CN104098650B - 阿托西班的中间体的合成与应用 - Google Patents
阿托西班的中间体的合成与应用 Download PDFInfo
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- CN104098650B CN104098650B CN201310129543.9A CN201310129543A CN104098650B CN 104098650 B CN104098650 B CN 104098650B CN 201310129543 A CN201310129543 A CN 201310129543A CN 104098650 B CN104098650 B CN 104098650B
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- Prior art keywords
- benzyl
- proline
- asparagine
- acid
- cysteinyl
- Prior art date
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- VWXRQYYUEIYXCZ-OBIMUBPZSA-N atosiban Chemical compound C1=CC(OCC)=CC=C1C[C@@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CCCN)C(=O)NCC(N)=O)CSSCCC(=O)N1 VWXRQYYUEIYXCZ-OBIMUBPZSA-N 0.000 title claims abstract description 25
- 229960002403 atosiban Drugs 0.000 title claims abstract description 25
- 108700007535 atosiban Proteins 0.000 title claims abstract description 25
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 13
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 13
- 239000000543 intermediate Substances 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000007791 liquid phase Substances 0.000 claims abstract description 13
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims abstract description 7
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229960001230 asparagine Drugs 0.000 claims abstract description 5
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims abstract description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims abstract description 4
- GHBAYRBVXCRIHT-VIFPVBQESA-N S-benzyl-L-cysteine zwitterion Chemical compound OC(=O)[C@@H](N)CSCC1=CC=CC=C1 GHBAYRBVXCRIHT-VIFPVBQESA-N 0.000 claims abstract description 4
- 235000009582 asparagine Nutrition 0.000 claims abstract description 4
- 229960000310 isoleucine Drugs 0.000 claims abstract description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims abstract description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 135
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 65
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 63
- 239000007787 solid Substances 0.000 claims description 38
- 108010060199 cysteinylproline Proteins 0.000 claims description 37
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- -1 acyl ammonia Chemical compound 0.000 claims description 33
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 33
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 28
- 238000003756 stirring Methods 0.000 claims description 25
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 24
- 229940093499 ethyl acetate Drugs 0.000 claims description 21
- 235000019439 ethyl acetate Nutrition 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000000706 filtrate Substances 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 17
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 16
- 230000006837 decompression Effects 0.000 claims description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 15
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 15
- 239000002253 acid Substances 0.000 claims description 14
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- 125000006239 protecting group Chemical group 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- 239000007864 aqueous solution Substances 0.000 claims description 13
- 229910021529 ammonia Inorganic materials 0.000 claims description 12
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 12
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 12
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 11
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 claims description 11
- 229960002317 succinimide Drugs 0.000 claims description 10
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 claims description 9
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 9
- 238000001704 evaporation Methods 0.000 claims description 9
- 238000013019 agitation Methods 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 6
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 claims description 6
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 5
- 229940024606 amino acid Drugs 0.000 claims description 5
- 235000001014 amino acid Nutrition 0.000 claims description 5
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 5
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 3
- ONOURAAVVKGJNM-SCZZXKLOSA-N (2s,3r)-2-azaniumyl-3-phenylmethoxybutanoate Chemical compound [O-]C(=O)[C@@H]([NH3+])[C@@H](C)OCC1=CC=CC=C1 ONOURAAVVKGJNM-SCZZXKLOSA-N 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 2
- 239000005864 Sulphur Substances 0.000 claims description 2
- 230000000903 blocking effect Effects 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- XNROFTAJEGCDCT-UHFFFAOYSA-N 1-benzylpyrrolidine-2-carboxylic acid Chemical compound OC(=O)C1CCCN1CC1=CC=CC=C1 XNROFTAJEGCDCT-UHFFFAOYSA-N 0.000 claims 1
- 244000248349 Citrus limon Species 0.000 claims 1
- 235000005979 Citrus limon Nutrition 0.000 claims 1
- 238000010189 synthetic method Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 9
- 238000006243 chemical reaction Methods 0.000 abstract description 6
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- 238000009776 industrial production Methods 0.000 abstract description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 abstract 2
- 239000004473 Threonine Substances 0.000 abstract 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 abstract 1
- 238000010647 peptide synthesis reaction Methods 0.000 abstract 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 abstract 1
- 238000001308 synthesis method Methods 0.000 abstract 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 19
- 150000001875 compounds Chemical class 0.000 description 18
- 238000005160 1H NMR spectroscopy Methods 0.000 description 16
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 14
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 14
- 239000003921 oil Substances 0.000 description 13
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
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- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 5
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 125000002252 acyl group Chemical group 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- GSYSFVSGPABNNL-UHFFFAOYSA-N methyl 2-dimethoxyphosphoryl-2-(phenylmethoxycarbonylamino)acetate Chemical group COC(=O)C(P(=O)(OC)OC)NC(=O)OCC1=CC=CC=C1 GSYSFVSGPABNNL-UHFFFAOYSA-N 0.000 description 4
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- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 3
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 3
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
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- TURCVFSKRYADID-GOSISDBHSA-N methyl (2R)-2-(3-benzylsulfanylpropanoylamino)-3-(4-hydroxyphenyl)propanoate Chemical class COC([C@H](NC(CCSCC1=CC=CC=C1)=O)CC1=CC=C(C=C1)O)=O TURCVFSKRYADID-GOSISDBHSA-N 0.000 description 2
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- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 1
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- 239000012964 benzotriazole Substances 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
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- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
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- 238000005336 cracking Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 210000003785 decidua Anatomy 0.000 description 1
- 235000015177 dried meat Nutrition 0.000 description 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 1
- VXYFARNRGZWHTJ-SBSPUUFOSA-N hydron;methyl (2r)-2-amino-3-(4-hydroxyphenyl)propanoate;chloride Chemical compound Cl.COC(=O)[C@H](N)CC1=CC=C(O)C=C1 VXYFARNRGZWHTJ-SBSPUUFOSA-N 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- HVTICUPFWKNHNG-UHFFFAOYSA-N iodoethane Chemical compound CCI HVTICUPFWKNHNG-UHFFFAOYSA-N 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 1
- 229960001723 oxytocin Drugs 0.000 description 1
- 235000015320 potassium carbonate Nutrition 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- IVRIRQXJSNCSPQ-UHFFFAOYSA-N propan-2-yl carbonochloridate Chemical compound CC(C)OC(Cl)=O IVRIRQXJSNCSPQ-UHFFFAOYSA-N 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 150000004756 silanes Chemical class 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229960001866 silicon dioxide Drugs 0.000 description 1
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种阿托西班关键中间体的液相合成方法,即H‑Ile‑Thr(Bzl)‑Asn‑Cys(Bzl)‑Pro‑OH的制备方法。采用此液相接肽合成方法,反应易监测,较固相合成法成本低,更适合大规模工业化生产。主要合成步骤:采用BSA保护脯氨酸,通过N‑羟基琥珀酰亚胺酯法在脯氨酸N端逐个连接S‑苄基半胱氨酸、天冬酰胺、苄基保护的苏氨酸和异亮氨酸,得到五肽中间体。
Description
技术领域
本发明涉及一种合成阿托西班五肽中间体的液相合成方法,以及该关键中间体在制备阿托西班中的应用。
背景技术
阿托西班是由辉凌公司(Ferring AB)研制,其结构如下(Ⅱ),是子宫内及蜕膜、胎膜上受体的环状催产素抑制剂,于2000年3月首次在奥地利上市。使用后可剂量相关性地抑制宫缩,并使环状肽催产素介导的前列腺素分泌减少,达到保胎目的。
目前阿托西班的合成多采用固相合成方法,例如使用Rink Amide树脂为载体,脱Fmoc-保护后得到H2N-Rink Amide树脂,以HOBt、DIPCI等为缩合剂,将Fmoc-Gly-OH的羧基与树脂氨基相连,得到Fmoc-Gly。再采用Fmoc策略依次固相合成序列剩余氨基酸。用裂解试剂切割得到阿托西班。相似固相合成方法见专利EP0710243、CN101357937A、CN101696236A。固相合成方法存在这样几个问题:制备量少、设备复杂、需要使用大量的树脂和洗涤溶剂等。
本发明使用液相合成法制备阿托西班的关键中间体:H-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-OH(结构I),可用于阿托西班的制备。进而解决了阿托西班通过液相合成法生产的技术问题。其中使用BSA作为脯氨酸羧基部分的保护基,使得氨基酸在依次接肽过程中,产率高,易处理,利于大规模工业生产。
发明内容
本发明的目的在于提供阿托西班五肽中间体的一种液相合成方法,进而实现阿托西班的液相合成,解决目前采用固相合成法成本高,反应不易监测,不适宜大规模化生产等问题。
本发明的技术解决方案是:
1、阿托西班五肽中间体的液相合成方法:
a.以脯氨酸为原料,使用BSA保护其羧基,再与N端保护的S-苄基半胱氨酸的活性酯连接,制得N-叔丁氧羰酰基-S-苄基半胱氨酰-脯氨酸,再于酸性条件下脱去N端保护基得到S-苄基半胱酰氨-脯氨酸;
b.以S-苄基半胱酰氨-脯氨酸为原料,使用BSA保护脯氨酰的羧基部分,再与N端保护的天冬酰胺的活性酯连接,制得N-叔丁氧羰酰基-天冬酰胺-S-苄基半胱氨酰-脯氨酸,再于酸性条件下脱去N端保护基得到天冬酰胺-S-苄基半胱氨酰-脯氨酸;
c.以天冬酰胺-S-苄基半胱氨酰-脯氨酸为原料,使用BSA保护脯氨酰的羧基部分,再与N端保护的O-苄基苏氨酸的活性酯连接,制得N-叔丁氧羰酰基-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸,再于酸性条件下脱去N端保护基得到O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸;
d.以O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸为原料,使用BSA保护脯氨酰的羧基部分,再与N端保护的异亮氨酸的活性酯连接,制得N-叔丁氧羰酰基-异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸,再于酸性条件下脱去N端保护基得到异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸,即H-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-OH。
合成路线如下:
其中R1=Boc、Cbz、Trt、Mz
R2=OSu、ONb、OBt、OOBt、OAt。
步骤a、c、d中所述的N端保护基团优选的选自叔丁氧羰基(BOC)、苄氧羰基(Cbz)、三苯甲基(Trt)、对甲氧苄氧羰基(Mz)。
用酸脱除N端保护基时优选的酸选自包括三氟乙酸、盐酸、氢氟酸;所用溶剂包括:二氯甲烷、四氢呋喃、1,4-二氧六环。
步骤a、b、c、d中所述的氨基酸的相应活性酯优选的选自N-羟基琥珀酰亚胺酯、N-羟基-5-降冰片烯-2,3-二甲酰亚胺酯、1-羟基苯骈三氮唑酯、3-羟基-4-氧-3,4-二氢-1,2,3-苯骈三氮唑酯、1-羟基-7-偶氮苯并三氮唑酯。
2、本发明优选的制备方法,其步骤如下:
(1)制备S-苄基半胱酰氨-脯氨酸
将BSA和L-脯氨酸加到无水二氯甲烷中,10~35℃下搅拌2~4h,滴入N-叔丁氧羰酰基-S-苄基半胱氨酸-(N-羟基)琥珀酰亚胺酯的二氯甲烷溶液,10~35℃下反应5~8h,减压蒸除溶剂,加乙醚,用5%的碳酸氢钠水溶液萃取;合并水相,加入柠檬酸调至PH5~7,用乙酸乙酯萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得淡黄色油状物,加入无水乙醚析出白色固体;将白色固体加至无水二氯甲烷中,0~15℃下搅拌滴加三氟乙酸至VCH2Cl2:VCF3COOH=1~5:1,搅拌反应2~5h,减压浓缩至干,得到淡黄色油状物,用乙酸乙酯/乙醚重结晶,得到白色固体;
(2)制备天冬酰胺-S-苄基半胱氨酰-脯氨酸
将S-苄基半胱酰氨-脯氨酸和BSA加到无水二氯甲烷中,10~35℃下搅拌3~4h,滴入N-叔丁氧羰酰基-天冬酰胺-(N-羟基)琥珀酰亚胺酯的二氯甲烷溶液,10~35℃下搅拌8~10h,减压蒸除溶剂,加乙醚,用5%的碳酸氢钠水溶液萃取;合并水相,加入柠檬酸调至PH5~7,用乙酸乙酯萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得淡黄色油状物,乙酸乙酯重结晶,得到的固体加至无水二氯甲烷中,0~15℃下搅拌滴加三氟乙酸至VCH2Cl2:VCF3COOH=1~5:1,10~35℃下搅拌反应2~5h,减压浓缩至干,得到淡黄色油状物,用乙酸乙酯/乙醚重结晶,得到白色固体;
(3)制备O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸
将天冬酰胺-S-苄基半胱氨酰-脯氨酸和BSA加到无水二氯甲烷中,10~35℃下搅拌3~4h,滴入N-叔丁氧羰酰基-O-苄基-苏氨酸-(N-羟基)琥珀酰亚胺酯的二氯甲烷溶液,10~35℃下搅拌6~8h,减压蒸除溶剂,加乙醚,用5%的碳酸氢钠水溶液萃取;合并水相,加入柠檬酸调至PH5~8,用乙酸乙酯萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得淡黄色油状物,乙酸乙酯重结晶,得到的固体加至无水二氯甲烷中,0~15℃下搅拌滴加三氟乙酸至VCH2Cl2:VCF3COOH=1~5:1,10~35℃下搅拌反应2~5h,减压浓缩至干,用甲醇/乙醚重结晶,得到白色固体;
(4)制备异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸
将O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸和BSA加到无水二氯甲烷中,10~35℃下搅拌3~4h,滴入N-叔丁氧羰酰基-异亮氨酸-(N-羟基)琥珀酰亚胺酯的二氯甲烷溶液,10~35℃下搅拌6~8h,减压蒸除溶剂,加乙醚,用5%的碳酸氢钠水溶液萃取;合并水相,加入柠檬酸调至PH5~8,用乙酸乙酯萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,得到的白色固体加至无水二氯甲烷中,0~15℃下搅拌滴加三氟乙酸至VCH2Cl2:VCF3COOH=1~5:1,10~35℃下搅拌反应2~5h,减压浓缩至干,用甲醇/乙醚重结晶,得到白色固体。
3、采用BSA作为脯氨酸羧基的保护基,与氨基酸反应迅速且完全,由于是中性硅烷化试剂,故不改变反应体系中的酸碱性;同时此保护基易于脱去,加入水即可分解且副产物溶于水,易与主产物进行分离;另外BSA来源广泛,价格便宜,常用于工业生产。
4、五肽中间体在制备阿托西班中的应用:五肽中间体在BSA的保护下与Mpa(Bzl)-D-Tyr(Et)-N3连接,再与H-Orn(z)-Gly-NH2在缩合剂DCC、添加剂HOBt作用下进行反应,得到的产物经液氨\钠脱去苄基,最后通过I2氧化,形成二硫键得到阿托西班。
有益技术效果:
本发明采用液相合成法,使用BSA作为脯氨酸保护基,从而制得阿托西班五肽中间体,所需设备简单,与固相合成法相比,具有很多优点:(1)不需要树脂和大大过量的保护氨基酸及缩合剂;(2)不需要大量的洗涤溶剂;(3)反应易于监测;(4)选取BSA作为保护基,其来源广,反应选择性好且易于脱除;(5)易于实现规模化生产,大大降低生产成本。
术语和简称:
Boc:叔丁氧羰基
Cbz:苄氧羰基
Trt:三苯甲基
Mz:对甲氧苄氧羰基
BSA:N,O-双(三甲基硅基)乙酰胺
IBCF:氯甲酸异丙酯
NMM:N-甲基吗啉
DCC:N,N’-二环己基碳二亚胺
EDCI:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐
DIAD:偶氮二甲酸二异丙酯
TPP:三苯基膦
DIPCI:N,N′-二异丙基碳二亚胺
Mpa(Bzl)-D-Tyr(Et)-N3:N-[3-(苄巯基)丙酰]-O-乙基-D-酪氨酸酰叠氮
H-Orn(z)-Gly-NH2:N-苄氧羰基-鸟氨酰-甘氨酰胺
H-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-OH:异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸
DMF:N,N-二甲基甲酰胺
HOBt:1-羟基苯并三唑
HOSu:N-羟基琥珀酰亚胺
HONb:N-羟基-5-降冰片烯-2,3-二甲酰亚胺
HOOBt:3-羟基-4-氧-3,4-二氢-1,2,3-苯骈三氮唑
HOAt:1-羟基-7-偶氮苯并三氮唑
DIPEA:N,N’-二异丙基乙胺
TFA:三氟乙酸
THF:四氢呋喃
具体实施方式
下面结合具体的实施例来对本发明进行阐述。
制备例
制备例1 N-叔丁氧羰酰基-S-苄基半胱氨酸-(N-羟基)琥珀酰亚胺酯(a)
N-叔丁氧羰酰基-S-苄基半胱氨酸(20.0g,64.2mmol)和N-羟基琥珀酰亚胺(7.3g,64.2mmol)加至1,4-二氧六环(300ml)内,室温搅拌。控温于5℃左右,缓慢滴入DCC(14.5g,70.6mmol)的二氯甲烷(50ml)溶液,。室温搅拌2.5h,过滤,滤液减压浓缩至干。所得黄色油状物,用异丙醇重结晶,得类白色固体20.2g,收率77.0%。1H NMR(CDCl3)δ:1.49(s,9H);2.81(s,4H);
2.9(m,2H);3.80(s,2H);4.80(m,1H);5.24(d,1H,J=6.7Hz);7.32(m,5H)
制备例2 N-叔丁氧羰酰基-天冬酰胺-(N-羟基)琥珀酰亚胺酯(b)
N-叔丁氧羰酰基-天冬酰胺(10.0g,43.1mmol)和N-羟基琥珀酰亚胺(5.0g,43.1mmol)加至1,4-二氧六环(100ml)内。控温于10℃左右搅拌,缓慢滴入DCC(8.9g,43.16mmol)的二氯甲烷(10ml)溶液,室温搅拌12h,过滤,滤液减压浓缩至干。所得油状物,用异丙醇重结晶,得类白色固体10.4g,收率73.3%。1H NMR(DMSO-d6)δ:1.36(s,9H);2.64(m,2H);2.77(s,4H);4.72(m,1H);7.01(s,1H);7.47(s,1H);7.51(d,1H,J=8.8Hz)
制备例3 N-叔丁氧羰酰基-O-苄基-苏氨酸-(N-羟基)琥珀酰亚胺酯(c)
N-叔丁氧羰酰基-O-苄基-苏氨酸(14.6g,47.3mmol)和N-羟基琥珀酰亚胺(5.4g,47.3mmol)加至1,4-二氧六环(130ml)内。控温于10℃左右搅拌,缓慢滴入DCC(9.8g,47.3mmol)的二氯甲烷(20ml)溶液,。室温搅拌4h,过滤,滤液减压浓缩至干。所得油状物,用乙酸乙酯-石油醚(4:1)重结晶,得类白色固体16.3g,收率84.8%。1H NMR(CDCl3)δ:1.31(d,3H,J=6.4Hz);1.45(s,9H);2.85(s,4H);4.30(q,1H,J=6.8Hz)4.56(dd,2H,J=5.8Hz,12.8Hz);
4.71(d,1H,J=8.7Hz);5.35(d,1H,J=8.6Hz);7.30(m,5H)
制备例4 N-叔丁氧羰酰基-异亮氨酸-(N-羟基)琥珀酰亚胺酯(d)
N-叔丁氧羰酰基-异亮氨酸(9.6g,41.6mol)和N-羟基琥珀酰亚胺(4.8g,41.6mmol)加至1,4-二氧六环(100ml)内,搅拌至全溶。控温于10℃左右,缓慢滴入DCC(8.8g,42.5mmol)的二氯甲烷(10ml)溶液,。室温搅拌6h,过滤,滤液减压浓缩至干。所得油状物,用异丙醇重结晶,得类白色固体8.7g,收率63.7%。1H NMR(CDCl3)δ:0.97(t,3H,J=7.4Hz);1.04(d,3H,J=6.7Hz);1.26~1.33(m,1H);1.57(s,9H);1.59(m,1H);1.92(m,1H);2.90(s,4H);4.63(m,1H);5.01(d,1H,J=8.6Hz)
实施例
实施例1 S-苄基半胱酰氨-脯氨酸三氟乙酸盐(2)
步骤1 N-叔丁氧羰酰基-S-苄基半胱氨酰-脯氨酸(1)
BSA(18.7ml,76.4mmol)和L-脯氨酸(3.7g,31.8mmol)加到无水二氯甲烷(200ml)中,室温搅拌2h,滴入化合物a(13.0g,31.8mmol)的二氯甲烷(30ml)溶液,室温搅拌,反应6h,减压蒸除溶剂,加乙醚(50ml),用5%的碳酸氢钠(50ml×3)水溶液萃取。合并水相,加入5%柠檬酸水溶液调至PH6,用乙酸乙酯(100ml×3)萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得淡黄色油状物,加入无水乙醚(20ml)析出白色固体7.0g,收率54.2%。ESI-MS(m/z):409.3[M+H]+;1H NMR(CDCl3)δ:1.45(s,9H);2.01(m,2H);2.16(m,2H);2.61~2.82(m,2H);3.42(m,1H);3.63(m,1H);3.72(s,2H);4.54(m,2H);5.39(d,1H,J=6Hz);7.32(m,5H)
步骤2 S-苄基半胱酰氨-脯氨酸三氟乙酸盐(2)
化合物1(7.0g,17.1mmol)加至无水二氯甲烷(25ml)中,搅拌下滴加三氟乙酸(12ml),控温于25℃下。减压浓缩至干,加入无水乙醚(20ml),析出类白色固体8.3g,收率92.9%。ESI-MS(m/z):309.3[M+H-CF3COOH]+;1H NMR(DMSO-d6)δ:1.86(m,3H);2.19(m,1H);2.72(m,1H);2.83(m,1H);3.43(m,1H);3.66(m,1H);3.83(s,2H);4.28(m,2H);7.33(m,5H);8.25(br,2H)。
实施例2 天冬酰胺-S-苄基半胱氨酰-脯氨酸三氟乙酸盐(4)
步骤1 N-叔丁氧羰酰基-天冬酰胺-S-苄基半胱氨酰-脯氨酸(3)
化合物2(7.3g,22.4mmol)和BSA(11.9ml,48.8mmol)加到无水二氯甲烷(100ml)中,室温搅拌3h,滴入化合物b(8.6g,20.4mmol)的二氯甲烷(10ml)溶液,室温搅拌6h,减压蒸除溶剂,加乙醚(50ml),用5%的碳酸氢钠(50ml×3)水溶液萃取。合并水相,加入5%柠檬酸水溶液调至PH6,用乙酸乙酯(100ml×3)萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得淡黄色油状物,加入无水乙醚(10ml)析出类白色固体5.6g,收率52.6%。ESI-MS(m/z):523.5[M+H]+;1H NMR(DMSO-d6)δ:1.39(s,9H);1.86(m,3H);2.10(m,1H);2.34~2.47(m,2H);2.53(m,1H);2.71(m,1H);3.38(m,1H);3.52(m,1H);3.75(s,2H);4.02(m,1H);4.18~4.26(m,2H);4.68(m,1H);6.82(m,1H);7.23(m,1H);7.33(m,5H);7.96(d,1H,J=8Hz)
步骤2 天冬酰胺-S-苄基半胱氨酰-脯氨酸三氟乙酸盐(4)
化合物3(5.0g,9.6mmol)加至无水二氯甲烷(20ml)中,搅拌下滴加三氟乙酸(9ml),控温于25℃下。减压浓缩至干,加入无水乙醚(20ml),析出浅黄色固体5.4g,收率88.5%。ESI-MS(m/z):423.3[M+H-CF3COOH]+;1H NMR(DMSO-d6)δ:1.86(m,3H);2.10(m,1H);2.34~2.47(m,2H);2.53(m,1H);2.71(m,1H);3.38(m,1H);3.52(m,1H);3.75(s,2H);4.02(m,1H);4.18~4.26(m,2H);4.68(m,1H);6.82(m,1H);7.23(m,1H);7.33(m,5H);8.13(br,2H)。
实施例3 O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸三氟乙酸盐(6)
步骤1 N-叔丁氧羰酰基-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸(5)
化合物4(4.1g,7.6mmol)和BSA(4.5ml,1.84mmol)加到无水二氯甲烷(40ml)中,室温搅拌3h,滴入化合物c(3.4g,8.4mmol)的二氯甲烷(10ml)溶液,室温搅拌5h,减压蒸除溶剂,加乙醚(40ml),用5%的碳酸氢钠(30ml×3)水溶液萃取。合并水相,加入5%柠檬酸水溶液调至PH6,用乙酸乙酯(60ml×3)萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得淡黄色油状物,加入无水乙醚(20ml)析出浅黄色固体2.3g,收率42.4%。ESI-MS(m/z):714.5[M+H]+;
步骤2 O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸三氟乙酸盐(6)
化合物5(2.0g,2.8mmol)加至无水二氯甲烷(10ml)中,搅拌下滴加三氟乙酸(5ml),控温于25℃下。减压浓缩至干,加入无水乙醚(10ml),析出浅黄色固体2.1g,收率90.9%。ESI-MS(m/z):614.3[M+H-CF3COOH]+。
实施例4 异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸三氟乙酸盐(8)
步骤1 N-叔丁氧羰酰基-异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸(7)
化合物6(2.0g,2.8mmol)和BSA(1.5ml,6.2mmol)加到无水二氯甲烷(20ml)中,室温搅拌3h,滴入化合物物d(0.8g,3.6mmol)的二氯甲烷(5ml)溶液,搅拌至全溶,反应6h,减压蒸除溶剂,加乙醚(20ml)和5%的碳酸氢钠(20ml×3)水溶液,析出白色固体,过滤,得黄褐色固体1.3g,收率56.3%。ESI-MS(m/z):827.4[M+1]+;
步骤2 异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸三氟乙酸盐(8)
化合物7(1.0g,1.2mmol)加至无水二氯甲烷(10ml)中,搅拌下滴加三氟乙酸(5ml),控温于25℃下。减压浓缩至干,加入无水乙醚(10ml),析出黄褐色固体10.9g,收率87.2%。ESI-MS(m/z):727.1[M+H-CF3COOH]+。
应用实例
应用实例1 N-[3-(苄巯基)丙酰]-O-乙基-D-酪氨酸酰肼(vi)
步骤1 3-(苄巯基)丙酸(i)
巯基丙酸(10.0ml,114.6mmol)加至1M NaOH(250ml)和EtOH(200ml)中,降温至0℃,缓慢滴加溴苄(14.3ml,120.5mmol),搅拌10min,自然升温,室温搅拌3h。用1M HCl调节PH=1,析出大量白色固体,二氯甲烷CH2Cl2萃取(200ml×3),弃去水相。用1M NaOH调节有机相至PH=12,析出白色固体,水萃取。再用1M HCl调节水相PH=1,过滤得到大量白色结晶,干燥,得到白色晶体14.3g,收率63.6%。ESI-MS(m/z):197.11[M+H]+;1H NMR(CDCl3)δ:2.59(t,2H,J=6.8Hz);2.67(t,2H,J=6.8Hz);3.74(s,2H);7.29(m,5H)
步骤2 3-(苄巯基)丙酰氯(ii)
化合物i(10.0g,51.0mmol)加至甲苯(300ml)内,滴加SOCl2(8.6ml,118.8mmol),加热至80℃,搅拌2h(TLC检测),减压浓缩,得到黄色油状物10.0g,收率91%。
步骤3 D-酪氨酸甲酯盐酸盐(iii)
D-酪氨酸(10.0g,55.0mmol)加至无水甲醇(200ml)内,通入干燥的HCl气体1h,室温搅拌3h(TLC检测),减压浓缩,得类白色固体11.0g,收率86.6%。ESI-MS(m/z):196.20[M+H]+;1H NMR(DMSO-d6)δ:1.85(br,2H);2.71(m,2H);3.48(m,1H);3.56(s,3H);6.82(dd,4H,J=8.6Hz,7.2Hz);9.17(s,1H)。
步骤4 N-[3-(苄巯基)丙酰]-D-酪氨酸甲酯(iv)
化合物iii(9.0g,38.7mmol)加至CH2Cl2(250ml)中,室温搅拌下,加入吡啶(9.2g,116.1mmol)和TMSCl(6.4ml,50.4mmol)。降温至0℃,滴加化合物ii(9.0g,41.8mmol)室温搅拌4h,减压浓缩,得黄色油状物,加入乙酸乙酯(100ml),用0.5M HCl调节PH=7,弃去水层。有机相用饱和食盐水洗涤,无水硫酸钠干燥,过滤,滤液减压浓缩至干,得黄色油状物,硅胶柱层析纯化(VPE:VEA=2:1)得到白色结晶8.4g,收率58.6%。ESI-MS(m/z):374.28[M+H]+;1H NMR(CDCl3)δ:2.36(t,2H,J=6.8Hz);2.68(t,2H,J=6.8Hz);3.05(m,2H);3.71(s,2H);3.74(s,3H);4.84(dd,1H,J=5.9Hz,13.5Hz);6.12(d,1H,J=7.8Hz);6.83(dd,4H,J=8.6Hz,7.2Hz);7.30(m,5H).
步骤5 N-[3-(苄巯基)丙酰]-O-乙基-D-酪氨酸甲酯(v)
化合物iv(3.0g,8.0mmol)加至无水DMF(30ml)和K2CO3(2.2g,16.0mmol)中,氮气保护下,再加入碘乙烷(1.28ml,16.0mmol),室温搅拌20h,加入乙酸乙酯(100ml)和水(80ml×3)萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得到白色结晶2.6g,收率78.1%。ESI-MS(m/z):402.16[M+H]+;1H NMR(CDCl3)δ:1.34(t,3H,J=8Hz);2.41(t,2H,J=6.8Hz);2.55(t,2H,J=6.8Hz);2.91(m,2H);3.70(s,2H);3.72(s,3H);
3.98(q,2H,J=8Hz);4.82(dd,1H,J=5.9Hz,13.5Hz);6.23(d,1H,J=7.8Hz);6.84(dd,4H,J=J=8.6Hz,7.2Hz);7.32(m,5H).
步骤6 N-[3-(苄巯基)丙酰]-O-乙基-D-酪氨酸酰肼(vi)
化合物v(4.0g,10.0mmol)加至MeOH(24.5ml)内,滴加85%的水合肼(8.23ml),室温搅拌,5min后产生大量白色沉淀,继续搅拌1h,放入冰箱冷藏过夜。过滤,滤饼用水洗涤三次,得到白色固体3.5g,收率87.5%。
ESI-MS(m/z):402.54[M+H]+;1H NMR(DMSO-d6)δ:1.27(t,3H,J=8Hz);
2.33(t,2H,J=6.8Hz);2.43(t,2H,J=6.8Hz);2.65(m,1H);2.81(m,1H);3.66(s,2H);
3.93(q,2H,J=8Hz);4.23(br,1H);4.36(m,1H);6.23(d,1H,J=7.8Hz);6.87(dd,4H,
J=8.6Hz,7.2Hz);7.32(m,5H);8.14(d,1H,J=8.7Hz);9.14(s,1H)
应用实例2 N-苄氧羰基-鸟氨酰-甘氨酰胺盐酸盐(x)
步骤1 N-叔丁氧羰基-N’-苄氧羰基-鸟氨酸(viii)
N-苄氧羰基-鸟氨酸(5.3g,20.0mmol)与碳酸钾(3.0g,22.0mmol)加入溶液H2O(60ml)与1,4-二氧六环(25ml)中,冰浴冷却至0℃后。将Boc2O(4.6g,21.0mmol)溶于1,4-二氧六环(50ml)中,再将其缓慢滴加至上述含cbz的混合溶液中,滴毕后撤去冰浴,室温下搅拌过夜。减压蒸除1,4-二氧六环。再加入H2O(10ml),用1mol/L盐酸调至pH=2~3。用乙酸乙酯萃取(80ml×3),合并有机相,再分别用10mmol/L盐酸(24ml),饱和氯化铵溶液(50ml)和H2O(30ml)洗涤,无水硫酸钠干燥,过滤旋干透明粘状物6.9g,收率94.5%。ESI-MS(m/z):367.25。1H-NMR(300MHz,DMSO)δ:12.37(br,1H),7.32(s,4H),7.21(s,1H),7.03(d,1H,J=7.7Hz),4.98(s,2H),3.81(s,1H),3.31(s,1H),2.95(d,2H,J=5.4Hz),1.63(m,1H),1.44(m,3H),1.35(s,9H)。
步骤2 N-叔丁氧羰基-N’-苄氧羰基-鸟氨酰-甘氨酰胺(ix)
化合物viii(3.9g,10.6mmol)溶于无水THF(40ml)中,再加入NMM(1.1g,10.6mmol),N2保护,降温至-18℃搅拌,30min后升温至-15℃再缓慢滴加IBCF(1.15g,10.6mmol)的无水THF(15ml)溶液,保持此温度搅拌30min,再加入甘氨酰胺盐酸盐(1.2g,10.6mmol)和NMM(1.1g,10.6mmol)后,自然升温至室温,搅拌反应18h。减压蒸除溶剂后加入乙酸乙酯(40ml),再分别用5%NaHCO3溶液(50ml×3),H2O(50ml)和5%柠檬酸溶液(50ml×3)洗涤。用无水MgSO4干燥,过滤,将滤液减压旋干,得到淡黄色固体,用少量乙酸乙酯洗去杂质,抽滤,得到白色固体2.3g,收率52.4%。ESI-MS(m/z):423.33,1H-NMR(300MHz,DMSO)δ:8.04(s,1H),7.34(d,5H,J=5.0Hz),7.20(d,2H,J=16.7Hz),7.10(s,1H),7.02(d,1H,J=7.3Hz),5.00(s,2H),3.85(s,1H),3.62(d,2H,J=6.0Hz),2.97(d,2H,J=5.4Hz),1.60(s,1H),1.42(s,2H),1.38(s,9H)。
步骤3 N-苄氧羰基-鸟氨酰-甘氨酰胺盐酸盐(x)
化合物ix(0.42g,1.0mmol)加入到4mol/L HCl的1,4-二氧六环溶液(12.5ml)中,室温搅拌,约6min后有大量白色固体析出,反应约3.5h(TCL检测),抽滤,烘干得白色固体0.3g,收率84.4%。ESI-MS(m/z):323.16,1H-NMR(300MHz,DMSO)δ:8.79(m,1H),8.32(s,3H),7.48(s,1H),7.36(dd,6H,J=11.9Hz,6.9Hz),7.14(s,1H),5.01(s,2H),3.92–3.54(m,3H),3.00(d,2H,J=6.0Hz),1.70(s,2H),1.48(s,2H)。
应用实例3 阿托西班(atosiban)
步骤1 N-[3-(苄巯基)丙酰]-O-乙基-D-酪氨酰-异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸(vii)
化合物8(6.3ml,7.5mmol)加至无水DMF(6ml)中,再加入BSA(4.39ml),室温搅拌4h,备用。化合物vi(3.0g,7.5mmol)加至无水DMF(6ml)中,降温至-15℃搅拌至全溶。再加入4M HCl的THF溶液(9.4ml),缓慢滴加亚硝酸叔丁酯(0.9ml,0.9mmol),在-10℃下搅拌3h。加入三乙胺(5.2ml),溶液变浑浊后再加入备用的化合1与BSA的混合液。温度保持在-10℃下,搅拌过夜,过滤,弃去滤饼。将滤液减压浓缩,加入乙醚,有大量固体析出。过滤得到白色固体5.3g,收率64.7%。ESI-MS(m/z):1096.98[M+H]+
步骤2 N-[3-(苄巯基)丙酰]-O-乙基-D-酪氨酰-异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酰-N-苄氧羰基-鸟氨酰-甘氨酰胺(xi)
化合物x(1.7g,4.7mmol)加至DMF(10ml)中,室温搅拌,加入HOBt(0.7g,5.5mmol),EDCI(1.0g,5.5mmol)和DIPEA(1.4ml,8.0mmol),反应4h后,再加入化合物vii(4.0g,3.6mmol),搅拌过夜。减压蒸除溶剂,加入乙醚(30ml)和H2O(30ml),析出大量固体,过滤,烘干得白色固体2.4g,收率47.2%,ESI-MS(m/z):1041.09[M+H]+。
步骤3 阿托西班(atosiban)
Na(46mg,2.00mmol)加至液氨(15ml)中,温度控制在-78℃以下,溶液变蓝,加入化合物xi(250mg,0.18mmol),温度保持在-78℃下搅拌4h,加入氯化铵,用氮气吹干液氨。加入0.5M HCl(20ml)和THF(20ml)萃取,弃去水层,在有机层中加入碘(22.8mg,0.18mmol),室温搅拌3h,用硫代硫酸钠水溶液萃取(15ml×3),合并有机相,减压浓缩,得到黄色油壮物,通过反相制备HPLC纯化得到atosiban,ESI-MS(m/z):994.65[M+H]+。
Claims (5)
1.阿托西班五肽中间体的液相合成方法,其特征在于,包括如下步骤:
a.以脯氨酸为原料,使用BSA保护其羧基,再与N端保护的S-苄基半胱氨酸的活性酯连接,制得N-叔丁氧羰酰基-S-苄基半胱氨酰-脯氨酸,再于酸性条件下脱去N端保护基得到S-苄基半胱酰氨-脯氨酸;
b.以S-苄基半胱酰氨-脯氨酸为原料,使用BSA保护脯氨酰的羧基部分,再与N端保护的天冬酰胺的活性酯连接,制得N-叔丁氧羰酰基-天冬酰胺-S-苄基半胱氨酰-脯氨酸,再于酸性条件下脱去N端保护基得到天冬酰胺-S-苄基半胱氨酰-脯氨酸;
c.以天冬酰胺-S-苄基半胱氨酰-脯氨酸为原料,使用BSA保护脯氨酰的羧基部分,再与N端保护的O-苄基苏氨酸的活性酯连接,制得N-叔丁氧羰酰基-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸,再于酸性条件下脱去N端保护基得到O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸;
d.以O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸为原料,使用BSA保护脯氨酰的羧基部分,再与N端保护的异亮氨酸的活性酯连接,制得N-叔丁氧羰酰基-异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸,再于酸性条件下脱去N端保护基得到异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸,即H-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-OH,其结构(Ⅰ)如下
e.具体合成路线如下:
其中R1=Boc、Cbz、Trt、Mz
R2=OSu、ONb、OBt、OOBt、OAt。
2.根据权利要求1所述的阿托西班五肽中间体的液相合成方法,其特征在于:步骤a、c、d中所述的N端保护基团选自叔丁氧羰基(BOC)、苄氧羰基(Cbz)、三苯甲基(Trt)、对甲氧苄氧羰基(Mz)。
3.根据权利要求1所述的阿托西班五肽中间体的液相合成方法,其特征在于:用酸脱除N端保护基时所述的酸选自包括三氟乙酸、盐酸、氢氟酸;所用溶剂包括:二氯甲烷、四氢呋喃、1,4-二氧六环。
4.根据权利要求1所述的阿托西班五肽中间体的液相合成方法,其特征在于:步骤a、b、c、d中所述的氨基酸的相应活性酯选自N-羟基琥珀酰亚胺酯、N-羟基-5-降冰片烯-2,3-二甲酰亚胺酯、1-羟基苯骈三氮唑酯、3-羟基-4-氧-3,4-二氢-1,2,3-苯骈三氮唑酯、1-羟基-7-偶氮苯并三氮唑酯。
5.根据权利要求1-4中任一项所述的合成方法,其特征在于:包括如下步骤:
(a)制备S-苄基半胱酰氨-脯氨酸
将BSA和L-脯氨酸加到无水二氯甲烷中,10~35℃下搅拌2~4h,滴入N-叔丁氧羰酰基-S-苄基半胱氨酸-(N-羟基)琥珀酰亚胺酯的二氯甲烷溶液,10~35℃下反应5~8h,减压蒸除溶剂,加乙醚,用5%的碳酸氢钠水溶液萃取;合并水相,加入柠檬酸调至PH5~7,用乙酸乙酯萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得淡黄色油状物,加入无水乙醚析出白色固体;将白色固体加至无水二氯甲烷中,0~15℃下搅拌滴加三氟乙酸至VCH2Cl2:VCF3COOH=1~5:1,搅拌反应2~5h,减压浓缩至干,得到淡黄色油状物,用乙酸乙酯/乙醚重结晶,得到白色固体;
(b)制备天冬酰胺-S-苄基半胱氨酰-脯氨酸
将S-苄基半胱酰氨-脯氨酸和BSA加到无水二氯甲烷中,10~35℃下搅拌3~4h,滴入N-叔丁氧羰酰基-天冬酰胺-(N-羟基)琥珀酰亚胺酯的二氯甲烷溶液,10~35℃下搅拌8~10h,减压蒸除溶剂,加乙醚,用5%的碳酸氢钠水溶液萃取;合并水相,加入柠檬酸调至PH5~7,用乙酸乙酯萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得淡黄色油状物,乙酸乙酯重结晶,得到的固体加至无水二氯甲烷中,0~15℃下搅拌滴加三氟乙酸至VCH2Cl2:VCF3COOH=1~5:1,10~35℃下搅拌反应2~5h,减压浓缩至干,得到淡黄色油状物,用乙酸乙酯/乙醚重结晶,得到白色固体;
(c)制备O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸
将天冬酰胺-S-苄基半胱氨酰-脯氨酸和BSA加到无水二氯甲烷中,10~35℃下搅拌3~4h,滴入N-叔丁氧羰酰基-O-苄基-苏氨酸-(N-羟基)琥珀酰亚胺酯的二氯甲烷溶液,10~35℃下搅拌6~8h,减压蒸除溶剂,加乙醚,用5%的碳酸氢钠水溶液萃取;合并水相,加入柠檬酸调至PH5~8,用乙酸乙酯萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,滤液减压浓缩至干,得淡黄色油状物,乙酸乙酯重结晶,得到的固体加至无水二氯甲烷中,0~15℃下搅拌滴加三氟乙酸至VCH2Cl2:VCF3COOH=1~5:1,10~35℃下搅拌反应2~5h,减压浓缩至干,用甲醇/乙醚重结晶,得到白色固体;
(d)制备异亮氨酰-O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸
将O-苄基-苏氨酰-天冬酰胺-S-苄基半胱氨酰-脯氨酸和BSA加到无水二氯甲烷中,10~35℃下搅拌3~4h,滴入N-叔丁氧羰酰基-异亮氨酸-(N-羟基)琥珀酰亚胺酯的二氯甲烷溶液,10~35℃下搅拌6~8h,减压蒸除溶剂,加乙醚,用5%的碳酸氢钠水溶液萃取;合并水相,加入柠檬酸调至PH5~8,用乙酸乙酯萃取,合并有机相,用饱和食盐水洗涤后经无水硫酸钠干燥,过滤,得到的白色固体加至无水二氯甲烷中,0~15℃下搅拌滴加三氟乙酸至VCH2Cl2:VCF3COOH=1~5:1,10~35℃下搅拌反应2~5h,减压浓缩至干,用甲醇/乙醚重结晶,得到白色固体。
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