs in full liquid phase provided by the invention comprises the following steps:
S1、液相合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH；S1. Compound 1 synthesized in liquid phase: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH;
S2、液相合成化合物2：R
1-R
5-Leu-OR
2；
S2, liquid phase synthesis of compound 2: R 1 -R 5 -Leu-OR 2 ;
S3、液相合成化合物3：H-R
5-Leu-OR
2；
S3, liquid phase synthesis of compound 3: HR 5 -Leu-OR 2 ;
S4、液相合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R
5-Leu-OR
2；
S4, liquid phase synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;
S5、液相合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R
5-Leu-OR
2；
S5, liquid phase synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;
S6、液相合成化合物6：H-His(R
3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R
5-Leu-OR
2；
S6, liquid phase synthesis of compound 6: H-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;
S7、液相合成化合物7：S7, liquid phase synthesis of compound 7:
R
4-Pyr-His(R
3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R
5-Leu-OR
2；
R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;
S8、液相合成化合物8：S8, liquid phase synthesis of compound 8:
R
4-Pyr-His(R
3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R
5-Leu-OH；
R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OH;
S9、液相合成化合物9：H-Arg(pbf)-Pro-Gly-NH
2；
S9, liquid phase synthesis of compound 9: H-Arg(pbf)-Pro-Gly-NH 2 ;
S10、液相合成化合物10：S10, liquid phase synthesis of compound 10:
R
4-Pyr-His(R
3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R
5-Leu-Arg(pbf)-Pro-Gly-NH
2；
R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-Arg(pbf)-Pro-Gly-NH 2 ;
S11、瑞林类药物粗品的制备；S11, the preparation of the crude product of ralin drugs;
其中，R
1为氨基保护基团，包括Fmoc、Z、Boc中的任意一种；R
2为羧基保护基团，包括甲酯Me，乙酯Et，苄酯Bzl，三苯甲酯Tr中的任意一种；R
3包括Boc或Trt中的任意一种；R
4为氨基保护基团，包括Fmoc、Z、Boc中的任意一 种；所述R
5为D-Trp、Gly、D-2-Nal中的任意一种。例如R
5为D-Trp时，所得瑞林类药物为曲普瑞林；R
5为Gly时，所得瑞林类药物为戈那瑞林；R
5为D-2-Nal时，所得瑞林类药物为那法瑞林。
Wherein, R 1 is an amino protecting group, including any one of Fmoc, Z, Boc; R 2 is a carboxyl protecting group, including methyl ester Me, ethyl ester Et, benzyl ester Bzl, trityl ester Tr Any one; R 3 includes any one of Boc or Trt; R 4 is an amino protecting group, including any one of Fmoc, Z, Boc; the R 5 is D-Trp, Gly, D-2 Any of -Nal. For example, when R is D-Trp, the obtained ralin drug is triptorelin; when R is Gly , the obtained ralin drug is gonadorelin; when R is D-2-Nal, the obtained ralin is triptorelin. The class drug is nafarelin.
在较优的技术方案中，步骤S1具体包括以下步骤：In a preferred technical solution, step S1 specifically includes the following steps:
以Fmoc-Trp(Boc)-Ser(tBu)-OSu和H-Tyr(tBu)-OH为反应单元进行缩合反应，在溶剂中反应得到化合物1；所述Fmoc-Trp(Boc)-Ser(tBu)-OSu和所述H-Tyr(tBu)-OH的摩尔比为1∶1.05-2，所述H-Tyr(tBu)-OH与所述有机碱的摩尔比为1∶1，所述溶剂包括DMF、THF、甲醇、乙醇、NMP中的任意一种。Take Fmoc-Trp(Boc)-Ser(tBu)-OSu and H-Tyr(tBu)-OH as reaction units to carry out condensation reaction, react in a solvent to obtain compound 1; said Fmoc-Trp(Boc)-Ser(tBu )-OSu and the H-Tyr(tBu)-OH molar ratio is 1:1.05-2, the H-Tyr(tBu)-OH and the organic base molar ratio is 1:1, the solvent Including any one of DMF, THF, methanol, ethanol, and NMP.
在较优的技术方案中，步骤S2具体包括以下步骤：In a preferred technical solution, step S2 specifically includes the following steps:
以R
1-R
5-OH、H-Leu-OR
2为反应单元进行缩合反应，R
1-R
5-OH与H-Leu-OR
2的摩尔比为1∶1.05-2，加入活化剂、有机碱、缩合剂，H-Leu-OR
2与活化剂、缩合剂、有机碱的比为1∶1∶1∶1，反应完全后，过滤、析出、洗涤、干燥，收集固体得化合物2；
Condensation reaction is carried out with R 1 -R 5 -OH, H-Leu-OR 2 as the reaction unit, the molar ratio of R 1 -R 5 -OH to H-Leu-OR 2 is 1:1.05-2, adding activator, Organic base, condensing agent, the ratio of H-Leu-OR 2 to activator, condensing agent, and organic base is 1:1:1:1, after the reaction is complete, filter, precipitate, wash, dry, and collect the solid to obtain compound 2;
所述活化剂为多肽合成常用的活化剂，包括HOSu、HOBt、HOAt、HOOBt中的任意一种；所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt; the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU; the organic base includes any one of DIEA, TEA, NMM; the solvent includes any one of THF, DCM, DMF, NMP, dioxane.
在较优的技术方案中，步骤S3具体包括以下步骤：In a preferred technical solution, step S3 specifically includes the following steps:
以步骤S2制得的化合物2为底物，加入脱保护试剂和溶剂，浓缩至少量，析出，过滤，真空干燥得化合物3；Using compound 2 prepared in step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, separating out, filtering, and drying in vacuo to obtain compound 3;
所述脱保护试剂包括三氟乙酸、二乙胺、哌嗪、哌啶中的任意一种；所述溶剂为DMF、甲醇、乙醇、DCM、THF中的任意一种。The deprotection reagent includes any one of trifluoroacetic acid, diethylamine, piperazine, and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM, and THF.
在较优的技术方案中，步骤S4具体包括以下步骤：In a preferred technical solution, step S4 specifically includes the following steps:
以步骤S1合成的化合物1和步骤S3合成的化合物3为反应单元进行缩合反应，其中化合物1和化合物3的摩尔比为1∶1.05-2，加入有机碱、缩合剂，其中化合物3与有机碱、缩合剂的摩尔比为1∶1∶1，在溶剂中反应完全后，浓缩、过滤、洗涤、干燥，得化合物4；Condensation reaction is carried out with compound 1 synthesized in step S1 and compound 3 synthesized in step S3 as reaction units, wherein the molar ratio of compound 1 and compound 3 is 1: 1.05-2, adding organic base and condensing agent, wherein compound 3 and organic base 1. The molar ratio of the condensing agent is 1:1:1. After the reaction in the solvent is complete, concentrate, filter, wash, and dry to obtain compound 4;
所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的 任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
在较优的技术方案中，步骤S5中具体包括以下步骤：In a preferred technical solution, step S5 specifically includes the following steps:
取化合物4，加入脱保护试剂反应脱去Fmoc基团，浓缩至少量，析出固体，过滤，真空干燥得化合物5；所述脱保护试剂包括二乙胺、哌嗪、哌啶溶液的任意一种。Take compound 4, add a deprotection reagent to react to remove the Fmoc group, concentrate to a certain amount, precipitate a solid, filter, and vacuum-dry to obtain compound 5; the deprotection reagent includes any one of diethylamine, piperazine, and piperidine solutions .
在较优的技术方案中，步骤S6具体包括以下步骤：In a preferred technical solution, step S6 specifically includes the following steps:
以Fmoc-His(R
3)-OH、步骤S5中合成的化合物5为反应单元进行缩合反应，其中化合物5与Fmoc-His(R
3)-OH的摩尔比为1∶1.05-2，加入活化剂、有机碱、缩合剂，其中Fmoc-His(R
3)-OH与活化剂、缩合剂、有机碱的比为1∶1∶1∶1，在溶剂中反应完全，浓缩、过滤、洗涤、干燥，脱保护得化合物6；
Condensation reaction is carried out with Fmoc-His(R 3 )-OH and compound 5 synthesized in step S5 as the reaction unit, wherein the molar ratio of compound 5 to Fmoc-His(R 3 )-OH is 1:1.05-2, adding activating agent, organic base, condensing agent, wherein the ratio of Fmoc-His(R 3 )-OH to activator, condensing agent, and organic base is 1:1:1:1, the reaction is complete in the solvent, concentrated, filtered, washed, Drying, deprotection to obtain compound 6;
所述活化剂为多肽合成常用的活化剂，包括HOSu、HOBt、HOAt、HOOBt中的任意一种；所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt; the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU; the organic base includes any one of DIEA, TEA, NMM; the solvent includes any one of THF, DCM, DMF, NMP, dioxane.
在较优的技术方案中，步骤S7具体包括以下步骤：In a preferred technical solution, step S7 specifically includes the following steps:
以R
4-Pyr-OH和步骤S6合成的化合物6进行缩合反应，其中化合物6与R
4-Pyr-OH的摩尔比为1∶1.05-2；加入有机碱、缩合剂，其中R4-Pyr-OH与缩合剂、有机碱的摩尔比为1∶1∶1，反应完全后，过滤、洗涤、干燥，得化合物7；
Perform condensation reaction with R 4 -Pyr-OH and compound 6 synthesized in step S6, wherein the molar ratio of compound 6 to R 4 -Pyr-OH is 1:1.05-2; add organic base and condensing agent, wherein R4-Pyr- The molar ratio of OH to condensing agent and organic base is 1:1:1. After the reaction is complete, filter, wash, and dry to obtain compound 7;
所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
在较优的技术方案中，步骤S8具体包括以下步骤：In a preferred technical solution, step S8 specifically includes the following steps:
取甲醇和化合物7反应，缓慢加入2MNaOH，反应2-4h，过滤、洗涤、干燥得化合物8；Take methanol to react with compound 7, slowly add 2M NaOH, react for 2-4h, filter, wash and dry to obtain compound 8;
其中，NaOH和化合物7的摩尔比为1.5∶1-20∶1；Wherein, the molar ratio of NaOH and compound 7 is 1.5:1-20:1;
步骤S9具体包括以下步骤：Step S9 specifically includes the following steps:
以R
1-Arg(pbf)-OH、H-Pro-Gly-NH
2为反应单元进行缩合反应，其中R
1-Arg(pbf)-OH和H-Pro-Gly-NH
2的摩尔比为1∶1.05-2，加入有机碱、缩合剂， 其中R
1-Arg(pbf)-OH与有机碱、缩合剂的摩尔比为1∶1∶1，在溶剂中反应完全后，析出固体，过滤，干燥，脱保护，浓缩，析出固体，过滤，真空干燥得化合物9；
Condensation reaction with R 1 -Arg(pbf)-OH, H-Pro-Gly-NH 2 as reaction units, wherein the molar ratio of R 1 -Arg(pbf)-OH and H-Pro-Gly-NH 2 is 1 : 1.05-2, add organic base and condensing agent, wherein the molar ratio of R 1 -Arg(pbf)-OH to organic base and condensing agent is 1:1:1, after complete reaction in the solvent, a solid is precipitated, filtered, Drying, deprotection, concentration, precipitation of solid, filtration, and vacuum drying to obtain compound 9;
所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
在较优的技术方案中，步骤S10具体包括以下步骤：In a preferred technical solution, step S10 specifically includes the following steps:
以化合物8和化合物9为反应单元进行缩合反应，其中化合物8和化合物9的摩尔比为1∶1.05-2，加入缩合剂、有机碱，其中化合物9和缩合剂、有机碱的摩尔比为1∶1∶1，反应完全后，过滤、洗涤、干燥得化合物10；Carry out condensation reaction with compound 8 and compound 9 as the reaction unit, wherein the molar ratio of compound 8 and compound 9 is 1: 1.05-2, add condensing agent, organic base, wherein the molar ratio of compound 9 and condensing agent, organic base is 1 : 1:1, after the reaction is complete, filter, wash, and dry to obtain compound 10;
所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种；The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM A kind; Described solvent comprises any one in THF, DCM, DMF, NMP, dioxane;
步骤S11具体包括以下步骤：Step S11 specifically includes the following steps:
取化合物10于反应器中，加入裂解液反应完成后，用冷冻乙醚沉淀，过滤，收集固体，得到瑞林类药物粗品；Take compound 10 in the reactor, add the lysate, after the reaction is completed, precipitate with frozen ether, filter, collect the solid, and obtain the crude product of ralin;
所述裂解液的组分，按体积比计，包括：TFA∶TIS∶H
2O＝95∶2.5∶2.5。
The components of the lysate, by volume ratio, include: TFA:TIS:H 2 O=95:2.5:2.5.
需要说明的是，上述技术方案中所用试剂均为普通市售药剂；在上述技术方案中，析出或者析出固体操作通常采用醚类试剂，包括石油醚、异丙醚、乙醚中的任意一种或任意组合，优选地，为石油醚。It should be noted that the reagents used in the above-mentioned technical schemes are common commercially available medicaments; in the above-mentioned technical schemes, ether reagents are usually used for precipitation or solid precipitation operations, including any one of petroleum ether, isopropyl ether, diethyl ether or Any combination, preferably, petroleum ether.
本发明技术方案，具有如下优点：The technical solution of the present invention has the following advantages:
本发明通过全液相合成法，创造性地发明绿色、温和的生产工艺，没有使用任何剧毒、易制毒试剂，成本极大降低，非常适合大规模生产。The invention creatively invents a green and mild production process through the full liquid phase synthesis method, without using any highly toxic and precursor reagents, greatly reducing the cost, and is very suitable for large-scale production.
采用本发明的合成方法生产的戈那瑞林粗品收率在82％以上，纯度可达95％以上；曲普瑞林粗品收率在90％左右，纯度可达90％以上；那法瑞林粗品收率在86％以上，纯度可达81％以上。The yield of the crude product of gonadorelin produced by the synthetic method of the present invention is more than 82%, and the purity can reach more than 95%; the yield of the crude product of triptorelin is about 90%, and the purity can reach more than 90%; The yield of the crude product is above 86%, and the purity can reach above 81%.
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地， 下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.
图1是本发明实施例1制备的戈那瑞林粗品的HPLC谱图；Fig. 1 is the HPLC spectrogram of the crude product of gonadorelin prepared in Example 1 of the present invention;
图2是本发明实施例3制备的曲普瑞林粗品的HPLC谱图；Fig. 2 is the HPLC spectrogram of the triptorelin crude product that the embodiment of the present invention 3 prepares;
图3是本发明实施例4制备的那法瑞林粗品的HPLC谱图。Fig. 3 is the HPLC spectrogram of the nafarelin crude product prepared in Example 4 of the present invention.
本发明权利要求书和说明书中出现物质的英文缩写对应的中文名称见表1。See Table 1 for the Chinese names corresponding to the English abbreviations of the substances appearing in the claims of the present invention and the description.
表1Table 1
| 英文缩写English abbreviations | 中文名称Chinese name |
| FmocFmoc | 9-芴甲氧羰基9-fluorenylmethoxycarbonyl |
| TrpTrp | 色氨酸Tryptophan |
| BocBoc | 叔丁氧羰基tert-butoxycarbonyl |
| SerSer | 丝氨酸serine |
| tButB | 叔丁基tert-butyl |
| TyrTyr | 酪氨酸Tyrosine |
| LeuLeu | 亮氨酸Leucine |
|
Arg | 精氨酸arginine |
| pbfpbf | 2，2，4，6，7-五甲基二氢苯并呋喃基2,2,4,6,7-Pentamethyldihydrobenzofuryl |
| ProPro | 脯氨酸proline |
| GlyGly | 甘氨酸Glycine |
| EtEt | 乙基Ethyl |
| PyrPyr | 焦谷氨酸pyroglutamic acid |
| HisHis | 组氨酸Histidine |
| DMFDMF | N，N-二甲基甲酰胺N,N-Dimethylformamide |
| THFTHF | 四氢呋喃Tetrahydrofuran |
| HOSUHOSU | 琥珀酰亚胺Succinimide |
| DCCDCC | 二环己基碳二亚胺Dicyclohexylcarbodiimide |
| TEATEA | 三乙胺Triethylamine |
| DEADEA | 二乙胺Diethylamine |
| TFATFA | 三氟乙酸Trifluoroacetate |
| TrTr | 三苯甲酯Trityl |
| HOBtHOB | 1-羟基苯并三唑1-Hydroxybenzotriazole |
| HOAtHOAt |
N-羟基-7-氮杂苯并三氮唑N-Hydroxy-7- |
| HOOBtHOOB | 3，4-二氢-3-羟基-4-氧-1，2，3-苯并三嗪3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine |
| DICDIC | N，N′-二异丙基碳二亚胺N,N'-Diisopropylcarbodiimide |
| EDCEDC | 1，2-二氯乙烷1,2-Dichloroethane |
| BOPBOP | 卡特缩合剂Carter condensation agent |
| pyBOPpyBOP | 1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐1H-Benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate |
| AOPAOP | 7-氮杂苯并三唑-1-基氧基三(二甲胺基)膦六氟磷酸盐7-Azabenzotriazol-1-yloxytris(dimethylamino)phosphine hexafluorophosphate |
| TBTUTBTU | 2-(1H-苯并三偶氮L-1-基)-1，1，3，3-四甲基脲四氟硼酸酯2-(1H-Benzotrisazo L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate |
| HBTUHBTU | 0-(苯并三唑-1-基)-N，N，N′，N′-四甲基脲六氟磷酸酯0-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate |
| HATUHATU | 0-(7-氮杂苯并三唑-1-基)-N，N，N′，N′-四甲基脲六氟磷酸盐0-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate |
| DIEADIEA | N，N-二异丙基乙胺N,N-Diisopropylethylamine |
| NMMNMM | N-甲基吗啡林N-methylmorphine |
| DCMDCM | 二氯甲烷Dichloromethane |
| NMPNMP | N-甲基-2-吡咯烷酮N-methyl-2-pyrrolidone |
| ZZ | 苄氧酰基Benzyloxyacyl |
| NalNal | 萘基naphthyl |
实施例1Example 1
一种全液相合成戈那瑞林的方法，其步骤如下：A method for synthesizing gonadorelin in full liquid phase, the steps are as follows:
1、液相合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH1. Liquid phase synthesis of compound 1: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH
1.1投料：1.1 Feeding:
按照表2的物料用量进行投料。Feed according to the amount of materials in Table 2.
表2Table 2
| 物料materials | 用量Dosage |
| Fmoc-Trp(Boc)-Ser(tBu)-OSUFmoc-Trp(Boc)-Ser(tBu)-OSU | 100mmol100mmol |
| H-Tyr(tBu)-OHH-Tyr(tBu)-OH | 110mmol110mmol |
| TEATEA | 110mmol110mmol |
| DMFDMF | 400ml400ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
1.2操作过程1.2 Operation process
将Fmoc-Trp(Boc)-Ser(tBu)-OSu用DMF完全溶解后，加入H-Tyr(tBu)-OH，再加TEA开始反应，HPLC检测反应完全。After Fmoc-Trp(Boc)-Ser(tBu)-OSu was completely dissolved in DMF, H-Tyr(tBu)-OH was added, and then TEA was added to start the reaction, which was detected by HPLC.
将反应液分两次倒入三角瓶中，再加入0.5M盐酸快速搅拌析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至容器中，称重。化合物1的HPLC检测条件为：The reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to stir and precipitate rapidly. The filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids into containers and weigh. The HPLC detection condition of compound 1 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
收率：95.8％，纯度：87.6％。Yield: 95.8%, purity: 87.6%.
2、液相合成化合物2：Fmoc-Gly-Leu-OMe.HCl2. Liquid phase synthesis of compound 2: Fmoc-Gly-Leu-OMe.HCl
2.1投料2.1 Feeding
按照表3的物料进行投料。Feeding was carried out according to the materials in Table 3.
表3table 3
| 物料materials | 用量Dosage |
| Fmoc-Gly-OHFmoc-Gly-OH | 150mmol150mmol |
| HOSUHOSU | 165mmol165mmol |
| TEATEA | 165mmol165mmol |
| DCCDCC | 165mmol165mmol |
| H-Leu-OMe.HClH-Leu-OMe.HCl | 165mmol165mmol |
| DMFDMF | 500ml500ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
2.2操作过程：2.2 Operation process:
准确称取Fmoc-Gly-OH、HOSU于反应瓶中，用DMF完全溶解后，再称取H-Leu-OMe.HCl于三角瓶中，用DMF完全溶解后冷浴10min，加入TEA，快速摇匀，加入上述反应瓶中，继续冷浴5min后加入DCC开始反应，HPLC检测反应完全。待完全反应后过滤反应液，用0.5M盐酸水溶液析出，过滤的固体，然后用 纯化水洗至中性，30℃干燥，收集固体装至三角瓶中，称重。Accurately weigh Fmoc-Gly-OH and HOSU in a reaction flask, dissolve them completely with DMF, then weigh H-Leu-OMe.HCl in a Erlenmeyer flask, dissolve them completely in DMF, then cool in a bath for 10 minutes, add TEA, and shake quickly Mix well, add to the above-mentioned reaction bottle, continue the cold bath for 5 minutes, then add DCC to start the reaction, and the reaction is complete by HPLC detection. After the reaction is complete, filter the reaction solution, precipitate with 0.5M hydrochloric acid aqueous solution, filter the solid, then wash with purified water until neutral, dry at 30°C, collect the solid and put it in a conical flask, and weigh it.
化合物2的HPLC检测条件为：The HPLC detection condition of compound 2 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，50％B-90％B。Gradient: 0-30min, 50%B-90%B.
收率：107.2％，纯度：95.7％。Yield: 107.2%, purity: 95.7%.
3、液相合成化合物3：H-Gly-Leu-OMe3. Liquid phase synthesis of compound 3: H-Gly-Leu-OMe
3.1投料：3.1 Feeding:
按照表4的物料进行投料。Feeding was carried out according to the materials in Table 4.
表4Table 4
| 物料materials | 用量Dosage |
| Fmoc-Gly-Leu-OMe.HClFmoc-Gly-Leu-OMe.HCl | 150mmol150mmol |
| 二乙胺Diethylamine | 400ml400ml |
| 石油醚petroleum ether | 1L1L |
3.2操作过程3.2 Operation process
准确称取Fmoc-Gly-Leu-OMe.HCl于反应瓶中，加入二乙胺反应，HPLC检测反应完全。浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物3的HPLC检测条件为：Accurately weigh Fmoc-Gly-Leu-OMe.HCl in a reaction flask, add diethylamine to react, and HPLC detects that the reaction is complete. Concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and dry in vacuo. The HPLC detection condition of compound 3 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，3％B-90％B。Gradient: 0-30min, 3%B-90%B.
收率：91.8％，纯度：94.6％。Yield: 91.8%, purity: 94.6%.
4、液相合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe4. Liquid phase synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe
4.1投料：4.1 Feeding:
按照表5的物料进行投料。Feeding was carried out according to the materials in Table 5.
表5table 5
| 物料materials | 用量Dosage |
| Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OHFmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH | 95.8mmol95.8mmol |
| BOPBOP | 105.4mmol105.4mmol |
| TEATEA | 105.4mmol105.4mmol |
| H-Gly-Leu-OMeH-Gly-Leu-OMe | 105.4mmol105.4mmol |
| DMFDMF | 200ml200ml |
| DCMDCM | 200ml200ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
4.2操作过程4.2 Operation process
准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH、BOP于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA，再将H-Gly-Leu-OMe用DCM溶解后加入反应中开始反应，HPLC检测反应完全，浓缩，用0.5M盐酸沉淀，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，称重。化合物4的HPLC检测条件为：Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH and BOP in the reaction bottle, dissolve them completely in DMF, cool in the bath for 10min, add DIEA, then use H-Gly-Leu-OMe After DCM was dissolved, it was added to the reaction to start the reaction. The reaction was detected by HPLC, concentrated, precipitated with 0.5M hydrochloric acid, filtered to collect the solid, washed with purified water until neutral (pH test paper detection), and weighed. The HPLC detection condition of compound 4 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，80％B-90％B。Gradient: 0-30min, 80%B-90%B.
收率：92.3％，纯度：90％Yield: 92.3%, Purity: 90%
5、液相合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe5. Liquid phase synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe
5.1投料5.1 Feeding
按照表6的物料投料。Feeding according to the materials in Table 6.
表6Table 6
| 物料materials | 用量Dosage |
| Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMeFmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe | 88.4mmol88.4mmol |
| 二乙胺Diethylamine | 500ml500ml |
| 石油醚petroleum ether | 1L1L |
5.2操作过程5.2 Operation process
准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe于反应瓶中，加入二乙胺反应，HPLC检测反应完全，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe in a reaction bottle, add diethylamine to react, HPLC detects that the reaction is complete, concentrate to a certain amount, add petroleum ether to precipitate a solid , filtered, and dried in vacuo.
化合物5的HPLC检测条件为：The HPLC detection condition of compound 5 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，50％B-90％B。Gradient: 0-30min, 50%B-90%B.
收率：96.2％，纯度：88.6％。Yield: 96.2%, purity: 88.6%.
6、液相合成化合物6：6. Liquid phase synthesis of compound 6:
H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMeH-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe
6.1投料6.1 Feeding
按照表7的物料进行投料。Feed according to the materials in Table 7.
表7Table 7
6.2操作过程6.2 Operation process
准确称取H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe、HOBt、Fmoc-His(Trt)-OH于反应瓶中，用DMF完全溶解后冷浴10min后加入DCC开始反应，过滤反应液，倒入反应瓶中，再加入二乙胺反应20min，浓缩至少量，加入0.5M盐酸溶液析出固体，过滤，烘干。Accurately weigh H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe, HOBt, Fmoc-His(Trt)-OH in the reaction flask, completely dissolve in DMF and cool in the bath for 10min Add DCC to start the reaction, filter the reaction solution, pour it into a reaction bottle, add diethylamine to react for 20 minutes, concentrate to a small amount, add 0.5M hydrochloric acid solution to precipitate a solid, filter, and dry.
取Fmoc-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)--Leu-OMe于反应瓶中，加入二乙胺反应20min，HPLC检测反应完全，浓缩至少量，加入石油醚析出固体，过滤，烘干。化合物6的HPLC检测条件为：Take Fmoc-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)--Leu-OMe in a reaction bottle, add diethylamine to react for 20min, HPLC detects that the reaction is complete, concentrate to a certain amount, add petroleum A solid precipitated from ether was filtered and dried. The HPLC detection condition of compound 6 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
收率：82.1％，纯度：92.8％。Yield: 82.1%, purity: 92.8%.
7、液相合成化合物7：7. Liquid phase synthesis of compound 7:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMeBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe
7.1投料7.1 Feeding
按照表8的物料进行投料。Feeding was carried out according to the materials in Table 8.
表8Table 8
| 物料materials | 用量Dosage |
| Boc-Pyr-OHBoc-Pyr-OH | 76.9mmol76.9mmol |
| BOPBOP | 76.9mmol76.9mmol |
| TEATEA | 76.9mmol76.9mmol |
| H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMeH-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe | 69.9mmol69.9mmol |
| DMFDMF | 350ml350ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
7.2操作过程7.2 Operation process
准确称取Boc-Pyr-OH、BOP于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA，再将H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe用DMF溶解后加入反应中开始反应，HPLC检测反应完全；过滤反应液，滤渣用DMF洗涤两次，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。Accurately weigh Boc-Pyr-OH and BOP in the reaction bottle, dissolve them completely in DMF, cool in the bath for 10 minutes, add DIEA, then add H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu) -Gly-Leu-OMe was dissolved in DMF and added to the reaction to start the reaction. HPLC detected that the reaction was complete; filtered the reaction solution, washed the filter residue twice with DMF, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, and washed it with purified water until neutral (pH test paper detection), dry, and weigh.
化合物7的HPLC检测条件为：The HPLC detection condition of compound 7 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
收率：85％，纯度：80.1％。Yield: 85%, purity: 80.1%.
8、液相合成化合物8：8. Liquid phase synthesis of compound 8:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OHBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OH
称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe(59.4mmol)，置于1110ml甲醇中搅拌5min完全溶解后，缓慢加入110ml 2M NaOH反应，开始反应，HPLC检测反应完全，加入0.1M盐酸溶液，沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。化合物8的HPLC检测条件为：Weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe(59.4mmol), put it in 1110ml methanol and stir for 5min to completely dissolve, then slowly add 110ml 2M NaOH reaction, start the reaction, HPLC detects that the reaction is complete, add 0.1M hydrochloric acid solution, precipitate, collect the solid after filtration, then wash with purified water to neutrality (pH test paper detection), dry, and weigh. The HPLC detection condition of compound 8 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
收率：83.9％，纯度：94.07％。Yield: 83.9%, purity: 94.07%.
9、液相合成化合物9：H-Arg(pbf)-Pro-Gly-NH
2
9. Liquid phase synthesis of compound 9: H-Arg(pbf)-Pro-Gly-NH 2
9.1投料9.1 Feeding
按照表9的物料进行投料。Feed according to the materials in Table 9.
表9Table 9
| 物料materials | 用量Dosage |
| H-Pro-Gly-NH2H-Pro-Gly-NH2 | 150mmol150mmol |
| BOPBOP | 157.5mmol157.5mmol |
| DIEADIEA | 157.5mmol157.5mmol |
| Fmoc-Arg(pbf)-OHFmoc-Arg(pbf)-OH | 157.5mmol157.5mmol |
| TEATEA | 157.5mmol157.5mmol |
| DMFDMF | 400ml400ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
| 二乙胺Diethylamine | 500ml500ml |
9.2操作过程9.2 Operation process
准确称取Fmoc-Arg(pbf)-OH、BOP于反应瓶中，用DMF完全溶解后冷浴10min，加入DIEA，撤掉冷浴，反应20min。Accurately weigh Fmoc-Arg(pbf)-OH and BOP in a reaction flask, dissolve them completely in DMF, then cool them for 10 minutes, add DIEA, remove the cooling bath, and react for 20 minutes.
称取H-Pro-Gly-NH2于三角瓶中，用DMF完全溶解后加入TEA，快速混匀后加入上反应中开始反应。HPLC检测反应完全，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，加入二乙胺反应，HPLC检测反应完全，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。HPLC检测条件为：Weigh H-Pro-Gly-NH2 in a Erlenmeyer flask, dissolve it completely with DMF, add TEA, mix quickly and add to the above reaction to start the reaction. HPLC detected that the reaction was complete, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, then washed with purified water until neutral (pH test paper detection), dried, added diethylamine to react, HPLC detected that the reaction was complete, concentrated to a certain amount, added petroleum ether to precipitate The solid was filtered and dried in vacuo. HPLC detection conditions are:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，5％B-30％B。Gradient: 0-30min, 5%B-30%B.
收率93％，纯度：95.5％。Yield: 93%, purity: 95.5%.
10、液相合成化合物10：10. Liquid phase synthesis of compound 10:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-Arg(pbf)-Pro-Gly-NH
2
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-Arg(pbf)-Pro-Gly-NH 2
10.1投料10.1 Feeding
按照表10的物料进行投料。Feeding was carried out according to the materials in Table 10.
表10Table 10
| 物料materials | 用量Dosage |
| Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OHBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OH | 49.8mmol49.8mmol |
| BOPBOP | 54.8mmol54.8mmol |
| TEATEA | 54.8mmol54.8mmol |
| H-Arg(pbf)-Pro-Gly-NH2H-Arg(pbf)-Pro-Gly-NH2 | 54.8mmol54.8mmol |
| DMFDMF | 200ml200ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 500ml500ml |
10.2操作过程10.2 Operation process
准确称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OH、BOP于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA，再将H-Arg(pbf)-Pro-Gly-NH
2用DMF溶解后加入反应中开始反应。HPLC检测反应完全，加入0.5M盐酸析出固体，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。HPLC检测条件为：
Accurately weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OH and BOP in the reaction flask, dissolve completely in DMF, cool in the bath for 10min, and then add DIEA , Then H-Arg(pbf)-Pro-Gly-NH 2 was dissolved in DMF and added to the reaction to start the reaction. HPLC detected that the reaction was complete, and 0.5M hydrochloric acid was added to precipitate a solid, which was collected by filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed. HPLC detection conditions are:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
其结果参照图10，收率98.1％，纯度：94.8％The result is referring to Fig. 10, yield 98.1%, purity: 94.8%
11、合成戈那瑞林粗品11. Synthetic crude gonadorelin
将48.6mmol的化合物10加入反应瓶中，加入300ml裂解液(TFA∶TIS∶H
2O＝95∶2.5∶2.5)中反应30min后，用冷冻乙醚沉淀，过滤，收集固体，得到产物粗品，产物用水溶解后HPLC检测分析。HPLC检测条件为：
Add 48.6mmol of compound 10 into the reaction flask, add 300ml of lysate (TFA:TIS: H2O =95:2.5:2.5) to react for 30min, precipitate with frozen ether, filter, collect the solid to obtain the crude product, the product After dissolving in water, it was detected and analyzed by HPLC. HPLC detection conditions are:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，25％B-25％B。Gradient: 0-30min, 25%B-25%B.
其结果参照图1，收率：82.4％，纯度95.2％。Referring to Figure 1 for the results, the yield is 82.4%, and the purity is 95.2%.
实施例2Example 2
一种全液相合成戈那瑞林的方法，其步骤如下：A method for synthesizing gonadorelin in full liquid phase, the steps are as follows:
1、液相合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH1. Liquid phase synthesis of compound 1: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH
其过程同实施例1。Its process is with embodiment 1.
2、液相合成化合物2：Boc-Gly-Leu-OEt·HCl2. Liquid phase synthesis of compound 2: Boc-Gly-Leu-OEt·HCl
2.1投料2.1 Feeding
按照表11的物料进行投料。Feed according to the materials in Table 11.
表11Table 11
| 物料materials | 用量Dosage |
| Boc-Gly-OHBoc-Gly-OH | 150mmol150mmol |
| HOSUHOSU | 300mmol300mmol |
| TEATEA | 300mmol300mmol |
| DCCDCC | 300mmol300mmol |
| H-Leu-OEt·HClH-Leu-OEt·HCl | 300mmol300mmol |
| DMFDMF | 500ml500ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
操作过程同实施例1。收率：106.2％，纯度：95.2％。Operation process is with embodiment 1. Yield: 106.2%, purity: 95.2%.
3、液相合成化合物3：H-Gly-Leu-OEt3. Liquid phase synthesis of compound 3: H-Gly-Leu-OEt
3.1投料：3.1 Feeding:
按照表12的物料进行投料。Feed according to the materials in Table 12.
表12Table 12
| 物料materials | 用量Dosage |
| Boc-Gly-Leu-OEt.HClBoc-Gly-Leu-OEt.HCl | 150mmol150mmol |
| 50％TFA/DCM50% TFA/DCM | 400ml400ml |
| 石油醚petroleum ether | 1L1L |
操作过程同实施例1。收率：91.2％，纯度：90.5％。Operation process is with embodiment 1. Yield: 91.2%, purity: 90.5%.
4、液相合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt4. Liquid phase synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt
4.1投料：4.1 Feeding:
按照表13的物料进行投料。Feed according to the materials in Table 13.
表13Table 13
| 物料materials | 用量Dosage |
| Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OHFmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH | 95.8mmol95.8mmol |
| BOPBOP | 191.6mmol191.6mmol |
| TEATEA | 191.6mmol191.6mmol |
| H-Gly-Leu-OEtH-Gly-Leu-OEt | 191.6mmol191.6mmol |
| DMFDMF | 200ml200ml |
| DCMDCM | 200ml200ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
操作过程同实施例1。收率：91.8％，纯度：80％Operation process is with embodiment 1. Yield: 91.8%, Purity: 80%
5、液相合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt5. Liquid phase synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt
5.1投料5.1 Feeding
按照表14的物料投料。Feed according to the materials in Table 14.
表14Table 14
| 物料materials | 用量Dosage |
| Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEtFmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt | 88.4mmol88.4mmol |
| 二乙胺Diethylamine | 500ml500ml |
| 石油醚petroleum ether | 1L1L |
操作过程同实施例1。收率：95.8％，纯度：84.8％。Operation process is with embodiment 1. Yield: 95.8%, purity: 84.8%.
6、液相合成化合物6：6. Liquid phase synthesis of compound 6:
H-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEtH-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt
6.1投料6.1 Feeding
按照表15的物料进行投料。Feed according to the materials in Table 15.
表15Table 15
操作过程同实施例1。收率：82.0％，纯度：81.5％。Operation process is with embodiment 1. Yield: 82.0%, purity: 81.5%.
7、液相合成化合物7：7. Liquid phase synthesis of compound 7:
Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEtFmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt
7.1投料7.1 Feeding
按照表16的物料进行投料。Feed according to the materials in Table 16.
表16Table 16
| 物料materials | 用量Dosage |
| Fmoc-Pyr-OHFmoc-Pyr-OH | 139.8mmol139.8mmol |
| BOPBOP | 139.8mmol139.8mmol |
| 二乙胺Diethylamine | 139.8mmol139.8mmol |
| H-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEtH-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt | 69.9mmol69.9mmol |
| DMFDMF | 350ml350ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
操作过程同实施例1。收率：85％，纯度：85.3％。Operation process is with embodiment 1. Yield: 85%, purity: 85.3%.
8、液相合成化合物8：8. Liquid phase synthesis of compound 8:
Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OHFmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OH
8.1投料8.1 Feeding
Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt：59.4mmolFmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OEt: 59.4mmol
称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe(59.4mmol)，置于1110ml甲醇中搅拌5min完全溶解后，缓慢加入1110ml2MNaOH反应，开始反应，HPLC检测反应完全，加入0.1M盐酸溶液，沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。其余同实施例1。收率：83.5％，纯度：88.6％。Weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OMe(59.4mmol), put it in 1110ml of methanol and stir for 5min to dissolve completely, then slowly add 1110ml of 2M NaOH React, start the reaction, HPLC detects that the reaction is complete, add 0.1M hydrochloric acid solution, precipitate, collect the solid after filtration, then wash with purified water to neutrality (pH test paper detection), dry, and weigh. All the other are with embodiment 1. Yield: 83.5%, purity: 88.6%.
9、液相合成化合物9：H-Arg-Pro-Gly-NH
2
9. Liquid phase synthesis of compound 9: H-Arg-Pro-Gly-NH 2
9.1投料9.1 Feeding
按照表17的物料进行投料。Feed according to the materials in Table 17.
表17Table 17
| 物料materials | 用量Dosage |
| H-Pro-Gly-NH 2 H-Pro-Gly-NH 2 | 150mmol150mmol |
| BOPBOP | 300mmol300mmol |
| DIEADIEA | 300mmol300mmol |
| Fmoc-Arg(pbf)-OHFmoc-Arg(pbf)-OH | 300mmol300mmol |
| TEATEA | 300mmol300mmol |
| DMFDMF | 400ml400ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
| 50％TFA/DCM50% TFA/DCM | 500ml500ml |
操作过程同实施例1。收率93％，纯度：82％。Operation process is with embodiment 1. Yield: 93%, purity: 82%.
10、液相合成化合物10：10. Liquid phase synthesis of compound 10:
Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-Arg-Pro-Gly-NH2Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-Arg-Pro-Gly-NH2
10.1投料10.1 Feeding
按照表18的物料进行投料。Feed according to the materials in Table 18.
表18Table 18
| 物料materials | 用量Dosage |
| Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OHFmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-Gly-Leu-OH | 49.8mmol49.8mmol |
| BOPBOP | 99.6mmol99.6mmol |
| TEATEA | 99.6mmol99.6mmol |
| H-Arg-Pro-Gly-NH 2 H-Arg-Pro-Gly- NH2 | 99.6mmol99.6mmol |
| DMFDMF | 200ml200ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 500ml500ml |
操作过程同实施例1。收率97.8％，纯度：81.1％Operation process is with embodiment 1. Yield 97.8%, purity: 81.1%
11、合成戈那瑞林粗品11. Synthetic crude gonadorelin
11.1投料11.1 Feeding
化合物10：48.7mmolCompound 10: 48.7 mmol
裂解液(TFA∶TIS∶H2O＝95∶2.5∶2.5)：300mlLysis solution (TFA:TIS:H2O=95:2.5:2.5): 300ml
其操作过程同实施例1。收率：82.1％，纯度95.1％。Its operation process is with embodiment 1. Yield: 82.1%, purity 95.1%.
实施例3Example 3
一种全液相合成曲普瑞林的方法，其步骤如下：A method for synthesizing triptorelin in full liquid phase, the steps are as follows:
1、液相合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH1. Liquid phase synthesis of compound 1: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH
1.1投料：1.1 Feeding:
按照表19的物料进行投料。Feed according to the materials in Table 19.
表19Table 19
| 物料materials | 用量Dosage |
| Fmoc-Trp(Boc)-Ser(tBu)-OSUFmoc-Trp(Boc)-Ser(tBu)-OSU | 100mmol100mmol |
| H-Tyr(tBu)-OHH-Tyr(tBu)-OH | 110mmol110mmol |
| TEATEA | 110mmol110mmol |
| DMFDMF | 400ml400ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
1.2操作过程1.2 Operation process
将Fmoc-Trp(Boc)-Ser(tBu)-OSu用DMF完全溶解后，再准确称取H-Tyr(tBu)-OH加入上述反应瓶，加入TEA反应，HPLC检测反应完全后，将反应液分两次倒入三角瓶中，再加入0.5M盐酸快速搅拌析出，过滤的固体，然后用纯化水洗至中性，35℃干燥。收集固体装至容器中，称重。收率为96％。化合物1的HPLC检测条件为：After completely dissolving Fmoc-Trp(Boc)-Ser(tBu)-OSu in DMF, accurately weigh H-Tyr(tBu)-OH and add it to the above reaction bottle, add TEA to react, after HPLC detects that the reaction is complete, the reaction solution Pour it into the Erlenmeyer flask twice, then add 0.5M hydrochloric acid to stir and precipitate rapidly, and filter the solid, then wash it with purified water until it is neutral, and dry it at 35°C. Collect the solids into containers and weigh. The yield was 96%. The HPLC detection condition of compound 1 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，50％B-90％B。Gradient: 0-30min, 50%B-90%B.
收率：96％；纯度：87.6％。Yield: 96%; Purity: 87.6%.
2、液相合成化合物2：Fmoc-D-Trp-Leu-OMe.HCl2. Liquid phase synthesis of compound 2: Fmoc-D-Trp-Leu-OMe.HCl
2.1投料2.1 Feeding
按照表20的物料进行投料。Feed according to the materials in Table 20.
表20Table 20
| 物料materials | 用量Dosage |
| Fmoc-D-Trp-OHFmoc-D-Trp-OH | 150mmol150mmol |
| HOSUHOSU | 165mmol165mmol |
| TEATEA | 165mmol165mmol |
| DCCDCC | 165mmol165mmol |
| H-Leu-OEt.HClH-Leu-OEt.HCl | 165mmol165mmol |
| DMFDMF | 500ml500ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
2.2操作过程：2.2 Operation process:
准确称取Fmoc-D-Trp-OH、HOSU于反应瓶中，用DMF完全溶解后，再称取H-Leu-OEt.HCl于三角瓶中，用DMF完全溶解后冷浴10min，加入TEA，快速摇匀，加入上述反应瓶中，继续冷浴5min后加入DCC开始反应，HPLC检测反应完全后，过滤反应液，用0.5M盐酸水溶液析出，过滤的固体，然后用纯化水洗至中性，35℃干燥。收集固体装至三角瓶中，称重。化合物2的HPLC检测条件为：Accurately weigh Fmoc-D-Trp-OH and HOSU in a reaction flask, dissolve them completely with DMF, then weigh H-Leu-OEt. Shake quickly, add to the above-mentioned reaction bottle, continue the cold bath for 5 minutes, then add DCC to start the reaction. After the reaction is complete by HPLC, filter the reaction solution, precipitate with 0.5M hydrochloric acid aqueous solution, filter the solid, and then wash with purified water until neutral, 35 ℃ dry. Collect the solids and put them in a Erlenmeyer flask and weigh them. The HPLC detection condition of compound 2 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，50％B-90％B。Gradient: 0-30min, 50%B-90%B.
收率：108.1％，纯度：95.7％。Yield: 108.1%, purity: 95.7%.
3、液相合成化合物3：H-D-Trp-Leu-OMe3. Liquid phase synthesis of compound 3: H-D-Trp-Leu-OMe
3.1投料：3.1 Feeding:
按照表21的物料进行投料。Feed according to the materials in Table 21.
表21Table 21
| 物料materials | 用量Dosage |
| Fmoc-D-Trp-Leu-OMe.HClFmoc-D-Trp-Leu-OMe.HCl | 150mmol150mmol |
| 二乙胺Diethylamine | 400ml400ml |
| 石油醚petroleum ether | 1L1L |
3.2操作过程3.2 Operation process
准确称取Fmoc-D-Trp-Leu-OEt.HCl于反应瓶中，加入二乙胺反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物3的HPLC检测条件为：Accurately weigh Fmoc-D-Trp-Leu-OEt.HCl in a reaction flask, add diethylamine to react for 20 minutes, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and vacuum dry. The HPLC detection condition of compound 3 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，30％B-50％B。Gradient: 0-30min, 30%B-50%B.
收率：87.2％；纯度：93.3％。Yield: 87.2%; Purity: 93.3%.
4、液相合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe4. Liquid phase synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe
4.1投料：4.1 Feeding:
按照表22的物料进行投料。Feed according to the materials in Table 22.
表22Table 22
| 物料materials | 用量Dosage |
| Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OHFmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH | 96mmol96mmol |
| BOPBOP | 105.6mmol105.6mmol |
| TEATEA | 105.6mmol105.6mmol |
| H-D-Trp-Leu-OMeH-D-Trp-Leu-OMe | 105.6mmol105.6mmol |
| DMFDMF | 200ml200ml |
| DCMDCM | 200ml200ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
4.2操作过程4.2 Operation process
准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH、BOP于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA，再将H-D-Trp-Leu-OMe用DCM溶解后加入反应中开始反应，HPLC检测反应完全，浓缩，用0.5M盐酸沉淀，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，称重。化合物4的HPLC检测条件为：Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH and BOP in the reaction bottle, dissolve them completely in DMF, cool in the bath for 10min, add DIEA, then use H-D-Trp-Leu-OMe After DCM was dissolved, it was added to the reaction to start the reaction. The reaction was detected by HPLC, concentrated, precipitated with 0.5M hydrochloric acid, filtered to collect the solid, washed with purified water until neutral (pH test paper detection), and weighed. The HPLC detection condition of compound 4 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，80％B-90％B。Gradient: 0-30min, 80%B-90%B.
收率：92％，纯度：80％。Yield: 92%, purity: 80%.
5、合成H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe5. Synthesis of H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe
5.1投料5.1 Feeding
按照表23的物料投料。According to the material feeding in Table 23.
表23Table 23
| 物料materials | 用量Dosage |
| Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEtFmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OEt | 88.3mmol88.3mmol |
| 二乙胺Diethylamine | 500ml500ml |
| 石油醚petroleum ether | 1L1L |
5.2操作过程5.2 Operation process
准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe于反应瓶中， 加入二乙胺反应脱去Fmoc基团，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。化合物5的HPLC检测条件为：Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe in a reaction flask, add diethylamine to remove the Fmoc group, concentrate to a certain amount, add petroleum ether A solid precipitated, was filtered, and dried in vacuo. The HPLC detection condition of compound 5 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
收率：92.1％，纯度：90.3％。Yield: 92.1%, purity: 90.3%.
6、合成H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe6. Synthesis of H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe
6.1投料6.1 Feeding
按照表24的物料进行投料。Feed according to the materials in Table 24.
表24Table 24
6.2操作过程6.2 Operation process
准确称取H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe、HOBt、Fmoc-His(Trt)-OH于反应瓶中，用DMF完全溶解后冷浴10min后加入DCC开始反应，HPLC检测反应完全，过滤反应液，倒入反应瓶中，再加入二乙胺反应20min，浓缩至少量，加入0.5M盐酸溶液析出固体，过滤，烘干，加入二乙胺反应20min，浓缩至少量，加入石油醚析出固体，过滤，烘干，称重。化合物6的HPLC检测条件为：Accurately weigh H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe, HOBt, Fmoc-His(Trt)-OH in the reaction flask, dissolve completely in DMF and cool in the bath After 10 minutes, add DCC to start the reaction. HPLC detects that the reaction is complete. Filter the reaction solution and pour it into a reaction bottle. Then add diethylamine to react for 20 minutes. Concentrate to a small amount. The amine was reacted for 20 minutes, concentrated to a certain amount, and petroleum ether was added to precipitate a solid, which was filtered, dried, and weighed. The HPLC detection condition of compound 6 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
收率：86％，纯度：90.6％。Yield: 86%, purity: 90.6%.
7、液相合成化合物7：7. Liquid phase synthesis of compound 7:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMeBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe
7.1投料7.1 Feeding
按照表25的物料进行投料。Feed according to the materials in Table 25.
表25Table 25
| 物料materials | 用量Dosage |
| Boc-Pyr-OHBoc-Pyr-OH | 76.9mmol76.9mmol |
| BOPBOP | 76.9mmol76.9mmol |
| TEATEA | 76.9mmol76.9mmol |
| H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMeH-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe | 69.9mmol69.9mmol |
| DMFDMF | 350ml350ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
7.2操作过程7.2 Operation process
准确称取Boc-Pyr-OH、BOP于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA，再将H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe用DMF溶解后加入反应中开始反应，HPLC检测反应完全，过滤反应液，滤渣用DMF洗涤两次，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。化合物7的HPLC检测条件为：Accurately weigh Boc-Pyr-OH and BOP in the reaction bottle, dissolve them completely in DMF, cool in the bath for 10 minutes, add DIEA, then add H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu) -D-Trp-Leu-OMe is dissolved in DMF and then added to the reaction to start the reaction. HPLC detects that the reaction is complete. The reaction solution is filtered. The filter residue is washed twice with DMF and precipitated with 0.5M hydrochloric acid. After filtration, the solid is collected and washed with purified water until Neutral (pH test paper detection), dry, weigh. The HPLC detection condition of compound 7 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
收率：80％，纯度：78.5％。Yield: 80%, purity: 78.5%.
8、液相合成化合物8：8. Liquid phase synthesis of compound 8:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OHBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OH
称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe(56mmol)，置于1110ml甲醇中搅拌使完全溶解，缓慢加入2MNaOH110ml，开始反应，HPLC检测反应完全，加入盐酸溶液，沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。化合物7的HPLC检测条件为：Weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OMe (56mmol), put it in 1110ml of methanol and stir to dissolve completely, slowly add 2MNaOH110ml , start the reaction, HPLC detects that the reaction is complete, add hydrochloric acid solution, precipitate, collect the solid after filtration, then wash with purified water to neutrality (pH test paper detection), dry, and weigh. The HPLC detection condition of compound 7 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
收率：72％，纯度：84.7％。Yield: 72%, purity: 84.7%.
9、液相合成化合物9：H-Arg(pbf)-Pro-Gly-NH
2
9. Liquid phase synthesis of compound 9: H-Arg(pbf)-Pro-Gly-NH 2
9.1投料9.1 Feeding
按照表26的物料进行投料。Feed according to the materials in Table 26.
表26Table 26
| 物料materials | 用量Dosage |
| H-Pro-Gly-NH 2 H-Pro-Gly-NH 2 | 150mmol150mmol |
| BOPBOP | 157.5mmol157.5mmol |
| DIEADIEA | 157.5mmol157.5mmol |
| Fmoc-Arg(pbf)-OHFmoc-Arg(pbf)-OH | 157.5mmol157.5mmol |
| TEATEA | 157.5mmol157.5mmol |
| DMFDMF | 400ml400ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 1L1L |
| 二乙胺Diethylamine | 500ml500ml |
9.2操作过程9.2 Operation process
准确称取Fmoc-Arg(pbf)-OH(150mmol)、BOP(157.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min，加入DIEA(157.5mmol)，撤掉冷浴，反应20min。Accurately weigh Fmoc-Arg(pbf)-OH (150mmol) and BOP (157.5mmol) in a reaction flask, dissolve them completely in DMF, then cool in a bath for 10min, add DIEA (157.5mmol), remove the cooling bath, and react for 20min.
称取H-Pro-Gly-NH
2(150mmol)于三角瓶中，用DMF完全溶解后加入TEA(157.5mmol)，快速混匀后加入上反应中开始反应，HPLC检测反应完全，用0.5M盐酸1L沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥。加入二乙胺500mL反应20min，浓缩、石油醚析出固体，过滤，真空干燥。化合物9的HPLC检测条件为：
Weigh H-Pro-Gly-NH 2 (150mmol) in a Erlenmeyer flask, dissolve it completely with DMF, add TEA (157.5mmol), mix quickly and add to the above reaction to start the reaction, HPLC detects that the reaction is complete, and use 0.5M hydrochloric acid 1L of precipitate was collected by filtration, then washed with purified water until neutral (detected by pH test paper), and dried. Add 500 mL of diethylamine to react for 20 min, concentrate, and precipitate solid with petroleum ether, filter and dry in vacuo. The HPLC detection condition of compound 9 is:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，5％B-30％BGradient: 0-30min, 5%B-30%B
收率93％，纯度：95.5％。Yield: 93%, purity: 95.5%.
10、液相合成化合物10：10. Liquid phase synthesis of compound 10:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-Arg(pbf)-Pro-Gly-NH
2
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-Arg(pbf)-Pro-Gly-NH 2
10.1投料10.1 Feeding
按照表10的物料进行投料。Feeding was carried out according to the materials in Table 10.
表27Table 27
| 物料materials | 用量Dosage |
| Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OHBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OH | 40.3mmol40.3mmol |
| BOPBOP | 44.3mmol44.3mmol |
| TEATEA | 44.3mmol44.3mmol |
| H-Arg(pbf)-Pro-Gly-NH 2 H-Arg(pbf)-Pro-Gly-NH 2 | 44.3mmol44.3mmol |
| DMFDMF | 200ml200ml |
| 0.5M盐酸溶液0.5M hydrochloric acid solution | 500ml500ml |
10.2操作过程10.2 Operation process
准确称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OH、BOP于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA，再将H-Arg(pbf)-Pro-Gly-NH
2用DMF溶解后加入反应中开始反应；HPLC检测反应完全后，加入0.5M盐酸析出固体，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。HPLC分析条件为：
Accurately weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp-Leu-OH and BOP in the reaction bottle, dissolve them completely in DMF and cool in the bath for 10 minutes Add DIEA, then dissolve H-Arg(pbf)-Pro-Gly- NH with DMF and add to the reaction to start the reaction; after the HPLC detection reaction is complete, add 0.5M hydrochloric acid to precipitate a solid, collect the solid by filtration, and then wash it with purified water until Neutral (pH test paper detection), dry, weigh. HPLC analysis conditions are:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
梯度：0-30min，70％B-90％B。Gradient: 0-30min, 70%B-90%B.
收率：95％，纯度：81.1％。Yield: 95%, purity: 81.1%.
11、合成曲普瑞林粗品11. Synthetic crude triptorelin
将化合物10(42.1mmol)加入反应瓶中，加入300ml裂解液(TFA∶TIS∶H
2O＝95∶2.5∶2.5)反应30min后用冷冻乙醚沉淀，过滤，收集固体，得到产物粗品，产物用水溶解后HPLC检测分析。HPLC检测分析条件如下：
Add compound 10 (42.1mmol) into the reaction flask, add 300ml of lysate (TFA:TIS:H 2 O = 95:2.5:2.5) for 30min, precipitate with frozen diethyl ether, filter, collect the solid to obtain the crude product, the product is water After dissolution, HPLC detection and analysis. HPLC detection and analysis conditions are as follows:
安捷伦1260型HPLC，Krosmail100-5C18column4.6*250mmAgilent 1260 HPLC, Krosmail100-5C18column4.6*250mm
A：5％乙腈/H
2O溶液，0.1％TFA；B：乙腈/H
2O，0.1％TFA(HPLC)；
A: 5% acetonitrile/H 2 O solution, 0.1% TFA; B: acetonitrile/H 2 O, 0.1% TFA (HPLC);
梯度洗脱：0～30min；70％B～75％B；Gradient elution: 0~30min; 70%B~75%B;
λ＝210nm；流速＝1.0ml/min。λ = 210 nm; flow rate = 1.0 ml/min.
结果参照图2，收率：89.4％，纯度91.3％。Referring to Figure 2 for the results, the yield: 89.4%, purity 91.3%.
实施例4Example 4
需要说明的是，该实施例中，各化合物及产品采用HPLC进行纯度分析，如无特殊说明，HPLC检测条件为：It should be noted that, in this embodiment, the purity analysis of each compound and product is carried out by HPLC. If there is no special instruction, the detection conditions of HPLC are as follows:
流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈；Mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
检测波长：210nm；流速：1ml/min；固定相：C18色谱柱，5μ，
Detection wavelength: 210nm; flow rate: 1ml/min; stationary phase: C18 column, 5μ,
一种全液相合成那法瑞林的方法，其步骤如下所示。A method for synthesizing nafarelin in full liquid phase, the steps are as follows.
1、液相合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH1. Liquid phase synthesis of compound 1: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH
将Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol)用DMF完全溶解后，再准确称取H-Tyr(tBu)-OH(110mmol)加入上述反应瓶，加TEA(110mmol)开始反应。搅拌反应60min后，HPLC检测反应完全。Dissolve Fmoc-Trp(Boc)-Ser(tBu)-OSu(100mmol) completely in DMF, then accurately weigh H-Tyr(tBu)-OH(110mmol) into the above reaction flask, add TEA(110mmol) to start the reaction . After stirring and reacting for 60 min, HPLC detected that the reaction was complete.
将反应液分两次倒入三角瓶中，再加入0.5M盐酸快速搅拌析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至容器中，称重；The reaction solution was poured into the Erlenmeyer flask twice, and then 0.5M hydrochloric acid was added to rapidly stir and precipitate, and the filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solids and put them in containers and weigh them;
收率：96％，纯度：87.6％。Yield: 96%, purity: 87.6%.
2、液相合成化合物2：Fmoc-D-2-Nal-Leu-OMe.HCl2. Liquid phase synthesis of compound 2: Fmoc-D-2-Nal-Leu-OMe.HCl
准确称取Fmoc-D-2-Nal-OH(150mmol)、HOSU(165mmol)于反应瓶中，用DMF完全溶解后，再称取H-Leu-OMe.HCl(165mmol)于三角瓶中，用DMF完全溶解后冷浴10min，加入TEA(165mmol)，快速摇匀，加入上述反应瓶中，继续冷浴5min后加入DCC(165mmol)开始反应。反应1.5h后HPLC检测反应完全。待完全反应后过滤反应液，用0.5M盐酸水溶液析出，过滤的固体，然后用纯化水洗至中性，30℃干燥。收集固体装至三角瓶中，称重；Accurately weigh Fmoc-D-2-Nal-OH (150mmol) and HOSU (165mmol) in the reaction flask, dissolve it completely with DMF, then weigh H-Leu-OMe.HCl (165mmol) in the Erlenmeyer flask, and use After the DMF was completely dissolved, the solution was cooled for 10 minutes, TEA (165 mmol) was added, shaken up quickly, added to the above reaction bottle, and DCC (165 mmol) was added to start the reaction after continuing the cold bath for 5 minutes. After 1.5 h of reaction, HPLC detected that the reaction was complete. After the reaction was complete, the reaction solution was filtered and precipitated with 0.5M hydrochloric acid aqueous solution. The filtered solid was then washed with purified water until neutral, and dried at 30°C. Collect the solid and put it in the Erlenmeyer flask and weigh it;
收率：108.1％，纯度：95.7％。Yield: 108.1%, purity: 95.7%.
3、液相合成化合物3：H-D-2-Nal-Leu-OMe3. Liquid phase synthesis of compound 3: H-D-2-Nal-Leu-OMe
准确称取Fmoc-D-2-Nal-Leu-OMe.HCl(150mmol)于反应瓶中，加入400ml二乙胺反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥。Accurately weigh Fmoc-D-2-Nal-Leu-OMe.HCl (150mmol) into a reaction flask, add 400ml of diethylamine to react for 20min, concentrate to a small amount, add petroleum ether to precipitate a solid, filter, and vacuum dry.
收率：87.2％，纯度：91％。Yield: 87.2%, purity: 91%.
4、液相合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe4. Liquid phase synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe
准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(96mmol)、BOP(105.6mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(105.6mmol)，再将H-D-2-Nal-Leu-OMe(105.6mmol)用DCM溶解后加入反应中开始反应。Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH(96mmol) and BOP(105.6mmol) in the reaction flask, dissolve them completely in DMF, and add DIEA(105.6mmol) in the cooling bath for 10min , and then H-D-2-Nal-Leu-OMe (105.6mmol) was dissolved in DCM and added to the reaction to start the reaction.
反应1.0h后HPLC检测反应完全，浓缩，用0.5M盐酸沉淀，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，称重；After reacting for 1.0 h, HPLC detected that the reaction was complete, concentrated, precipitated with 0.5M hydrochloric acid, collected the solid by filtration, washed with purified water until neutral (pH test paper detection), and weighed;
收率：92％，纯度：80％。Yield: 92%, purity: 80%.
5、液相合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe5. Liquid phase synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe
准确称取Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe(88.3mmol)于反应瓶中，加入二乙胺500ml反应20min，浓缩至少量，加入石油醚析出固 体，过滤，真空干燥。Accurately weigh Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe (88.3mmol) in a reaction flask, add 500ml of diethylamine to react for 20min, concentrate to a certain amount, Petroleum ether was added to precipitate a solid, which was filtered and dried in vacuo.
收率：92.1％，纯度：85.4％Yield: 92.1%, Purity: 85.4%
6、液相合成化合物6：6. Liquid phase synthesis of compound 6:
H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMeH-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe
准确称取H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe(81.3mmol)、HOBt(89.5mmol)、Fmoc-His(Trt)-OH(89.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DCC(89.5mmol)开始反应。Accurately weigh H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe(81.3mmol), HOBt(89.5mmol), Fmoc-His(Trt)-OH(89.5 mmol) in a reaction flask, was completely dissolved with DMF, cooled in a bath for 10 min, and then DCC (89.5 mmol) was added to start the reaction.
反应1.5h后HPLC检测反应完全，过滤反应液，倒入反应瓶中，再加入500ml二乙胺反应20min，浓缩至少量，加入0.5M盐酸溶液析出固体，过滤，烘干；得Fmoc-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe，加入二乙胺脱保护反应20min，浓缩至少量，加入石油醚析出固体，过滤，烘干。After reacting for 1.5h, HPLC detected that the reaction was complete, filtered the reaction solution, poured it into a reaction flask, added 500ml of diethylamine to react for 20min, concentrated to a small amount, added 0.5M hydrochloric acid solution to precipitate a solid, filtered, and dried; to obtain Fmoc-His ( Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe, adding diethylamine for deprotection reaction for 20min, concentrating to a small amount, adding petroleum ether to precipitate solid, filtering and drying Dry.
收率：86％，纯度：81.8％。Yield: 86%, purity: 81.8%.
7、液相合成化合物7：7. Liquid phase synthesis of compound 7:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMeBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe
准确称取Boc-Pyr-OH(76.9mmol)、BOP(76.9mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入TEA(76.9mmol)，再将化合物6(69.9mmol)用DMF溶解后加入反应中开始反应。Accurately weigh Boc-Pyr-OH (76.9mmol) and BOP (76.9mmol) in a reaction flask, dissolve them completely in DMF, and then add TEA (76.9mmol) in a cold bath for 10min, then dissolve compound 6 (69.9mmol) in DMF After adding to the reaction to start the reaction.
反应1.5h后HPLC检测反应完全，过滤反应液，滤渣用DMF洗涤两次，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重；After reacting for 1.5 hours, HPLC detected that the reaction was complete, filtered the reaction solution, washed the filter residue twice with DMF, precipitated with 0.5M hydrochloric acid, collected the solid after filtration, washed it with purified water until neutral (detected by pH test paper), dried, and weighed;
收率：80％，纯度：85.1％。Yield: 80%, purity: 85.1%.
8、液相合成化合物8：8. Liquid phase synthesis of compound 8:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OHBoc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OH
取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe(56mmol)，置于1110ml甲醇中搅拌5min完全溶解后，取2MNaOH110ml缓慢加入反应，开始反应。反应3h后HPLC检测反应完全，加入盐酸溶液，沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。Take Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OMe(56mmol), put it in 1110ml of methanol and stir for 5min to dissolve completely, then take 110ml of 2MNaOH was slowly added to the reaction to start the reaction. After reacting for 3 hours, HPLC detected that the reaction was complete, added hydrochloric acid solution, precipitated, collected the solid after filtration, washed it with purified water until neutral (detected by pH test paper), dried, and weighed.
收率：72％，纯度：88.9％。Yield: 72%, purity: 88.9%.
9、液相合成化合物9：H-Arg(pbf)-Pro-Gly-NH
2
9. Liquid phase synthesis of compound 9: H-Arg(pbf)-Pro-Gly-NH 2
准确称取Fmoc-Arg(pbf)-OH(157.5mmol)、BOP(157.5mmol)于反应瓶中，用DMF完全溶解后冷浴10min，加入DIEA(157.5mmol)，撤掉冷浴，反应20min。Accurately weigh Fmoc-Arg(pbf)-OH (157.5mmol) and BOP (157.5mmol) in a reaction flask, dissolve them completely in DMF, then cool them for 10 minutes, add DIEA (157.5mmol), remove the cooling bath, and react for 20 minutes.
称取H-Pro-Gly-NH
2(150mmol)于三角瓶中，用DMF完全溶解后加入TEA(157.5mmol)，快速混匀后加入上反应中开始反应。反应2h后，用0.5M盐酸沉淀，过滤后收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，得Fmoc-Arg(pbf)-Pro-Gly-NH
2，加入二乙胺反应20min，浓缩至少量，加入石油醚析出固体，过滤，真空干燥，即得化合物9。
Weigh H-Pro-Gly-NH 2 (150mmol) in a Erlenmeyer flask, dissolve it completely with DMF, add TEA (157.5mmol), mix quickly and add to the above reaction to start the reaction. After reacting for 2 hours, precipitate with 0.5M hydrochloric acid, collect the solid after filtration, then wash with purified water until neutral (detected by pH test paper), and dry to obtain Fmoc-Arg(pbf)-Pro-Gly-NH 2 , which is reacted by adding diethylamine After 20 minutes, concentrate to a certain amount, add petroleum ether to precipitate a solid, filter, and dry in vacuo to obtain compound 9.
收率：91％，纯度：95.6％。Yield: 91%, purity: 95.6%.
10、液相合成化合物10：10. Liquid phase synthesis of compound 10:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-Arg(pbf)-Pro-Gly-NH
2
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-Arg(pbf)-Pro-Gly-NH 2
称取Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OH(40.3mmol)、BOP(44.3mmol)于反应瓶中，用DMF完全溶解后冷浴10min后加入DIEA(44.3mmol)，再将H-Arg(pbf)-Pro-Gly-NH
2(44.3mmol)用DMF溶解后加入反应中开始反应。
Weigh Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-2-Nal-Leu-OH (40.3mmol), BOP (44.3mmol) in the reaction flask, After completely dissolving in DMF, DIEA (44.3 mmol) was added in a cold bath for 10 min, and then H-Arg(pbf)-Pro-Gly-NH 2 (44.3 mmol) was dissolved in DMF and added to the reaction to start the reaction.
反应1.0h后HPLC检测反应完全，加入0.5M盐酸析出固体，过滤收集固体，然后用纯化水洗至中性(pH试纸检测)，干燥，称重。After 1.0 h of reaction, HPLC detected that the reaction was complete, and 0.5M hydrochloric acid was added to precipitate a solid, which was collected by filtration, washed with purified water until neutral (detected by pH test paper), dried, and weighed.
收率95％，纯度：74.9％。Yield 95%, purity: 74.9%.
11、合成那法瑞林粗品11. Synthesis of crude nafarelin
将化合物10(42.1mmol)加入反应瓶中，加入300ml裂解液(TFA∶TIS∶H
2O＝95∶2.5∶2.5)反应30min后用冷冻乙醚沉淀，过滤，收集固体，得到产物粗品，产物用水溶解后HPLC检测分析。结果如图3所示，收率：86％，纯度83.5％。
Add compound 10 (42.1mmol) into the reaction flask, add 300ml of lysate (TFA:TIS:H 2 O = 95:2.5:2.5) for 30min, precipitate with frozen diethyl ether, filter, collect the solid to obtain the crude product, the product is water After dissolution, HPLC detection and analysis. The results are shown in Figure 3, yield: 86%, purity 83.5%.
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Apparently, the above-mentioned embodiments are only examples for clear description, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. And the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.
Claims (10)
- 一种全液相合成瑞林类药物的方法，其特征在于，包括如下步骤：A method for synthesizing ralin drugs in full liquid phase is characterized in that it comprises the steps of:S1、液相合成化合物1：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH；S1. Compound 1 synthesized in liquid phase: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH;S2、液相合成化合物2：R 1-R 5-Leu-OR 2； S2, liquid phase synthesis of compound 2: R 1 -R 5 -Leu-OR 2 ;S3、液相合成化合物3：H-R 5-Leu-OR 2； S3, liquid phase synthesis of compound 3: HR 5 -Leu-OR 2 ;S4、液相合成化合物4：Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； S4, liquid phase synthesis of compound 4: Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;S5、液相合成化合物5：H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； S5, liquid phase synthesis of compound 5: H-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;S6、液相合成化合物6：H-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； S6, liquid phase synthesis of compound 6: H-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;S7、液相合成化合物7：S7, liquid phase synthesis of compound 7:R 4-Pyr-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OR 2； R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OR 2 ;S8、液相合成化合物8：S8, liquid phase synthesis of compound 8:R 4-Pyr-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-OH； R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-OH;S9、液相合成化合物9：H-Arg(pbf)-Pro-Gly-NH 2； S9, liquid phase synthesis of compound 9: H-Arg(pbf)-Pro-Gly-NH 2 ;S10、液相合成化合物10：S10, liquid phase synthesis of compound 10:R 4-Pyr-His(R 3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5-Leu-Arg(pbf)-Pro-Gly-NH 2； R 4 -Pyr-His(R 3 )-Trp(Boc)-Ser(tBu)-Tyr(tBu)-R 5 -Leu-Arg(pbf)-Pro-Gly-NH 2 ;S11、瑞林类药物粗品的制备；S11, the preparation of the crude product of ralin drugs;其中，R 1为氨基保护基团，包括Fmoc、Z、Boc中的任意一种；R 2为羧基保护基团，包括甲酯Me，乙酯Et，苄酯Bzl，三苯甲酯Tr中的任意一种；R 3包括Boc或Trt中的任意一种；R 4为氨基保护基团，包括Fmoc、Z、Boc中的任意一种；所述R 5为D-Trp、Gly、D-2-Nal中的任意一种。 Wherein, R 1 is an amino protecting group, including any one of Fmoc, Z, Boc; R 2 is a carboxyl protecting group, including methyl ester Me, ethyl ester Et, benzyl ester Bzl, trityl ester Tr Any one; R 3 includes any one of Boc or Trt; R 4 is an amino protecting group, including any one of Fmoc, Z, Boc; the R 5 is D-Trp, Gly, D-2 Any of -Nal.
- 根据权利要求1所述的一种全液相合成瑞林类药物的方法，其特征在于，步骤S1具体包括以下步骤：A method for synthesizing ralin drugs in full liquid phase according to claim 1, wherein step S1 specifically comprises the following steps:以Fmoc-Trp(Boc)-Ser(tBu)-OSu和H-Tyr(tBu)-OH为反应单元进行缩合反应，在溶剂中反应得到化合物1；所述Fmoc-Trp(Boc)-Ser(tBu)-OSu和所述H-Tyr(tBu)-OH的摩尔比为1∶1.05-2，所述H-Tyr(tBu)-OH与所述有机碱的摩尔比为1∶1，所述溶剂包括DMF、THF、甲醇、乙醇、NMP中的任意一种。Take Fmoc-Trp(Boc)-Ser(tBu)-OSu and H-Tyr(tBu)-OH as reaction units to carry out condensation reaction, react in a solvent to obtain compound 1; said Fmoc-Trp(Boc)-Ser(tBu )-OSu and the H-Tyr(tBu)-OH molar ratio is 1:1.05-2, the H-Tyr(tBu)-OH and the organic base molar ratio is 1:1, the solvent Including any one of DMF, THF, methanol, ethanol, and NMP.
- 根据权利要求1所述的一种全液相合成瑞林类药物的方法，其特征在于，步骤S2具体包括以下步骤：A method for full liquid phase synthesis of ralin drugs according to claim 1, wherein step S2 specifically comprises the following steps:以R 1-R 5-OH、H-Leu-OR 2为反应单元进行缩合反应，R 1-R 5-OH与H-Leu-OR 2的 摩尔比为1∶1.05-2，加入活化剂、有机碱、缩合剂，H-Leu-OR 2与活化剂、缩合剂、有机碱的比为1∶1∶1∶1，反应完全后，过滤、析出、洗涤、干燥，收集固体得化合物2； Condensation reaction is carried out with R 1 -R 5 -OH, H-Leu-OR 2 as the reaction unit, the molar ratio of R 1 -R 5 -OH to H-Leu-OR 2 is 1:1.05-2, adding activator, Organic base, condensing agent, the ratio of H-Leu-OR 2 to activator, condensing agent, and organic base is 1:1:1:1, after the reaction is complete, filter, precipitate, wash, dry, and collect the solid to obtain compound 2;所述活化剂为多肽合成常用的活化剂，包括HOSu、HOBt、HOAt、HOOBt中的任意一种；所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt; the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU; the organic base includes any one of DIEA, TEA, NMM; the solvent includes any one of THF, DCM, DMF, NMP, dioxane.
- 根据权利要求1所述的一种全液相合成瑞林类药物的方法，其特征在于，步骤S3具体包括以下步骤：A method for synthesizing ralin drugs in full liquid phase according to claim 1, wherein step S3 specifically comprises the following steps:以步骤S2制得的化合物2为底物，加入脱保护试剂和溶剂，浓缩至少量，析出，过滤，真空干燥得化合物3；Using compound 2 prepared in step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, separating out, filtering, and drying in vacuo to obtain compound 3;所述脱保护试剂包括三氟乙酸、二乙胺、哌嗪、哌啶中的任意一种；所述溶剂为DMF、甲醇、乙醇、DCM、THF中的任意一种。The deprotection reagent includes any one of trifluoroacetic acid, diethylamine, piperazine, and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM, and THF.
- 根据权利要求1所述的一种全液相合成瑞林类药物的方法，其特征在于，步骤S4具体包括以下步骤：A method for synthesizing ralin drugs in full liquid phase according to claim 1, wherein step S4 specifically comprises the following steps:以步骤S1合成的化合物1和步骤S3合成的化合物3为反应单元进行缩合反应，其中化合物1和化合物3的摩尔比为1∶1.05-2，加入有机碱、缩合剂，其中化合物3与有机碱、缩合剂的摩尔比为1∶1∶1，在溶剂中反应完全后，浓缩、过滤、洗涤、干燥，得化合物4；Condensation reaction is carried out with compound 1 synthesized in step S1 and compound 3 synthesized in step S3 as reaction units, wherein the molar ratio of compound 1 and compound 3 is 1: 1.05-2, adding organic base and condensing agent, wherein compound 3 and organic base 1. The molar ratio of the condensing agent is 1:1:1. After the reaction in the solvent is complete, concentrate, filter, wash, and dry to obtain compound 4;所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
- 根据权利要求1所述的一种全液相合成瑞林类药物的方法，其特征在于，步骤S5中具体包括以下步骤：A method for synthesizing ralin drugs in full liquid phase according to claim 1, wherein step S5 specifically comprises the following steps:取化合物4，加入脱保护试剂反应脱去Fmoc基团，浓缩至少量，析出固体，过滤，真空干燥得化合物5；所述脱保护试剂包括二乙胺、哌嗪、哌啶溶液的任意一种。Take compound 4, add a deprotection reagent to react to remove the Fmoc group, concentrate to a certain amount, precipitate a solid, filter, and vacuum-dry to obtain compound 5; the deprotection reagent includes any one of diethylamine, piperazine, and piperidine solutions .
- 根据权利要求1所述的一种全液相合成瑞林类药物的方法，其特征在于，步骤S6具体包括以下步骤：A method for synthesizing ralin drugs in full liquid phase according to claim 1, wherein step S6 specifically comprises the following steps:以Fmoc-His(R 3)-OH、步骤S5中合成的化合物5为反应单元进行缩合反应，其中化合物5与Fmoc-His(R 3)-OH的摩尔比为1∶1.05-2，加入活化剂、有机碱、缩合剂，其中Fmoc-His(R 3)-OH与活化剂、缩合剂、有机碱的比为1∶1∶1∶1，在溶剂中反应完全，浓缩、过滤、洗涤、干燥，脱保护得化合物6； Condensation reaction is carried out with Fmoc-His(R 3 )-OH and compound 5 synthesized in step S5 as the reaction unit, wherein the molar ratio of compound 5 to Fmoc-His(R 3 )-OH is 1:1.05-2, adding activating agent, organic base, condensing agent, wherein the ratio of Fmoc-His(R 3 )-OH to activator, condensing agent, and organic base is 1:1:1:1, the reaction is complete in the solvent, concentrated, filtered, washed, Drying, deprotection to obtain compound 6;所述活化剂为多肽合成常用的活化剂，包括HOSu、HOBt、HOAt、HOOBt中的任意一种；所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The activator is a commonly used activator for polypeptide synthesis, including any one of HOSu, HOBt, HOAt, HOOBt; the condensing agent is a commonly used condensing agent for polypeptide synthesis, including DCC, DIC, EDC, BOP, pyBOP, AOP , TBTU, HBTU, HATU; the organic base includes any one of DIEA, TEA, NMM; the solvent includes any one of THF, DCM, DMF, NMP, dioxane.
- 根据权利要求1所述的一种全液相合成瑞林类药物的方法，其特征在于，步骤S7具体包括以下步骤：A method for synthesizing ralin drugs in full liquid phase according to claim 1, wherein step S7 specifically comprises the following steps:以R 4-Pyr-OH和步骤S6合成的化合物6进行缩合反应，其中化合物6与R 4-Pyr-OH的摩尔比为1∶1.05-2；加入有机碱、缩合剂，其中R4-Pyr-OH与缩合剂、有机碱的摩尔比为1∶1∶1，反应完全后，过滤、洗涤、干燥，得化合物7； Perform condensation reaction with R 4 -Pyr-OH and compound 6 synthesized in step S6, wherein the molar ratio of compound 6 to R 4 -Pyr-OH is 1:1.05-2; add organic base and condensing agent, wherein R4-Pyr- The molar ratio of OH to condensing agent and organic base is 1:1:1. After the reaction is complete, filter, wash, and dry to obtain compound 7;所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
- 根据权利要求1所述的一种全液相合成瑞林类药物的方法，其特征在于，步骤S8具体包括以下步骤：A method for synthesizing ralin drugs in full liquid phase according to claim 1, wherein step S8 specifically comprises the following steps:取甲醇和化合物7反应，缓慢加入2MNaOH，反应2-4h，过滤、洗涤、干燥得化合物8；Take methanol to react with compound 7, slowly add 2M NaOH, react for 2-4h, filter, wash and dry to obtain compound 8;其中，NaOH和化合物7的摩尔比为1.5∶1-20∶1；Wherein, the molar ratio of NaOH and compound 7 is 1.5:1-20:1;步骤S9具体包括以下步骤：Step S9 specifically includes the following steps:以R 1-Arg(pbf)-OH、H-Pro-Gly-NH 2为反应单元进行缩合反应，其中R 1-Arg(pbf)-OH和H-Pro-Gly-NH 2的摩尔比为1∶1.05-2，加入有机碱、缩合剂，其中R 1-Arg(pbf)-OH与有机碱、缩合剂的摩尔比为1∶1∶1，在溶剂中反应完全后，析出固体，过滤，干燥，脱保护，浓缩，析出固体，过滤，真空干燥得化合物9； Condensation reaction with R 1 -Arg(pbf)-OH, H-Pro-Gly-NH 2 as reaction units, wherein the molar ratio of R 1 -Arg(pbf)-OH and H-Pro-Gly-NH 2 is 1 : 1.05-2, add organic base and condensing agent, wherein the molar ratio of R 1 -Arg(pbf)-OH to organic base and condensing agent is 1:1:1, after complete reaction in solvent, precipitate solid, filter, Drying, deprotection, concentration, precipitation of solid, filtration, and vacuum drying to obtain compound 9;所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、 AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种。The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, HATU; the organic base includes any one of DIEA, TEA, NMM species; the solvent includes any one of THF, DCM, DMF, NMP, and dioxane.
- 根据权利要求1所述的一种全液相合成瑞林类药物的方法，其特征在于，步骤S10具体包括以下步骤：A method for synthesizing ralin drugs in full liquid phase according to claim 1, wherein step S10 specifically comprises the following steps:以化合物8和化合物9为反应单元进行缩合反应，其中化合物8和化合物9的摩尔比为1∶1.05-2，加入缩合剂、有机碱，其中化合物9和缩合剂、有机碱的摩尔比为1∶1∶1，反应完全后，过滤、洗涤、干燥得化合物10；Carry out condensation reaction with compound 8 and compound 9 as the reaction unit, wherein the molar ratio of compound 8 and compound 9 is 1: 1.05-2, add condensing agent, organic base, wherein the molar ratio of compound 9 and condensing agent, organic base is 1 : 1:1, after the reaction is complete, filter, wash, and dry to obtain compound 10;所述缩合剂为多肽合成常用的缩合剂，包括DCC、DIC、EDC、BOP、pyBOP、AOP、TBTU、HBTU、HATU中的任意一种；所述有机碱包括DIEA、TEA、NMM中的任意一种；所述溶剂包括THF、DCM、DMF、NMP、二氧六环中的任意一种；The condensing agent is a commonly used condensing agent for polypeptide synthesis, including any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU; the organic base includes any one of DIEA, TEA, and NMM A kind; Described solvent comprises any one in THF, DCM, DMF, NMP, dioxane;步骤S11具体包括以下步骤：Step S11 specifically includes the following steps:取化合物10于反应器中，加入裂解液反应完成后，用冷冻乙醚沉淀，过滤，收集固体，得到瑞林类药物粗品；Take compound 10 in the reactor, add the lysate, after the reaction is completed, precipitate with frozen ether, filter, collect the solid, and obtain the crude product of ralin;所述裂解液的组分，按体积比计，包括：TFA∶TIS∶H 2O＝95∶2.5∶2.5。 The components of the lysate, by volume ratio, include: TFA:TIS:H 2 O=95:2.5:2.5.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111006172.6A CN113603750A (en) | 2021-08-30 | 2021-08-30 | Method for synthesizing nafarelin in full liquid phase |
| CN202111006172.6 | 2021-08-30 | ||
| CN202111007820.XA CN113603752A (en) | 2021-08-30 | 2021-08-30 | Method for synthesizing gonadorelin in full liquid phase |
| CN202111007822.9 | 2021-08-30 | ||
| CN202111007822.9A CN113698458A (en) | 2021-08-30 | 2021-08-30 | Method for synthesizing triptorelin in full liquid phase |
| CN202111007820.X | 2021-08-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023030278A1 true WO2023030278A1 (en) | 2023-03-09 |
Family
ID=85411927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/115637 WO2023030278A1 (en) | 2021-08-30 | 2022-08-29 | Full-liquid-phase synthesis method for reelin drug |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2023030278A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113603752A (en) * | 2021-08-30 | 2021-11-05 | 湖南三太药业有限公司 | Method for synthesizing gonadorelin in full liquid phase |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5977302A (en) * | 1997-11-20 | 1999-11-02 | Ortho-Mcneil Pharmaceutical, Inc. | Liquid phase process for the preparation of GnRH peptides |
| US20090005535A1 (en) * | 2005-11-25 | 2009-01-01 | Nanokem S.A. | Solution-Phase Synthesis of Leuprolide and Its Intermediates |
| CN107540727A (en) * | 2016-06-28 | 2018-01-05 | 深圳翰宇药业股份有限公司 | The preparation method of Buserelin or Goserelin |
| CN107602668A (en) * | 2017-10-10 | 2018-01-19 | 合肥师范学院 | A kind of fully synthetic method for preparing Buserelin of liquid phase |
| CN109879935A (en) * | 2019-03-04 | 2019-06-14 | 南京工业大学 | Liquid phase synthesis method of polypeptide |
| CN111233980A (en) * | 2020-03-13 | 2020-06-05 | 南京礼威生物医药有限公司 | Method for synthesizing goserelin by fragment method |
| CN112175046A (en) * | 2020-10-12 | 2021-01-05 | 湖南津安生物科技有限公司 | Method for synthesizing triptorelin by polypeptide solid-liquid combination |
| CN113527438A (en) * | 2021-08-30 | 2021-10-22 | 湖南三太药业有限公司 | Method for synthesizing alarelin in full liquid phase |
| CN113603750A (en) * | 2021-08-30 | 2021-11-05 | 湖南三太药业有限公司 | Method for synthesizing nafarelin in full liquid phase |
| CN113603752A (en) * | 2021-08-30 | 2021-11-05 | 湖南三太药业有限公司 | Method for synthesizing gonadorelin in full liquid phase |
| CN113698458A (en) * | 2021-08-30 | 2021-11-26 | 湖南三太药业有限公司 | Method for synthesizing triptorelin in full liquid phase |
-
2022
- 2022-08-29 WO PCT/CN2022/115637 patent/WO2023030278A1/en unknown
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5977302A (en) * | 1997-11-20 | 1999-11-02 | Ortho-Mcneil Pharmaceutical, Inc. | Liquid phase process for the preparation of GnRH peptides |
| US20090005535A1 (en) * | 2005-11-25 | 2009-01-01 | Nanokem S.A. | Solution-Phase Synthesis of Leuprolide and Its Intermediates |
| CN107540727A (en) * | 2016-06-28 | 2018-01-05 | 深圳翰宇药业股份有限公司 | The preparation method of Buserelin or Goserelin |
| CN107602668A (en) * | 2017-10-10 | 2018-01-19 | 合肥师范学院 | A kind of fully synthetic method for preparing Buserelin of liquid phase |
| CN109879935A (en) * | 2019-03-04 | 2019-06-14 | 南京工业大学 | Liquid phase synthesis method of polypeptide |
| CN111233980A (en) * | 2020-03-13 | 2020-06-05 | 南京礼威生物医药有限公司 | Method for synthesizing goserelin by fragment method |
| CN112175046A (en) * | 2020-10-12 | 2021-01-05 | 湖南津安生物科技有限公司 | Method for synthesizing triptorelin by polypeptide solid-liquid combination |
| CN113527438A (en) * | 2021-08-30 | 2021-10-22 | 湖南三太药业有限公司 | Method for synthesizing alarelin in full liquid phase |
| CN113603750A (en) * | 2021-08-30 | 2021-11-05 | 湖南三太药业有限公司 | Method for synthesizing nafarelin in full liquid phase |
| CN113603752A (en) * | 2021-08-30 | 2021-11-05 | 湖南三太药业有限公司 | Method for synthesizing gonadorelin in full liquid phase |
| CN113698458A (en) * | 2021-08-30 | 2021-11-26 | 湖南三太药业有限公司 | Method for synthesizing triptorelin in full liquid phase |
Non-Patent Citations (1)
| Title |
|---|
| SONG, YUN; WEI, SHAO-CHUAN; WANG, YONG-FENG; JIAO, SHU-QING: "Progress in the Synthesis of Anti-cancer Oligopeptide Drugs-Relins", CHINESE JOURNAL OF SYNTHETIC CHEMISTRY, CHENGDU YUJI HUAXUESUO, CN, vol. 18, no. 2, 31 December 2010 (2010-12-31), CN , pages 141 - 147, 167, XP009544249, ISSN: 1005-1511, DOI: 10.15952/j.cnki.cjsc.2010.02.035 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113603752A (en) * | 2021-08-30 | 2021-11-05 | 湖南三太药业有限公司 | Method for synthesizing gonadorelin in full liquid phase |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20190177392A1 (en) | Synthesis of glp-1 peptides | |
| FI60859B (en) | FOERFARANDE FOER FRAMSTAELLNING AV OVULATIONSFRAMKALLANDE DEKAPEPTIDAMIDDERIVAT | |
| EP1773870A2 (en) | Methods for the production of peptide having a c-terminal amide | |
| NO143218B (en) | ANALOGY PROCEDURE FOR THE PREPARATION OF NONAPEPTIDAMIDES WITH LH-RH ACTIVITY | |
| NO321786B1 (en) | LH-RH peptide analogs, peptide agonist or antagonist of LH-RH and their applications and pharmaceutical compositions containing them. | |
| WO2019113872A1 (en) | Method for synthesizing linaclotide | |
| JPS5962556A (en) | Hormone antagonistic peptide, manufacture and drug | |
| WO2023030278A1 (en) | Full-liquid-phase synthesis method for reelin drug | |
| US8377891B2 (en) | Process for synthesis of cyclic octapeptide | |
| CN104610433A (en) | Preparation method of cetrorelix | |
| US20100184952A1 (en) | Method for selective removal of dibenzofulvene derivative | |
| CN104371008B (en) | The method that fragment condensation prepares terlipressin | |
| ZA200107753B (en) | Novel LHRH antagonists with improved solubility characteristics. | |
| CN109575109A (en) | The method that fragment condensation prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 | |
| CN112279894A (en) | Method for synthesizing alarelin by polypeptide solid-liquid combination | |
| CN113698458A (en) | Method for synthesizing triptorelin in full liquid phase | |
| CN113603752A (en) | Method for synthesizing gonadorelin in full liquid phase | |
| CN113603750A (en) | Method for synthesizing nafarelin in full liquid phase | |
| WO2020057088A1 (en) | Method for synthesizing goserelin | |
| WO2023030277A1 (en) | Method for fully liquid-phase synthesis of grnh nonapeptide amide analog | |
| CN113527438A (en) | Method for synthesizing alarelin in full liquid phase | |
| AU2001260110B2 (en) | Novel LHRH-antagonists, production and use thereof as medicament | |
| Arabanian et al. | Synthesis of novel Gn-RH analogues using Ugi-4MCR | |
| CN103554229B (en) | The method of solid phase synthesis buserelin | |
| CN106589072B (en) | Synthesis method of goserelin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22863413 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |