CN114805480A - Preparation method of octreotide - Google Patents
Preparation method of octreotide Download PDFInfo
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- CN114805480A CN114805480A CN202110065785.0A CN202110065785A CN114805480A CN 114805480 A CN114805480 A CN 114805480A CN 202110065785 A CN202110065785 A CN 202110065785A CN 114805480 A CN114805480 A CN 114805480A
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- Prior art keywords
- octreotide
- resin
- reagent
- producing
- fmoc
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- DEQANNDTNATYII-OULOTJBUSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-benzyl-n-[(2r,3r)-1,3-dihydroxybutan-2-yl]-7-[(1r)-1-hydroxyethyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxa Chemical compound C([C@@H](N)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)C1=CC=CC=C1 DEQANNDTNATYII-OULOTJBUSA-N 0.000 title claims abstract description 99
- 108010016076 Octreotide Proteins 0.000 title claims abstract description 99
- 229960002700 octreotide Drugs 0.000 title claims abstract description 96
- 238000002360 preparation method Methods 0.000 title claims abstract description 42
- 229920005989 resin Polymers 0.000 claims abstract description 132
- 239000011347 resin Substances 0.000 claims abstract description 132
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 53
- 150000001875 compounds Chemical class 0.000 claims abstract description 28
- 125000006239 protecting group Chemical group 0.000 claims abstract description 27
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 19
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 17
- 239000000047 product Substances 0.000 claims abstract description 16
- 238000005336 cracking Methods 0.000 claims abstract description 14
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 13
- 230000008569 process Effects 0.000 claims abstract description 10
- 239000012043 crude product Substances 0.000 claims abstract description 9
- 229920001184 polypeptide Polymers 0.000 claims abstract description 9
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 9
- 238000005520 cutting process Methods 0.000 claims abstract description 8
- 239000007790 solid phase Substances 0.000 claims abstract description 7
- 238000001308 synthesis method Methods 0.000 claims abstract description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 57
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical group [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims description 32
- 238000009833 condensation Methods 0.000 claims description 29
- 230000005494 condensation Effects 0.000 claims description 29
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 28
- 239000000203 mixture Substances 0.000 claims description 27
- 150000001413 amino acids Chemical class 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 18
- NROKBHXJSPEDAR-UHFFFAOYSA-M potassium fluoride Chemical compound [F-].[K+] NROKBHXJSPEDAR-UHFFFAOYSA-M 0.000 claims description 14
- 238000010511 deprotection reaction Methods 0.000 claims description 13
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 13
- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 claims description 12
- 238000006482 condensation reaction Methods 0.000 claims description 12
- 238000005886 esterification reaction Methods 0.000 claims description 11
- 239000012279 sodium borohydride Substances 0.000 claims description 11
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 11
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 10
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- MUVQIIBPDFTEKM-IUYQGCFVSA-N (2r,3s)-2-aminobutane-1,3-diol Chemical compound C[C@H](O)[C@H](N)CO MUVQIIBPDFTEKM-IUYQGCFVSA-N 0.000 claims description 9
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 9
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- HBAHZZVIEFRTEY-UHFFFAOYSA-N 2-heptylcyclohex-2-en-1-one Chemical compound CCCCCCCC1=CCCCC1=O HBAHZZVIEFRTEY-UHFFFAOYSA-N 0.000 claims description 7
- 229920001367 Merrifield resin Polymers 0.000 claims description 7
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000012071 phase Substances 0.000 claims description 7
- 239000011698 potassium fluoride Substances 0.000 claims description 7
- 235000003270 potassium fluoride Nutrition 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 238000003776 cleavage reaction Methods 0.000 claims description 6
- 230000007017 scission Effects 0.000 claims description 6
- 230000032050 esterification Effects 0.000 claims description 5
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 claims description 5
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 4
- 239000007821 HATU Substances 0.000 claims description 4
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 claims description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 3
- 239000012448 Lithium borohydride Substances 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 239000011575 calcium Substances 0.000 claims description 3
- 229910052791 calcium Inorganic materials 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 238000004007 reversed phase HPLC Methods 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims 1
- 238000002156 mixing Methods 0.000 claims 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 4
- 238000007086 side reaction Methods 0.000 abstract description 3
- 125000001433 C-terminal amino-acid group Chemical group 0.000 abstract 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 28
- 235000001014 amino acid Nutrition 0.000 description 23
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 10
- 238000001914 filtration Methods 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 description 7
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 6
- -1 Fmoc amino acid Chemical class 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 125000004122 cyclic group Chemical group 0.000 description 5
- 229920005990 polystyrene resin Polymers 0.000 description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 5
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 4
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 238000006722 reduction reaction Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- YDJXDYKQMRNUSA-UHFFFAOYSA-N tri(propan-2-yl)silane Chemical compound CC(C)[SiH](C(C)C)C(C)C YDJXDYKQMRNUSA-UHFFFAOYSA-N 0.000 description 4
- XZXYQEHISUMZAT-UHFFFAOYSA-N 2-[(2-hydroxy-5-methylphenyl)methyl]-4-methylphenol Chemical compound CC1=CC=C(O)C(CC=2C(=CC=C(C)C=2)O)=C1 XZXYQEHISUMZAT-UHFFFAOYSA-N 0.000 description 3
- KXDHJXZQYSOELW-UHFFFAOYSA-N Carbamic acid Chemical compound NC(O)=O KXDHJXZQYSOELW-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 3
- 239000004473 Threonine Substances 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 229940107816 ammonium iodide Drugs 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 229960001494 octreotide acetate Drugs 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 125000004066 1-hydroxyethyl group Chemical group [H]OC([H])([*])C([H])([H])[H] 0.000 description 2
- 206010000599 Acromegaly Diseases 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- 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 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 229920000361 Poly(styrene)-block-poly(ethylene glycol) Polymers 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 125000004218 chloromethyl group Chemical group [H]C([H])(Cl)* 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 125000002653 sulfanylmethyl group Chemical group [H]SC([H])([H])[*] 0.000 description 2
- AQRLNPVMDITEJU-UHFFFAOYSA-N triethylsilane Chemical compound CC[SiH](CC)CC AQRLNPVMDITEJU-UHFFFAOYSA-N 0.000 description 2
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 2
- LKRXXARJBFBMCE-BDAKNGLRSA-N (2s,3r)-3-[(2-methylpropan-2-yl)oxy]-2-[(2-methylpropan-2-yl)oxycarbonylamino]butanoic acid Chemical compound CC(C)(C)O[C@H](C)[C@@H](C(O)=O)NC(=O)OC(C)(C)C LKRXXARJBFBMCE-BDAKNGLRSA-N 0.000 description 1
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-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
- DFPYXQYWILNVAU-UHFFFAOYSA-N 1-hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1.C1=CC=C2N(O)N=NC2=C1 DFPYXQYWILNVAU-UHFFFAOYSA-N 0.000 description 1
- RULWBFLPUAFFGY-UHFFFAOYSA-N 2-(hydroxymethyl)benzamide Chemical compound NC(=O)C1=CC=CC=C1CO RULWBFLPUAFFGY-UHFFFAOYSA-N 0.000 description 1
- IIVWHGMLFGNMOW-UHFFFAOYSA-N 2-methylpropane Chemical compound C[C](C)C IIVWHGMLFGNMOW-UHFFFAOYSA-N 0.000 description 1
- ZHGNHOOVYPHPNJ-UHFFFAOYSA-N Amigdalin Chemical compound FC(F)(F)C(=O)OCC1OC(OCC2OC(OC(C#N)C3=CC=CC=C3)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C2OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C1OC(=O)C(F)(F)F ZHGNHOOVYPHPNJ-UHFFFAOYSA-N 0.000 description 1
- 101000632994 Homo sapiens Somatostatin Proteins 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 102000005157 Somatostatin Human genes 0.000 description 1
- 108010056088 Somatostatin Proteins 0.000 description 1
- 239000003875 Wang resin Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- VILAVOFMIJHSJA-UHFFFAOYSA-N dicarbon monoxide Chemical compound [C]=C=O VILAVOFMIJHSJA-UHFFFAOYSA-N 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 102000045305 human SST Human genes 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 229960000553 somatostatin Drugs 0.000 description 1
- 238000003786 synthesis reaction 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
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- OHSJPLSEQNCRLW-UHFFFAOYSA-N triphenylmethyl radical Chemical compound C1=CC=CC=C1[C](C=1C=CC=CC=1)C1=CC=CC=C1 OHSJPLSEQNCRLW-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a preparation method of octreotide, which comprises the following steps: (1) resin is adopted as a solid phase carrier, and is esterified with the C-terminal amino acid of octreotide to prepare an amino acid-resin compound; (2) preparing a protected octreotide-resin compound by adopting an Fmoc strategy solid-phase polypeptide synthesis method; (3) removing side chain protecting groups by using a cutting reagent to prepare a deprotected octreotide-resin compound; (4) adding a reducing agent to crack octreotide from resin, and cyclizing a disulfide bond while cracking to obtain an octreotide crude product; (5) and purifying the crude product by a high performance liquid chromatography to obtain the refined octreotide product. The invention provides a simple and convenient preparation method of octreotide with low cost and higher yield and purity, which solves the problems of high cost, more side reactions, complex process or lower yield of the prior octreotide preparation.
Description
The technical field is as follows:
the invention belongs to the field of polypeptide chemistry, and particularly relates to a preparation method of octreotide.
Background art:
octreotide (formula 1) is a cyclic octapeptide containing a disulfide bond in the molecule, is an octapeptide derivative of artificially synthesized human somatostatin, and has similar action to natural somatostatin. The chemical name is D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophanyl-L-lysyl-L-threonyl-L-cysteinyl-L-threonine-cyclo- (2 → 7) -disulfide; the English name is Octreotide Acetate. The company Sandoz, switzerland (Sandoz) was synthesized in 1982, and in 1988 the FDA approved octreotide acetate for the treatment of gastrointestinal secretory tumors and pituitary tumors and acromegaly; octreotide acetate was re-approved by the U.S. FDA in 1998 for the treatment of acromegaly.
The preparation methods of octreotide are mainly disclosed as two methods: solid phase methods and liquid phase methods. The patent ZL00134258.4 is synthesized by a liquid phase method, and has the disadvantages of complicated steps and long production period. At present, solid phase methods are still the mainstream methods for preparing octreotide, and a class of methods, such as patent applications CN201010165270.X, patent ZL200510029221.2 and ZL201210352668.3, adopts chlorine resin and chlorine resin analogues, and synthesizes the octreotide after the connection with the threoninol. The disadvantage of this type of process is the low efficiency of the conjugation of the chlorinated resin to the threoninol, resulting in excessive threoninol and resin waste, with an increase in the solvent consumption and ultimately the cost required for subsequent washing of the resin. Another approach is to use the solution of linker addition to solve the problem of low efficiency of threoninol-to-resin binding, as in patent applications cn2016109844. x, patent ZL200510024502.9 and ZL 2004100108332. The method can be used for preparing octreotide only after the sunol and the linker are synthesized and purified through a liquid phase reaction, so that the process period is increased, the quality control of a raw material is additionally increased, and the process is complicated. The two methods both need expensive threoninol as a raw material, and the utilization rate of the threoninol is not high, so that the production cost is high. The third solid phase synthesis method adopts ester reduction to obtain octreotide containing threoninol, so as to solve the problem of high cost, for example, in patent application CN02137521.6, Boc-Thr (Ac) -Wang resin is adopted, TFA is used for removing Boc protecting group, then Fmoc amino acid is condensed in sequence, and piperidine is used for removing Fmoc protecting group, and then condensation is carried out. After the synthesis is finished, sodium borohydride is used for solid-phase reduction of ester to obtain the octreotide, and the yield is only 10.7%. Further problems exist in that: when TFA is used for removing Boc protecting group, Thr of amino acid can be cut off, and the yield is reduced; in addition, when piperidine is used for removing Fmoc, the side chain protecting group Ac of Thr is easy to fall off, and side reaction is generated. Patent ZL201310333598.1 uses Boc-Thr (tBu) -OH to link to chloromethyl resin, hydrochloric acid/isopropanol to remove Boc, Pd/C hydrogenation cutting and reduction to alcohol, the main problems are: the Pd/C poisoning failure can be caused by a mode of firstly removing the sulfhydryl protecting group Trt and then hydrogenating; and needs to cyclize in the liquid phase to form disulfide bonds, resulting in large amounts of waste.
Disclosure of Invention
The invention aims to provide a simple and convenient preparation method of octreotide with low cost and higher yield and purity, which solves the problems of high cost, more side reactions, complex process or lower yield of the prior octreotide preparation.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a preparation method of octreotide, which comprises the following steps:
(1) adding Fmoc-Thr (tBu) -OH and an esterification reagent into resin for esterification reaction to obtain an Fmoc-Thr (tBu) -resin compound;
(2) according to the solid-phase polypeptide synthesis method, according to the sequence of octreotide, the protected amino acids are coupled to the resin obtained in the step (1) one by one according to the sequence from C end to N end to obtain a protected octreotide-resin compound;
(3) adding a cutting reagent into the protected octreotide-resin compound, and removing a polypeptide side chain protecting group to obtain a deprotected octreotide-resin compound;
(4) adding a reducing agent into the deprotected octreotide-resin compound to reduce an ester bond formed by the esterification reaction in the step (1) into threoninol, simultaneously cracking octreotide from the resin, and cyclizing a disulfide bond while cracking to obtain a crude octreotide product;
(5) and separating and purifying the crude product of octreotide to obtain octreotide.
The invention also provides a preparation method of octreotide, which comprises the following steps:
(1) adding Fmoc-Thr (tBu) -OH and an esterification reagent into PAM resin, HMBA resin or Merrifield resin for esterification reaction to obtain an Fmoc-Thr (tBu) -resin compound;
(2) adding an Fmoc deprotection reagent into the Fmoc-Thr (tBu) -resin compound obtained in the step (1) to remove an Fmoc protective group, washing the resin after the reaction is finished to obtain Thr (tBu) -resin, then adding a second amino acid at the C end of octreotide and a condensation reagent into the Thr (tBu) -resin compound to carry out a condensation reaction, and washing the resin after the condensation reaction is finished; replacing amino acids according to the sequence of octreotide from C end to N end, and repeating Fmoc deprotection reaction and condensation reaction to obtain a protected octreotide-resin compound;
(3) adding a cutting reagent into the protected octreotide-resin compound, and removing a polypeptide side chain protecting group to obtain a deprotected octreotide-resin compound;
(4) adding a reducing agent into the deprotected octreotide-resin compound to reduce bonds formed by the esterification reaction in the step (1) into threoninol, and simultaneously cracking octreotide from the resin, wherein the reducing agent provides a weak alkaline environment, so that a disulfide bond is cyclized while the octreotide is cracked from the resin, and a crude octreotide product is obtained;
(5) and separating and purifying the crude octreotide product by using a reverse phase high performance liquid chromatography to obtain a refined octreotide product.
In some embodiments of the present invention, the resin type in the step (1) includes a polystyrene resin, a polyethylene glycol resin or a polystyrene-polyethylene glycol copolymerized resin.
In some embodiments of the invention, the resin in step (1) is a PAM resin, a HMBA resin, or a Merrifield resin.
In some embodiments of the invention, the esterifying reagent in step (1) is one or more of DIC, HOBt, DMAP, potassium fluoride.
In some embodiments of the invention, the ratio between the substances in step (1) is n [ Fmoc-Thr (tBu) -OH ]: n (DIC): n (HOBt): n (DMAP): n (potassium fluoride): n (resin) is 4 to 1: 4-1: 4-1: 0.5-0.1: 4-1: 1.
in some embodiments of the present invention, the reaction time in the step (1) is 1 to 24 hours.
In some embodiments of the invention, the Fmoc deprotecting reagent in step (2) is a mixture of reagents having a volume ratio of 1: 1-4 of piperidine and dimethylformamide. The Fmoc deprotection reaction temperature is 20-60 ℃, and the reaction time is 10-60 min.
In some embodiments of the present invention, the protecting groups of the amino acid in the step (2) are: the alpha amino groups of all amino acids are Fmoc protecting groups, the threonine side chain hydroxyl protecting group is tert-butyl, the cysteine side chain protecting group is trityl, and the lysine and D-tryptophan side chain protecting groups are tert-butoxycarbonyl.
In some embodiments of the present invention, the condensing agent in step (2) is one or more of a mixture of condensing agent 1, condensing agent 2 and condensing agent 3; wherein, the condensation reagent 1 is one of DCC, DIC, HBTU, HATU, BOP and PyBOP, the condensation reagent 2 is one of HOBt and HOAt, and the condensation reagent 3 is DIEA.
In some embodiments of the invention, the ratio of the amounts of the substances of the reagents in step (2) is: amino acids: condensation reagent 1: condensation reagent 2: condensation reagent 3: and (3) resin is 6-2: 6-2: 6-2: 12-4: 1.
in some embodiments of the invention, the DCC or DIC is not used in combination with DIEA in step (2).
In some embodiments of the present invention, the condensation reaction in step (2) is performed at a temperature of 20 to 60 ℃ for 1 to 4 hours.
In some embodiments of the present invention, the cleavage reagent in step (3) is a TFA mixture, and the volume ratio of TFA in the mixture is not less than 80%.
In some embodiments of the invention, the TFA mixture comprises, in addition to TriTFA, redistilled water, TES, DTT, Phenol, Thioalisole, NH 4 I. One or more of TIS, Anisole and EDT.
In some embodiments of the present invention, the temperature for adding the cleavage reagent to the octreotide-resin complex protected in step (3) is 0 to 60 ℃ and the reaction time is 1 to 4 hours.
In some embodiments of the present invention, the reducing agent in the step (4) is a lithium chloride mixed solution.
In some embodiments of the present invention, the lithium chloride mixed solution further includes one or more of sodium borohydride, lithium borohydride, calcium borohydride, potassium borohydride, and sodium borohydride in addition to lithium chloride.
In some embodiments of the present invention, the amount of lithium chloride in the lithium chloride mixed solution is 50%.
In some embodiments of the present invention, the solution in the step (4) is weakly alkaline and has a pH of 8 to 10.
In some embodiments of the present invention, the solvent in step (4) is a mixture of tetrahydrofuran and ethanol.
In some embodiments of the present invention, the volume ratio of tetrahydrofuran to ethanol in the step (4) is 2-4: 1.
in some embodiments of the invention, the ratio of reducing agent to resin in step (4) is n (reducing agent): n (resin) is 20 to 5: 1, the concentration of the reducing agent solution is 5-20 mg/mL, and the reaction is carried out for 5-24 hours at room temperature.
In some embodiments of the present invention, the crude octreotide obtained in step (5) is separated and purified by reverse phase high performance liquid chromatography to obtain a refined octreotide.
In some embodiments of the invention, the purification conditions in step (5) are: the column packing is prepared in a type of carbon-supported silica gel.
In some embodiments of the present invention, the mobile phase in step (5) is one or more of water, acetonitrile or methanol solution with 0.1% trifluoroacetic acid by mass ratio.
In some embodiments of the invention, the preparative column in step (5) is C 4 /C 8 /C 18 Type of reverse phase chromatography column.
The detailed steps of the preferred technical scheme of the invention are as follows:
(1) preparation of Fmoc-Thr (tBu) -resin Complex
The resin was added to a reaction vessel with a filtration device. The solvent dichloromethane was added to the vessel, the resin was immersed, allowed to stand for 1 hour, and filtered.
Dissolving Fmoc-Thr (tBu) -OH and an esterification reagent in DMF, adding the mixture into resin, and reacting for 1-24 h at room temperature. After the reaction is finished, adding a solvent DMF, washing the resin for six times, and filtering. Wherein the resin type comprises polystyrene resin, polyethylene glycol resin or polystyrene-polyethylene glycol copolymerized resin, including but not limited to PAM resin, HMBA resin or Merrifield resin, the esterifying reagent is one or more of DIC, HOBt, DMAP and potassium fluoride, and the ratio among the substances is n [ Fmoc-Thr (tBu) -OH ]: n (DIC): n (HOBt): n (DMAP): n (potassium fluoride): n (resin) is 4 to 1: 4-1: 4-1: 0.5-0.1: 1.
(2) preparation of protected octreotide-resin complexes
The Fmoc-Thr (tBu) -resin complex described above was added with a solvent in a volume ratio of 1: 1-4 PIP and DMF solution, performing deprotection reaction, and reacting for 10-60 min at 20-60 ℃. After the reaction, filtering. The resin was then washed with DMF and filtered.
The second amino acid at the C-terminus of octreotide [ Fmoc-Cys (Trt) -OH ] and a condensation reagent were added to the reactor. Reacting for 1-4 h at 20-60 ℃. After the reaction, DMF was added to wash the resin, and the mixture was filtered. The condensation reagent is one or a mixture of a condensation reagent 1, a condensation reagent 2 and a condensation reagent 3; wherein, the condensation reagent 1 is one of DCC, DIC, HBTU, HATU, BOP and PyBOP, the condensation reagent 2 is one of HOBt and HOAt, the condensation reagent 3 is DIEA, and DIC or DCC is not combined with DIEA for application. Wherein the ratio of the amounts of the reactant materials is: n [ Fmoc-Cys (Trt) -OH ]: n (condensation reagent 1): n (condensing agent 2): n (condensing agent 3): n (resin) is 6-2: 6-2: 6-2: 12-4: 1.
and (3) replacing amino acids according to the sequence from the C end to the N end of the octreotide, and carrying out cyclic deprotection reaction and condensation reaction to synthesize the protected octreotide-resin compound. Wherein the ratio of the amounts of the reactant materials and the reaction conditions are the same as Fmoc-Cys (Trt) -OH.
(3) Preparation of deprotected octreotide-resin complexes
Adding a cutting reagent into the resin, and reacting for 1-4 hours at the temperature of 0-60 ℃. The cutting reagent is TFA, TES, DTT, Phenol, Thioanisole, NH 4 I. And a mixture of at least one of TIS, Anisole and EDT and TFA, wherein the volume ratio of TFA in the mixture is not less than 80%. After the reaction was completed, methylene chloride was added to wash the resin.
(4) Preparation of crude octreotide
Adding a reducing agent into the deprotected octreotide-resin compound, reacting for 5-24 h, cracking octreotide from resin, and cyclizing a disulfide bond while cracking to obtain an octreotide crude product; the reducing agent is a mixed solution of lithium chloride and at least one of sodium borohydride, lithium borohydride, calcium borohydride, potassium borohydride and sodium borohydride, the quantity proportion of lithium chloride in the mixed solution of lithium chloride is 50%, and the proportion of the reducing agent to resin is n (reducing agent): n (resin) is 20 to 5: 1, the concentration of the reducing agent solution is 5-20 mg/mL, and the reaction is carried out for 5-24 h at room temperature.
(5) Preparation of octreotide extract
And dissolving the crude octreotide product by using 5 percent methanol or acetonitrile aqueous solution by volume ratio, and purifying by adopting preparative high performance liquid chromatography to obtain a refined octreotide product. Preparation column is C 4 /C 8 /C 18 The type of reversed phase chromatographic column has a mobile phase of one or more of water containing 0.1% TFA, acetonitrile containing 0.1% TFA and methanol containing 0.1% TFA.
The invention has the beneficial effects that: PAM resin, HMBA resin or Merrifield resin is adopted as a solid phase carrier, and Fmoc is adopted to protect threonine, octreotide is not cut off from the resin when a side chain protecting group is removed, so that the removal of the side chain protecting group on the solid phase carrier can be realized, the separation of the side chain protecting group and octreotide can be completed only by simple washing, and the process is simplified. The threonine with low price is adopted as the raw material, so that the use of expensive threonine raw material is avoided, and the production cost is greatly reduced; the process of synchronously carrying out reduction, cutting and cyclization is adopted, and the volume of a reducing agent is small, so that the concentration of polypeptide can reach 25mmol/L, the solvent required for forming disulfide bonds is greatly reduced (the concentration of octreotide in the traditional process is 1-2mmol/L), the waste liquid of tetrahydrofuran and ethanol is greatly reduced, the process is simplified, and the yield and the purity are high.
Some abbreviations commonly used in the present invention have the following meanings:
fmoc
tBu: tert-butyl radical
Trt: trityl radical
TFA: trifluoroacetic acid
EDT (electro-thermal transfer coating): ethanedithiol
Phenol: phenol and its preparation
Thioanisole: phenylmethyl sulfide
TES: triethylsilane
And (3) TIS: tri-isopropyl silane
DTT: 1,2 dithiothreitol
Anisole: phenylmethyl ether
NH 4 I: ammonium iodide
MeOH: methanol
DCM: methylene dichloride
DMF: n, N-dimethylformamide
PIP: piperidine derivatives
DMAP: dimethylaminopyridine
DIEA: n, N-diisopropylethylamine
BOP: katt condensation agent
HOBt 1-hydroxybenzotriazole
DIC: n, N-diisopropylcarbodiimide
DCC: n, N-dicyclohexylcarbodiimide
HOAt: n-hydroxy-7-azobenzotriazol
HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate
HATU: 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate
PyBOP: benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
Fmoc-Thr (tBu) -OH: n- (9-fluorenylmethoxycarbonyl) -O-tert-butyl-threonine
Fmoc-Cys (Trt) -OH: n- (9-fluorenylmethoxycarbonyl) -S-trityl-cysteine
PAM resin: 4- (hydroxymethyl) phenylacetamidomethyl-polystyrene resin
HMBA resin: hydroxymethyl benzamide polystyrene resin
Merrifield resin: chloromethyl polystyrene resin
As used herein, a "condensation reaction" is a reaction in which two or more organic molecules interact to covalently bind together a macromolecule, often with the concomitant loss of small molecules (e.g., water, hydrogen chloride, alcohol, etc.).
As used herein, "protecting group" or "protecting group" has its ordinary meaning in the art. A protecting group includes a chemical moiety that is attached to or configured to attach to a reactive group (i.e., a protected group) within a molecule (e.g., a peptide) such that the protecting group prevents or otherwise inhibits the protected group from participating in a reaction. Protection may be performed by attaching a protecting group to the molecule. Deprotection can occur when a protecting group is removed from a molecule, for example, by chemical transformation to remove the protecting group.
As used herein, a "polypeptide" or "peptide" has its ordinary meaning in the art and may refer to an amide from two or more aminocarboxylic acid molecules (the same or different) that forms a covalent bond by formally losing water from the carbonyl carbon of one aminocarboxylic acid molecule and the nitrogen atom of another aminocarboxylic acid molecule. "amino acid residue" also has its ordinary meaning in the art and refers to the composition of an amino acid (as a single amino acid or as part of a peptide) after it is combined with a peptide, another amino acid, or an amino acid residue. Generally, when an amino acid is combined with another amino acid or amino acid residue, water is removed, and the remaining amino acid is referred to as an amino acid residue. The term "amino acid" also has its ordinary meaning in the art and can include both proteinogenic amino acids and non-proteinogenic amino acids. The abbreviations for amino acid residues in the present invention are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
Detailed Description
The invention is further illustrated by the following examples, which should not be construed as limiting the invention. In the following examples, the alpha amino groups of all amino acids are Fmoc protecting groups, the threonine side chain hydroxyl protecting group is t-butyl, the cysteine side chain protecting group is trityl, and the lysine and D-tryptophan side chain protecting groups are t-butoxycarbonyl.
Example 1: preparation of octreotide
(1) Preparation of Fmoc-Thr (tBu) -resin Complex
A reactor equipped with a filtration apparatus was charged with 1g of PAM resin (1.0mmol/g), 10ml of methylene chloride was injected, and the mixture was allowed to stand for 1 hour and filtered.
4mmol of Fmoc-Thr (tBu) -OH, 4mmol of DIC, 4mmol of HOBt and 0.5mmol of DMAP were weighed out, dissolved in 10ml of DMF and added to the resin and reacted at room temperature for 24 h. After the reaction was completed, the mixture was filtered. Then 10ml DMF was added, the resin was washed six times and filtered.
(2) Preparation of protected octreotide-resin complexes
To the reactor was added 10ml of a 1: PIP of 1 was reacted with DMF solution at 20 ℃ for 30 min. After the reaction, filtering. The resin was then washed six times with 10ml DMF and filtered.
6mmol of Fmoc-Cys (Trt) -OH, 6mmol of DIC and 6mmol of HOBt are dissolved in 10ml of DMF, and the resulting solution is added to a reactor and reacted at 30 ℃ for 4 hours. After the reaction was completed, the mixture was filtered. Then 10ml DMF was added, the resin was washed six times and filtered.
And (3) replacing amino acids according to the sequence from the C end to the N end of the octreotide, and carrying out cyclic deprotection reaction and condensation reaction to synthesize the protected octreotide-resin compound.
(3) Preparation of deprotected octreotide-resin complexes
To the above-protected octreotide-resin complex, 10ml of a cleavage reagent (TFA: phenol: thioansole: aqueous ammonium iodide solution: 87.5%: 5%: 2.5%: 5%, concentration of aqueous ammonium iodide solution: 10mg/ml) was added and reacted at 25 ℃ for 2 hours. After the reaction was completed, dichloromethane was added to wash the resin 6 times.
(4) Preparation of crude octreotide
Adding 20mmol, the concentration of 10mg/mL and the mass ratio of substances of 1: 1, and the solvent is a solution of sodium borohydride and lithium chloride with a volume ratio of 3: 1 of tetrahydrofuran and ethanol, reacting for 24 hours, cracking octreotide from resin, and cyclizing a disulfide bond while cracking to obtain an octreotide crude product.
(5) Preparation of octreotide competitive products
Dissolving the crude octreotide product in acetonitrile water solution with the volume ratio of 5%, purifying by high performance liquid chromatography to obtain refined octreotide product, and freeze-drying to obtain 0.55g, the purity of 99.3% and the yield of 67.5%. The preparation conditions are as follows: c 18 The column was prepared with two mobile phases of water and acetonitrile in a mass ratio of 0.1% TFA.
Example 2: preparation of octreotide
(1) Preparation of Fmoc-Thr (tBu) -resin Complex
To a reactor equipped with a filtration apparatus was added 1g of HMBA resin (1.0mmol/g), 10ml of methylene chloride was injected, and the mixture was allowed to stand for 1 hour and filtered.
3mmol of Fmoc-Thr (tBu) -OH, 3mmol of DIC, 3mmol of HOBt and 0.3mmol of DMAP were weighed out, dissolved in 10ml of DMF and added to the resin and reacted at room temperature for 10 h. After the reaction was completed, the mixture was filtered. Then 10ml DMF was added, the resin was washed six times and filtered.
(2) Preparation of protected octreotide-resin complexes
To the reactor was added 10ml of a 1: 3 PIP was reacted with DMF solution at 30 ℃ for 60 min. After the reaction, filtering. The resin was then washed six times with 10ml DMF and filtered.
4mmol of Fmoc-Cys (Trt) -OH, 4mmol of DIC and 4mmol of HOBt are dissolved in 10ml of DMF, and the obtained solution is added to a reactor and reacted at 40 ℃ for 2 hours. After the reaction was completed, the mixture was filtered. Then 10ml DMF was added, the resin was washed six times and filtered.
And (3) replacing amino acids according to the sequence from the C end to the N end of the octreotide, and carrying out cyclic deprotection reaction and condensation reaction to synthesize the protected octreotide-resin compound.
(3) Preparation of deprotected octreotide-resin complexes
To the above protected octreotide-resin complex, 10ml of a cleavage reagent (TFA: phenol: thioansole: EDT: 87.5%: 5%: 2.5%: 5%) was added and reacted at 40 ℃ for 4 hours. After the reaction was completed, dichloromethane was added to wash the resin 6 times.
(4) Preparation of crude octreotide
Adding 10mmol of octreotide-resin complex with the concentration of 5mg/mL and the mass ratio of 1: 1, sodium borohydride and lithium chloride solution, wherein the solvent is a mixture of sodium borohydride and lithium chloride in a volume ratio of 2: 1 of tetrahydrofuran and ethanol, reacting for 10 hours, cracking octreotide from resin, and cyclizing a disulfide bond while cracking to obtain an octreotide crude product.
(5) Preparation of octreotide extract
And dissolving the crude octreotide product in acetonitrile water solution with the volume ratio of 5%, purifying by high performance liquid chromatography to obtain a refined octreotide product, and freeze-drying to obtain 0.407g, wherein the purity is 98.9% and the yield is 50%. The preparation conditions are as follows: c 8 The column was prepared with two mobile phases, water and acetonitrile, both in 0.1% TFA by mass.
Example 3: preparation of octreotide
(1) Preparation of Fmoc-Thr (tBu) -resin Complex
To a reactor equipped with a filtration device was added 1g of Merrifield resin (1.0mmol/g), 10ml of methylene chloride was injected, and the mixture was allowed to stand for 1 hour and filtered.
1mmol of Fmoc-Thr (tBu) -OH, 1mmol of potassium fluoride were weighed out, dissolved in 10ml of DMF, added to the resin and reacted at room temperature for 10 h. After the reaction was completed, the mixture was filtered. Then 10ml DMF was added, the resin was washed six times and filtered.
(2) Preparation of protected octreotide-resin complexes
To the reactor was added 10ml of a 1: 4 PIP was reacted with DMF solution at 20 ℃ for 10 min. After the reaction, filtering. The resin was then washed six times with 10ml DMF and filtered.
2mmol of Fmoc-Cys (Trt) -OH, 2mmol of HBTU and 4mmol of DIEA were dissolved in 10ml of DMF, and the resulting solution was added to a reactor and reacted at 20 ℃ for 1 hour. After the reaction was completed, the mixture was filtered. Then 10ml DMF was added, the resin was washed six times and filtered.
And (3) replacing amino acids according to the sequence from the C end to the N end of the octreotide, and carrying out cyclic deprotection reaction and condensation reaction to synthesize the protected octreotide-resin compound.
(3) Preparation of deprotected octreotide-resin complexes
To the above protected octreotide-resin complex, 10ml of a cleavage reagent (TFA: TIS: thioansole: EDT: 87.5%: 5%: 2.5%: 5%) was added and reacted at 0 ℃ for 1 hour. After the reaction was completed, dichloromethane was added to wash the resin 6 times.
(4) Preparation of crude octreotide
Adding 5mmol of sodium borohydride solution with the concentration of 20mg/mL into the deprotected octreotide-resin complex, wherein the volume ratio of the solvent is 4: 1, reacting for 5 hours, cracking octreotide from resin, and cyclizing a disulfide bond while cracking to obtain an octreotide crude product.
(5) Preparation of octreotide extract
Dissolving the crude octreotide product in acetonitrile water solution with the volume ratio of 5%, purifying by high performance liquid chromatography to obtain refined octreotide product, and freeze-drying to obtain 0.203g, the purity of 98.3% and the yield of 40%. The preparation conditions are as follows: c 4 The column was prepared with two mobile phases of 0.1% TFA in water and methanol, respectively.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (17)
1. A preparation method of octreotide is characterized by comprising the following steps: the method comprises the following steps:
(1) adding Fmoc-Thr (tBu) -OH and an esterification reagent into resin for esterification reaction to obtain an Fmoc-Thr (tBu) -resin compound;
(2) according to the solid-phase polypeptide synthesis method, according to the sequence of octreotide, the protected amino acids are coupled to the resin obtained in the step (1) one by one according to the sequence from C end to N end to obtain a protected octreotide-resin compound;
(3) adding a cutting reagent into the protected octreotide-resin compound, and removing a polypeptide side chain protecting group to obtain a deprotected octreotide-resin compound;
(4) adding a reducing agent into the deprotected octreotide-resin compound to reduce ester bonds formed in the esterification reaction in the step (1) into threoninol, simultaneously cracking octreotide from the resin, and cyclizing disulfide bonds while cracking to obtain an octreotide crude product;
(5) and separating and purifying the crude product of octreotide to obtain octreotide.
2. The method for producing octreotide according to claim 1, wherein: the resin in the step (1) is PAM resin, HMBA resin or Merrifield resin.
3. The method for producing octreotide according to claim 1 or 2, characterized in that: the esterification reagent in the step (1) is one or more of DIC, HOBt, DMAP and potassium fluoride.
4. The method for producing octreotide according to claim 3, wherein: the proportion of each substance in the step (1) is n [ Fmoc-Thr (tBu) -OH ]: n (DIC): n (HOBt): n (DMAP): n (potassium fluoride): n (resin) is 4 to 1: 4-1: 4-1: 0.5-0.1: 4-1: 1.
5. the method for producing octreotide according to claim 1, wherein: adding an Fmoc deprotection reagent into the Fmoc-Thr (tBu) -resin compound obtained in the step (1) to remove an Fmoc protecting group, washing the resin after the reaction is finished to obtain Thr (tBu) -resin, then adding a second amino acid at the C end of octreotide and a condensation reagent into the Thr (tBu) -resin compound to carry out condensation reaction, and washing the resin after the condensation reaction is finished; and (3) replacing amino acids according to the sequence of the octreotide from C end to N end, and repeating Fmoc deprotection reaction and condensation reaction to obtain the protected octreotide-resin compound.
6. The method for producing octreotide according to claim 5, wherein: the Fmoc deprotection reagent is prepared by the following steps of 1: 1-4 of piperidine and dimethylformamide, wherein the temperature of Fmoc deprotection reaction is 20-60 ℃.
7. The method for producing octreotide according to claim 5, wherein: the condensation reagent is one or a mixture of a condensation reagent 1, a condensation reagent 2 and a condensation reagent 3; wherein, the condensation reagent 1 is one of DCC, DIC, HBTU, HATU, BOP and PyBOP, the condensation reagent 2 is one of HOBt and HOAt, and the condensation reagent 3 is DIEA.
8. The method for producing octreotide according to claim 7, wherein the ratio of the amounts of the substances of the respective reagents is: amino acids: condensation reagent 1: condensation reagent 2: condensation reagent 3: and (3) resin is 6-2: 6-2: 6-2: 12-4: 1.
9. the process for the preparation of octreotide according to claim 7 or 8, wherein DCC or DIC is not used in combination with DIEA.
10. The method for producing octreotide according to claim 1, wherein: the cleavage reagent in step (3) is a TFA mixture, and preferably, the TFA volume ratio of the TFA mixture is not less than 80%.
11. The method for producing octreotide according to claim 10, wherein: the TFA mixture also comprises redistilled water, TES, DTT, Phenol, Thioanisole and NH besides the tri-TFA 4 I、TIS、Mixing one or more of Anisole and EDT.
12. The method for producing octreotide according to claim 1, wherein: and (4) the reducing agent is a lithium chloride mixed solution.
13. The method for producing octreotide according to claim 12, wherein: the lithium chloride mixed solution comprises one or more of sodium borohydride, lithium borohydride, calcium borohydride, potassium borohydride and sodium borohydride besides lithium chloride.
14. The method for producing octreotide according to claim 12 or 13, wherein: the amount ratio of lithium chloride in the lithium chloride mixed solution is 50%.
15. The method for producing octreotide according to claim 1, wherein: the ratio of the reducing agent to the resin in the step (4) is n (reducing agent): n (resin) is 20 to 5: 1, the concentration of the reducing agent solution is 5-20 mg/mL.
16. The method for producing octreotide according to claim 1, wherein: and (5) separating and purifying the crude octreotide product in the step (5) by using reverse-phase high performance liquid chromatography to obtain a refined octreotide product.
17. The method for producing octreotide according to claim 16, wherein: the purification conditions in the step (5) are as follows: the preparation column filler is carbon-supported silica gel, and the mobile phase is one or a mixture of water, acetonitrile or methanol solution of trifluoroacetic acid with the mass ratio of 0.1%.
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