CN113603751A - Method for synthesizing histrelin in full liquid phase - Google Patents
Method for synthesizing histrelin in full liquid phase Download PDFInfo
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- CN113603751A CN113603751A CN202111006173.0A CN202111006173A CN113603751A CN 113603751 A CN113603751 A CN 113603751A CN 202111006173 A CN202111006173 A CN 202111006173A CN 113603751 A CN113603751 A CN 113603751A
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- compound
- tbu
- condensing agent
- liquid phase
- boc
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- 239000007791 liquid phase Substances 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 46
- HHXHVIJIIXKSOE-QILQGKCVSA-N histrelin Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)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)CC(N=C1)=CN1CC1=CC=CC=C1 HHXHVIJIIXKSOE-QILQGKCVSA-N 0.000 title claims abstract description 29
- 229960002193 histrelin Drugs 0.000 title claims abstract description 29
- 108700020746 histrelin Proteins 0.000 title claims abstract description 29
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 19
- 150000001875 compounds Chemical class 0.000 claims abstract description 57
- 125000000031 ethylamino group Chemical group [H]C([H])([H])C([H])([H])N([H])[*] 0.000 claims abstract description 14
- 238000010511 deprotection reaction Methods 0.000 claims abstract description 13
- 229940125904 compound 1 Drugs 0.000 claims abstract description 11
- 229940125773 compound 10 Drugs 0.000 claims abstract description 7
- 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 abstract description 7
- 229940125782 compound 2 Drugs 0.000 claims abstract description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 94
- 238000006243 chemical reaction Methods 0.000 claims description 77
- 239000003795 chemical substances by application Substances 0.000 claims description 54
- 230000015572 biosynthetic process Effects 0.000 claims description 40
- 150000007530 organic bases Chemical class 0.000 claims description 40
- 238000003786 synthesis reaction Methods 0.000 claims description 40
- 239000002904 solvent Substances 0.000 claims description 35
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 34
- 239000007787 solid Substances 0.000 claims description 33
- 238000001914 filtration Methods 0.000 claims description 31
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 30
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 26
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 19
- 238000001035 drying Methods 0.000 claims description 19
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 17
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims description 16
- 229920001184 polypeptide Polymers 0.000 claims description 16
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 16
- 238000006482 condensation reaction Methods 0.000 claims description 14
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 14
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 13
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 12
- 239000007821 HATU Substances 0.000 claims description 12
- 239000012317 TBTU Substances 0.000 claims description 12
- 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 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 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 9
- PTSTYNLPDWVGLZ-RGMNGODLSA-N (2s)-n-ethylpyrrolidine-2-carboxamide;hydrochloride Chemical compound Cl.CCNC(=O)[C@@H]1CCCN1 PTSTYNLPDWVGLZ-RGMNGODLSA-N 0.000 claims description 8
- 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
- 229940126214 compound 3 Drugs 0.000 claims description 8
- 125000006239 protecting group Chemical group 0.000 claims description 8
- 239000012190 activator Substances 0.000 claims description 7
- 229940125898 compound 5 Drugs 0.000 claims description 7
- 230000001376 precipitating effect Effects 0.000 claims description 7
- 230000003213 activating effect Effects 0.000 claims description 6
- -1 benzyl ester Chemical class 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 5
- 230000009089 cytolysis Effects 0.000 claims description 5
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- 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 4
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 4
- 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 4
- 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 4
- 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 4
- 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 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- GVIXTVCDNCXXSH-AWEZNQCLSA-N (2s)-2-amino-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]pentanoic acid Chemical compound OC(=O)[C@@H](N)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C GVIXTVCDNCXXSH-AWEZNQCLSA-N 0.000 claims description 2
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 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
- 239000004480 active ingredient Substances 0.000 claims 1
- 239000013543 active substance Substances 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 239000012634 fragment Substances 0.000 abstract 2
- 238000007127 saponification reaction Methods 0.000 abstract 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 42
- 239000000463 material Substances 0.000 description 23
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 16
- 238000004128 high performance liquid chromatography Methods 0.000 description 14
- 238000005303 weighing Methods 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 239000003208 petroleum Substances 0.000 description 8
- 239000008213 purified water Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 6
- 230000007935 neutral effect Effects 0.000 description 6
- 238000001816 cooling Methods 0.000 description 4
- 238000001291 vacuum drying Methods 0.000 description 4
- SIRPVCUJLVXZPW-LJQANCHMSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1h-imidazol-5-yl)propanoic acid Chemical compound C([C@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CNC=N1 SIRPVCUJLVXZPW-LJQANCHMSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000011031 large-scale manufacturing process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- OUHPNBGKEMHUCQ-OAHLLOKOSA-N (2r)-3-(1-benzylimidazol-4-yl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound C1=NC(C[C@@H](NC(=O)OC(C)(C)C)C(O)=O)=CN1CC1=CC=CC=C1 OUHPNBGKEMHUCQ-OAHLLOKOSA-N 0.000 description 2
- 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 2
- AZBCBZPTQNYCPF-UHFFFAOYSA-N 3-(1-benzylimidazol-4-yl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)NC(C(=O)O)CC(N=C1)=CN1CC1=CC=CC=C1 AZBCBZPTQNYCPF-UHFFFAOYSA-N 0.000 description 2
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 2
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 2
- 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 2
- 238000003287 bathing Methods 0.000 description 2
- 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 2
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 2
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 2
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 2
- 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 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 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 1
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical group CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 208000006155 precocious puberty Diseases 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003746 solid phase reaction Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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Classifications
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- 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
-
- 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
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- 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)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a method for synthesizing histrelin in a full liquid phase, and relates to the technical field of medicines. The method comprises the following steps: synthesizing compound 1Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH and compound 2R by liquid phase method1‑D‑His(Bzl)‑Leu‑OR2The compound 2 is subjected to deprotection and then condensed with the compound 1 to obtain a compound 4, and the compound 4 is respectively inoculated into His (R) after deprotection3)‑、R4the-Pyr-fragment is used for obtaining a compound 7, the compound 7 is inoculated with an-Arg (pbf) -Pro-NHEt fragment after saponification to obtain a compound 10, and the compound 10 is cracked to obtain crude histrelin. The purity of the histrelin prepared by the method provided by the invention can reach more than 80%.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for synthesizing histrelin in a full liquid phase.
Background
Histrelin, having the formula: c66H86N18O12Molecular weight: 1323.5, respectively; is a nonapeptide analogue of artificially synthesized gonadotropin releasing hormone (GnRH), is used for treating central sexual precocity, and has certain inhibiting effect on advanced prostate cancer.
In the prior art, the synthesis of the histrelin is mainly a solid-phase method or a solid-liquid combination method, for example, the synthesis method of the histrelin with the application number of CN201210334868.6 needs to carry out peptide grafting operation on resin, has low product yield and low purity, and is not suitable for large-scale production. At present, no reports on the synthesis of histrelin by a full liquid phase method exist.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defects of high cost, more solvents, easy-to-prepare toxic reagents, high environmental protection pressure and low purity of crude products of the current mainstream solid-phase reaction, thereby providing a method for synthesizing the histrelin in a full liquid phase. The purity of the crude product can reach more than 80 percent, thereby being beneficial to large-scale production.
The invention provides a method for synthesizing histrelin in a full liquid phase, which comprises the following steps:
s1, synthesis of compound 1: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH;
s2, synthesis of compound 2: r1-D-His(Bzl)-Leu-OR2；
S3, synthesis of compound 3: H-D-His (Bzl) -Leu-OR2；
S4, synthesis of compound 4: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OR2；
S5, synthesis of compound 5: H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OR2；
S6, synthesis of compound 6: H-His (R)3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OR2；
S7, synthesis of compound 7:R4-Pyr-His(R3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OR2；
s8, synthesis of compound 8: r4-Pyr-His(R3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OH；
S9, synthesis of compound 9: H-Arg (pbf) -Pro-NHEt;
s10, synthesis of compound 10:
R4-Pyr-His(R3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-Arg(pbf)-Pro-NHEt；
s11, preparing a crude product of histrelin;
wherein R is1Is an amino protecting group, and comprises any one of Fmoc, Z and Boc; r2Is a carboxyl protecting group, and comprises any one of methyl ester Me, ethyl ester Et, benzyl ester Bzl and trityl ester Tr; r3Including any of Boc or Trt; r4The amino protecting group comprises any one of Fmoc, Z and Boc.
Preferably, step S1 specifically includes the following steps:
carrying out condensation reaction by taking Fmoc-Trp (Boc) -Ser (tBu) -OSu and H-Tyr (tBu) -OH as reaction units, and reacting in a solvent to obtain a compound 1; the molar ratio of Fmoc-Trp (Boc) -Ser (tBu) -OSu to H-Tyr (tBu) -OH is 1: 1.05-2, the molar ratio of H-Tyr (tBu) -OH to the organic base is 1:1, and the solvent comprises any one of DMF, THF, methanol, ethanol and NMP.
More preferably, the organic base is TEA, the molar ratio of Fmoc-Trp (Boc) -Ser (tBu) -OSu to H-Tyr (tBu) -OH is 1:1.1, the molar ratio of H-Tyr (tBu) -OH to the organic base is 1:1, the solvent comprises any one of DMF, THF, methanol, ethanol and NMP, and preferably, the solvent is DMF.
Preferably, step S2 specifically includes the following steps:
with R1-D-His(Bzl)-OH、H-Leu-OR2Carrying out a condensation reaction for the reaction unit, R1-D-His (Bzl) -OH and H-Leu-OR2The mol ratio of the components is 1: 1.05-2, an activating agent, organic alkali and condensation are addedThe ratio of H-Leu-OR2 to activator, condensing agent and organic base is 1:1, after the reaction is completed, the compound is filtered, separated out, washed and dried, and the solid is collected to obtain a compound 2;
the activator is an activator commonly used for polypeptide synthesis, and comprises any one of HOSu, HOBt, HOAt and HOOBt, and more preferably, the activator is HOSu; the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU, and more preferably, the condensing agent is DCC; the organic base comprises any one of DIEA, TEA and NMM, and more preferably, the organic base is TEA; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane, and more preferably the solvent is DMF.
Preferably, step S3 specifically includes the following steps:
taking the compound 2 prepared in the step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, precipitating, filtering, and drying in vacuum to obtain a compound 3;
the deprotection reagent comprises any one of trifluoroacetic acid, hydrogen chloride solution, diethylamine, piperazine and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM and THF.
Further, R1The amino protecting group is preferably Fmoc or Boc.
Further, R2Is a carboxyl protecting group, preferably Me or Et; more preferably Me.
When R is1In Fmoc, the deprotection reagent in step S3 is preferably diethylamine; when R is1In the case of Boc, the deprotecting reagent is preferably TFA.
Preferably, step S4 specifically includes the following steps:
carrying out condensation reaction by taking the compound 1 synthesized in the step S1 and the compound 3 synthesized in the step S3 as reaction units, wherein the molar ratio of the compound 1 to the compound 3 is 1: 1.05-2, adding an organic base and a condensing agent, wherein the molar ratio of the compound 3 to the organic base to the condensing agent is 1:1, and after the reaction in a solvent is completed, concentrating, filtering, washing and drying to obtain a compound 4;
the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane. More preferably, the solvent is DMF.
Preferably, step S5 specifically includes the following steps:
taking a compound 4, adding a deprotection reagent to react and remove Fmoc groups, concentrating to a small amount, separating out a solid, filtering, and drying in vacuum to obtain a compound 5; the deprotection reagent comprises any one of diethylamine, piperazine and piperidine solution.
Preferably, step S6 specifically includes the following steps:
with Fmoc-His (R)3) -OH, compound 5 synthesized in step S5 as a reaction unit, and carrying out a condensation reaction, wherein compound 5 and Fmoc-His (R)3) The molar ratio of-OH is 1: 1.05-2, and activating agent, organic base and condensing agent are added, wherein Fmoc-His (R) is3) The ratio of-OH to activating agent, condensing agent and organic base is 1:1, the reaction is completed in solvent, and the compound 6 is obtained by concentration, filtration, washing and drying;
the activator is commonly used for polypeptide synthesis and comprises any one of HOSu, HOBt, HOAt and HOOBt; the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane. More preferably, the solvent is DMF.
Preferably, step S7 specifically includes the following steps:
with R4A condensation reaction of-Pyr-OH and Compound 6 synthesized in step S6, wherein Compound 6 is reacted with R4The molar ratio of Pyr-OH is 1: 1.05-2; adding organic base and condensing agent, wherein R4The mol ratio of-Pyr-OH to the condensing agent to the organic base is 1:1, after the reaction is completed, the compound is obtained by filtering, washing and dryingAn object 7;
the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane. More preferably, the solvent is DMF.
Preferably, step S8 specifically includes the following steps:
taking methanol to react with the compound 7, slowly adding 2M NaOH, reacting for 2-4h, filtering, washing and drying to obtain a compound 8;
wherein the molar ratio of NaOH to the compound 7 is 1.5: 1-20: 1;
step S9 specifically includes the following steps:
with R1The condensation reaction is carried out by taking Arg (pbf) -OH and H-Pro-NHEt.HCl as reaction units, wherein R is1The mol ratio of-Arg (pbf) -OH to H-Pro-NHEt.HCl is 1: 1.05-2, organic base and condensing agent are added, wherein the mol ratio of H-Pro-NHEt.HCl to organic base and condensing agent is 1:1, after the reaction in solvent is completed, solid is separated out, and the compound 9 is obtained by filtering, drying, deprotection, concentration, solid separation, filtering and vacuum drying;
the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane. More preferably, the solvent is DMF.
When R is in the specification1R in-Arg (pbf) -OH1For the Boc group, 50% TFA/DCM was used as the deprotecting reagent, and pbf group was absent in compounds 9 and 10.
Preferably, step S10 specifically includes the following steps:
carrying out condensation reaction by taking a compound 8 and a compound 9 as reaction units, wherein the molar ratio of the compound 8 to the compound 9 is 1: 1.05-2, adding a condensing agent and an organic base, wherein the molar ratio of the compound 9 to the condensing agent to the organic base is 1:1, and filtering, washing and drying after the reaction is completed to obtain a compound 10;
the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane; more preferably, the solvent is DMF.
Step S11 specifically includes the following steps:
putting the compound 10 into a reactor, adding a lysis solution, precipitating with frozen ether, filtering, and collecting solid to obtain a crude product of histrelin; the components of the lysis solution comprise, by volume: TFA, TIS, H2O＝95∶2.5∶2.5。
The reagents used in the technical scheme are all common commercially available reagents; in the above technical solution, ether reagent is usually used for the operation of separating out or separating out solid, and includes any one or any combination of petroleum ether, isopropyl ether and diethyl ether, preferably petroleum ether.
The technical scheme of the invention has the following advantages:
the invention creatively invents a green and mild production process by a full liquid phase synthesis method, does not use any reagent which is extremely toxic and easy to produce toxic, produces the product with the yield of more than 85 percent, can achieve the purity of the crude product of more than 80 percent, greatly reduces the cost, and is very suitable for large-scale production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is an HPLC chromatogram of crude histrelin prepared in accordance with the present invention.
Detailed Description
The Chinese names corresponding to the English abbreviations of the substances appearing in the claims and the specification of the present invention are shown in Table 1.
TABLE 1
In the following examples, the purity of each compound and product was analyzed by HPLC, and unless otherwise specified, the HPLC conditions were as follows:
mobile phase A: 0.1% TFA/water, mobile phase B: 0.1% TFA/acetonitrile;
detection wavelength: 210 nm; flow rate: 1 ml/min; stationary phase: c18 column, 5 μ,
example 1
A method for synthesizing histrelin by a full liquid phase method comprises the following steps:
1. liquid phase synthesis of compound 1: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH
1.1 feeding:
the materials were dosed as per table 2.
TABLE 2
| Material(s) | Dosage of |
| Fmoc-Trp(Boc)-Ser(tBu)-OSU | 100mmol |
| H-Tyr(tBu)-OH | 110mmol |
| TEA | 110mmol |
| DMF | 400ml |
| 0.5M hydrochloric acid solution | 1L |
1.2 procedure
After Fmoc-Trp (Boc) -Ser (tBu) -OSu was completely dissolved in DMF, H-Tyr (tBu) -OH was accurately weighed and added to the reaction flask, and TEA (110 mmol) was added to start the reaction.
After stirring the reaction for 60min, the reaction was complete as detected by HPLC.
Pouring the reaction solution into a triangular flask twice, adding 0.5M hydrochloric acid, quickly stirring to separate out, filtering to obtain a solid, washing with purified water to be neutral, and drying at 30 ℃. The solid was collected, filled into a container and weighed. Yield: 96.2 percent; purity: 87.6 percent.
2. Liquid phase synthesis of compound 2: Fmoc-D-His (Bzl) -Leu-OMe
2.1 charging
The materials were dosed as per table 3.
TABLE 3
| Material(s) | Dosage of |
| Fmoc-D-His(Bzl)-OH | 150mmol |
| HOSU | 165mmol |
| TEA | 165mmol |
| DCC | 165mmol |
| H-Leu-OMe.HCl | 165mmol |
| DMF | 500ml |
| 0.5M hydrochloric acid solution | 1L |
2.2, operation process:
accurately weighing Fmoc-D-His (Bzl) -OH and HOSU in a reaction bottle, completely dissolving with DMF, then weighing H-Leu-OMe.HCl in a triangular flask, completely dissolving with DMF, then carrying out cold bath for 10min, adding TEA, rapidly shaking up, adding into the reaction bottle, continuing to carry out cold bath for 5min, then adding DCC to start reaction.
The reaction was complete after 1.5h by HPLC.
After complete reaction, the reaction solution was filtered, precipitated with 0.5M aqueous hydrochloric acid, and the filtered solid was washed with purified water to neutrality and dried at 30 ℃. Collecting the solid, filling into a triangular flask, and weighing; yield: 108.1%, purity: 93.4 percent.
3. Liquid phase synthesis of compound 3: H-D-His (Bzl) -Leu-OMe
3.1 feeding:
the materials were dosed as per table 4.
TABLE 4
| Material(s) | Dosage of |
| Fmoc-D-His(Bzl)-Leu-OMe.HCl | 150mmol |
| Diethylamine | 400ml |
| Petroleum ether | 1L |
3.2 procedure
Accurately weighing Fmoc-D-His (Bzl) -Leu-OMe.HCl in a reaction bottle, adding diethylamine to react for 20min, concentrating to a small amount, adding petroleum ether to precipitate a solid, filtering, and vacuum drying. Yield: 82.4%, purity: 87.2 percent.
4. Synthesis of the Compound 4Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OMe
4.1 feeding:
the materials were dosed as per table 5.
TABLE 5
| Material(s) | Dosage of |
| Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH | 96.2mmol |
| BOP | 105.8mmol |
| TEA | 105.8mmol |
| H-D-His(Bzl)-Leu-OMe | 105.8mmol |
| DMF | 200ml |
| DCM | 200ml |
| 0.5M hydrochloric acid solution | 1L |
4.2 procedure
Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH and BOP are accurately weighed into a reaction bottle, the reaction bottle is completely dissolved by DMF, then is cooled for 10min, then DIEA is added, H-D-His (Bzl) -Leu-OMe is dissolved by DCM, and then added into the reaction to start the reaction.
After the reaction is carried out for 1.0h, HPLC (high performance liquid chromatography) detection shows that the reaction is complete, concentration is carried out, 0.5M hydrochloric acid is used for precipitation, solid is collected by filtration, then purified water is used for washing until the solution is neutral (pH test paper detection), and weighing is carried out; yield of compound 4: 94.1%, purity: 88.6 percent.
5. Liquid phase synthesis of compound 5: H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OMe
5.1 charging
The materials were dosed as per table 6.
TABLE 6
| Material(s) | Dosage of |
| Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe | 90.5mmol |
| Diethylamine | 500ml |
| Petroleum ether | 1L |
5.2 procedure
Accurately weighing Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OMe in a reaction bottle, adding diethylamine to react for 20min, concentrating to a small amount, adding petroleum ether to precipitate a solid, filtering, and vacuum drying. Yield of compound 5: 93%, purity: 89 percent.
6. Liquid phase synthesis of compound 6: H-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OMe
6.1 charging
The materials were dosed as per table 7.
TABLE 7
6.2 procedure
Weighing Fmoc-His (Trt) -OH, HOBt, H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OMe in a reaction bottle, completely dissolving with DMF, cooling for 10min, and adding DCC to start reaction.
After reacting for 1.5h, detecting by HPLC to complete the reaction, filtering the reaction solution, pouring into a reaction bottle, adding diethylamine for reacting for 20min, concentrating to a small amount, adding 0.5M hydrochloric acid solution to precipitate a solid, filtering, and drying.
Putting Fmoc-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OMe into a reaction bottle, adding diethylamine for reaction for 20min, concentrating to a small amount, adding petroleum ether to precipitate a solid, filtering and drying.
Yield of compound 6: 89%, purity: 85 percent.
7. Liquid phase synthesis of compound 7:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe
7.1 charging
The material was dosed according to the material amounts of table 8.
TABLE 8
| Material(s) | Dosage of |
| Boc-Pyr-OH | 76.3mmol |
| BOP | 76.3mmol |
| TEA | 76.3mmol |
| H-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe | 69.4mmol |
| DMF | 350ml |
| 0.5M hydrochloric acid solution | 1L |
7.2 procedure
Accurately weighing Boc-Pyr-OH and BOP into a reaction bottle, completely dissolving the Boc-Pyr-OH and the BOP with DMF, cooling the reaction bottle for 10min, adding DIEA, dissolving H-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OMe with DMF, and adding the solution into the reaction to start the reaction.
After 1.5h of reaction, HPLC detection shows that the reaction is complete, the reaction solution is filtered, the filter residue is washed twice with DMF, precipitated with 0.5M hydrochloric acid, the solid is collected after filtration, washed with purified water to neutral (pH test paper detection), and dried.
Yield: 80%, purity: 85.1 percent.
8. Liquid phase synthesis of compound 8:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OH
8.1 charging
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OMe：55.5mmol
Methanol: 1110ml
2M NaOH：110ml
0.1M hydrochloric acid solution: 2L of
8.2 procedure
Weighing Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OMe, putting the mixture into methanol, stirring for 5min, completely dissolving, slowly adding 2M NaOH into the mixture for reaction, and starting the reaction.
After reacting for 3h, detecting the reaction is complete by HPLC, adding a hydrochloric acid solution, precipitating, filtering, collecting a solid, washing with purified water to be neutral (detected by a pH test paper), drying and weighing.
Yield: 83%, purity: 84 percent.
9. Liquid phase synthesis of compound 9: H-Arg (pbf) -Pro-NHEt
9.1 charging
The materials were dosed as per table 9.
TABLE 9
| Material(s) | Dosage of |
| Fmoc-Arg(pbf)-OH | 150mmol |
| BOP | 157.5mmol |
| TEA | 157.5mmol |
| H-Pro-NHEt.HCl | 157.5mmol |
| TEA | 157.5mmol |
| DMF | 400ml |
| 0.5M hydrochloric acid solution | 1L |
| Diethylamine | 500ml |
9.2 procedure
Fmoc-Arg (pbf) -OH and BOP are accurately weighed in a reaction flask, and are cooled for 10min after being completely dissolved by DMF, DIEA is added, the cooling bath is removed, and the reaction is carried out for 20 min.
Weighing H-Pro-NHEt.HCl in a triangular flask, completely dissolving with DMF, adding TEA, rapidly mixing, adding into the mixture, and starting the reaction.
After reacting for 2h, detecting the reaction is complete by HPLC, precipitating by using 0.5M hydrochloric acid, filtering, collecting a solid, washing by using purified water until the solid is neutral (detecting by using pH test paper), and drying; adding diethylamine to react for 20min, concentrating to a small amount, adding petroleum ether to precipitate solid, filtering, and vacuum drying. Yield 94%, purity: 96.3 percent.
10. Liquid phase synthesis of compound 10:
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-Arg(pbf)-Pro-NHEt
10.1 charging
The materials were dosed as in table 10.
Watch 10
| Material(s) | Dosage of |
| Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OH | 46.1mmol |
| BOP | 50.7mmol |
| TEA | 50.7mmol |
| H-Arg(pbf)-Pro-NHEt | 50.7mmol |
| DMF | 200ml |
| 0.5M hydrochloric acid solution | 500ml |
10.2 procedure
Accurately weighing Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OH and BOP into a reaction bottle, completely dissolving the mixture by DMF, cooling the solution for 10min, adding DIEA, dissolving H-Arg (pbf) -Pro-NHEt by DMF, and adding the solution into the reaction to start the reaction.
After 1.0h of reaction, HPLC detects the reaction is complete, 0.5M hydrochloric acid is added to precipitate a solid, the solid is collected by filtration, and then the solid is washed to be neutral by purified water (detected by pH test paper), dried and weighed.
Yield 104.2%, purity: 80.5 percent.
11. Synthesis of crude Himalathion
11.1 charging
Boc-Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-Arg(pbf)-Pro-NHEt：46.1mmol
Lysis buffer (TFA: TIS: H2O ═ 95: 2.5): 300ml
11.2 procedure
Adding Boc-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-Arg (pbf) -Pro-NHEt into a reaction bottle, adding lysate to react for 30min, precipitating with frozen ether, filtering, collecting solid to obtain a crude product, dissolving the crude product with water, and analyzing by HPLC detection. The yield thereof is as follows: 85.8 percent and the purity is 86.6 percent.
Example 2
A method for synthesizing histrelin by a full liquid phase method comprises the following steps:
1. liquid phase synthesis of compound 1: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH
The procedure is as in example 1.
2. Liquid phase synthesis of compound 2: Boc-D-His (Bzl) -Leu-OEt. HCl
2.1 charging
The materials were dosed as in table 11.
TABLE 11
| Material(s) | Dosage of |
| Boc-D-His(Bzl)-OH | 150mmol |
| HOSU | 165mmol |
| TEA | 165mmol |
| DCC | 165mmol |
| H-Leu-OEt.HCl | 300mmol |
| DMF | 500ml |
| 0.5M hydrochloric acid solution | 1L |
2.2, operation process:
accurately weighing Boc-D-His (Bzl) -OH and HOSU in a reaction bottle, completely dissolving with DMF, then weighing H-Leu-OEt.HCl in a triangular flask, completely dissolving with DMF, then cold-bathing for 10min, adding TEA, quickly shaking up, adding into the reaction bottle, continuing cold-bathing for 5min, adding DCC, and starting reaction. After 1h the reaction was complete as determined by HPLC. After complete reaction, the reaction solution was filtered, precipitated with 0.5M aqueous hydrochloric acid, and the filtered solid was washed with purified water to neutrality and dried at 30 ℃. The solid was collected, filled into a flask, and weighed. The yield thereof is as follows: 107.6%, purity: 92.8 percent.
3. Liquid phase synthesis of compound 3: H-D-His (Bzl) -Leu-OEt
150mmol of Compound 2 was weighed out into a reaction flask and 500ml of 50% TFA/DCM was added for 20min, and the rest was the same as in example 1. Yield: 82.1%, purity: 87.1 percent.
4. Synthesis of the Compound 4Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OEt
4.1 feeding:
the amounts charged are shown in Table 12.
TABLE 12
| Material(s) | Dosage of |
| Fmoc-Trp(Boc)-Ser(tBu)-Tyr(tBu)-OH | 96.2mmol |
| BOP | 105.8mmol |
| TEA | 105.8mmol |
| H-D-His(Bzl)-Leu-OEt | 192.4mmol |
| DMF | 200ml |
| DCM | 200ml |
| 0.5M hydrochloric acid solution | 1L |
The procedure was as in example 1.
The yield thereof is as follows: 94.5%, purity: 88.3 percent.
5. Liquid phase synthesis of compound 5: H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OEt
The procedure is as in example 1.
6. Liquid phase synthesis of compound 6: H-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OEt
6.1 charging
The materials were dosed as in table 13.
Watch 13
The procedure was as in example 1. Yield: 89.3%, purity: 84.5 percent.
7. Liquid phase synthesis of compound 7:
Boc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OEt
7.1 charging
The batch was carried out according to the contents of Table 14.
TABLE 14
The procedure was as in example 1.
Yield: 80.1%, purity: 85.1 percent.
8. Liquid phase synthesis of compound 8:
Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OH
8.1 charging
Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OEt：55.5mmol
Methanol: 1110ml
2M NaOH：1110ml
0.1M hydrochloric acid solution: 2L of
The procedure was as in example 1.
Yield: 82.8%, purity: 83.5 percent.
9. Liquid phase synthesis of compound 9: H-Arg-Pro-NHEt
9.1 charging
The amounts charged are shown in Table 15.
The procedure was as in example 1.
Yield 94.1%, purity: 96.3 percent.
10. Liquid phase synthesis of compound 10:
Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-Arg-Pro-NHEt
10.1 charging
The amounts charged are shown in Table 16.
TABLE 16
| Material(s) | Dosage of |
| Fmoc-Pyr-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OH | 46.1mmol |
| BOP | 46.1mmol |
| TEA | 46.1mmol |
| H-Arg-Pro-NHEt | 92.2mmol |
| DMF | 200ml |
| 0.5M hydrochloric acid solution | 500ml |
The procedure was as in example 1.
Yield 103%, purity: 80.3 percent.
11. Synthesis of crude Himalathion
Compound 10: 42.1mmol
Lysis solution (TFA: TIS: H)2O＝95∶2.5∶2.5)：300ml。
The procedure was as in example 1. The yield thereof is as follows: 85.5 percent and the purity is 84.6 percent.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for synthesizing histrelin in a full liquid phase is characterized by comprising the following steps:
s1, liquid-phase synthesis of compound 1: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -OH;
s2, liquid-phase synthesis of compound 2: r1-D-His(Bzl)-Leu-OR2；
S3, liquid phase synthesis of compound 3: H-D-His (Bzl) -Leu-OR2；
S4, liquid phase synthesis of compound 4: Fmoc-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OR2；
S5, liquid phase synthesis of compound 5: H-Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-His (Bzl) -Leu-OR2；
S6, liquid phase synthesis of compound 6: H-His (R)3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OR2；
S7, liquid phase synthesis of compound 7:
R4-Pyr-His(R3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OR2；
s8, liquid phase synthesis of compound 8:
R4-Pyr-His(R3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-OH；
s9, liquid phase synthesis of compound 9: H-Arg (pbf) -Pro-NHEt;
s10, liquid phase synthesis of compound 10:
R4-Pyr-His(R3)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-His(Bzl)-Leu-Arg(pbf)-Pro-NHEt；
s11, preparing a crude product of histrelin;
wherein R is1Is an amino protecting group, and comprises any one of Fmoc, Z and Boc; r2Is a carboxyl protecting group, and comprises any one of methyl ester Me, ethyl ester Et, benzyl ester Bzl and trityl ester Tr; r3Including any of Boc or Trt; r4The amino protecting group comprises any one of Fmoc, Z and Boc.
2. The method for synthesizing histrelin in full liquid phase according to claim 1, wherein step S1 specifically comprises the following steps:
carrying out condensation reaction by taking Fmoc-Trp (Boc) -Ser (tBu) -OSu and H-Tyr (tBu) -OH as reaction units, and reacting in a solvent to obtain a compound 1; the molar ratio of Fmoc-Trp (Boc) -Ser (tBu) -OSu to H-Tyr (tBu) -OH is 1: 1.05-2, the molar ratio of H-Tyr (tBu) -OH to the organic base is 1:1, and the solvent comprises any one of DMF, THF, methanol, ethanol and NMP.
3. The method for synthesizing histrelin in full liquid phase according to claim 1, wherein step S2 specifically comprises the following steps:
with R1-D-His(Bzl)-OH、H-Leu-OR2Carrying out a condensation reaction for the reaction unit, R1-D-His (Bzl) -OH and H-Leu-OR2The molar ratio of the active ingredients is 1: 1.05-2, and an activating agent, an organic base and a condensing agent are added into the mixture, H-Leu-OR2The ratio of the active agent to the condensing agent to the organic base is 1:1, after the reaction is completed, the mixture is filtered, separated out, washed and dried, and the solid is collected to obtain a compound 2;
the activator is commonly used for polypeptide synthesis and comprises any one of HOSu, HOBt, HOAt and HOOBt; the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.
4. The method for synthesizing histrelin in full liquid phase according to claim 1, wherein step S3 specifically comprises the following steps:
taking the compound 2 prepared in the step S2 as a substrate, adding a deprotection reagent and a solvent, concentrating to a small amount, precipitating, filtering, and drying in vacuum to obtain a compound 3;
the deprotection reagent comprises any one of trifluoroacetic acid, diethylamine, piperazine and piperidine; the solvent is any one of DMF, methanol, ethanol, DCM and THF.
5. The method for synthesizing histrelin in full liquid phase according to claim 1, wherein step S4 specifically comprises the following steps:
carrying out condensation reaction by taking the compound 1 synthesized in the step S1 and the compound 3 synthesized in the step S3 as reaction units, wherein the molar ratio of the compound 1 to the compound 3 is 1: 1.05-2, adding an organic base and a condensing agent, wherein the molar ratio of the compound 3 to the organic base to the condensing agent is 1:1, and after the reaction in a solvent is completed, concentrating, filtering, washing and drying to obtain a compound 4;
the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.
6. The method for synthesizing histrelin in full liquid phase according to claim 1, wherein step S5 comprises the following steps:
taking a compound 4, adding a deprotection reagent to react and remove Fmoc groups, concentrating to a small amount, separating out a solid, filtering, and drying in vacuum to obtain a compound 5; the deprotection reagent comprises any one of diethylamine, piperazine and piperidine solution.
7. The method for synthesizing histrelin in full liquid phase according to claim 1, wherein step S6 specifically comprises the following steps:
with Fmoc-His (R)3) -OH, compound 5 synthesized in step S5 as a reaction unit, and carrying out a condensation reaction, wherein compound 5 and Fmoc-His (R)3) The molar ratio of-OH is 1: 1.05-2, and activating agent, organic base and condensing agent are added, wherein Fmoc-His (R) is3) The ratio of-OH to an activating agent, a condensing agent and organic base is 1:1, the reaction is completed in a solvent, and a compound 6 is obtained after concentration, filtration, washing, drying and deprotection;
the activator is commonly used for polypeptide synthesis and comprises any one of HOSu, HOBt, HOAt and HOOBt; the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.
8. The method for synthesizing histrelin in full liquid phase according to claim 1, wherein step S7 specifically comprises the following steps:
with R4A condensation reaction of-Pyr-OH and Compound 6 synthesized in step S6, wherein Compound 6 is reacted with R4The molar ratio of Pyr-OH is 1: 1.05-2; adding organic base and a condensing agent, wherein the molar ratio of R4-Pyr-OH to the condensing agent to the organic base is 1:1, and after the reaction is completed, filtering, washing and drying to obtain a compound 7;
the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.
9. The method for synthesizing histrelin in full liquid phase according to claim 1, wherein step S8 specifically comprises the following steps:
taking methanol to react with the compound 7, slowly adding 2M NaOH, reacting for 2-4h, filtering, washing and drying to obtain a compound 8;
wherein the molar ratio of NaOH to the compound 7 is 1.5: 1-20: 1;
step S9 specifically includes the following steps:
with R1The condensation reaction is carried out by taking Arg (pbf) -OH and H-Pro-NHEt.HCl as reaction units, wherein R is1The mol ratio of-Arg (pbf) -OH to H-Pro-NHEt.HCl is 1: 1.05-2, organic base and condensing agent are added, wherein the mol ratio of H-Pro-NHEt.HCl to organic base and condensing agent is 1:1, solid is precipitated after the reaction in solvent is completed, the solid is filtered, dried, the diethylamine is dissolved and reacted, the solid is precipitated, the solid is filtered and dried in vacuum, and the compound 9 is obtained;
the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane.
10. The method for synthesizing histrelin in full liquid phase according to claim 1, wherein step S10 specifically comprises the following steps:
carrying out condensation reaction by taking a compound 8 and a compound 9 as reaction units, wherein the molar ratio of the compound 8 to the compound 9 is 1: 1.05-2, adding a condensing agent and an organic base, wherein the molar ratio of the compound 9 to the condensing agent to the organic base is 1:1, and filtering, washing and drying after the reaction is completed to obtain a compound 10;
the condensing agent is a condensing agent commonly used for polypeptide synthesis and comprises any one of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU and HATU; the organic base comprises any one of DIEA, TEA and NMM; the solvent comprises any one of THF, DCM, DMF, NMP and dioxane;
step S11 specifically includes the following steps:
putting the compound 10 into a reactor, adding a lysis solution, precipitating with frozen ether, filtering, and collecting solid to obtain a crude product of histrelin;
the components of the lysis solution comprise, by volume: TFA, TIS, H2O＝95∶2.5∶2.5。
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| PCT/CN2022/115636 WO2023030277A1 (en) | 2021-08-30 | 2022-08-29 | Method for fully liquid-phase synthesis of grnh nonapeptide amide analog |
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| EP0889901A1 (en) * | 1996-03-19 | 1999-01-13 | Abbott Laboratories | Process and intermediates for the synthesis of lhrh antagonists |
| CN102850437A (en) * | 2012-09-12 | 2013-01-02 | 上海吉尔多肽有限公司 | Histrelin synthesizing method |
| CN112279894A (en) * | 2020-10-12 | 2021-01-29 | 湖南津安生物科技有限公司 | Method for synthesizing alarelin by polypeptide solid-liquid combination |
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- 2021-08-30 CN CN202111006173.0A patent/CN113603751A/en active Pending
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|---|---|---|---|---|
| EP0889901A1 (en) * | 1996-03-19 | 1999-01-13 | Abbott Laboratories | Process and intermediates for the synthesis of lhrh antagonists |
| CN102850437A (en) * | 2012-09-12 | 2013-01-02 | 上海吉尔多肽有限公司 | Histrelin synthesizing method |
| CN112279894A (en) * | 2020-10-12 | 2021-01-29 | 湖南津安生物科技有限公司 | Method for synthesizing alarelin by polypeptide solid-liquid combination |
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