WO2024093907A1 - 向眼部和中枢神经系统递送sirna的配体 - Google Patents
向眼部和中枢神经系统递送sirna的配体 Download PDFInfo
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- WO2024093907A1 WO2024093907A1 PCT/CN2023/127728 CN2023127728W WO2024093907A1 WO 2024093907 A1 WO2024093907 A1 WO 2024093907A1 CN 2023127728 W CN2023127728 W CN 2023127728W WO 2024093907 A1 WO2024093907 A1 WO 2024093907A1
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- Prior art keywords
- compound
- nhc
- alkyl
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- mmol
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- 108020004459 Small interfering RNA Proteins 0.000 title claims description 41
- 239000003446 ligand Substances 0.000 title description 8
- 210000003169 central nervous system Anatomy 0.000 title description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 213
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 113
- 239000002773 nucleotide Substances 0.000 claims abstract description 93
- 150000003839 salts Chemical class 0.000 claims abstract description 70
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 66
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims abstract description 59
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 32
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 4
- 125000000217 alkyl group Chemical group 0.000 claims description 124
- -1 thiophosphate diester Chemical class 0.000 claims description 83
- 230000000692 anti-sense effect Effects 0.000 claims description 62
- 108091081021 Sense strand Proteins 0.000 claims description 54
- 239000004055 small Interfering RNA Substances 0.000 claims description 49
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 36
- 229910052739 hydrogen Inorganic materials 0.000 claims description 35
- 125000000171 (C1-C6) haloalkyl group Chemical group 0.000 claims description 34
- 239000007787 solid Substances 0.000 claims description 31
- 125000000304 alkynyl group Chemical group 0.000 claims description 30
- 230000000295 complement effect Effects 0.000 claims description 30
- 239000000126 substance Substances 0.000 claims description 28
- 125000001424 substituent group Chemical group 0.000 claims description 28
- 229910052799 carbon Inorganic materials 0.000 claims description 26
- 125000004432 carbon atom Chemical group C* 0.000 claims description 26
- 229910052736 halogen Inorganic materials 0.000 claims description 26
- 150000002367 halogens Chemical class 0.000 claims description 26
- 125000006239 protecting group Chemical group 0.000 claims description 26
- 125000003342 alkenyl group Chemical group 0.000 claims description 23
- 125000003118 aryl group Chemical group 0.000 claims description 23
- 125000001165 hydrophobic group Chemical group 0.000 claims description 22
- 229910052757 nitrogen Inorganic materials 0.000 claims description 21
- 229910052805 deuterium Inorganic materials 0.000 claims description 20
- 108020004999 messenger RNA Proteins 0.000 claims description 20
- 229920006395 saturated elastomer Polymers 0.000 claims description 19
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 claims description 18
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 claims description 18
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 16
- 125000005647 linker group Chemical group 0.000 claims description 16
- 229910019142 PO4 Inorganic materials 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 125000002947 alkylene group Chemical group 0.000 claims description 13
- 125000005842 heteroatom Chemical group 0.000 claims description 13
- 229910052760 oxygen Inorganic materials 0.000 claims description 13
- 229910052717 sulfur Inorganic materials 0.000 claims description 13
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 12
- 125000000025 triisopropylsilyl group Chemical group C(C)(C)[Si](C(C)C)(C(C)C)* 0.000 claims description 12
- 125000000739 C2-C30 alkenyl group Chemical group 0.000 claims description 11
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 claims description 11
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 11
- 125000004184 methoxymethyl group Chemical group [H]C([H])([H])OC([H])([H])* 0.000 claims description 11
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 claims description 10
- 125000004092 methylthiomethyl group Chemical group [H]C([H])([H])SC([H])([H])* 0.000 claims description 10
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 9
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 9
- 125000002743 phosphorus functional group Chemical group 0.000 claims description 9
- 239000007790 solid phase Substances 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 8
- 239000010452 phosphate Substances 0.000 claims description 8
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 7
- 229910052698 phosphorus Inorganic materials 0.000 claims description 7
- 239000000651 prodrug Chemical group 0.000 claims description 7
- 229940002612 prodrug Drugs 0.000 claims description 7
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 7
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 claims description 6
- DCERHCFNWRGHLK-UHFFFAOYSA-N C[Si](C)C Chemical compound C[Si](C)C DCERHCFNWRGHLK-UHFFFAOYSA-N 0.000 claims description 6
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims description 6
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 claims description 6
- 150000008300 phosphoramidites Chemical class 0.000 claims description 6
- ILMRJRBKQSSXGY-UHFFFAOYSA-N tert-butyl(dimethyl)silicon Chemical compound C[Si](C)C(C)(C)C ILMRJRBKQSSXGY-UHFFFAOYSA-N 0.000 claims description 6
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 6
- 125000000037 tert-butyldiphenylsilyl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1[Si]([H])([*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 6
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 6
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 6
- 125000000923 (C1-C30) alkyl group Chemical group 0.000 claims description 5
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 5
- LJCZNYWLQZZIOS-UHFFFAOYSA-N 2,2,2-trichlorethoxycarbonyl chloride Chemical compound ClC(=O)OCC(Cl)(Cl)Cl LJCZNYWLQZZIOS-UHFFFAOYSA-N 0.000 claims description 5
- UTQNKKSJPHTPBS-UHFFFAOYSA-N 2,2,2-trichloroethanone Chemical group ClC(Cl)(Cl)[C]=O UTQNKKSJPHTPBS-UHFFFAOYSA-N 0.000 claims description 5
- WSNDAYQNZRJGMJ-UHFFFAOYSA-N 2,2,2-trifluoroethanone Chemical compound FC(F)(F)[C]=O WSNDAYQNZRJGMJ-UHFFFAOYSA-N 0.000 claims description 5
- 125000001313 C5-C10 heteroaryl group Chemical group 0.000 claims description 5
- 125000002668 chloroacetyl group Chemical group ClCC(=O)* 0.000 claims description 5
- HRDXJKGNWSUIBT-UHFFFAOYSA-N methoxybenzene Chemical group [CH2]OC1=CC=CC=C1 HRDXJKGNWSUIBT-UHFFFAOYSA-N 0.000 claims description 5
- DVFXLNFDWATPMW-IWOKLKJTSA-N tert-butyldiphenylsilyl Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO[Si](C=2C=CC=CC=2)(C=2C=CC=CC=2)C(C)(C)C)[C@@H](OP(O)(=O)OC[C@@H]2[C@H](C[C@@H](O2)N2C3=C(C(NC(N)=N3)=O)N=C2)OP(O)(=O)OC[C@@H]2[C@H](C[C@@H](O2)N2C3=C(C(NC(N)=N3)=O)N=C2)OP(O)(=O)OC[C@@H]2[C@H](C[C@@H](O2)N2C3=C(C(NC(N)=N3)=O)N=C2)OP(O)(=O)OC[C@@H]2[C@H](CC(O2)N2C3=NC=NC(N)=C3N=C2)OP(O)(=O)OC[C@@H]2[C@H](C[C@@H](O2)N2C3=C(C(NC(N)=N3)=O)N=C2)O)C1 DVFXLNFDWATPMW-IWOKLKJTSA-N 0.000 claims description 5
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 claims description 5
- 125000006376 (C3-C10) cycloalkyl group Chemical group 0.000 claims description 4
- 125000006714 (C3-C10) heterocyclyl group Chemical group 0.000 claims description 4
- 125000004450 alkenylene group Chemical group 0.000 claims description 4
- 125000004419 alkynylene group Chemical group 0.000 claims description 4
- 125000006538 C11 alkyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 3
- 125000006539 C12 alkyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 3
- 125000005600 alkyl phosphonate group Chemical group 0.000 claims description 3
- 239000001177 diphosphate Substances 0.000 claims description 3
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 3
- 235000011180 diphosphates Nutrition 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 3
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
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- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 3
- 239000001226 triphosphate Substances 0.000 claims description 3
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- 125000006708 (C5-C14) heteroaryl group Chemical group 0.000 claims description 2
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 claims description 2
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 claims description 2
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 2
- 125000006245 phosphate protecting group Chemical group 0.000 claims description 2
- 125000004437 phosphorous atom Chemical group 0.000 claims description 2
- 230000003637 steroidlike Effects 0.000 claims description 2
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 claims 2
- 230000002401 inhibitory effect Effects 0.000 claims 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 claims 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 4
- 230000008499 blood brain barrier function Effects 0.000 abstract description 3
- 210000001218 blood-brain barrier Anatomy 0.000 abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 257
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 28
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- WPWRNMZBLPVTAW-UHFFFAOYSA-N dichloromethane;2,2,2-trichloroacetic acid Chemical compound ClCCl.OC(=O)C(Cl)(Cl)Cl WPWRNMZBLPVTAW-UHFFFAOYSA-N 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- BJVWCKXHSNBHGB-UHFFFAOYSA-L disodium;chloride;hydroxide Chemical compound [OH-].[Na+].[Na+].[Cl-] BJVWCKXHSNBHGB-UHFFFAOYSA-L 0.000 description 1
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- 238000001647 drug administration Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 1
- 239000003885 eye ointment Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
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- 125000004404 heteroalkyl group Chemical group 0.000 description 1
- UQEAIHBTYFGYIE-UHFFFAOYSA-N hexamethyldisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)C UQEAIHBTYFGYIE-UHFFFAOYSA-N 0.000 description 1
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 125000006038 hexenyl group Chemical group 0.000 description 1
- 125000005980 hexynyl group Chemical group 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
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- 125000001921 locked nucleotide group Chemical group 0.000 description 1
- 229960001929 meloxicam Drugs 0.000 description 1
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- TUDYPXFSYJRWDP-UHFFFAOYSA-N methoxy methyl carbonate Chemical compound COOC(=O)OC TUDYPXFSYJRWDP-UHFFFAOYSA-N 0.000 description 1
- 150000005217 methyl ethers Chemical class 0.000 description 1
- CPZBTYRIGVOOMI-UHFFFAOYSA-N methylsulfanyl(methylsulfanylmethoxy)methane Chemical compound CSCOCSC CPZBTYRIGVOOMI-UHFFFAOYSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- ONKSSDKXDIVIHK-UHFFFAOYSA-N n,n-didecyldodecanamide Chemical group CCCCCCCCCCCC(=O)N(CCCCCCCCCC)CCCCCCCCCC ONKSSDKXDIVIHK-UHFFFAOYSA-N 0.000 description 1
- DSWNRHCOGVRDOE-UHFFFAOYSA-N n,n-dimethylmethanimidamide Chemical compound CN(C)C=N DSWNRHCOGVRDOE-UHFFFAOYSA-N 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- HGASFNYMVGEKTF-UHFFFAOYSA-N octan-1-ol;hydrate Chemical compound O.CCCCCCCCO HGASFNYMVGEKTF-UHFFFAOYSA-N 0.000 description 1
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 1
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- 125000004817 pentamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 125000003538 pentan-3-yl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000002255 pentenyl group Chemical group C(=CCCC)* 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 125000005981 pentynyl group Chemical group 0.000 description 1
- TYZYRCHEVXXLSJ-UHFFFAOYSA-N phenylmethoxymethoxymethoxymethylbenzene Chemical compound C=1C=CC=CC=1COCOCOCC1=CC=CC=C1 TYZYRCHEVXXLSJ-UHFFFAOYSA-N 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
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- 238000003753 real-time PCR Methods 0.000 description 1
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- 238000010839 reverse transcription Methods 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 210000003786 sclera Anatomy 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
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- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- FGTJJHCZWOVVNH-UHFFFAOYSA-N tert-butyl-[tert-butyl(dimethyl)silyl]oxy-dimethylsilane Chemical compound CC(C)(C)[Si](C)(C)O[Si](C)(C)C(C)(C)C FGTJJHCZWOVVNH-UHFFFAOYSA-N 0.000 description 1
- 125000001973 tert-pentyl group Chemical group [H]C([H])([H])C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 125000000383 tetramethylene group Chemical group [H]C([H])([*:1])C([H])([H])C([H])([H])C([H])([H])[*:2] 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000003354 tissue distribution assay Methods 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-M toluene-4-sulfonate Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-M 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- PLPCIGCUDCHUFB-UHFFFAOYSA-N tri(propan-2-yl)-[tri(propan-2-yl)silyloxymethoxymethoxy]silane Chemical compound CC(C)[Si](C(C)C)(C(C)C)OCOCO[Si](C(C)C)(C(C)C)C(C)C PLPCIGCUDCHUFB-UHFFFAOYSA-N 0.000 description 1
- LGSAOJLQTXCYHF-UHFFFAOYSA-N tri(propan-2-yl)-tri(propan-2-yl)silyloxysilane Chemical compound CC(C)[Si](C(C)C)(C(C)C)O[Si](C(C)C)(C(C)C)C(C)C LGSAOJLQTXCYHF-UHFFFAOYSA-N 0.000 description 1
- 125000005039 triarylmethyl group Chemical group 0.000 description 1
- 150000005691 triesters Chemical class 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 125000003774 valeryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
- C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
- C07D265/30—1,4-Oxazines; Hydrogenated 1,4-oxazines not condensed with other rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
Definitions
- the present invention belongs to the field of medicine, and specifically relates to a hydrophobic group having the ability to enhance the ability of double-stranded RNA to pass through the blood-brain barrier and/or be delivered to the eye, such as the R group in formula (I), and a compound of formula (I) in which the hydrophobic group is connected to a nucleotide, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
- RNA interference is a phenomenon in which double-stranded RNA (dsRNA, also known as siRNA) induces efficient and specific degradation of target mRNA.
- dsRNA double-stranded RNA
- siRNA siRNA
- siRNA due to the presence of the blood-brain barrier, it is difficult to deliver siRNA to the central nervous system to exert its effect, which limits the application of siRNA.
- Some attempts have been made in the art to deliver siRNA to the central nervous system such as WO2004094595A2, which discloses the use of a single lipid ligand (such as cholesterol or long-chain alkane) at the end of the chain to deliver siRNA, WO2019217459A1, which discloses the use of a single lipid ligand to deliver siRNA inside the chain, and WO2021092371A2, which discloses a series of new lipid ligand structures.
- the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
- the present invention provides an oligonucleotide comprising one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
- the present invention provides a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and/or the antisense strand comprises one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
- hydrophobic group (R group in formula (I)) provided by the present invention can be linked to the siRNA via an existing linker structure, such as a biodegradable linker structure.
- the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned double-stranded RNA.
- the present invention provides a cell containing the aforementioned double-stranded RNA or the aforementioned vector.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cell, and optionally a pharmaceutically acceptable carrier or excipient.
- the present invention provides a kit comprising the aforementioned double-stranded RNA, the aforementioned vector, or the aforementioned cell.
- FIG1 shows the reduction in the expression of SOD1 in the cervical spinal cord, thoracic spinal cord, cerebellum, brainstem, hippocampus and frontal cortex after puncture and injection of the siRNA of the present invention in SD rats.
- FIG. 2 shows the reduction in the expression of the TTR gene in the eyes of C57BL/6 mice after bilateral intravitreal injection of the siRNA of the present invention.
- C 1-6 alkyl includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.
- C 1-30 alkyl refers to a straight or branched saturated hydrocarbon group having 1 to 30 carbon atoms. In some embodiments, C 5-25 alkyl, C 10-20 alkyl, C 1-20 alkyl, C 1-10 alkyl and C 1-6 alkyl are preferred.
- C 1-6 alkyl examples include: methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), isopropyl (C 3 ), n-butyl (C 4 ), tert-butyl (C 4 ), sec-butyl (C 4 ), isobutyl (C 4 ), n-pentyl (C 5 ), 3-pentyl (C 5 ), pentyl (C 5 ), neopentyl (C 5 ), 3-methyl-2-butyl (C 5 ), tert-pentyl (C 5 ) and n-hexyl (C 6 ).
- C 1-6 alkyl also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus).
- the alkyl group may be optionally substituted by one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
- alkyl abbreviations include: Me(-CH 3 ), Et(-CH 2 CH 3 ), iPr(-CH(CH 3 ) 2 ), nPr(-CH 2 CH 2 CH 3 ), n-Bu(-CH 2 CH 2 CH 2 CH 3 ) or i-Bu(-CH 2 CH(CH 3 ) 2 ).
- C2-30 alkenyl refers to a straight or branched hydrocarbon group having 2 to 30 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C10-25 alkenyl, C2-10 alkenyl, C2-6 alkenyl and C2-4 alkenyl are preferred. Examples of C2-6 alkenyl include: vinyl ( C2 ), 1-propenyl ( C3 ), 2-propenyl ( C3 ), 1-butenyl ( C4 ), 2-butenyl (C4), butadienyl ( C4 ), pentenyl ( C5 ), pentadienyl ( C5 ), hexenyl ( C6 ), and the like.
- C2-6 alkenyl also includes heteroalkenyl, wherein one or more (e.g., 1 , 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus).
- An alkenyl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
- C 2-30 alkynyl refers to a straight or branched hydrocarbon group having 2 to 30 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 10-25 alkynyl, C 2-10 alkynyl, C 2-6 alkynyl and C 2-4 alkynyl are preferred.
- C 2-6 alkynyl examples include, but are not limited to, ethynyl (C 2 ), 1-propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ), pentynyl (C 5 ), hexynyl (C 6 ), and the like.
- C 2-6 alkynyl also includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus).
- An alkynyl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
- C 1-10 alkylene refers to a divalent group formed by removing another hydrogen of a C 1-10 alkyl, a C 2-10 alkenyl and a C 2-10 alkynyl, respectively, and may be substituted or unsubstituted.
- C 2-8 alkylene, C 3-7 alkylene, C 4-6 alkylene, C 1-4 alkylene, C 2-4 alkylene and C 1-3 alkylene are preferred.
- Unsubstituted alkylene includes, but is not limited to, methylene (—CH 2 —), ethylene (—CH 2 CH 2 —), propylene (—CH 2 CH 2 CH 2 —), butylene (—CH 2 CH 2 CH 2 CH 2 —), pentylene (— CH 2 CH 2 CH 2 CH 2 CH 2 —), hexylene (—CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 —), and the like.
- substituted alkylene groups for example, substituted alkylene groups with one or more alkyl(methyl) groups, include, but are not limited to, substituted methylene groups (—CH(CH 3 )—, —C(CH 3 ) 2 —), substituted ethylene groups (—CH(CH 3 )CH 2 —, —CH 2 CH(CH 3 )—, —C(CH 3 ) 2 CH 2 —, —CH 2 C(CH 3 ) 2 — ), substituted propylene groups (—CH(CH 3 )CH 2 CH 2 —, —CH 2 CH(CH 3 )CH 2 —, —CH 2 CH 2 CH(CH 3 )—, —C(CH 3 ) 2 CH 2 CH 2 —, —CH 2 C(CH 3 ) 2 CH 2 —, —CH 2 CH 2 C(CH 3 ) 2 —), and the like.
- substituted methylene groups —CH(CH 3 )—, —C(CH 3 ) 2 —
- C 0-10 alkylene group means a chemical bond as well as the above-mentioned "C 1-10 alkylene group”.
- Halo or "halogen” refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
- C 1-20 haloalkyl refers to the above-mentioned “C 1-20 alkyl”, “C 1-6 alkyl” and “C 1-4 alkyl", respectively, which are substituted with one or more halogen groups.
- C 1-4 haloalkyl is particularly preferred, and C 1-2 haloalkyl is more preferred.
- haloalkyl groups include, but are not limited to, -CF 3 , -CH 2 F, -CHF 2 , -CHFCH 2 F, -CH 2 CHF 2 , -CF 2 CF 3 , -CCl 3 , -CH 2 Cl, -CHCl 2 , 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like.
- the haloalkyl group can be substituted at any available attachment point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
- hydrophobic group refers broadly to any chemical group that has an affinity for lipids.
- One way to characterize the hydrophobicity of a hydrophobic group is by the octanol-water partition coefficient logKow , where Kow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase when a two-phase system is at equilibrium.
- logKow of the hydrophobic moiety is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10.
- the hydrophobic moiety is the R group in the compound of formula I.
- Alkyl, alkenyl, alkynyl, etc. as defined herein are optionally substituted groups.
- each of Raa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two Ra groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
- each of R bb is independently selected from the group consisting of hydrogen, -OH, -OR aa , -N(R cc ) 2 , -CN, -C( ⁇ O)R aa , -C( ⁇ O)N(R cc ) 2 , -CO 2 R aa , -SO 2 R aa , -C( ⁇ NR cc )OR aa , -C( ⁇ NR cc )N(R cc ) 2 , -SO 2 N(R cc ) 2 , -SO 2 R cc , -SO 2 OR cc , -SOR aa , -C( ⁇ S)N(R cc ) 2 , -C( ⁇ O)SR cc , -C( ⁇ S)SR cc , -P( ⁇ O) 2 R aa , -P( ⁇ O)(R aa ) 2
- each of R cc is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R cc groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R dd groups;
- Each of R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;
- each of Rff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two Rff groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 Rgg groups;
- siRNA refers to a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary thereto.
- a target RNA e.g., mRNA, e.g., a transcript of a gene encoding a protein
- siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA, and a sense strand complementary to the antisense strand.
- mRNA is also referred to herein as mRNA to be silenced.
- a gene is also referred to as a target gene.
- the RNA to be silenced is an endogenous gene or a pathogen gene.
- RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.
- antisense strand refers to a strand of an siRNA that includes a region that is completely, fully or substantially complementary to a target sequence.
- sense strand refers to a strand of an siRNA that includes a region that is completely, fully or substantially complementary to a region that is the antisense strand as defined herein.
- complementary region refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence.
- mispairing can be located in the interior or terminal regions of the molecule.
- the most tolerated mispairing is located in the terminal regions, for example, within 5, 4, 3, 2 or 1 nucleotides at 5' and/or 3' ends.
- the antisense strand portion that is most sensitive to mispairing is referred to as a "seed region".
- seed region For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.
- complementary refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions.
- stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50°C or 70°C for 12-16 hours.
- “complementary” sequences may also include or be formed entirely from non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides.
- Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.
- a polynucleotide that is "at least partially complementary,” “fully complementary,” or “substantially complementary” to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest.
- mRNA messenger RNA
- a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding PCSK9.
- Fully complementary refers to the extent to which the sense strand only needs to be complementary to the antisense strand in order to maintain the overall double-stranded characteristics of the molecule.
- perfect complementarity is generally required, in some cases, particularly in the antisense strand, one or more, such as 6, 5, 4, 3, 2 or 1 mismatches (relative to the target mRNA) may be included, but the sense strand and the antisense strand can still maintain the overall double-stranded characteristics of the molecule.
- shRNA refers to short hairpin RNA.
- shRNA includes two short inverted repeat sequences.
- the shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences separated by a stem-loop sequence in the middle, forming a hairpin structure controlled by the polIII promoter. Then 5-6 Ts are connected as the transcription terminator of RNA polymerase III.
- Nucleoside is a compound composed of two substances, a purine base or a pyrimidine base, and ribose or deoxyribose
- nucleotide is a compound composed of three substances, a purine base or a pyrimidine base, ribose or deoxyribose, and phosphate
- oligonucleotide refers to a nucleic acid molecule (RNA or DNA) with a length of, for example, less than 100, 200, 300 or 400 nucleotides.
- Base is the basic unit of synthesis of nucleosides, nucleotides and nucleic acids. It contains nitrogen and is also called “nitrogenous base”.
- capital letters A, U, T, G and C represent the base composition of nucleotides, which are adenine, uracil, thymine, guanine and cytosine respectively.
- the "modification" of the nucleotides described herein includes, but is not limited to, methoxy modification, fluorination modification, phosphorothioate linkage, or conventional protecting group protection, etc.
- the fluorination-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced by fluorine
- the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
- Modified nucleotides herein include, but are not limited to, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, nucleotides containing thiophosphate groups, vinyl phosphate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases containing nucleotides, and terminal nucleotides, deoxyribonucleotides or conventional protective groups connected to cholesterol derivatives or dodecanoic acid didecylamide groups.
- the 2'-fluoro modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine.
- the 2'-deoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
- Ligand moiety refers to a chemical moiety conjugated to a siRNA that is capable of altering the distribution, targeting or lifetime of the siRNA.
- a ligand provides enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cell or organ compartment, a tissue, an organ, or a region of the body) compared to, for example, a siRNA in the absence of such a ligand.
- Reactive phosphorus group refers to a phosphorus-containing group contained in a nucleotide unit or a nucleotide analog unit, which can react with a hydroxyl or amine group contained in another molecule, especially in another nucleotide unit or in another nucleotide analog, by a nucleophilic attack reaction. Typically, such a reaction produces an ester-type internucleoside bond connecting the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit.
- the reactive phosphorus group can be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, including but not limited to: natural phosphates, thiophosphates, dithiophosphates, borane phosphates, borane thiophosphates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, thiophosphorodiesters, thiophosphorothioates, diphosphates and triphosphates, preferably -P(OCH 2 CH 2 CN)(N(iPr) 2 ).
- Protecting group refers to any atom or group of atoms added to a molecule to prevent an existing group in the molecule from undergoing an undesirable chemical reaction.
- a “protecting group” may be an unstable chemical moiety known in the art that is used to protect reactive groups, such as hydroxyl, amino, and thiol groups, to prevent undesirable or inappropriate reactions during chemical synthesis.
- Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group intact or available for further reactions.
- a non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butyloxycarbonyl (BOC), acetyl or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy] methyl or tert-butyldiphenylsilyl); esters (e.g.
- Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), ⁇ -methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, neopentyl (V), benzyloxymethyl acetal (BOM), benzyloxymethyl ether (BOM ...
- Hydro protecting group refers to a group that can protect the hydroxyl group from chemical reactions and can be removed under specific conditions to restore the hydroxyl group. It mainly includes silane type protecting groups, acyl type protecting groups or ether type protecting groups, preferably the following:
- TMS trimethylsilyl
- TES triethylsilyl
- DMIPS dimethylisopropylsilyl
- DEIPS diethylisopropylsilyl
- TDMS tert-butyldimethylsilyl
- TDPS tert-butyldiphenylsilyl
- TIPS triisopropylsilyl
- acetyl (Ac) chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-meth
- pharmaceutically acceptable salt refers to those carboxylates, amino acid addition salts of the compounds of the present invention which are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.
- the present invention includes tautomers, which are functional isomers produced by rapid movement of an atom in two positions in a molecule.
- tautomers which are functional isomers produced by rapid movement of an atom in two positions in a molecule.
- Compounds that exist in different tautomeric forms are not limited to any specific tautomer, but are intended to cover all tautomeric forms.
- the compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and/or diastereoisomeric forms.
- the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers.
- Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.
- HPLC high pressure liquid chromatography
- the present invention also includes isotopically labeled compounds (isotopic variants), which are equivalent to those described in formula (I), but one or more atoms are replaced by atoms having atomic masses or mass numbers different from the atomic masses or mass numbers commonly found in nature.
- isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl, respectively.
- isotopically labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotopically labeled reagents with readily available isotopically labeled reagents when carrying out the processes disclosed in the following schemes and/or the Examples and Preparations.
- the present invention specifically relates to a compound of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
- L1 and L2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;
- Rs is selected from H, D, halogen, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated;
- n 0, 1, 2, 3, 4, 5 or 6;
- R is -C(O)-C 0-10 alkylene-LR 1 , -C(O)-C 2-10 alkenylene-LR 1 or -C(O)-C 2-10 alkynylene-LR 1 ;
- L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -NHC(O)-CH(OR 1 )CH 2 O-, -C(O)NH-CH(OR 1 )CH 2 O-, -OC(O)-CH(OR 1 )CH 2 O-, -C(O)O-CH(OR 1 )CH 2 O-, -NHC(O)-CH(R 1 )-, -C(O)NH-CH(R 1 )-, -OC(O)-CH(R 1 )- , -C(O)O-CH(R 1 )- , -CH(OR 1 )CH 2 O- , -O-CH 2 CH(R 1 )O-,
- R 1 is independently C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH 2 CH 2 - group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring;
- hydrogen atoms in the C0-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, C1-30 alkyl, C2-30 alkenyl and C2-30 alkynyl groups may be optionally replaced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogens, C1-6 alkyl or C1-6 haloalkyl groups, and are optionally deuterated until fully deuterated.
- the present invention also relates to an oligonucleotide comprising one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
- L 2 represents H or a solid support, or represents the position of connection with the adjacent nucleotide
- the present invention also relates to a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence that is sufficiently complementary to the sense strand and the target mRNA, wherein the sense strand and/or the antisense strand comprises one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
- L 2 represents H or a hydroxyl protecting group, or represents the position for connection with the adjacent nucleotide
- L1 is H; in another embodiment, L1 is a reactive phosphorus group; in another embodiment, L1 is a hydroxyl protecting group; in another embodiment, L1 is a solid support.
- L2 is H; in another embodiment, L2 is a reactive phosphorus group; in another embodiment, L2 is a hydroxyl protecting group; in another embodiment, L2 is a solid support; in another embodiment, L2 represents the position of attachment to the adjacent nucleotide.
- one of L 1 and L 2 is -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl; in another more specific embodiment, one of L 1 and L 2 is -C(O)CH 2 CH 2 C(O)OH.
- Rs is H; in another embodiment, Rs is D; in another embodiment, Rs is halogen; in another embodiment, Rs is C 1-6 alkyl; in another embodiment, Rs is C 1-6 haloalkyl; in another embodiment, when Rs is C 1-6 alkyl or C 1-6 haloalkyl, the group is optionally deuterated until fully deuterated;
- R is -C(O)-C 0-10 alkylene-LR 1 ; in another embodiment, R is -C(O)-C 2-10 alkenylene-LR 1 ; in another embodiment, R is -C(O)-C 2-10 alkynylene-LR 1 .
- R is -C(O)-LR 1 ; in another more specific embodiment, R is -C(O)-C 2-8 alkylene-LR 1 ; in another more specific embodiment, R is -C(O)-C 3-7 alkylene-LR 1 ; in another more specific embodiment, R is -C(O)-C 4-6 alkylene-LR 1 ; in another more specific embodiment, R is -C(O)-C 1-3 alkylene-LR 1 .
- L is a chemical bond; in another embodiment, L is -NHC(O)-; in another embodiment, L is -C(O)NH-; in another embodiment, L is -OC(O)-; in another embodiment, L is -C(O)O-; in another embodiment, L is -SS-; in another embodiment, L is -NHC(O)O-; in another embodiment, L is -NHC(O)NH-; in another embodiment, L is -OC(O)O-; in another embodiment, L is -OC(O)NH-; in another embodiment, L is -NHC(O)-CH(OR 1 )CH 2 O-; in another embodiment, L is -C(O)NH-CH(OR 1 )CH 2 O-; in another embodiment, L is -OC(O)-CH(OR 1 )CH 2 O-; in another embodiment, L is -C(O)CH(OR 1 )CH 2 O-; in another embodiment, L is -OC(O)-CH(OR 1
- L is a bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O) O- , -OC(O)NH-, -O-CH(CH(OH)CH2OH)-, -O - CH(CH(NH2)CH2OH)-, -O-CH(CH2OH ) CH ( OH)-, -NH-CH ( CH2OH)CH(OH) - , -O-CH2CH(OH)CH( OH ) - , -NHC(O)-CH2- O -CH(CH(OH)CH2OH)-, -NHC(O) -CH2 -O-CH(CH( NH2 ) CH2OH )-, -NHC(O)-CH2- O -CH(CH(OH)CH2OH)-
- L is -NHC(O)-CH(OR 1 )CH 2 O-, -C(O)NH-CH(OR 1 )CH 2 O-, -OC(O)-CH(OR 1 )CH 2 O-, -C(O)O-CH(OR 1 )CH 2 O-, -NHC(O)-CH(R 1 )-, -C(O)NH-CH(R 1 )-, -OC(O)-CH(R 1 )-, -C(O)O-CH(R 1 )-, -CH(OR 1 )CH 2 O-, -O-CH(R 1 )CH 2 O-, or -O-CH 2 CH(R 1 )O-; in another more specific embodiment, L is -NHC(O)-CH(OR 1 )CH 2 O-, -NHC(O)-CH(R 1 )-, or -CH(OR 1 )CH 2 O-; in another more specific embodiment, L
- R 1 is C 1-30 alkyl; in another embodiment, R 1 is C 2-30 alkenyl; in another embodiment, R 1 is C 2-30 alkynyl; in another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group in R 1 may be replaced by heteroatoms selected from O, S and N; in another embodiment, the -CH 2 CH 2 - group in R 1 may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-; in another embodiment, the substituents on one or more carbon atoms in R 1 may be connected to form a saturated or unsaturated ring.
- R 1 is independently C 1-30 alkyl or C 2-30 alkenyl, wherein 1, 2, 3, 4, 5, 6, 7 or 8 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or a -CH 2 CH 2 - group may be replaced by -NHC(O)- or -C(O)NH-, or substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring; in another more specific embodiment, R 1 is independently C 5-25 alkyl, a C 10-25 alkenyl containing 1, 2, 3, 4, 5 or 6 double bonds, a C 5-25 alkyl in which 1, 2, 3, 4 or 5 carbon atoms are replaced by N heteroatoms and/or 1, 2 or 3 -CH 2 CH 2 - groups are replaced by -C(O)NH-, or a C 5-25 alkyl in which substituents on one or more carbon atoms are linked to form a steroidal ring; in another
- any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments.
- any technical solution or any combination thereof of L1 can be combined with any technical solution or any combination thereof of L2 , Rs , m, R, L and R1 , etc.
- the present invention is intended to include all combinations of these technical solutions, which are not listed one by one due to space limitations.
- the present invention also provides a vector, which comprises a nucleotide sequence encoding the siRNA of the present invention.
- the vector of the present invention can amplify or express the nucleotide sequence encoding the siRNA of the present invention connected thereto.
- siRNA targeting the PCSK9 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be short-lived (within hours to weeks) or continuous (weeks to months or longer), depending on the specific construct used and the target tissue or cell type.
- the coding nucleotides of the siRNA can be introduced into a linear construct, a circular plasmid or a viral vector.
- the nucleotides of the siRNA can be integrated into the cell genome for stable expression, or expressed in a stable extrachromosomal inheritance.
- siRNA expression vectors are typically DNA plasmids or viral vectors.
- Viral vector systems containing siRNA coding sequences include but are not limited to: (a) adenovirus vectors; (b) retrovirus vectors; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenovirus or gut-free adenovirus.
- the present invention also provides a cell containing the siRNA or vector of the present invention, wherein the siRNA or vector of the present invention can be transcribed in the cell.
- L1 and L2 are independently selected from H, a reactive phosphorus group, a hydroxyl protecting group or a solid support;
- Rs is selected from H, D, halogen, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated;
- n 0, 1, 2, 3, 4, 5 or 6;
- R is -C(O)-C 0-10 alkylene-LR 1 , -C(O)-C 2-10 alkenylene-LR 1 or -C(O)-C 2-10 alkynylene-LR 1 ;
- L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -NHC(O)-CH(OR 1 )CH 2 O-, -C(O)NH-CH(OR 1 )CH 2 O-, -OC(O)-CH(OR 1 )CH 2 O-, -C(O)O-CH(OR 1 )CH 2 O-, -NHC(O)-CH(R 1 )-, -C(O)NH-CH(R 1 )-, -OC(O)-CH(R 1 )- , -C(O)O-CH(R 1 )- , -CH(OR 1 )CH 2 O- , -O-CH 2 CH(R 1 )O-,
- R 1 is independently C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms in the group may be replaced by heteroatoms selected from O, S and N, or the -CH 2 CH 2 - group may be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms may be linked to form a saturated or unsaturated ring;
- hydrogen atoms in the C0-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, C1-30 alkyl, C2-30 alkenyl and C2-30 alkynyl groups may be optionally replaced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogens, C1-6 alkyl or C1-6 haloalkyl groups, and are optionally deuterated until fully deuterated.
- Technical solution 10 The compound of any one of technical solutions 1-9, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the compound is selected from the following:
- An oligonucleotide comprising one or more compounds of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
- L 2 represents H or a solid support, or represents the position of connection with the adjacent nucleotide
- Rs is selected from H, D, halogen, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated;
- n 0, 1, 2, 3, 4, 5 or 6;
- R is a hydrophobic group
- L 2 represents H or a solid support, or represents the position of connection with the adjacent nucleotide
- R s , m and R are as defined in any one of technical solutions 1-5.
- L 2 represents H or a solid support, or represents the position of connection with the adjacent nucleotide
- H or a hydroxyl protecting group or represents the position of attachment to the adjacent nucleotide
- the other represents H or a solid phase support, or indicates the position of connection with the adjacent nucleotide.
- Technical Solution 15 An oligonucleotide according to any one of Technical Solutions 11-14, which comprises a compound of formula (I’) according to any one of Technical Solutions 11-13 at the 5’ end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
- An oligonucleotide according to any one of Technical Solutions 11-15 which comprises a compound of formula (I’) according to any one of Technical Solutions 11-13 at its 3’ end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
- Technical Solution 17 An oligonucleotide according to any one of Technical Solutions 11-16, which comprises a compound of formula (I’) according to any one of Technical Solutions 11-13 at its 5’ end and 3’ end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
- Technical Solution 18 An oligonucleotide according to any one of Technical Solutions 11-17, which contains one or more compounds of formula (I’) according to any one of Technical Solutions 11-13, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, inside the oligonucleotide.
- An oligonucleotide which comprises two or more hydrophobic groups inside the oligonucleotide, at the 5' end and/or the 3' end; preferably, the hydrophobic group is as defined as the R group in the compound of formula (I); preferably, the hydrophobic group is connected to the oligonucleotide via a linker, such as a biodegradable linker.
- L 2 represents H or a hydroxyl protecting group, or represents the position for connection with the adjacent nucleotide
- Rs is selected from H, D, halogen, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated;
- n 0, 1, 2, 3, 4, 5 or 6;
- R is a hydrophobic group
- L 2 represents H or a hydroxyl protecting group, or represents the position for connection with the adjacent nucleotide
- R s , m and R are as defined in any one of technical solutions 1-5.
- L 2 represents H or a hydroxyl protecting group, or represents the position for connection with the adjacent nucleotide
- R s , m and R are as defined in technical solutions 1-5.
- One of them represents H, or represents the position of connection with the adjacent nucleotide, and the other indicates H or a hydroxyl protecting group, or the position of attachment to the adjacent nucleotide.
- Technical Solution 23 A double-stranded RNA according to any one of Technical Solutions 20-22, wherein the positive strand comprises a compound of formula (I’) according to any one of Technical Solutions 11-13 at the 5’ end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
- Technical Solution 24 A double-stranded RNA according to any one of Technical Solutions 20-23, wherein the positive strand comprises a compound of formula (I’) according to any one of Technical Solutions 11-13 at the 3’ end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
- Technical Solution 25 A double-stranded RNA according to any one of Technical Solutions 20-24, wherein the positive strand comprises a compound of formula (I’) according to any one of Technical Solutions 11-13 at the 5’ end and the 3’ end, respectively, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
- Technical Solution 26 A double-stranded RNA according to any one of Technical Solutions 20-25, wherein the positive strand comprises one or more compounds of formula (I’) according to any one of Technical Solutions 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, inside the oligonucleotide.
- Technical Solution 28 A double-stranded RNA according to any one of Technical Solutions 20-27, wherein the antisense strand comprises a compound of formula (I’) according to any one of Technical Solutions 11-13 at the 3’ end, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
- Technical Solution 30 A double-stranded RNA according to any one of Technical Solutions 20-29, wherein the antisense strand comprises one or more compounds of formula (I’) according to any one of Technical Solutions 11-13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, inside the oligonucleotide.
- Technical solution 31 A double-stranded RNA according to any one of technical solutions 20-30, wherein two or more of the sense strand and/or antisense strand There are at least 5 to 30 nucleotides between each compound of formula (I'), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.
- Double-stranded RNA according to any one of Technical Solutions 20-31, wherein the double-stranded RNA comprises two compounds of formula (I’), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which are located at any two of the following sites: the 5’ end of the sense strand, the 3’ end of the sense strand, the 5’ end of the antisense strand and the 3’ end of the antisense strand; preferably located at the 5’ end of the sense strand and the 3’ end of the sense strand.
- formula (I’) pharmaceutically acceptable salts, tautomers or stereoisomers thereof
- a double-stranded RNA according to any one of Technical Solutions 20 to 32, wherein the double-stranded RNA comprises three compounds of formula (I’), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which are located at any three of the following sites: the 5’ end of the sense strand, the 3’ end of the sense strand, the 5’ end of the antisense strand and the 3’ end of the antisense strand; preferably located at the 5’ end of the sense strand, the 3’ end of the sense strand and the 3’ end of the antisense strand.
- formula (I’) pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which are located at any three of the following sites: the 5’ end of the sense strand, the 3’ end of the sense strand, the 5’ end of the antisense strand and the 3’ end of the antisense strand; preferably located at the 5’ end of the sense strand, the 3’ end of the sense strand and the 3’ end
- Double-stranded RNA according to any one of Technical Solutions 20-33, wherein the double-stranded RNA comprises four compounds of formula (I’), or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, which are located at the following sites: the 5’ end of the sense chain, the 3’ end of the sense chain, the 5’ end of the antisense chain and the 3’ end of the antisense chain.
- the double-stranded RNA of any one of Technical Solutions 20-33 further comprising a terminal phosphate protecting group or a prodrug protecting group coupled to the 5' end of the antisense strand, preferably a vinyl phosphate ester group or a prodrug protecting group represented by formula (X):
- X1 is selected from OH or
- R a is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated;
- R b and R c are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl, and said R b and R c may be optionally substituted by D, C 6-10 aryl or 5-10 membered heteroaryl until fully deuterated;
- X2 is a chemical bond connected to the first nucleotide at the 5' end of the antisense strand, preferably connected through a hydroxyl group;
- X3 is independently selected from O or S;
- T is selected from
- each R T1 is independently selected from H, D, halogen, CN, C 1-6 alkyl , C 1-6 haloalkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, or a chain comprising GalNAc, which is optionally deuterated, up to fully deuterated;
- Each R T2 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated;
- Each R T3 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated;
- Each R T4 is independently selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated;
- n 0, 1, 2, 3, 4 or 5;
- n 0, 1, 2, 3, 4 or 5;
- p 0, 1, 2, 3, 4 or 5;
- X is selected from a chemical bond, -O-, -S-, -C(O)-, -C(O)O-, -OC(O) -, -OC(O)NRX1-, -NRX1C(O)O-, -NRX1C ( O )- or -C(O) NRX1- ;
- RX1 is selected from H, C1-6 alkyl or C1-6 haloalkyl, which is optionally deuterated until fully deuterated;
- L is -Ar-(CH 2 ) 1-6 -O-, wherein each CH 2 may be optionally substituted by R#, R# is selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated until fully deuterated;
- Ar in L is connected to X, and the oxygen atom is connected to the phosphorus atom;
- Ar is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-14 membered heteroaryl, wherein the C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl may be optionally substituted with 1, 2, 3, 4 or 5 R*;
- R* is selected from H, D, halogen, CN, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl or C 2-6 alkynyl, which is optionally deuterated up to fully deuterated;
- P1 is selected from a protecting group, preferably a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Allo c), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxy
- the double-stranded RNA of any one of Technical Solutions 20-35 which is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA), and is preferably used to inhibit genes expressed in the eye.
- siRNA small interfering RNA
- shRNA short hairpin RNA
- LL6, LL7 and LL8 are selected from the following compounds:
- nucleic acid chain means connected to the 3' carbon or corresponding position of the previous nucleotide or nucleotide analog via a phosphate group, a phosphorothioate group or other linking group, It means that it is connected to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog through a phosphate group, a thiophosphate group or other linking group; when the corresponding structure is located at the terminal position of the nucleic acid chain, Correspondingly, this refers to attachment to hydrogen, terminal modifications, terminal protecting groups, or other structures that may be used at the ends of nucleic acid chains.
- Technical solution 40 A vector comprising a nucleotide sequence encoding the double-stranded RNA described in any one of the aforementioned technical solutions 20-39.
- Technical solution 41 A cell containing the double-stranded RNA as described in any one of Technical Solutions 20-39 or the vector as described in Technical Solution 40.
- Technical Solution 42 A pharmaceutical composition comprising the double-stranded RNA as described in any one of Technical Solutions 20-39, the vector as described in Technical Solution 40, or the cell as described in Technical Solution 41, and optionally a pharmaceutically acceptable carrier or excipient.
- Technical solution 43 A kit comprising the double-stranded RNA as described in any one of Technical Solutions 20-39, the vector as described in Technical Solution 40, or the cell as described in Technical Solution 41.
- nucleic acid chain means connected to the 3' carbon or corresponding position of the previous nucleotide or nucleotide analog via a phosphate group, a phosphorothioate group or other linking group, It means that it is connected to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog through a phosphate group, a phosphorothioate group or other linking group; when the corresponding structure is located at the terminal position of the nucleic acid chain, Correspondingly, this refers to attachment to hydrogen, terminal modifications, terminal protecting groups, or other structures that may be used at the ends of nucleic acid chains.
- oligonucleotides are as follows, wherein the sequence of 5'->3' from the to connect.
- the corresponding structure is located in the middle of the nucleic acid chain, means connected to the 3' carbon or corresponding position of the previous nucleotide or nucleotide analog via a phosphate group, a phosphorothioate group or other linking group, It means that it is connected to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog through a phosphate group, a thiophosphate group or other linking group; if the corresponding structure is located at the terminal position of the nucleic acid chain, Correspondingly, this refers to attachment to hydrogen, terminal modifications, terminal protecting groups, or other structures that may be used at the ends of nucleic acid chains.
- the organic phase was washed with a 1:1 mixed solution of saturated NaHCO 3 and saturated saline (20 mL x 3), and then the organic phase was dried over Na 2 SO 4 and spun dry.
- the crude product was purified by prep-HPLC (chromatographic column: Waters Xbridge BEH C18 150*25mm*5um; mobile phase: TEAA-ACN; gradient: 55%-95%/16min; flow rate: 15ml/min) to give a yellow oily compound DL0082 (170mg, yield 35.34%, purity 97.26%).
- reaction solution was spin-dried, and the crude product was purified by prep-HPLC (column: 01-Waters Xbridge BEH C18 19 ⁇ 150 mm, 5 ⁇ m; conditions: TEAA-ACN; Begin B 65-95; gradient time: 15 minutes; 100% B holding time: 2 minutes; flow rate: 15 ml/min) to obtain a yellow oily product DL0084 (80.0 mg, yield: 36.27%).
- the combined organic phase was washed with ammonium chloride solution (20.0 mL x 1), sodium bicarbonate solution (20.0 mL x 1), and saturated brine (20.0 mL x 1), dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain a crude product.
- reaction solution was dried by spin drying, and the crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*25mm*5um; condition: TEAA-ACN; Begin B 55-95; gradient time: 15 minutes; 100% B holding time: 6 minutes; flow rate: 15 ml/min), to obtain a yellow oily product DL0127 (120 mg, 0.133 mmol, 35.56%).
- the reaction solution was diluted with DCM (50.0 mL), washed with saturated citric acid aqueous solution (10.0 mL x 3), the organic phase was washed with saturated NaHCO 3 aqueous solution (20.0 mL x 3) and saturated saline aqueous solution (20.0 mL x 3), and then the organic phase was dried with Na 2 SO 4 , and the organic phase was spin-dried to obtain a crude product.
- the reaction solution was diluted with DCM (20.0 mL), washed with saturated citric acid aqueous solution (5.00 mL x 3), saturated NaHCO 3 aqueous solution (5.00 mL x 3) and saturated saline solution (5.00 mL x 3), and then the organic phase was dried over anhydrous Na 2 SO 4 , and the organic phase was spin-dried to obtain a crude product.
- the reaction solution was diluted with dichloromethane (30 mL) and washed with saturated citric acid solution (15 mL x 3), saturated sodium bicarbonate solution (15 mL x 3) and saturated sodium chloride solution (15 mL x 3) in sequence.
- the product 3 was obtained as a white solid (630 mg, yield 30.87%).
- reaction solution was spin-dried, and the crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*25mm*5um; conditions: TEAA-CAN; Begin B 55-95; gradient time: 15 minutes; 100% B holding time: 6 minutes; flow rate: 15 ml/min) to obtain a yellow oily product DL0135 (140 mg, yield 31.91%, purity 99.54%).
- the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated citric acid solution (30 mL x 3), saturated sodium bicarbonate solution (30 mL x 3) and saturated sodium chloride solution (30 mL x 3) in sequence.
- the product 3 was obtained as a white solid (1530 mg, yield 81.02%).
- reaction solution was diluted with dichloromethane (10 mL), and washed with saturated citric acid solution (10 mLX3), saturated sodium bicarbonate solution (10 mLX3) and saturated sodium chloride solution (10 mLX3) in sequence.
- the crude product was purified by Prep-HPLC (chromatographic column: Waters Xbridge BEH C18 100*25mm*5um; mobile phase: TEAA-ACN; gradient: 65%-95%/15min; flow rate: 15ml/min).
- a colorless oily product DL0136 (163mg, yield 40.63%, purity 97.57%) was obtained.
- the crude product was purified by prep-HPLC (chromatographic column: Waters Xbridge BEH C18 150*25mm*5um; mobile phase: TEAA-ACN; gradient: 65%-95%/15min; flow rate: 15ml/min) to obtain a white solid compound DL0137 (45.0mg, yield 33.69%, purity 95.06%).
- the crude product was prepared and separated by a reverse phase column (chromatographic column: Waters Xbridge BEH C18 100*25mm*5um; mobile phase: TEAA-CAN; B%: 60%-95%, 10min; flow rate: 15ml/min) to obtain a colorless oily liquid DL0138 (178 mg, yield 52.93%, purity 97.55%).
- reaction solution was diluted with dichloromethane (50.0 mL), washed with citric acid (10 mL x 3), sodium bicarbonate (10 mL x 3), and saturated sodium chloride (10 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered and dried to obtain a yellow solid product compound 3 (250 mg, 0.613 mmol, 86.00%).
- aqueous phase was extracted with dimethyltetrahydrofuran (20 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and dried to obtain a brown solid product, compound 4 (350 mg, 0.889 mmol, 109.84%).
- reaction solution was diluted with dichloromethane (100 mL), washed with saturated sodium bicarbonate aqueous solution (30 mL x 3) and saturated sodium chloride aqueous solution (30 mL x 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain a brown solid crude product compound 6 (600 mg, 0.727 mmol).
- reaction solution was spin-dried, and the crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*25mm*5um; conditions: TEAA-ACN; Begin B 55-95; gradient time: 15 minutes; 100% B holding time: 6 minutes; flow rate: 15 ml/min) to obtain a yellow oily product DL0140 (120 mg, yield 35.89%, purity 97.34%).
- reaction solution was dried by spin drying, 10.0 mL of water was added, the pH of the aqueous phase was adjusted to ⁇ 4 with 1M HCl, the aqueous phase was extracted with dimethyltetrahydrofuran (10.0 mL x 2), the combined organic phase was washed with saturated brine (10.0 mL x 1), dried over anhydrous sodium sulfate, filtered, and spin dried to obtain a white solid crude product 7 (460 mg, 0.55 mmol, 73.6%).
- reaction solution was spin-dried, and the crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 19*150 mm; conditions: TEAA-ACN; Begin B 65-95; gradient time: 15 minutes; 100% B holding time: 2 minutes; flow rate: 15 ml/min) to obtain a white solid product DL0142 (106 mg, 0.08 mmol, 53.9%).
- reaction liquid was directly suspended under reduced pressure to obtain a crude product, which was prepared and separated by a reverse phase column (chromatographic column: 01-Waters Xbridge BEH C18 19*150 mm; mobile phase: TEAA-ACN; gradient: 75%-95%/18 minutes; flow rate: 15 ml/minute) to obtain a colorless oily compound DL0143 (110 mg, yield 55.49%, purity 99.49%).
- a reverse phase column chromatographic column: 01-Waters Xbridge BEH C18 19*150 mm; mobile phase: TEAA-ACN; gradient: 75%-95%/18 minutes; flow rate: 15 ml/minute
- the reaction solution was diluted with DCM (50.0 mL), washed with saturated NaHCO 3 aqueous solution (20.0 mL x 3), the organic phase was washed with saturated saline solution (20.0 mL x 3), and then the organic phase was dried with Na 2 SO 4 , and the organic phase was spin-dried to obtain a crude product.
- the reaction solution was diluted with DCM (50.0 mL), washed with saturated NaHCO 3 aqueous solution (20.0 mL X 3), and the organic phase was washed with saturated saline solution (20.0 mL X 3), and then the organic phase was dried with Na 2 SO 4 , and the organic phase was spin-dried to obtain a crude product.
- the reaction solution was diluted with DCM (20.0 mL), washed with saturated citric acid aqueous solution (5.00 mL X 3), saturated NaHCO 3 aqueous solution (5.00 mL X 3) and saturated saline solution (5.00 mL X 3), and then the organic phase was dried over anhydrous Na 2 SO 4 , and the organic phase was spin-dried to obtain a crude product.
- Compound 8 500 mg).
- the crude product was prepared and separated by a reverse phase column (chromatographic column: 01-Waters Xbridge BEH C18 19*150 mm; mobile phase: TEAA-ACN; B%: 75%-95%, 15 min; flow rate: 15 ml/min) to obtain a white solid compound DL0144 (218 mg, yield 65.91%, purity 91.17%).
- the crude product was purified by Prep-HPLC (chromatographic column: Waters Xbridge BEH C18 100*25mm*5um; mobile phase: TEAA-ACN; gradient: 45%-95%/16min; flow rate: 15ml/min) to obtain a colorless oily product DL0145 (84.0mg, yield 36.26%, purity 98.70%).
- the siRNA of the present invention is prepared using the solid phase phosphoramidite method well known in the art.
- the specific method can be referred to, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and is briefly described as follows.
- nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement order of the positive chain nucleotides.
- Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or thiolation, and the synthesis scale is 5umol of oligonucleotides.
- the synthesis conditions are as follows:
- the nucleoside monomer was provided in a 0.05 mol/L acetonitrile solution, and the reaction conditions for each step were the same, i.e., the temperature was 25 degrees.
- a 3% trichloroacetic acid-dichloromethane solution was used for deprotection, and the deprotection was repeated 3 times.
- the activating agent used in the coupling reaction was a 0.25 mol/L 5-ethylthiotetrazolyl (ETT)-acetonitrile solution, and the coupling was repeated 2 times.
- ETT 5-ethylthiotetrazolyl
- the capping reaction used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/v) and the capping was repeated 2 times.
- the oxidation reaction used 0.05 mol/L iodine in tetrahydrofuran/pyridine/water (70/20/10, v/v/v) and the oxidation was repeated 2 times.
- the thiolation reaction used 0.2 mol/L phenylacetyl disulfide (PADS) in acetonitrile/3-methylpyridine (1/1, v/v) and the thiolation was repeated 2 times.
- PADS phenylacetyl disulfide
- nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement order of the antisense chain nucleotides.
- Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or thiolation.
- the synthesis conditions of 5umol oligonucleotides of the antisense chain are the same as those of the sense chain.
- a column filled with strong anion fillers can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification, and the product can be collected and piped.
- a gel filler purification column can be used for desalination, and the elution system is pure water.
- Example 3 In vivo activity testing (CNS delivery)
- A, U, G and C represent the natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide and cytosine ribonucleotide, respectively.
- d indicates that the adjacent nucleotide on the right is a deoxyribonucleotide.
- dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.
- i inosine ribonucleotide
- m indicates that the adjacent nucleotide on its left is a 2'-OCH 3 modified nucleotide.
- Am, Um, Gm and Cm represent 2'-OCH 3 modified A, U, G and C.
- f indicates that the adjacent nucleotide on its left side is a 2'-F modified nucleotide.
- Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.
- s indicates that the two adjacent nucleotides and/or delivery vectors are linked by phosphorothioate.
- VP indicates that the adjacent nucleotide on the right is a vinyl phosphate-modified nucleotide.
- Ib represents an inverted abasic deoxyribonucleotide, which may include the following three structures depending on its position/connection mode in siRNA.
- mice SD rats, male, 6-8 weeks old, 2-3 rats per group;
- the residual inhibition rate was calculated by the following formula:
- ⁇ Ct [(target gene in Ct experimental group-internal reference in Ct experimental group)-(target gene in Ct control group-internal reference in Ct control group)].
- the target gene was SOD1
- the internal reference was GAPDH
- the control group was injected with artificial cerebrospinal fluid (aCSF).
- DR005713, DR005714, DR005715, DR005717, DR005718, DR005716, and DR005735 all reduced the expression of SOD1 in the central nervous system.
- the knockdown effect of DR005735 was better than that of other sequences.
- Example 4 In vivo activity testing (ocular delivery)
- sequence information used in this example is as follows:
- C57BL/6 mice male, 6-8 weeks were randomly divided into groups and administered a single dose of 2 ⁇ g per eye by bilateral intravitreal injection.
- the siRNA conjugate was administered in a 5 mg/mL solution (phosphate buffer solution as solvent); specifically, before the experiment, the siRNA conjugate was dissolved in phosphate buffer solution and diluted to the required solution concentration and volume, and the administration volume of phosphate buffer solution and siRNA conjugate was 1.5 ⁇ L/eye.
- the eyeballs were removed and separated into three parts: 1 retinal pigment epithelium (RPE) + choroid + sclera; 2 retina; 3 cornea + iris + ciliary body; the separated samples were immediately frozen in liquid nitrogen and then stored at -80°C for detection of mTTR mRNA.
- RPE retinal pigment epithelium
- the 2 - ⁇ Ct value was calculated and converted into a percentage to obtain the residual inhibition rate
- ⁇ Ct [(target gene in Ct experimental group-internal reference in Ct experimental group)-(target gene in Ct control group-internal reference in Ct control group)].
- the target gene was mTTR and the internal reference was mGAPDH.
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Abstract
本发明提供式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体。所述式(I)化合物包含在核苷酸内部,或者在5'端和/或3'端,用于提高双链RNA穿过血脑屏障的能力。本发明还涉及包含式(I)化合物的寡核苷酸、双链RNA、载体、细胞、药物组合物和试剂盒。
Description
本申请要求提交于2022年10月31日的中国申请202211347517.9的优先权,将其以其整体引入本文作为参考。
本发明属于医药领域,具体涉及具有提高双链RNA穿过血脑屏障和/或眼部递送的能力的疏水性基团,例如式(I)中的R基团,以及将该疏水性基团连接到核苷酸中的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
RNA干扰是一种由双链RNA(double-stranded RNA,dsRNA,也称siRNA)诱发的靶标mRNA高效特异性降解的现象。
然而,由于血脑屏障的存在,难以将siRNA递送到中枢神经系统继而发挥作用,这限制了siRNA的应用。本领域对于将siRNA递送到中枢神经系统进行了一些尝试,例如WO2004094595A2公开了在链末端使用单个脂质配体(例如胆固醇或长链烷烃)递送siRNA,WO2019217459A1公开了在链内部使用单个脂质配体递送siRNA,WO2021092371A2公开了一系列新的脂质配体结构。
本领域仍然需要开发更多的疏水性基团,以更有效地递送siRNA到眼部和/或中枢神经系统。
发明内容
在一个方面中,本发明提供了式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中各基团如下文所定义。
在另一个方面中,本发明提供了寡核苷酸,其包含一个或多个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中各基团如下文所定义。
在另一个方面中,本发明提供了双链RNA,其具有正义链和反义链,各链具有14至30个核苷酸,所述反义链包含与所述正义链和靶标mRNA充分互补的序列,其中所述正义链和/或反义链包含一个或多个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中各基团如下文所定义。
在另一个方面中,本发明提供的疏水性基团(式(I)中的R基团)可以通过现有的接头结构连接至siRNA,例如可生物降解的接头结构。
在另一个方面中,本发明提供了载体,其包含编码前述双链RNA的核苷酸序列。
在另一个方面中,本发明提供了细胞,其含有前述双链RNA或前述载体。
在另一个方面中,本发明提供了药物组合物,其包含前述双链RNA、前述载体、或前述细胞,以及任选的药学上可接受的载剂或赋形剂。
在另一个方面中,本发明提供了试剂盒,其包含前述双链RNA、前述载体、或前述细胞。
图1为在SD大鼠中经椎间孔穿刺推注本发明siRNA后,在颈段脊髓、胸段脊髓、小脑、脑干、海马和额叶皮层中SOD1的表达量的减少情况。
图2为在C57BL/6小鼠中经双侧眼玻璃体内注射本发明siRNA后,在眼部中TTR基因的表达量的减少情况。
发明详述
定义
化学定义
下面更详细地描述具体官能团和化学术语的定义。
当列出数值范围时,既定包括每个值和在所述范围内的子范围。例如“C1-6烷基”包括C1、C2、C3、C4、C5、C6、C1-6、C1-5、C1-4、C1-3、C1-2、C2-6、C2-5、C2-4、C2-3、C3-6、C3-5、C3-4、C4-6、C4-5和C5-6烷基。
“C1-30烷基”是指具有1至30个碳原子的直链或支链饱和烃基团。在一些实施方案中,C5-25烷基、C10-20烷基、C1-20烷基、C1-10烷基和C1-6烷基是优选的。C1-6烷基的例子包括:甲基(C1)、乙基(C2)、正丙基(C3)、异丙基(C3)、正丁基(C4)、叔丁基(C4)、仲丁基(C4)、异丁基(C4)、正戊基(C5)、3-戊基(C5)、戊基(C5)、新戊基(C5)、3-甲基-2-丁基(C5)、叔戊基(C5)和正己基(C6)。术语“C1-6烷基”还包括杂烷基,其中一或多个(例如,1、2、3或4个)碳原子被杂原子(例如,氧、硫、氮、硼、硅、磷)替代。烷基基团可以被一或多个取代基任选取代,例如,被1至5个取代基、1至3个取代基或1个取代基取代。常规烷基缩写包括:Me(-CH3)、Et(-CH2CH3)、iPr(-CH(CH3)2)、nPr(-CH2CH2CH3)、n-Bu(-CH2CH2CH2CH3)或i-Bu(-CH2CH(CH3)2)。
“C2-30烯基”是指具有2至30个碳原子和至少一个碳碳双键的直链或支链烃基团。在一些实施方案中,C10-25烯基、C2-10烯基、C2-6烯基和C2-4烯基是优选的。C2-6烯基的例子包括:乙烯基(C2)、1-丙烯基(C3)、2-丙烯基(C3)、1-丁烯基(C4)、2-丁烯基(C4)、丁二烯基(C4)、戊烯基(C5)、戊二烯基(C5)、己烯基(C6),等等。术语“C2-6烯基”还包括杂烯基,其中一或多个(例如,1、2、3或4个)碳原子被杂原子(例如,氧、硫、氮、硼、硅、磷)替代。烯基基团可以被一或多个取代基任选取代,例如,被1至5个取代基、1至3个取代基或1个取代基取代。
“C2-30炔基”是指具有2至30个碳原子、至少一个碳-碳叁键以及任选地一个或多个碳-碳双键的直链或支链烃基团。在一些实施方案中,C10-25炔基、C2-10炔基、C2-6炔基和C2-4炔基是优选的。C2-6炔基的例子包括但不限于:乙炔基(C2)、1-丙炔基(C3)、2-丙炔基(C3)、1-丁炔基(C4)、2-丁炔基(C4),戊炔基(C5)、己炔基(C6),等等。术语“C2-6炔基”还包括杂炔基,其中一或多个(例如,1、2、3或4个)碳原子被杂原子(例如,氧、硫、氮、硼、硅、磷)替代。炔基基团可以被一或多个取代基任选取代,例如,被1至5个取代基、1至3个取代基或1个取代基取代。
“C1-10亚烷基”、“C2-10亚烯基”和“C2-10亚炔基”分别是指除去C1-10烷基、C2-10烯基和C2-10炔基的另一个氢而形成的二价基团,并且可以是取代或未取代的。在一些实施方案中,C2-8亚烷基、C3-7亚烷基、C4-6亚烷基、C1-4亚烷基、C2-4亚烷基和C1-3亚烷基是优选的。未取代的所述亚烷基包括但不限于:亚甲基(-CH2-)、亚乙基(-CH2CH2-)、亚丙基(-CH2CH2CH2-)、亚丁基(-CH2CH2CH2CH2-)、亚戊基(- CH2CH2CH2CH2CH2-)、亚己基(-CH2CH2CH2CH2CH2CH2-),等等。示例性的取代的所述亚烷基,例如,被一个或多个烷基(甲基)取代的所述亚烷基,包括但不限于:取代的亚甲基(-CH(CH3)-、-C(CH3)2-)、取代的亚乙基(-CH(CH3)CH2-、-CH2CH(CH3)-、-C(CH3)2CH2-、-CH2C(CH3)2-)、取代的亚丙基(-CH(CH3)CH2CH2-、-CH2CH(CH3)CH2-、-CH2CH2CH(CH3)-、-C(CH3)2CH2CH2-、-CH2C(CH3)2CH2-、-CH2CH2C(CH3)2-),等等。
“C0-10亚烷基”是指化学键以及上述“C1-10亚烷基”。
“卤代”或“卤素”是指氟(F)、氯(Cl)、溴(Br)和碘(I)。
因此,“C1-20卤代烷基”、“C1-6卤代烷基”和“C1-4卤代烷基”分别是指上述“C1-20烷基”、“C1-6烷基”和“C1-4烷基”,其被一个或多个卤素基团取代。在一些实施方案中,C1-4卤代烷基是特别优选的,更优选C1-2卤代烷基。示例性的所述卤代烷基包括但不限于:-CF3、-CH2F、-CHF2、-CHFCH2F、-CH2CHF2、-CF2CF3、-CCl3、-CH2Cl、-CHCl2、2,2,2-三氟-1,1-二甲基-乙基,等等。卤代烷基基团可以在任何可用的连接点上被取代,例如,1至5个取代基、1至3个取代基或1个取代基。
术语“疏水性基团”广义上是指对脂质具有亲和力的任何化学基团。表征疏水性基团的疏水性的一种方式是通过辛醇-水分配系数logKow,其中Kow为二相系统在平衡时化学物质在辛醇相中的浓度与其在水相中的浓度的比率。通常,疏水性部分的logKow超过1、超过1.5、超过2、超过3、超过4、超过5或超过10。具体到本发明,疏水性部分即为式I化合物中的R基团。
本文定义的烷基、烯基和炔基等为任选取代的基团。
示例性的碳原子上的取代基包括但不局限于:卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORaa、-ON(Rbb)2、-N(Rbb)2、-N(Rbb)3
+X-、-N(ORcc)Rbb、-SH、-SRaa、-SSRcc、-C(=O)Raa、-CO2H、-CHO、-C(ORcc)2、-CO2Raa、-OC(=O)Raa、-OCO2Raa、-C(=O)N(Rbb)2、-OC(=O)N(Rbb)2、-NRbbC(=O)Raa、-NRbbCO2Raa、-NRbbC(=O)N(Rbb)2、-C(=NRbb)Raa、-C(=NRbb)ORaa、-OC(=NRbb)Raa、-OC(=NRbb)ORaa、-C(=NRbb)N(Rbb)2、-OC(=NRbb)N(Rbb)2、-NRbbC(=NRbb)N(Rbb)2、-C(=O)NRbbSO2Raa、-NRbbSO2Raa、-SO2N(Rbb)2、-SO2Raa、-SO2ORaa、-OSO2Raa、-S(=O)Raa、-OS(=O)Raa、-Si(Raa)3、-OSi(Raa)3、-C(=S)N(Rbb)2、-C(=O)SRaa、-C(=S)SRaa、-SC(=S)SRaa、-SC(=O)SRaa、-OC(=O)SRaa、-SC(=O)ORaa、-SC(=O)Raa、-P(=O)2Raa、-OP(=O)2Raa、-P(=O)(Raa)2、-OP(=O)(Raa)2、-OP(=O)(ORcc)2、-P(=O)2N(Rbb)2、-OP(=O)2N(Rbb)2、-P(=O)(NRbb)2、-OP(=O)(NRbb)2、-NRbbP(=O)(ORcc)2、-NRbbP(=O)(NRbb)2、-P(Rcc)2、-P(Rcc)3、-OP(Rcc)2、-OP(Rcc)3、-B(Raa)2、-B(ORcc)2、-BRaa(ORcc)、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基,其中,每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代;
或者在碳原子上的两个偕氢被基团=O、=S、=NN(Rbb)2、=NNRbbC(=O)Raa、=NNRbbC(=O)ORaa、=NNRbbS(=O)2Raa、=NRbb或=NORcc取代;
Raa的每个独立地选自烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基,或者两个Raa基团结合以形成杂环基或杂芳基环,其中,每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代;
Rbb的每个独立地选自:氢、-OH、-ORaa、-N(Rcc)2、-CN、-C(=O)Raa、-C(=O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(=NRcc)ORaa、-C(=NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(=S)N(Rcc)2、-C(=O)SRcc、-C(=S)SRcc、-P(=O)2Raa、-P(=O)(Raa)2、-P(=O)2N(Rcc)2、-P(=O)(NRcc)2、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基,或者两个Rbb基团结合以形成杂环基或杂芳基环,其中,每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代;
Rcc的每个独立地选自氢、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基,或者两个Rcc基团结合以形成杂环基或杂芳基环,其中,每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代;
Rdd的每个独立地选自:卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-ORee、-ON(Rff)2、-N(Rff)2,、-N(Rff)3
+X-、-N(ORee)Rff、-SH、-SRee、-SSRee、-C(=O)Ree、-CO2H、-CO2Ree、-OC(=O)Ree、-OCO2Ree、-C(=O)N(Rff)2、-OC(=O)N(Rff)2、-NRffC(=O)Ree、-NRffCO2Ree、-NRffC(=O)N(Rff)2、-C(=NRff)ORee、-OC(=NRff)Ree、-OC(=NRff)ORee、-C(=NRff)N(Rff)2、-OC(=NRff)N(Rff)2、-NRffC(=NRff)N(Rff)2、-NRffSO2Ree、-SO2N(Rff)2、-SO2Ree、-SO2ORee、-OSO2Ree、-S(=O)Ree、-Si(Ree)3、-OSi(Ree)3、-C(=S)N(Rff)2、-C(=O)SRee、-C(=S)SRee、-SC(=S)SRee、-P(=O)2Ree、-P(=O)(Ree)2、-OP(=O)(Ree)2、-OP(=O)(ORee)2、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基、杂芳基,其中,每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rgg基团取代,或者两个偕Rdd取代基可结合以形成=O或=S;
Ree的每个独立地选自烷基、卤代烷基、烯基、炔基、环烷基、芳基、杂环基和杂芳基,其中,每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rgg基团取代;
Rff的每个独立地选自氢、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基,或者两个Rff基团结合形成杂环基或杂芳基环,其中,每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rgg基团取代;
Rgg的每个独立地是:卤素、-CN、-NO2、-N3、-SO2H、-SO3H、-OH、-OC1-6烷基、-ON(C1-6烷基)2、-N(C1-6烷基)2、-N(C1-6烷基)3
+X-、-NH(C1-6烷基)2
+X-、-NH2(C1-6烷基)+X-、-NH3
+X-、-N(OC1-6烷基)(C1-6烷基)、-N(OH)(C1-6烷基)、-NH(OH)、-SH、-SC1-6烷基、-SS(C1-6烷基)、-C(=O)(C1-6烷基)、-CO2H、-CO2(C1-6烷基)、-OC(=O)(C1-6烷基)、-OCO2(C1-6烷基)、-C(=O)NH2、-C(=O)N(C1-6烷基)2、-OC(=O)NH(C1-6烷基)、-NHC(=O)(C1-6烷基)、-N(C1-6烷基)C(=O)(C1-6烷基)、-NHCO2(C1-6烷基)、-NHC(=O)N(C1-6烷基)2、-NHC(=O)NH(C1-6烷基)、-NHC(=O)NH2、-C(=NH)O(C1-6烷基)、-OC(=NH)(C1-
6烷基)、-OC(=NH)OC1-6烷基、-C(=NH)N(C1-6烷基)2、-C(=NH)NH(C1-6烷基)、-C(=NH)NH2、-OC(=NH)N(C1-6烷基)2、-OC(NH)NH(C1-6烷基)、-OC(NH)NH2、-NHC(NH)N(C1-6烷基)2、-NHC(=NH)NH2、-NHSO2(C1-6烷基)、-SO2N(C1-6烷基)2、-SO2NH(C1-6烷基)、-SO2NH2、-SO2C1-6烷基、-SO2OC1-6烷基、-OSO2C1-6烷基、-SOC1-6烷基、-Si(C1-6烷基)3、-OSi(C1-6烷基)3、-C(=S)N(C1-6烷基)2、C(=S)NH(C1-6烷基)、C(=S)NH2、-C(=O)S(C1-6烷基)、-C(=S)SC1-6烷基、-SC(=S)SC1-6烷基、-P(=O)2(C1-6烷基)、-P(=O)(C1-
6烷基)2、-OP(=O)(C1-6烷基)2、-OP(=O)(OC1-6烷基)2、C1-6烷基、C1-6卤代烷基、C2-C6烯基、C2-C6炔基、C3-C7环烷基、C6-C10芳基、C3-C7杂环基、C5-C10杂芳基;或者两个偕Rgg取代基可结合形成=O或=S;其中,X-为反离子。
示例性的氮原子上取代基包括但不局限于:氢、-OH、-ORaa、-N(Rcc)2、-CN、-C(=O)Raa、-C(=O)N(Rcc)2、-CO2Raa、-SO2Raa、-C(=NRbb)Raa、-C(=NRcc)ORaa、-C(=NRcc)N(Rcc)2、-SO2N(Rcc)2、-SO2Rcc、-SO2ORcc、-SORaa、-C(=S)N(Rcc)2、-C(=O)SRcc、-C(=S)SRcc、-P(=O)2Raa、-P(=O)(Raa)2、-P(=O)2N(Rcc)2、-P(=O)(NRcc)2、烷基、卤代烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基,或者连接至氮原子的两个Rcc基团结合形成杂环基或杂芳基环,其中,每个烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个Rdd基团取代,且其中Raa、Rbb、Rcc和Rdd如上所述。
其他定义
本文术语“siRNA”是一类双链RNA分子,其可以介导与其互补的靶RNA(例如mRNA,例如,编码蛋白质的基因的转录物)的沉默。siRNA通常是双链的,包括与靶RNA互补的反义链,和与该反义链互补的正义链。为方便起见,这样的mRNA在此也被称为有待被沉默的mRNA。这样的基因也称为靶基因。通常,有待被沉默的RNA是内源基因或病原体基因。另外,除了mRNA以外的RNA(例如tRNA)以及病毒RNA也可以被靶向。
术语“反义链”是指siRNA的这样一条链,所述链包含与靶序列完全、充分或基本互补的区域。术语“正义链”是指siRNA的这样一条链,所述链包括与作为在此定义的术语反义链的区域完全、充分或基本互补的区域。
术语“互补区域”是指反义链上与靶mRNA序列完全、充分或基本互补的区域。在互补区域与靶序列不完全互补的情况下,错配可以位于分子的内部或末端区域中。通常,最耐受的错配位于末端区域中,例如,在5’和/或3’端的5、4、3、2或1个核苷酸内。对错配最敏感的反义链部分被称为“种子区”。例如,在包含19nt的链的siRNA中,第19个位置(从5’向3’)可以耐受一些错配。
术语“互补”是指第一多核苷酸在某些条件例如严格条件下与第二多核苷酸杂交的能力。例如,严格条件可包括400mM NaCl、40mM PIPES pH 6.4、1mM EDTA在50℃或70℃下持续12-16小时。就满足以上相对于它们杂交的能力而言的要求来说,“互补”序列还可以包括或完全形成自非沃森-克里克碱基对和/或从非天然的以及经修饰的核苷酸形成的碱基对。此类非沃森-克里克碱基对包括但不限于G:U摇摆碱基配对或Hoogstein碱基配对。
与信使RNA(mRNA)的“至少部分互补”、“充分互补”或“基本上互补”的多核苷酸是指与感兴趣的mRNA的连续部分基本互补的多核苷酸。例如,如果序列与编码PCSK9的mRNA的非中断部分基本上互补,则多核苷酸与PCSK9mRNA的至少部分互补。在此的术语“互补”、“完全互补”、“充分互补”和“基本上互补”可以相对于siRNA的正义链与反义链之间,或siRNA试剂的反义链与靶序列之间的碱基配对使用。
“充分互补”是指为了维持分子的整体双链特征,正义链仅需要与反义链互补的程度。换言之,虽然通常需要完美的互补性,但在一些情况下,特别是在反义链中,可以包括一个或多个,例如6个、5个、4个、3个、2个或1个的错配(相对于靶标mRNA),但是正义链与反义链仍可以维持分子的整体双链特征。
“shRNA”是指短发夹RNA。shRNA包括两个短反向重复序列。克隆到shRNA表达载体中的shRNA包括两个短反向重复序列,中间由一茎环(loop)序列分隔的,组成发夹结构,由polⅢ启动子控制。随后再连上5-6个T作为RNA聚合酶Ⅲ的转录终止子。
“核苷”是由嘌呤碱或嘧啶碱、以及核糖或脱氧核糖两种物质组成的化合物,“核苷酸”则是由嘌呤碱或嘧啶碱、核糖或脱氧核糖以及磷酸三种物质组成的化合物,“寡核苷酸”是指例如具有少于100、200、300或400个核苷酸长度的核酸分子(RNA或DNA)。
“碱基”是合成核苷、核苷酸和核酸的基本组成单位,其组成元素中含有氮,也称“含氮碱基”。本文中,如无特别说明,大写字母A、U、T、G和C表示核苷酸的碱基组成,分别为腺嘌呤、尿嘧啶、胸腺嘧啶、鸟嘌呤和胞嘧啶。
本文中所述核苷酸的“修饰”包括但不限于甲氧基修饰、氟代修饰、硫代磷酸酯基连接或常规保护基保护等。例如,所述氟代修饰的核苷酸指核苷酸的核糖基2’位的羟基被氟取代形成的核苷酸,所述甲氧基修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。
本文中“修饰的核苷酸”包括但不限于2'-O-甲基修饰的核苷酸、2'-氟代修饰的核苷酸、2'-脱氧-修饰的核苷酸、肌苷核糖核苷酸、脱碱基核苷酸、反向无碱基脱氧核糖核苷酸、包含硫代磷酸酯基团的核苷酸、乙烯基磷酸酯修饰的核苷酸、锁核苷酸、2'-氨基-修饰的核苷酸、2'-烷基-修饰的核苷酸、吗啉代核苷酸、氨基磷酸酯、包含核苷酸的非天然碱基、以及连接到胆固醇基衍生物或十二烷酸二癸酰胺基团上的末端核苷酸、脱氧核糖核苷酸或常规保护基保护等。例如,所述2'-氟代修饰的核苷酸指核苷酸的核糖基2’位的羟基被氟取代形成的核苷酸。所述2'-脱氧-修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。
“配体部分”是指与siRNA缀合的化学部分,其能够改变siRNA的分布、靶向或寿命。在优选的实施方案中,与例如不存在这样一个配体的siRNA相比,这种配体为选择的靶标(例如分子、细胞或细胞类型、区室(例如细胞或器官区室、组织、器官或身体的区域)提供增强的亲和力。
“反应性磷基团”是指包含在核苷酸单元中或核苷酸类似物单元中的含磷基团,其可以通过亲核攻击反应,与包含在另一个分子中、尤其是另一个核苷酸单元中或另一个核苷酸类似物中的羟基或胺基反应。通常,这样的反应产生将所述第一核苷酸单元或所述第一核苷酸类似物单元与所述第二核苷酸单元或所述第二核苷酸类似物单元连接的酯型核苷间键。反应性磷基团可选自亚磷酰胺,H-膦酸酯,烷基-膦酸酯,磷酸酯或磷酸酯模拟物,包括但不限于:天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯和三磷酸酯,优选-P(OCH2CH2CN)(N(iPr)2)。
“保护基”是指被添加到分子中以防止分子中现有基团进行不期望的化学反应的任何原子或原子团。“保护基”可为本领域已知的不稳定的化学部分,其用于保护反应性基团,例如羟基、氨基和硫醇基团,以防止在化学合成过程中发生不期望的或不合时宜的反应。保护基通常在其它反应性位点的反应期间选择性地和/或正交地用于保护位点,然后可以被去除以留下未受保护的基团保持原样或可用于进一步的反应。
保护基团的非限制性列表包括苄基;取代的苄基;烷基羰基和烷氧基羰基(例如,叔丁氧基羰基(BOC)、乙酰基或异丁酰基);芳基烷基羰基和芳基烷氧基羰基(例如,苄基氧基羰基);取代的甲基醚(例如甲氧基甲基醚);取代的乙醚;取代的苄基醚;四氢吡喃基醚;甲硅烷基(例如,三甲基甲硅烷基、三乙基甲硅烷基、三异丙基甲硅烷基、叔丁基二甲基甲硅烷基、三-异丙基甲硅烷基氧基甲基、[2-(三甲基甲硅烷基)乙氧基]甲基或叔丁基二苯基甲硅烷基);酯类(例如苯甲酸酯);碳酸酯类(例如碳酸甲氧基甲基酯);磺酸酯类(例如甲苯磺酸酯或甲磺酸酯);非环缩酮(例如二甲基乙缩醛);环缩酮(例如,1,3-二噁烷、1,3-二氧戊环以及本文所述的那些);非环乙缩醛;环乙缩醛(例如,本文所述的那些);非环半缩醛;环半缩醛;环二硫缩酮(例如,1,3-二噻烷或1,3-二硫戊环);原酸酯(例如,本文所述的那些)以及三芳基甲基基团(例如,三苯甲基;单甲氧基三苯甲基(MMTr);4,4′-二甲氧基三苯甲基(DMTr);4,4′,4″-三甲氧基三苯甲基(TMTr);以及本文所述的那些)。优选的保护基团选自乙酰基(Ac)、苯甲酰基(Bzl)、苄基(Bn)、异丁酰基(iBu)、苯基乙酰基、苄基氧基甲基乙缩醛(BOM)、β-甲氧基乙氧基甲基醚(MEM)、甲氧基甲基醚(MOM)、对-甲氧基苄基醚(PMB)、甲基硫代甲基醚、新戊酰基(Piv)、四氢吡喃基(THP)、三苯基甲基(Trt)、甲氧基三苯甲基[(4-甲氧基苯基)二苯基甲基](MMT)、二甲氧基三苯甲基、[双-(4-甲氧基苯基)苯基甲基(DMT)、三甲基甲硅烷基醚(TMS)、叔丁基二甲基甲硅烷基醚(TBDMS)、三-异-丙基甲硅烷基氧基甲基醚(TOM)、三-异丙基甲硅烷基醚(TIPS)、甲基醚、乙氧基乙醚(EE)N,N-二甲基甲脒和2-氰基乙基(CE)。
“羟基保护基”是指能够避免羟基遭受化学反应,又可以在特定条件下脱除以恢复羟基的基团。主要包括硅烷型保护基、酰基型保护基或醚型保护基,优选以下:
三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、
对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基,优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基,更优选-C(O)CH2CH2C(O)OH。
本文所用的术语“药学上可接受的盐”表示本发明化合物的那些羧酸盐、氨基酸加成盐,它们在可靠的医学判断范围内适用于与患者组织接触,不会产生不恰当的毒性、刺激作用、变态反应等,与合理的益处/风险比相称,就它们的预期应用而言是有效的,包括(可能的话)本发明化合物的两性离子形式。
本发明包括互变异构体,其为分子中某一原子在两个位置迅速移动而产生的官能团异构体。在不同的互变异构形式存在的化合物,一个所述化合物并不局限于任何特定的互变异构体,而是旨在涵盖所有的互变异构形式。
本发明化合物可包括一个或多个不对称中心,且因此可以存在多种立体异构体形式,例如,对映异构体和/或非对映异构体形式。例如,本发明化合物可为单独的对映异构体、非对映异构体或几何异构体(例如顺式和反式异构体),或者可为立体异构体的混合物的形式,包括外消旋体混合物和富含一种或多种立体异构体的混合物。异构体可通过本领域技术人员已知的方法从混合物中分离,所述方法包括:手性高压液相色谱法(HPLC)以及手性盐的形成和结晶;或者优选的异构体可通过不对称合成来制备。
本发明还包括同位素标记的化合物(同位素变体),它们等同于式(I)所述的那些,但一个或多个原子被原子质量或质量数不同于自然界常见的原子质量或质量数的原子所代替。可以引入本发明化合物中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素,分别例如2H、3H、13C、11C、14C、15N、18O、17O、31P、32P、35S、18F和36Cl。含有上述同位素和/或其它原子的其它同位素的本发明化合物、其前体药物和所述化合物或所述前体药物的药学上可接受的盐都属于本发明的范围。某些同位素标记的本发明化合物、例如引入放射性同位素(例如3H和14C)的那些可用于药物和/或底物组织分布测定。氚、即3H和碳-14、即14C同位素是特别优选的,因为它们容易制备和检测。进而,被更重的同位素取代,例如氘、即2H,由于代谢稳定性更高可以提供治疗上的益处,例如延长体内半衰期或减少剂量需求,因而在有些情况下可能是优选的。同位素标记的本发明式(I)化合物及其前体药物一般可以这样制备,在进行下述流程和/或实施例与制备例所公开的工艺时,用容易得到的同位素标记的试剂代替非同位素标记的试剂。
本发明化合物
本发明具体涉及式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,
L1和L2独立地选自H、反应性磷基团、羟基保护基或固相载体;
Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基,其任选地被氘代,直至完全氘代;
m=0、1、2、3、4、5或6;
R为-C(O)-C0-10亚烷基-L-R1、-C(O)-C2-10亚烯基-L-R1或-C(O)-C2-10亚炔基-L-R1;
L为化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)-或-NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-;
R1独立地为C1-30烷基、C2-30烯基或C2-30炔基,其中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换,或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换,或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环;
其中C0-10亚烷基、C2-10亚烯基、C2-10亚炔基、C1-30烷基、C2-30烯基和C2-30炔基中的氢原子可以任选地被1、2、3、4、5、6、7、8、9、10或更多个卤素、C1-6烷基或C1-6卤代烷基替换,而且其任选地被氘代,直至完全氘代。
本发明还涉及寡核苷酸,其包含一个或多个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,
表示H或羟基保护基,或表示与相邻核苷酸连接的位置;
L2表示H或固相载体,或表示与相邻核苷酸连接的位置;
Rs、m和R如上所定义。
本发明还涉及双链RNA,其具有正义链和反义链,各链具有14至30个核苷酸,所述反义链包含与所述正义链和靶标mRNA充分互补的序列,其中所述正义链和/或反义链包含一个或多个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,
表示与相邻核苷酸连接的位置;
L2表示H或羟基保护基,或表示与相邻核苷酸连接的位置;
Rs、m和R如上所定义。
L1、L2和
在一个实施方案中,L1为H;在另一个实施方案中,L1为反应性磷基团;在另一个实施方案中,L1为羟基保护基;在另一个实施方案中,L1为固相载体。
在一个实施方案中,L2为H;在另一个实施方案中,L2为反应性磷基团;在另一个实施方案中,L2为羟基保护基;在另一个实施方案中,L2为固相载体;在另一个实施方案中,L2表示与相邻核苷酸连接的位置。
在更具体的实施方案中,L1和L2之一为-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基;在另一更具体的实施方案中,L1和L2之一为-C(O)CH2CH2C(O)OH。
在一个实施方案中,表示H;在另一个实施方案中,表示羟基保护基;在另一个实施方案中,表示与相邻核苷酸连接的位置。
Rs
在一个实施方案中,Rs为H;在另一个实施方案中,Rs为D;在另一个实施方案中,Rs为卤素;在另一个实施方案中,Rs为C1-6烷基;在另一个实施方案中,Rs为C1-6卤代烷基;在另一个实施方案中,Rs为C1-6烷基或C1-6卤代烷基时,该基团任选地被氘代,直至完全氘代;
m
在一个实施方案中,m=0;在另一个实施方案中,m=1;在另一个实施方案中,m=2;在另一个实施方案中,m=3;在另一个实施方案中,m=4;在另一个实施方案中,m=5;在另一个实施方案中,m=6。
R
在一个实施方案中,R为-C(O)-C0-10亚烷基-L-R1;在另一个实施方案中,R为-C(O)-C2-10亚烯基-L-R1;在另一个实施方案中,R为-C(O)-C2-10亚炔基-L-R1。
在更具体的实施方案中,R为-C(O)-L-R1;在另一更具体的实施方案中,R为-C(O)-C2-8亚烷基-L-R1;在另一更具体的实施方案中,R为-C(O)-C3-7亚烷基-L-R1;在另一更具体的实施方案中,R为-C(O)-C4-6亚烷基-L-R1;在另一更具体的实施方案中,R为-C(O)-C1-3亚烷基-L-R1。
L
在一个实施方案中,L为化学键;在另一个实施方案中,L为-NHC(O)-;在另一个实施方案中,L为-C(O)NH-;在另一个实施方案中,L为-OC(O)-;在另一个实施方案中,L为-C(O)O-;在另一个实施方案中,L为-S-S-;在另一个实施方案中,L为-NHC(O)O-;在另一个实施方案中,L为-NHC(O)NH-;在另一个实施方案中,L为-OC(O)O-;在另一个实施方案中,L为-OC(O)NH-;在另一个实施方案中,L为-NHC(O)-CH(OR1)CH2O-;在另一个实施方案中,L为-C(O)NH-CH(OR1)CH2O-;在另一个实施方案中,L为-OC(O)-CH(OR1)CH2O-;在另一个实施方案中,L为-C(O)O-CH(OR1)CH2O-;在另一个实施方案中,L为-NHC(O)-CH(R1)-;在另一个实施方案中,L为-C(O)NH-CH(R1)-;在另一个实施方案中,L为-OC(O)-CH(R1)-;在另一个实施方案中,L为-C(O)O-CH(R1)-;在另一个实施方案中,L为-CH(OR1)CH2O-;在另一个实施方案中,L为-O-CH(R1)CH2O-;在另一个实施方案中,L为-O-CH2CH(R1)O-;在另一个实施方案中,L为-O-CH(CH(OH)CH2OH)-;在另一个实施方案中,L为-O-CH(CH(NH2)CH2OH)-;在另一个实施方案中,L为-O-CH(CH2OH)CH(OH)-;在另一个实施方案中,L为-NH-CH(CH2OH)CH(OH)-;在另一个实施方案中,L为-O-CH2CH(OH)CH(OH)-;在另一个实施方案中,L为-O-CH2CH(NH2)CH(OH)-;在另一个实施方案中,L为-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-;在另一个实施方案中,L为-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-;在另一个实施方案中,L为-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-;在另一个实施方案中,L为-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-;在另一个实施方案中,L为-NHC(O)-CH2-O-CH2CH(OH)CH(OH)-;在另一个实施方案中,L为-NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-。
在更具体的实施方案中,L为化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)-或-NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-;在另一更具体的实施方案中,L为化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-或-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-;在另一更具体的实施方案中,L为化学键、-NHC(O)-、-S-S-、-NHC(O)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-或-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-;在另一更具体的实施方案中,L为化学键、-NHC(O)-、-S-S-或-NHC(O)O-;在另一更具体的实施方案中,L为-NHC(O)-。
在更具体的实施方案中,L为-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-或-O-CH2CH(R1)O-;在另一更具体的实施方案中,L为-NHC(O)-CH(OR1)CH2O-、-NHC(O)-CH(R1)-或-CH(OR1)CH2O-;在另一更具体的实施方案中,L为-NHC(O)-CH(OR1)CH2O-。
R1
在一个实施方案中,R1为C1-30烷基;在另一个实施方案中,R1为C2-30烯基;在另一个实施方案中,R1为C2-30炔基;在另一个实施方案中,R1中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换;在另一个实施方案中,R1中的-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换;在另一个实施方案中,R1中的一个或多个碳原子上的取代基可以连接成饱和或不饱和环。
在更具体的实施方案中,R1独立地为C1-30烷基或C2-30烯基,其中所述基团中不相邻的1、2、3、4、5、6、7或8个碳原子可以被选自O、S和N的杂原子替换,或者-CH2CH2-基团可以被-NHC(O)-或-C(O)NH-替换,或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环;在另一更具体的实施方案中,R1独立地为C5-25烷基、含有1、2、3、4、5或6个双键的C10-25烯基、其中1、2、3、4或5个碳原子被N杂原子替换和/或1、2或3个-CH2CH2-基团被-C(O)NH-替换的C5-25烷基、或者一个或多个碳原子上的取代基连接成甾体环的C5-25烷基;在另一更具体的实施方案中,R1选自以下基团:
C6烷基、C8烷基、C11烷基、C12烷基、C13烷基、C15烷基、C16烷基、C17烷基、C21烷基、
以上任一具体实施方案中的任一技术方案或其任意组合,可以与其它具体实施方案中的任一技术方案或其任意组合进行组合。例如,L1的任一技术方案或其任意组合,可以与L2、Rs、m、R、L和R1等的任一技术方案或其任意组合进行组合。本发明旨在包括所有这些技术方案的组合,限于篇幅,不再一一列出。
本发明还提供了载体,其包含编码本发明所述的siRNA的核苷酸序列。本发明的载体能够扩增或表达与其连接的编码本发明所述的siRNA的核苷酸。
例如,靶向PCSK9基因的siRNA可以从插入DNA或RNA载体中的转录单位表达。表达可以是短暂的(数小时至数星期内)或持续的(数星期至数个月或更久),取决于所使用的特定建构体及靶组织或细胞类型。可以将siRNA的编码核苷酸引入线性建构体、环状质体或病毒载体中。siRNA的核苷酸可以被整合到细胞基因组中稳定表达,或者在染色体外稳定遗传而表达。一般来说,siRNA表达载体通常是DNA质粒或病毒载体。
包含siRNA的编码序列的病毒载体系统包括但不局限于:(a)腺病毒载体;(b)逆转录病毒载体;(c)腺伴随病毒载体;(d)单纯疱疹病毒载体;(e)SV40载体;(f)多瘤病毒载体;(g)乳头瘤病毒载体;(h)微小核糖核酸病毒载体;(i)痘病毒载体;以及(j)辅助病毒依赖性腺病毒或无肠腺病毒。
本发明还提供了细胞,其含有本发明所述的siRNA或载体,其中本发明所述的siRNA或载体能够在细胞中转录。
本发明具体涉及以下技术方案:
技术方案1.式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,
L1和L2独立地选自H、反应性磷基团、羟基保护基或固相载体;
Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基,其任选地被氘代,直至完全氘代;
m=0、1、2、3、4、5或6;
R为-C(O)-C0-10亚烷基-L-R1、-C(O)-C2-10亚烯基-L-R1或-C(O)-C2-10亚炔基-L-R1;
L为化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)-或-NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-;
R1独立地为C1-30烷基、C2-30烯基或C2-30炔基,其中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换,或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换,或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环;
其中C0-10亚烷基、C2-10亚烯基、C2-10亚炔基、C1-30烷基、C2-30烯基和C2-30炔基中的氢原子可以任选地被1、2、3、4、5、6、7、8、9、10或更多个卤素、C1-6烷基或C1-6卤代烷基替换,而且其任选地被氘代,直至完全氘代。
技术方案2.技术方案1的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,R为-C(O)-C0-10亚烷基-L-R1,优选-C(O)-L-R1,优选-C(O)-C2-8亚烷基-L-R1,更优选-C(O)-C3-7亚烷基-L-R1,更优选-C(O)-C4-6亚烷基-L-R1,更优选-C(O)-C1-3亚烷基-L-R1。
技术方案3.技术方案1或2的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,L为化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)-或-NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-,优选化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-或-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-,优选化学键、-NHC(O)-、-S-S-、-NHC(O)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-或-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-,更优选化学键、-NHC(O)-、-S-S-或-NHC(O)O-,更优选-NHC(O)-。
技术方案4.技术方案1或2的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,L为-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-,优选-NHC(O)-CH(OR1)CH2O-、-NHC(O)-CH(R1)-或-CH(OR1)CH2O-,更优选-NHC(O)-CH(OR1)CH2O-。
技术方案5.技术方案1-4中任一项的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,R1独立地为C1-30烷基或C2-30烯基,其中所述基团中不相邻的1、2、3、4、5、6、7或8个碳原子可以被选自O、S和N的杂原子替换,或者-CH2CH2-基团可以被-NHC(O)-或-C(O)NH-替换,或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环;优选地,R1独立地为C5-25烷基、含有1、2、3、4、5或6个双键的C10-25烯基、其中1、2、3、4或5个碳原子被N杂原子替换和/或1、2或3个-CH2CH2-基团被-C(O)NH-替换的C5-25烷基、或者一个或多个碳原子上的取代基连接成甾体环的C5-25烷基;优选地,R1选自以下基团:
C6烷基、C8烷基、C11烷基、C12烷基、C13烷基、C15烷基、C16烷基、C17烷基、C21烷基、
技术方案6.技术方案1-5中任一项的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,L1和L2为H。
技术方案7.技术方案1-5中任一项的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,L1和L2之一为反应性磷基团,优选亚磷酰胺、H-膦酸酯、烷基-膦酸酯、磷酸酯或磷酸酯模拟物,例如天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯或三磷酸酯,优选-P(OCH2CH2CN)(N(iPr)2)。
技术方案8.技术方案1-5中任一项的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,L1和L2选自保护基,优选羟基保护基,例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基,优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基,更优选-C(O)CH2CH2C(O)OH。
技术方案9.技术方案1-8中任一项的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其选自以下通式:
其中各基团如技术方案1-8所定义。
技术方案10.技术方案1-9中任一项的化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,所述化合物选自以下:
技术方案11.寡核苷酸,其包含一个或多个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,
表示H或羟基保护基,或表示与相邻核苷酸连接的位置;
L2表示H或固相载体,或表示与相邻核苷酸连接的位置;
Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基,其任选地被氘代,直至完全氘代;
m=0、1、2、3、4、5或6;
R为疏水性基团;
优选地,
表示H或羟基保护基,或表示与相邻核苷酸连接的位置;
L2表示H或固相载体,或表示与相邻核苷酸连接的位置;
Rs、m和R如技术方案1-5中任一项所定义。
技术方案12.技术方案11的寡核苷酸,其中所述式(I’)化合物选自以下通式化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,
表示H或羟基保护基,或表示与相邻核苷酸连接的位置;
L2表示H或固相载体,或表示与相邻核苷酸连接的位置;
其他各基团如技术方案1-5所定义。
技术方案13.技术方案11的寡核苷酸,其中所述式(I’)化合物选自以下化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,所述化合物选自以下:
其中之一表示H或羟基保护基,或表示与相邻核苷酸连接的位置,并且另一个表示H或固相载体,或表示与相邻核苷酸连接的位置。
技术方案14.技术方案11-13中任一项的寡核苷酸,其具有14至30个核苷酸。
技术方案15.技术方案11-14中任一项的寡核苷酸,其在5’端包含一个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案16.技术方案11-15中任一项的寡核苷酸,其在3’端包含一个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案17.技术方案11-16中任一项的寡核苷酸,其在5’端和3’端分别包含一个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案18.技术方案11-17中任一项的寡核苷酸,其在寡核苷酸内部包含一个或多个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案19.寡核苷酸,其在寡核苷酸内部、5’端和/或3’端包含两个或多个疏水性基团;优选地,所述疏水性基团如式(I)化合物中R基团所定义;优选地,所述疏水性基团通过接头,例如可生物降解的接头,连接至寡核苷酸上。
技术方案20.双链RNA,其具有正义链和反义链,各链具有14至30个核苷酸,所述反义链包含与所述正义链和靶标mRNA充分互补的序列,其中所述正义链和/或反义链包含一个或多个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,
表示H,或表示与相邻核苷酸连接的位置;
L2表示H或羟基保护基,或表示与相邻核苷酸连接的位置;
Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基,其任选地被氘代,直至完全氘代;
m=0、1、2、3、4、5或6;
R为疏水性基团;
优选地,
表示H,或表示与相邻核苷酸连接的位置;
L2表示H或羟基保护基,或表示与相邻核苷酸连接的位置;
Rs、m和R如技术方案1-5中任一项所定义。
技术方案21.技术方案20的双链RNA,其中所述式(I’)化合物选自以下通式化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,
表示H,或表示与相邻核苷酸连接的位置;
L2表示H或羟基保护基,或表示与相邻核苷酸连接的位置;
Rs、m和R如技术方案1-5所定义。
技术方案22.技术方案20的双链RNA,其中所述式(I’)化合物选自以下化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,所述化合物选自以下:
其中之一表示H,或表示与相邻核苷酸连接的位置,并且另一个表示H或羟基保护基,或与相邻核苷酸连接的位置。
技术方案23.技术方案20-22中任一项的双链RNA,其中所述正义链在5’端包含一个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案24.技术方案20-23中任一项的双链RNA,其中所述正义链在3’端包含一个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案25.技术方案20-24中任一项的双链RNA,其中所述正义链在5’端和3’端分别包含一个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案26.技术方案20-25中任一项的双链RNA,其中所述正义链在寡核苷酸内部包含一个或多个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案27.技术方案20-26中任一项的双链RNA,其中所述反义链在5’端包含一个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案28.技术方案20-27中任一项的双链RNA,其中所述反义链在3’端包含一个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案29.技术方案20-28中任一项的双链RNA,其中所述反义链在5’端和3’端分别包含一个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案30.技术方案20-29中任一项的双链RNA,其中所述反义链在寡核苷酸内部包含一个或多个技术方案11-13中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
技术方案31.技术方案20-30中任一项的双链RNA,其中所述正义链和/或反义链上的两个或多
个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体之间间隔至少5-30个核苷酸。
技术方案32.技术方案20-31中任一项的双链RNA,其中所述双链RNA包含两个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其位于以下位点中的任两个:正义链的5’端、正义链的3’端、反义链的5’端和反义链的3’端;优选位于正义链的5’端和正义链的3’端。
技术方案33.技术方案20-32中任一项的双链RNA,其中所述双链RNA包含三个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其位于以下位点中的任三个:正义链的5’端、正义链的3’端、反义链的5’端和反义链的3’端;优选位于正义链的5’端、正义链的3’端和反义链的3’端。
技术方案34.技术方案20-33中任一项的双链RNA,其中所述双链RNA包含四个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其位于以下位点:正义链的5’端、正义链的3’端、反义链的5’端和反义链的3’端。
技术方案35.技术方案20-33中任一项的双链RNA,其进一步在所述反义链的5’端偶联末端磷酸保护基团或前药保护基团,优选乙烯基磷酸酯基或式(X)所示的前药保护基团:
其中,
X1选自OH或
Ra选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;
Rb和Rc独立地选自H、C1-6烷基或C1-6卤代烷基,所述Rb和Rc可任选地被D、C6-10芳基或5-10元杂芳基取代,直至完全氘代;
X2是与所述反义链的5’端的第一个核苷酸连接的化学键,优选通过羟基连接;
X3独立地选自O或S;
T选自
每个RT1独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C1-6卤代烷基、C2-6烯基、C2-
6炔基或包含GalNAc的链,其任选地被氘代,直至完全氘代;
每个RT2独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;
每个RT3独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;
每个RT4独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;
m为0、1、2、3、4或5;
n为0、1、2、3、4或5;
p为0、1、2、3、4或5;
X选自化学键、-O-、-S-、-C(O)-、-C(O)O-、-OC(O)-、-OC(O)NRX1-、-NRX1C(O)O-、-NRX1C(O)-或-C(O)NRX1-;
RX1选自H、C1-6烷基或C1-6卤代烷基,其任选地被氘代,直至完全氘代;
L为-Ar-(CH2)1-6-O-,其中每一个CH2可任选地被R#取代,R#选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;
L中的Ar与X相连,氧原子与磷原子相连;
Ar选自C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基,所述C3-10环烷基、3-10元杂环基、C6-10芳基或5-10元杂芳基可任选被1个、2个、3个、4个或5个R*取代;
R*选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;
其中P1选自保护基,优选羟基保护基,例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、4,4'-二甲氧基三苯甲基、-P(OCH2CH2CN)(N(iPr)2)或-C(O)CH2CH2C(O)OH,优选-P(OCH2CH2CN)(N(iPr)2)或-C(O)CH2CH2C(O)OH。
技术方案36.技术方案20-35中任一项的双链RNA,其选自小干扰RNA(siRNA)和短发夹RNA(shRNA),优选用于抑制在眼部表达的基因。
技术方案37.技术方案20-36中任一项的双链RNA,其中所述正义链包含以下核苷酸序列之一:
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL6、
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL7、
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL8、
LL6s-CmsAmUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-LL7、
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-dTdT-LL7、
CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-IbsIbsIbs IbsIbsIbs-LL7、
LL6s-IbsIbsIbs-CmsAmsUmUmUmUmAfAmUfCfCfUmCmAmCmUmCmUmAmAmsAms-IbsIbsIbs-LL7,
其中LL6、LL7和LL8分别选自以下化合物:
并且其中表示通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至上一个核苷酸或核苷酸类似物的3’碳或相应位置,表示通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至下一个核苷酸或核苷酸类似物的5’碳或相应位置;当相应结构位于核酸链末端位置时,相应地表示连接至氢、末端修饰、末端保护基团或其他可用于核酸链末端结构。
技术方案38.技术方案37的双链RNA,其中所述反义链包含以下核苷酸序列:
VPUmsUfsUmAmGmAfGmUfGfAmGmGmAmUfUmAfAmAmAmUmGmsAmsGm。
技术方案39.双链RNA,其具有正义链和反义链,各链具有14至30个核苷酸,所述反义链包含与所述正义链和靶标mRNA充分互补的序列,其中所述正义链和/或反义链在内部、5’端和/或3’端包含两个或多个疏水性基团;优选地,所述疏水性基团如式(I)化合物中R基团所定义;优选地,所述疏水性基团通过接头,例如可生物降解的接头,连接至正义链和/或反义链上。
技术方案40.载体,其包含编码前述技术方案20-39中任一项所述的双链RNA的核苷酸序列。
技术方案41.细胞,其含有如技术方案20-39中任一项所述的双链RNA或如技术方案40所述的载体。
技术方案42.药物组合物,其包含如技术方案20-39中任一项所述的双链RNA、如技术方案40所述的载体、或如技术方案41所述的细胞,以及任选的药学上可接受的载剂或赋形剂。
技术方案43.试剂盒,其包含如技术方案20-39中任一项所述的双链RNA、如技术方案40所述的载体、或如技术方案41所述的细胞。
技术方案44:双链RNA,其具有正义链和反义链,各链具有14至30个核苷酸,所述反义链包含与所述正义链和靶标mRNA充分互补的序列,其中所述正义链的3’端为下式化合物,或其药学上可接受的盐、互变异构体或立体异构体:
并且其中表示通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至上一个核苷酸或核苷酸类似物的3’碳或相应位置,表示通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至下一个核苷酸或核苷酸类似物的5’碳或相应位置;当相应结构位于核酸链末端位置时,相应地表示连接至氢、末端修饰、末端保护基团或其他可用于核酸链末端结构。
具体化合物列表
本发明化合物在寡核苷酸中的编号和结构如下,其中,以5’->3’的顺序从化合物的至连接。
具体而言,根据5’->3’的顺序,如相应结构位于核酸链中间位置,表示通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至上一个核苷酸或核苷酸类似物的3’碳或相应位置,表示通过磷酸酯基团、硫代磷酸酯基团或其他连接基团连接至下一个核苷酸或核苷酸类似物的5’碳或相应位置;如相应结构位于核酸链末端位置,相应地表示连接至氢、末端修饰、末端保护基团或其他可用于核酸链末端结构。
合成实施例
以下实施例用于例示本发明而非限制本发明的范围。
缩写
实施例1:中间体化合物制备实施例
实施例1.1 DL0066的制备
在室温下将化合物1溶解到DCM(20.0mL)中,向该溶液中加入DCI(0.06g,0.502mmol),5A分子筛和化合物2(0.33g,1.105mmol),置换3次氮气,反应混合物在25℃下搅拌1小时。反应液在冰盐浴下降温至0℃,向反应液中加入6mL饱和NaHCO3溶液和6mL饱和食盐水溶液,然后加入50mL DCM,有机相再用饱和NaHCO3和饱和食盐水的1:1混合溶液(20mL x 3)洗涤,然后将有机相用Na2SO4干燥旋干,所得的粗产品经柱层析纯化(石油醚/乙酸乙酯=10/3~10/5),得到白色固体DL0066(620mg,收率53.89%,纯度96.00%).1H NMR(400MHz,DMSO-d6)δ7.25-7.36(m,4H),7.13-
7.25(m,5H),7.01-7.03(m,1H),6.78-6.93(m,4H),5.30-5.32(m,1H),4.47-4.72(m,1H),4.24-4.34(m,1H),4.12-4.15(m,1H),3.69-3.71(m,8H),3.48-3.59(m,3H),3.35-3.47(m,1H),3.13-3.25(m,1H),2.86-3.06(m,3H),2.70-2.78(m,2H),2.09-2.31(m,5H),1.86-1.96(m,2H),1.70-1.85(m,3H),1.47-1.51(m,6H),1.22-1.42(m,11H),1.06-1.16(m,20H),0.93-0.95(m,4H),0.87-0.89(m,4H),0.82-0.85(m,7H),0.65(s,3H)
实施例1.2 DL0067的制备
将化合物1(250mg,0.380mmol)溶解在DCM(4.00mL)中,加入DIEA(0.377mL,2.28mmol),DMAP(11.6mg,0.095mmol)和化合物2(228mg,2.28mmol)。反应液在25℃下搅拌18小时。LCMS检测到产物该有的mass响应。TLC(PE/EA=3/1)有新点生成,反应液直接浓缩,用碱性硅胶正相柱(SiO2,PE/EA=1/0~3/1)纯化,得到黄色油状物DL0067(87.0mg,收率28.65%,纯度94.84%)。LCMS(ESI):m/z=756.4[M-H]-;1H NMR(400MHz,CD3OD)δ7.36-7.38(m,2H),7.24-7.30(m,6H),7.16-7.22(m,1H),6.81-6.87(m,4H),5.38-5.49(m,1H),4.24-4.29(m,1H),3.81-3.89(m,1H),3.77(d,J=2.0Hz,6H),3.67-3.70(m,1H),3.54-3.58(m,1H),3.08-3.25(m,1H),2.57-2.59(m,4H),2.30-2.39(m,2H),2.21-2.28(m,1H),2.07-2.16(m,1H),1.46-1.62(m,2H),1.22-1.35(m,24H),0.90(t,J=6.4Hz,3H).
实施例1.3 DL0082的制备
1.中间体3的制备
在25℃下将化合物1(2.00g,13.6mmol)溶于DCM(50.0mL)中,依次加入DIEA(6.74mL,40.8mmol),HATU(7.75g,20.4mmol),化合物2(3.48g,13.6mmol),反应液在25℃下搅拌12小时。LCMS
显示产物生成。TLC(DCM/MeOH=10/1,PMA)显示化合物2未反应完全,有若干新点生成。向其中加入乙酸乙酯(200mL),混合液体用盐水洗涤三次(15.0mL x 3),有机相经减压浓缩后得到粗产品化合物。粗品经柱层析纯化TLC(DCM/MeOH=10/1至5/1,PMA),得到黄色油状化合物3(1.15g,收率21.95%)。1H NMR(400MHz,CD3OD)δ3.79-3.91(m,2H),3.75-3.78(m,1H),3.57-3.69(m,4H),3.47-3.56(m,2H),3.27-3.34(m,1H),2.29-2.50(m,2H),1.53-1.66(m,2H),1.27-1.37(m,24H),0.84-0.95(m,3H)
2.中间体4的制备
在25℃下将化合物3(1.15g,2.98mmol)溶于吡啶(20.0mL)中,置换氮气三次。再将DMTrCl(1.11g,3.28mmol)的DCM(10.0mL)溶液加入到其中。混合液体在25℃下搅拌3小时。LCMS显示产物生成,原料未消耗完全。TLC(DCM/MeOH=10/1,PMA)显示原料未消耗完全,新点生成。向其中加入DCM(200mL),依次用饱和碳酸氢钠溶液(20.0mL x 3)和饱和食盐水洗涤三次(20.0mL x 3)。有机相经减压浓缩后得到粗产品化合物。粗品经柱层析纯化TLC(PE/EA=3/1至1/1,PMA),得到黄色油状化合物4(620mg,收率30.22%)。
3.DL0082的制备
在25℃下将化合物4(420mg,0.611mmol)溶于DCM(3.00mL)中,依次将DIEA(0.605mL,3.66mmol),DMAP(18.6mg,0.153mmol),化合物5(367mg,3.66mmol)加入到上述混合液体中。反应液体在25℃下搅拌3小时。LCMS显示产物生成,原料未消耗完全。向其中加入DCM(50.0mL),用饱和食盐水洗涤三次(50.0mL x 3)。有机相经减压浓缩后得到粗产品化合物。粗品经prep-HPLC(色谱柱:Waters Xbridge BEH C18 150*25mm*5um;流动相:TEAA-ACN;梯度:55%-95%/16min;流速:15ml/min)纯化,得到黄色油状化合物DL0082(170mg,收率35.34%,纯度97.26%)。
1H NMR(400MHz,CD3OD)δ7.43(d,J=8.0Hz,2H),7.18-7.34(m,7H),6.82-6.89(m,4H),4.16-4.25(m,1H),3.92-4.13(m,2H),3.85(d,J=5.20Hz,1H),3.78(s,6H),3.66-3.76(m,1H),3.53-3.64(m,2H),3.33-3.42(m,1H),3.28(s,1H),3.12-3.23(m,3H),2.48-2.65(m,4H),2.19-2.43(m,2H),1.52(s,2H),1.25-1.35(m,27H),0.86-0.93(m,3H)
实施例1.4 DL0084的制备
1.化合物3的制备
将化合物1(207mg,0.37mmol)溶入DCM(5.00mL)中,加入HATU(177mg,0.46mmol)和DIEA(0.15mL,0.93mmol),反应在25摄氏度下搅拌半小时,后加入化合物2(140mg,0.31mmol),反应在25摄氏度下反应16小时。TLC(DCM/MeOH=10/1)显示有新点生成。向反应液中加入10.0mL DCM,10.0mL H2O,分液,向有机相中加入碳酸氢钠溶液(10.0mL x 1),饱和食盐水洗涤(10.0mL x 1),无水硫酸钠干燥,过滤,旋干得到白色固体粗品产物化合物3(200mg,0.20mmol,65.10%)。
1H NMR(400MHz,CD3OD)δ7.45(d,J=8.0Hz,2H),7.16-7.38(m,7H),6.82-6.90(m,4H),4.39(d,J=8.0Hz,1H),3.93-4.17(m,2H),3.80(s,7H),3.39-3.71(m,8H),3.10-3.31(m,2H),2.84-3.03(m,2H),1.46-1.70(m,4H),1.16-1.45(m,51H),0.91(t,J=8.0Hz,6H)
2.DL0084的制备
将化合物3溶在DCM(7.0mL)中,加入DIEA(0.20mL,1.21mmol),DMAP(9.9mg,0.08mmol)和化合物4(121mg,1.21mmol),反应在25摄氏度下反应3小时。LCMS显示有所需产物的MS。反应液旋干,粗产品用prep-HPLC纯化(柱:01-Waters Xbridge BEH C18 19×150mm,5μm;条件:TEAA-ACN;Begin B 65-95;梯度时间:15分钟;100%B保持时间:2分钟;流速:15ml/分钟),得到黄色油状产物DL0084(80.0mg,收率:36.27%)。
1H NMR(400MHz,CDCl3)δ7.35(d,J=4.0Hz,2H),7.26-7.30(m,6H),7.09-7.25(m,1H),6.75(d,J=8.0Hz,4H),4.20-4.31(m,1H),3.75-4.16(m,5H),3.71(s,6H),3.32-3.56(m,6H),3.05-3.29(m,4H),2.99(q,J=8.0Hz,2H),2.50-2.60(m,4H),1.47(d,J=8.0Hz,4H),1.18(s,52H),0.77-0.84(m,6H)
实施例1.5 DL0127的制备
1.化合物3的制备
将化合物1(1.00g,3.90mmol)溶在DCM(20mL)中,加入HATU(1.48g,3.90mmol)和DIEA(3.86mL,23.3mmol),反应在25摄氏度下搅拌30分钟,后加入化合物2(0.68g,4.68mmol),反应在25摄氏度下搅拌16小时。LCMS显示有产物的ms值生成。TLC(PE/EA=1/1)显示有新点生成。向反应液中加入20mL水,反应液用DCM(15.0mL x 2)萃取。合并的有机相用氯化铵溶液(20.0mL x 1),碳酸氢钠溶液(20.0mL x 1),饱和食盐水洗涤(20.0mL x 1)洗涤,无水硫酸钠干燥,过滤,旋干得到粗产品。粗产品通过柱层析纯化(PE:EA=1/0-1/1),得到淡黄色固体产物化合物3(2.24g,5.839mmol,149.73%)。
1H NMR(400MHz,CD3OD)δ3.65(s,3H),3.15-3.19(m,2H),2.32(t,J=7.6Hz,2H),2.11-2.19(m,1H),1.59-1.68(m,2H),1.48-1.58(m,4H),1.37-1.40(m,2H),1.22-1.32(m,22H),0.89(t,J=6.8Hz,6H)
2.化合物4的制备
将化合物3溶在H2O(1.00mL)和THF(4.00mL)中,然后加入LiOH(218mg,5.21mmol),反应在25摄氏度下反应16小时。LCMS显示原料消失,TLC(PE/EA=1/1)显示有新点生成。向反应液中加入10.0mL水,反应液用EtOAc(10.0mL x 2)萃取。水相用3M HCl调节pH至~6,水相用EtOAc(10.0mL x 2)萃取,合并的有机相用饱和食盐水洗涤(10.0mL x 1),无水硫酸钠干燥,过滤,旋干得到白色固体产物化合物4(220mg,0.595mmol,45.67%)。
1H NMR(400MHz,CD3OD)δ3.18(t,J=8.0Hz,2H),2.29(t,J=8.0Hz,2H),2.15(m,1H),1.60-1.67(m,2H),1.48-1.59(m,4H),1.37-1.62(m,2H),1.17-1.35(m,22H),0.84-0.93(m,6H)
3.化合物6的制备
将化合物4(213mg,0.57mmol)溶在DCM(5.00mL)中,加入HATU(219mg,0.57mmol)和DIEA(0.22mL,1.335mmol),反应在25摄氏度下搅拌30分钟,后加入化合物5(200mg,0.44mmol),反应在25摄氏度下搅拌16小时。TLC(DCM/MeOH=10/1)显示有新点生成。LCMS显示原料消失,向反应液中加入10.0mL DCM稀释反应液,向反应液中加入碳酸氢钠溶液(10.0mL x 2),饱和食盐水洗涤(10.0mL x 2),无水硫酸钠干燥,过滤,旋干得到粗产品。粗产品通过柱层析纯化(DCM/MeOH=1/0-10/1),得到黄色油状产物化合物6(300mg,0.374mmol,84.15%)。1H NMR(400MHz,CD3OD)δ7.41-7.47(m,2H),7.20-7.36(m,7H),6.84-6.92(m,4H),3.85-4.06(m,2H),3.80(d,J=4.0Hz,6H),3.72-3.77(m,1H),3.42-3.71(m,5H),3.15-3.27(m,4H),2.13-2.43(m,3H),1.46-1.67(m,6H),1.20-1.38(m,23H),0.84-0.93(m,6H)
4.DL0127的制备
将化合物6(300mg,0.374mmol)溶在DCM(5.0mL)中,加入DIEA(0.371mL,2.247mmol),DMAP(18.30mg,0.150mmol)和化合物7(224.84mg,2.247mmol),反应在25摄氏度下反应16小时。LCMS显示有所需产物的MS。反应液旋干,粗产品用prep-HPLC纯化(柱:Waters Xbridge BEH C18
100*25mm*5um;条件:TEAA-ACN;Begin B 55-95;梯度时间:15分钟;100%B保持时间:6分钟;流速:15ml/分钟),得到黄色油状产物DL0127(120mg,0.133mmol,35.56%)。
1H NMR(400MHz,CD3OD)δ7.43(d,J=8.0Hz,2H),7.18-7.34(m,7H),6.86(dd,J=8.0,8.0Hz,4H),4.16-4.25(m,1H),3.92-4.14(m,2H),3.84-3.90(m,1H),3.78-3.82(m,6H),3.52-3.76(m,3H),3.34-3.46(m,1H),3.17-3.22(m,4H),2.49-2.62(m,4H),2.22-2.41(m,2H),2.10-2.17(m,1H),1.43-1.62(m,6H),1.25-1.35(m,24H),0.88(t,J=8.0Hz,6H)
实施例1.6 DL0133的制备
1化合物3的制备
在25℃下将化合物2(1.66g,8.26mmol)溶于DCM(50.0mL),依次加入HATU(3.93g,10.3mmol)和DIEA(3.42mL,20.7mmol),反应液在25℃下搅拌0.5小时,然后加入化合物1(1.00g,6.89mmol),反应液在25℃下搅拌14小时。LCMS显示有产物的mass值。薄层色谱(PE/EA=3/1)显示反应物消耗完全且有新点生成。将反应液用DCM(50.0mL)稀释,用饱和柠檬酸水溶液(10.0mL x 3)洗涤,有机相先后用饱和NaHCO3水溶液(20.0mL x 3)及饱和食盐水溶液(20.0mL x 3)洗涤,然后将有机相用Na2SO4干燥,有机相旋干得到粗品。粗品进行柱层析(PE/EA=1/0-3/1)纯化,得到白色固体化合物3(1.50g)。1H NMR(400MHz,CD3OD)δ3.65(s,3H),3.11-3.20(m,2H),2.33(t,J=7.2Hz,2H),2.16(t,J=7.6Hz,2H),1.57-1.68(m,4H),1.51(q,J=7.2Hz,2H),1.25-1.40(m,19H),0.86-0.93(m,3H).
2化合物4的制备
在25℃下将化合物3(1.00g,3.05mmol)溶于THF(5.00mL)和H2O(2.50mL)的混合溶剂中,加入LiOH(25.9mg,1.08mmol),反应液在25℃条件下搅拌反应18小时。薄层色谱(PE/EA=3/1)显示原料消耗完全且有新点生成。向反应液中加入1M HCl(1.00mL)溶液调酸性,然后加入二氯甲烷(20.0mL),分液。有机相用Na2SO4干燥,有机相旋干得到白色固体化合物4(900mg,收率94.04%)。1H NMR(400MHz,CDCl3)δ3.26(q,J=6.8Hz,2H),2.37(t,J=7.2Hz,2H),2.13-2.20(m,2H),1.80-1.90(m,1H),1.58-1.71(m,4H),1.53(q,J=7.2Hz,2H),1.36-1.43(m,2H),1.21-1.32(m,16H),0.84- 0.92(m,3H).
3化合物6的制备
在25℃下将化合物4(167mg,0.534mmol)溶于DCM(10.0mL),依次加入HATU(169mg,0.445mmol)和DIEA(0.074mL,0.445mmol),反应液在25℃下搅拌0.5小时,然后加入化合物5(200mg,0.445mmol),反应液在25℃下搅拌16小时。LCMS显示有产物的mass值。薄层色谱(DCM/MeOH=10/1)显示有新点。将反应液用DCM(20.0mL)稀释,先后用饱和柠檬酸水溶液(5.00mL x 3)、饱和NaHCO3水溶液(5.00mL x 3)和饱和食盐水溶液(5.00mL x 3)洗涤,然后将有机相用无水Na2SO4干燥,有机相旋干得到粗品。粗品进行柱层析(DCM/MeOH=1/0~10/1)纯化,得到化合物6(460mg)的粗品。1H NMR(400MHz,CD3OD)δ7.42(dd,J=7.6,2.0Hz,2H),7.17-7.33(m,6H),6.80-6.92(m,4H),3.89-3.98(m,1H),3.78(s,6H),3.36-3.75(m,8H),3.09-3.19(m,3H),2.11-2.40(m,4H),1.43-1.63(m,6H),1.28(s,18H),0.85-0.94(m,3H)
4 DL0133的制备
在25℃下将化合物6(460mg,0.617mmol)溶于DCM(10.0mL),依次加入DMAP(75.4mg,0.617mmol),DIEA(0.102mL,0.617mmol)和化合物7(371mg,3.71mmol),反应液在25℃下搅拌2小时。LCMS显示有产物的mass值。将反应液旋干得到粗品。粗品通过prep-HPLC(色谱柱:Waters Xbridge BEH C18 250*50mm*10um;流动相:TEAA-ACN;B%:55%-81%,10min;流速:15ml/min)分离,得到无色油状液体DL0133(180mg,收率34.52%,纯度98.01%)。1H NMR(400MHz,CD3OD)δ7.43(d,J=7.6Hz,2H),7.17-7.34(m,7H),6.86(dd,J=7.8,5.9Hz,4H),4.18-4.26(m,1H),3.93-4.15(m,2H),3.87(d,J=2.8Hz,1H),3.78(d,J=1.6Hz,6H),3.33-3.69(m,4H),3.10-3.23(m,5H),2.51-2.62(m,4H),2.23-2.41(m,2H),2.15(q,J=7.6Hz,2H),1.45-1.62(m,6H),1.21-1.39(m,22H),0.89(t,J=6.8Hz,3H).
实施例1.7 DL0134的制备
1化合物3的制备
在25℃下将化合物2(1.537mL,5.739mmol)溶于DCM(20.0mL)中,依次加入HATU(3.27g,8.609mmol)和DIEA(2.846mL,17.218mmol),混合液体在25℃下搅拌0.5小时后将化合物1(1g,6.887mmol)加入反应体系。反应液体在25℃下搅拌12小时。薄层色谱(PE/EA=2/1)显示原料反应完全,有新点生成。反应液用二氯甲烷稀释(30mL),依次用饱和柠檬酸溶液(15mLx3)、饱和碳酸氢钠溶液(15mLx3)和饱和氯化钠溶液(15mLx3)洗涤。有机相经无水硫酸钠干燥后减压浓缩得到粗品,粗品再经柱层析(PE/EA=50/50~40/60)纯化。得到白色固体产物3(630mg,收率30.87%)。1H NMR(400MHz,METHANOL-d4)δ3.65(s,3H),3.16(t,J=7.03Hz,2H),2.33(t,J=7.40Hz,2H),2.16(t,J=7.40Hz,2H),1.45-1.66(m,6H),1.25-1.36(m,22H),0.85-0.95(m,3H)
2化合物4的制备
在25℃将化合物3(630mg,1.772mmol)溶于THF(10mL)中,加入H2O(5mL)和KOH(149.13mg,2.658mmol),在25℃下反应12小时。T1:薄层色谱(PE/EA=2/1)和T2:薄层色谱(DCM:MeOH=10:1)显示原料消失,有新点生成。反应液加水稀释(15mL),用1moL/L的盐酸调pH至3~4,再用乙酸乙酯萃取3遍(15mL X 3),反应液减压浓缩得到白色固体产物4(610mg,收率100.80%)。1H NMR(400MHz,METHANOL-d4)δ3.11-3.21(m,2H),2.29(t,J=7.40Hz,2H),2.16(t,J=7.40Hz,2H),1.43-1.68(m,6H),1.22-1.43(m,22H),0.83-0.96(m,3H)
3化合物6的制备
在25℃将化合物4(227.92mg,0.667mmol)溶于DCM(10mL)中,加入HATU(317.18mg,0.834mmol)和DIEA(0.368mL,2.224mmol),反应0.5小时后加入化合物5,继续反应12小时。薄层色谱(DCM/MeOH=10/1)显示原料反应完全,有新点生成。反应液用二氯甲烷稀释(20mL),饱和食盐水洗涤三次(10mL X 3),有机相经减压浓缩后得到粗产品化合物。粗品经柱层析(DCM/MeOH=95/5~92/8)纯化。得到白色固体产物6(350mg,0.453mmol,81.41%)。1H NMR(400MHz,METHANOL-d4)δ7.42(dd,J=2.26,7.78Hz,2H),7.18-7.35(m,7H),6.78-6.94(m,4H),3.85-4.04(m,2H),3.78(d,J=2.51Hz,6H),3.66-3.75(m,1H),3.44-3.63(m,4H),3.33-3.42(m,1H),3.06-3.22(m,4H),2.26-2.42(m,2H),2.15(q,J=7.19Hz,2H),1.44-1.62(m,6H),1.28(s,22H),0.87-0.92(m,3H)
4.DL0134的制备
在25℃将化合物6(350mg,0.453mmol))溶于DCM(10mL)中,加入DMAP(13.83mg,0.113mmol)、DIEA(0.449mL,2.716mmol)和化合物7(271.83mg,2.716mmol)。反应液在25℃下搅拌12小时。LCMS显示原料反应完全,有产物生成。将反应液减压浓缩得粗品。粗品通过Prep-HPLC(色谱柱:Waters Xbridge BEH C18 100*25mm*5um;流动相:TEAA-ACN;梯度:55%-95%/15min;流速:15ml/min)纯化。得到无色油状产物DL0134(216mg,收率54.64%,纯度95.94%),1H NMR(400MHz,METHANOL-d4)δ7.41-7.47(m,2H),7.19-7.34(m,7H),6.81-6.91(m,4H),4.16-4.26(m,1H),3.84-4.14(m,3H),3.45-3.81(m,9H),3.35-3.43(m,1H),3.25-3.28(m,1H),3.11-3.23(m,8H),2.55-2.61(m,2H),2.48-2.54(m,2H),2.20-2.39(m,2H),2.11-2.18(m,2H),1.43-1.64(m,6H),1.21-1.37(m,30H),0.87-0.92(m,3H)
实施例1.8 DL0135的制备
1化合物3的制备
将化合物1(1.00g,3.90mmol)溶在DCM(20.0mL)中,加入HATU(1.78g,4.68mmol)和DIEA(3.87mL,23.4mmol),反应在25℃下反应0.5小时。然后加入化合物2(0.92g,5.07mmol),反应在25℃下反应16.5小时。薄层色谱(PE/EtOAc=2/1)显示有新点生成。向反应液中加入DCM(25.0mL)和水(20.0mL),反应液分液,水相用DCM(15.0mL)萃取。合并的有机相用饱和食盐水洗涤(20mL),无水硫酸钠干燥,过滤,旋干得到粗产品。粗产品通过柱层析纯化(PE/EtOAc=1/0~1/1),得到白色固体产物化合物3(1.20g,收率80.21%)。1H NMR(400MHz,CD3OD)δ3.67(s,3H),3.13-3.22(m,2H),2.35(t,J=7.6Hz,2H),2.18(t,J=7.6Hz,2H),1.47-1.71(m,6H),1.24-1.43(m,28H),0.87-0.97(m,3H)
2化合物4的制备
将化合物3(740mg,1.93mmol)溶在THF(2.00mL)和H2O(6.00mL)中,加入LiOH(324mg,7.72mmol),反应在20℃下反应16小时。薄层色谱(PE/EA=3/1,PMA)显示原料消耗完全,有新点生成。用1M HCl将反应液体调pH至5,用二甲基四氢呋喃将产物萃取(30.0mL X 3),有机相在减压条件下旋干,得到白色固体化合物4(650mg,收率91.17%)。
3化合物6的制备
将化合物4(267mg,0.723mmol)溶在DCM(5.00mL)中,加入HATU(275mg,0.723mmol)和DIEA(0.276mL,1.67mmol),反应在25℃下搅拌0.5小时,加入化合物5(250mg,0.556mmol),反应在25℃下搅拌16小时。薄层色谱(DCM/MeOH=10/1)显示有新点生成。向反应液中加入DCM(10.0mL)稀释反应液,向反应液中加入碳酸氢钠溶液(10.0mL X 2),饱和食盐水洗涤(10.0mL X 2)洗涤,无水硫酸钠干燥,过滤,旋干得到粗产品。粗产品通过柱层析纯化(DCM/MeOH=1/0-10/1),得到黄色油状产物化合物3(390mg,收率87.54%)。
4 DL0135的制备
将化合物6(390mg,0.487mmol)溶在DCM(7.00mL)中,加入DIEA(0.483mL,2.92mmol),DMAP(23.8mg,0.195mmol)和化合物7(292mg,2.92mmol),反应在25℃下反应3小时。LCMS显示有所需产物的MS。反应液旋干,粗产品用prep-HPLC纯化(柱:Waters Xbridge BEH C18 100*25mm*5um;条件:TEAA-CAN;Begin B 55-95;梯度时间:15分钟;100%B保持时间:6分钟;流速:15ml/分钟),得到黄色油状产物DL0135(140mg,收率31.91%,纯度99.54%)。1H NMR(400MHz,CD3OD)δ7.39-7.48(m,2H),7.17-7.36(m,7H),6.80-6.92(m,4H),4.18-4.26(m,1H),3.83-4.16(m,3H),3.78(m,6H),3.49-3.76(m,3H),3.33-3.45(m,1H),3.09-3.20(m,4H),2.50-2.64(m,4H),2.22-2.42(m,2H),2.15(q,J=8.0Hz,2H),1.42-1.65(m,6H),1.24-1.32(m,28H),0.84-0.96(m,3H)
实施例1.9 DL0136的制备
1化合物3的制备
在25℃下将化合物2(1305.00mg,4.587mmol)溶于DMF(50mL)中,依次加入HATU(2616.43mg,6.881mmol)和DIEA(2.275mL,13.762mmol),混合液体在25℃下搅拌0.5小时后将化合物1(1000mg,5.505mmol)加入反应体系。反应液体在25℃下搅拌11.5小时。薄层色谱(PE/EA=2/1)显示原料反应完全,有新点生成。反应液用乙酸乙酯稀释(50mL),依次用饱和柠檬酸溶液(30mLx3)、饱和碳酸氢钠溶液(30mLx3)和饱和氯化钠溶液(30mLx3)洗涤。有机相经无水硫酸钠干燥后减压浓缩得到粗品,粗品再经柱层析(PE/EA=70/30~60/40)纯化。得到白色固体产物3(1530mg,收率81.02%)。1H NMR(400MHz,METHANOL-d4)δ3.65(s,3H),3.15(br d,J=6.38Hz,2H),2.33(br t,J=7.25Hz,2H),2.09-2.24(m,2H),1.42-1.72(m,6H),1.29(br s,30H),0.90(br s,3H)
2化合物4的制备
在25℃下将化合物3(1.53g,3.717mmol)溶于THF(20mL)中,加入H2O(10mL)和LiOH(0.19g,4.460mmol),反应搅拌12小时。薄层色谱(PE/EA=2/1)显示原料消失,有新点生成。反应液减压浓缩,加水(10mL)和乙腈(5mL)溶解冻干。得到白色固体产物4(1313mg,收率87.54%)。1H NMR(400MHz,METHANOL-d4)δ3.16(t,J=6.94Hz,2H),2.29(t,J=7.38Hz,2H),2.16(t,J=7.50Hz,2H),1.45-1.63(m,6H),1.29(s,29H),0.90(br t,J=6.75Hz,3H)
3化合物6的制备
在25℃将化合物4(300mg,0.754mmol))溶于DCM(10mL)中,加入HATU(406.40mg,1.069mmol)和DIEA(0.416mL,2.515mmol),反应0.5小时后加入化合物5(282.64mg,0.629mmol),继续反应7.5小时。薄层色谱(DCM/MeOH=8/1)显示原料反应完全,有新点生成。反应液用二氯甲烷稀释(10mL),依次用饱和柠檬酸溶液(10mLX3)、饱和碳酸氢钠溶液(10mLX3)和饱和氯化钠溶液(10mLX3)洗涤。有机相经无水硫酸钠干燥后减压浓缩得到粗品,再经柱层析(DCM/MeOH=95/5~94/6)纯化。得到无色油状产物6(358mg,收率68.71%)。1H NMR(400MHz,METHANOL-d4)δ7.38-7.48(m,2H),7.20-7.35(m,7H),6.79-6.91(m,4H),3.86-4.08(m,2H),3.78(d,J=2.51Hz,6H),3.36-3.74(m,6H),3.06-3.24(m,4H),2.23-2.43(m,2H),2.15(q,J=6.94Hz,2H),1.44-1.64(m,6H),1.28(s,30H),0.86-0.92(m,3H)
4 DL0136的制备
在25℃将化合物6(358mg,0.432mmol)溶于DCM(5mL)中,加入DMAP(13.19mg,0.108mmol)、DIEA(0.428mL,2.591mmol)和化合物7(259.23mg,2.591mmol)。反应液在25℃下搅拌3小时。薄层色谱(DCM/MeOH=10/1)显示原料反应完全,有新点生成。将反应液用二氯甲烷稀释(10mL),依次用饱和NaHCO3溶液(10mLX 3)和饱和NaCl溶液(10mLx3)洗涤3遍,无水硫酸钠干燥后减压浓缩,经柱层析(DCM/MeOH=95/5~90/10)纯化得粗品。粗品通过Prep-HPLC(色谱柱:Waters Xbridge BEH C18 100*25mm*5um;流动相:TEAA-ACN;梯度:65%-95%/15min;流速:15ml/min)纯化。得到无色油状产物DL0136(163mg,收率40.63%,纯度97.57%),1H NMR(400MHz,METHANOL-d4)δ7.43(br d,J=8.03Hz,2H),7.18-7.34(m,7H),6.82-6.91(m,4H),4.08-4.26(m,2H),3.84-4.04(m,3H),3.78(d,J=2.01Hz,6H),3.54-3.65(m,2H),3.35-3.45(m,1H),3.13-3.22(m,4H),2.49-2.65(m,4H),2.23-2.44(m,2H),2.16(q,J=7.53Hz,2H),1.45-1.62(m,6H),1.25-1.36(m,33H),0.85-0.94(m,3H)
实施例1.10 DL0137的制备
1.化合物3的制备
在25℃下将化合物2(896mg,2.54mmol)溶于DCM(15.0mL)中,依次加入HATU(1205mg,3.17mmol),DIEA(1.05mL,6.34mmol)。混合液体在25℃下搅拌0.5小时后将化合物1(950mg,2.113mmol)加入到其中。混合液体在25℃下搅拌3小时。LCMS显示产物生成。TLC(DCM/MeOH=10/1)显示原料反应完全。向反应液体中加入二氯甲烷(60.0ml)后,用饱和食盐水洗涤三次(10.0ml x 3),有机相用无水硫酸钠干燥后在减压条件旋干得到粗品化合物。粗产品经柱层析纯化(PE/EA=5/1至1/1),得到无色油状化合物3(1.3g,1.66mmol,收率78.37%)。1H NMR(400MHz,CD3OD)δ7.78(d,J=7.6Hz,2H),7.57-7.69(m,2H),7.34-7.45(m,4H),7.24-7.32(m,7H),7.18-7.21(m,1H),6.93-7.10(m,1H),6.79-6.89(m,4H),4.29-4.39(m,2H),4.17(s,1H),3.86-4.01(m,1H),3.42-3.82(m,12H),3.33-3.40(m,1H),2.99-3.26(m,5H),2.13-2.43(m,2H),1.39-1.62(m,4H)
2.化合物4的制备
在25℃下将化合物3(650mg,0.828mmol)溶于MeCN(6.00mL)中,将Et2NH(1.00mL)加入到其中。混合液体在25℃下搅拌3小时。LCMS和TL(PE:EA=1:1)显示原料反应完全。反应液体直接在减压条件下浓缩旋干,得到化合物4(450mg,0.800mmol,收率96.57%)
3.化合物6的制备
在25℃下将化合物5(58.1mg,0.171mmol)溶于DMF(5.00mL)中,依次加入HATU(70.3mg,0.185mmol),DIEA(0.070mL,0.427mmol),搅拌0.5小时后将化合物4(80.0mg,0.142mmol)加入到其中,混合液体在25℃下搅拌12小时。TLC(DCM/MeOH=10/1)显示有新点生成。向反应液体中加入乙酸乙酯(60.0mL)后,用饱和碳酸氢钠溶液(10.0mL x 3),饱和食盐水洗涤三次(10.0mL x 3),有机相用无水硫酸钠干燥后在减压条件旋干,得到粗品化合物。粗产品经柱层析TLC(DCM/MeOH=10/1至5/1)纯化,得到黄色油状化合物6(120mg,收率95.34%)。1H NMR(400MHz,CD3OD)δ7.39-7.46(m,2H),7.16-7.34(m,7H),6.81-6.91(m,4H),3.71-3.78(m,8H),3.43-3.70(m,5H),3.33-3.43(m,2H),3.18-3.23(m,4H),3.12-3.17(m,2H),2.11-2.42(m,4H),1.44-1.64(m,6H),1.32-1.40(m,6H),1.25-1.32(m,38H),0.85-0.93(m,3H)
4..产品DL0137的制备
在25℃将化合物6(120mg,0.136mmol)和化合物7(81.4mg,0.813mmol)加入到DCM(3.00mL)中,依次加入DMAP(4.14mg,0.034mmol),DIEA(0.134mL,0.813mmol)。混合液体在25℃下搅拌12小时。TLC(DCM/MeOH=10/1,UV)显示原料反应完全,新点生成。向反应液体中加入二氯甲烷(60.0mL)后,用饱和碳酸氢钠溶液(10.0mL x 3),饱和食盐水洗涤三次(10.0mL x 3),有机相用无水硫酸钠干燥后在减压条件旋干,得到粗品化合物。粗产品经柱层析纯化(DCM/MeOH=10/1~8/1),得到无色油状化合物粗品,粗产品经prep-HPLC(色谱柱:Waters Xbridge BEH C18 150*25mm*5um;流动相:TEAA-ACN;梯度:65%-95%/15min;流速:15ml/min)纯化,得到白色固体化合物DL0137(45.0mg,收率33.69%,纯度95.06%)。1H NMR(400MHz,CD3OD)δ7.43(d,J=8.0Hz,2H),7.16-7.34(m,7H),6.84-6.87(m,4H),3.85-4.26(m,4H),3.67-3.78(m,7H),3.56-3.65(m,2H),3.33-3.46(m,1H),3.10-3.23(m,6H),2.48-2.63(m,4H),2.20-2.42(m,2H),2.16(q,J=7.6Hz,2H),1.45-1.63(m,6H),1.14-1.44(m,44H),0.85-0.94(m,3H)
实施例1.11 DL0138的制备
1化合物3的制备
在25℃下将化合物2(1.47mL,4.60mmol)溶于DCM(30.0mL),依次加入HATU(2.10g,5.52mmol)和DIEA(2.28mL,13.8mmol),搅拌0.5小时,然后加入化合物1(1.00g,5.52mmol),反应液在25℃下搅拌16小时。薄层色谱(PE/EA=3/1)显示原料消耗完全且有新点生成。向反应液中加入二氯甲烷(30.0mL)稀释,先后用饱和柠檬酸水溶液(10.0mL x 3)、饱和NaHCO3水溶液(10.0mL x 3)和饱和NaCl水溶液(10.0mL x 3)洗涤,然后有机相用无水Na2SO4干燥,在减压条件下浓缩得到粗品。粗产品通过柱层析(PE/EA=1/0~5/1)纯化,得到白色固体化合物3(1.30g,收率68.95%)。1H NMR(400MHz,CD3OD)δ5.35-5.41(m,2H),3.65(s,3H),3.16(t,J=6.8Hz,2H),2.33(t,J=7.2Hz,2H),2.16(t,J=7.6Hz,2H),1.98(d,J=4.8Hz,4H),1.56-1.68(m,4H),1.46-1.55(m,2H),1.27-1.40(m,22H),0.86-0.94(m,3H).
2化合物4的制备
在25℃下将化合物3(1.30g,3.17mmol)溶于THF(5.00mL)和H2O(2.50mL)的混合溶剂中,加入LiOH(0.150g,6.35mmol),反应液在25℃条件下搅拌反应18小时。薄层色谱(PE/EA=3/1)显示原料消耗完全且有新点生成。向反应液中加入1M HCl(10.0mL)溶液调酸性,然后加入二氯甲烷(20.0mL),分液。有机相用Na2SO4干燥,有机相旋干得到白色固体化合物4(800mg,收率63.72%)。1H NMR(400MHz,CDCl3)δ5.54-5.63(m,1H),5.38(dt,J=4.4,2.4Hz,2H),3.26(q,J=6.8Hz,2H),2.36(t,J=7.2Hz,2H),2.12-2.20(m,2H),1.91-2.02(m,4H),1.47-1.72(m,6H),1.24-1.42(m,22H),0.84-0.93(m,3H).
3化合物6的制备
在25℃下将化合物4(253mg,0.641mmol)溶于DCM(9.00mL),依次加入HATU(304mg,0.801mmol)和DIEA(0.529mL,3.20mmol),搅拌0.5小时,然后加入化合物5(240mg,0.534mmol),反应
液在25℃下搅拌16小时。薄层色谱(DCM/MeOH=10/1)显示原料消耗完全且有新点生成。向反应液中加入二氯甲烷(30.0mL)稀释,先后用饱和NaHCO3水溶液(10.0mL x 3)和饱和NaCl水溶液(10.0mL x 3)洗涤,然后有机相用无水Na2SO4干燥,在减压条件下浓缩得到粗品。粗产品通过柱层析(DCM/MeOH=1/0~10/1)纯化,得到淡黄色油状液体化合物6(300mg,收率67.92%)。
4 DL0138的制备
在25℃下将化合物6(300mg,0.363mmol)溶于DCM(10.0mL),依次加入DIEA(0.360mL,2.17mmol)、DMAP(17.7mg,0.145mmol)和化合物7(218mg,2.18mmol),反应液在25℃下搅拌2小时。LCMS显示有产物的mass值。将反应液旋干得到粗品。粗品通过反相柱(色谱柱:Waters Xbridge BEH C18 100*25mm*5um;流动相:TEAA-CAN;B%:60%-95%,10min;流速:15ml/min)制备分离,得到无色油状液体DL0138(178mg,收率52.93%,纯度97.55%)。1H NMR(400MHz,CD3OD)δ7.41-7.48(m,2H),7.20-7.35(m,7H),6.84-6.93(m,4H),5.39(t,J=3.6Hz,2H),3.93-4.26(m,3H),3.89(d,J=3.6Hz,1H),3.80(d,J=2.0Hz,6H),3.54-3.76(m,3H),3.35-3.48(m,1H),3.30(d,J=5.2Hz,1H),3.13-3.24(m,10H),2.49-2.63(m,4H),2.23-2.42(m,2H),2.17(q,J=7.6Hz,2H),1.98(d,J=4.8Hz,4H),1.46-1.64(m,6H),1.24-1.39(m,32H),0.88-0.95(m,3H).
实施例1.12 DL0139的制备
1化合物3的制备
将化合物1(200mg,0.713mmol),化合物2(647mg,3.566mmol),HOBt(144.55mg,1.070mmol),EDCI(205.06mg,1.070mmol),DIEA(0.943mL,5.705mmol)依次加入到DCM(5.00mL)中,在25℃反应14小时。TLC(石油醚:乙酸乙酯=2:1)显示原料化合物1消失,有新点生成。反应液用二氯甲烷(50.0mL)稀释,用柠檬酸(10mL x 3),碳酸氢钠(10mL x 3),饱和氯化钠(10mL x 3)洗涤,有机相用无水硫酸钠干燥,过滤旋干得到黄色固体产物化合物3(250mg,0.613mmol,86.00%)。1H NMR(400MHz,CDCl3)δ5.39-5.47(m,4H),3.65-3.70(m,3H),3.25(q,J=6.8Hz,2H),2.65-2.70(m,2H),2.32(t,J=7.2Hz,2H),2.15(t,J=7.6Hz,2H),1.94-2.03(m,4H),1.60-1.70(m,4H),1.52(q,J=7.2Hz,2H),1.26-1.38(m,16H),0.89(t,J=6.8Hz,3H).
2化合物4的制备
将化合物3(330mg,0.810mmol)溶解于THF(4.00mL)和H2O(2.00mL)中,加入LiOH(67.9mg,1.62mmol),在25℃反应14小时。TLC(石油醚:乙酸乙酯=1:1)显示原料消失,有新点生成。反应液降至0℃,用1M HCl调节反应液pH=5,水相用二甲基四氢呋喃(20毫升x 3)萃取,有机相用无水硫酸钠干燥,过滤,旋干得到棕色固体产物化合物4(350mg,0.889mmol,109.84%)。1H NMR(400MHz,CD3OD)δ5.32-5.46(m,4H),3.16(t,J=6.8Hz,2H),2.65(d,J=4.0Hz,2H),2.29(t,J=7.2Hz,2H),2.13-2.21(m,2H),1.96-2.03(m,4H),1.48-1.67(m,6H),1.26-1.38(m,16H),0.90(t,J=6.8Hz,3H).
3化合物6的制备
将化合物4(200mg,0.508mmol),HATU(263mg,0.693mmol),DIEA(0.229mL,1.38mmol)加入到DCM(3.00mL)中,在25℃反应30分钟,加入化合物5(207mg,0.462mmol),在25℃反应12小时。LCMS显示有83%产物生成。反应液用二氯甲烷(100毫升)稀释,用饱和碳酸氢钠水溶液(30毫升x 3)和饱和氯化钠水溶液(30毫升x 3)洗涤,有机相用无水硫酸钠干燥,过滤,旋干得到棕色固体粗品产物化合物6(600mg,0.727mmol)。
4 DL0139的制备
将化合物6(600mg,0.727mmol)和化合物7(436mg,4.36mmol)溶解在DCM(6.00mL)中,加入DIEA(0.721mL,4.36mmol)和DMAP(22.2mg,0.182mmol),氮气置换三次,反应液在25℃反应1小时,LCMS显示原料消失,有84.2%产物生成。反应液直接旋干得到粗产品,粗产品通过Prep-HPLC(色谱柱:01-Waters Xbridge BEH C18 19*150mm;流动相:TEAA-ACN;梯度:55%-95%/15min;流速:15ml/min;)纯化。得到无色油状产物DL0139(200mg,0.216mmol,29.73%)。MS:m/z=1971.7(M+H)+;1H NMR(400MHz,CD3OD)δ7.38-7.46(m,2H),7.18-7.35(m,7H),6.81-6.90(m,4H),5.32-5.45(m,4H),4.16-4.26(m,1H),3.85-4.13(m,3H),3.66-3.81(m,7H),3.56-3.65(m,2H),3.35-3.46(m,1H),3.14-3.22(m,9H),2.63-2.68(m,2H),2.54-2.61(m,2H),2.46-2.53(m,2H),2.23-2.41(m,2H),2.10-2.19(m,2H),1.94-2.02(m,4H),1.45-1.61(m,6H),1.22-1.38(m,26H),0.85-0.93
(m,3H).
实施例1.13 DL0140的制备
1化合物3的制备
将化合物1(500mg,1.52mmol)和HATU(752mg,1.97mmol)溶在DCM(5.00mL)中,加入DIEA(1.50mL,9.13mmol)。反应在40摄氏度下反应0.5小时。然后加入化合物2(106mg,0.731mmol)。反应在40摄氏度下反应16小时。TLC(PE/EtOAc=2/1)显示有新点生成。向反应液中加入25mL DCM和20mL水,反应液分液,水相用DCM(15mL x 1)萃取。合并的有机相用饱和食盐水洗涤(20mL x1),无水硫酸钠干燥,过滤,旋干得到粗产品。粗产品通过柱层析纯化(PE/EtOAc=1/0-1/1),得到化合物3(520mg,1.14mmol,74.97%)。1H NMR(400MHz,CD3OD)δ5.29-5.47(m,12H),3.67(s,3H),3.13-3.23(m,2H),2.80-2.95(m,10H),2.31-2.45(m,4H),2.19-2.27(m,2H),2.06-2.16(m,2H),1.58-1.70(m,2H),1.46-1.57(m,2H),1.32-1.42(m,2H),0.99(t,J=7.6Hz,3H)
2化合物4的制备
将化合物3(520mg,1.14mmol)溶在H2O(2mL)和THF(5mL)中,加入LiOH(191mg,4.56mmol)。反应在20摄氏度下反应16小时。TLC(PE/EtOAc=2/1)显示有新点生成。反应液旋干。向粗产品中加入15mL DCM和15mL水,反应液分液,水相用3M HCl调节pH到5。水相用DCM(15mL x 2)萃取。有机相用饱和食盐水洗涤(20mL x 1),无水硫酸钠干燥,过滤,旋干得到无色油状产品化合物4(420mg,0.951mmol,83.34%)。1H NMR(400MHz,CD3OD)δ5.21-5.48(m,12H),3.16(t,J=7.2Hz,2H),2.78-2.94(m,10H),2.34-2.43(m,2H),2.29(t,J=7.2Hz,2H),2.19-2.24(m,2H),2.04-2.15(m,2H),1.57-1.67(m,2H),1.46-1.56(m,2H),1.31-1.42(m,2H),0.97(t,J=7.6Hz,3H)。
3化合物6的制备
将化合物4(319mg,0.72mmol)溶在DCM(5mL)中,加入HATU(274mg,0.72mmol)和DIEA(0.27mL,1.66mmol),反应在25摄氏度下搅拌半小时,加入化合物5(250mg,0.556mmol),反应在25摄氏度下反应16小时。TLC(DCM/MeOH=10/1)显示有新点生成。向反应液中加入10mL DCM和15mL水,反应液分液,合并的有机相中加入碳酸氢钠溶液(10mL x 2),饱和食盐水洗涤(10mL x 2)洗涤,无水硫酸钠干燥,过滤,旋干得到粗产品。粗产品通过柱层析纯化(DCM/MeOH=1/0-10/1),得到黄色油状产物化合物6。
4 DL0140的制备
将化合物6(300mg,0.344mmol)溶在DCM(7mL)中,加入DIEA(0.34mL,2.06mmol),DMAP(16.7mg,0.13mmol)和化合物7(206mg,2.06mmol),反应在25摄氏度下反应3小时。反应液旋干,粗产品用prep-HPLC纯化(柱:Waters Xbridge BEH C18 100*25mm*5um;条件:TEAA-ACN;Begin B 55-95;梯度时间:15分钟;100%B保持时间:6分钟;流速:15ml/分钟),得到黄色油状产物DL0140(120mg,收率35.89%,纯度97.34%)。1H NMR(400MHz,CD3OD)δ7.43(d,J=8.0Hz,2H),7.17-7.36(m,7H),6.81-6.92(m,4H),5.19-5.46(m,11H),4.16-4.29(m,1H),3.84-4.15(m,3H),3.76-3.80(m,6H),3.35-3.73(m,4H),3.07-3.18(m,6H),2.76-2.93(m,10H),2.50-2.64(m,4H),2.16-2.44(m,6H),2.03-2.13(m,2H),1.42-1.66(m,4H),1.32-1.38(m,6H),0.96(t,J=8.0Hz,3H)
实施例1.14 DL0142的制备
1化合物2的制备
将化合物1(2.00g,10.9mmol)溶在THF(80.0mL)中,加入TBAB(0.71g,2.19mmol),化合物1A(13.4g,43.9mmol)和KOH(3.08g,54.8mmol),反应在25摄氏度下反应16小时。TLC(PE/EA=10/1)显示有新点生成。向反应液中加入15.0mL水,反应液用EtOAc(15.0mL x 2)萃取。合并的有机相用饱和食盐水洗涤(15.0mL x 1),无水硫酸钠干燥,过滤,旋干得到粗产品。粗产品通过柱层析纯化(PE/EA=1/0-10/1),得到无色油状产物2(5.5g,8.71mmol,79.40%)。1H NMR(400MHz,CD3OD)δ7.14-7.40(m,5H),4.41-4.55(m,2H),3.28-3.64(m,9H),0.26-1.08(m,56H),-0.58-0.00(m,6H)
2化合物3的制备
将化合物2(5.50g,8.75mmol)溶在EtOH(70.0mL)中,加入Pd(OH)2(0.20g,3.16mmol),反应在50摄氏度、H2(1atm)气压下反应16小时。TLC(PE/EA=10/1)显示有新点生成。过滤旋干得到粗产品。粗产品通过柱层析纯化(PE/EtOAC=1/0-10/1),得到白色固体产物3(2.45g,4.52mmol,51.9%)。1H NMR(400MHz,CD3OD)δ3.51-3.64(m,5H),3.47(s,4H),1.51-1.62(m,4H),1.29(s,52H),0.90(t,J=8.0Hz,6H)
3化合物4的制备
将化合物3(2.45g,4.52mmol)溶在DCM(15mL)和DMF(30.0mL)中,加入重铬酸吡啶盐(PDC)(5.96g,15.8mmol),反应在25摄氏度下反应16小时。TLC(PE/EA=3/1)显示有新点生成。向反应液中加入40.0mL H2O,后用DCM(40.0mL x 2)萃取,无水硫酸钠干燥,过滤,旋干得到粗产品。粗产品通过柱层析纯化(PE/EtOAC=1/0-5/1),得到白色固体产物4(1.1g,1.982mmol,43.77%)。1H NMR(400MHz,CDCl3)δ4.02-4.11(m,1H),3.77-3.87(m,1H),3.59-3.77(m,3H),3.44-3.56(m,2H),1.53-1.61(m,4H),1.26(s,52H),0.86-0.95(m,6H)
4化合物6的制备
将化合物4(665mg,1.198mmol)溶入DCM(15.0mL)中,加入HATU(592mg,1.55mmol),DIEA(1.18mL,7.19mmol)和化合物5(28.7mg,0.19mmol),反应在25摄氏度下反应16小时。TLC(PE/EA=3/1)显示有新点生成。向反应液中加入15.0mL DCM稀释反应液,向反应液中加入一水合柠檬酸(15.0mL x 1),碳酸氢钠溶液(15.0mL x 1),饱和食盐水洗涤(15.0mL x 1),无水硫酸钠干燥,过滤,旋干,得到白色固体粗品产物6(510mg,0.748mmol,62.39%)。1H NMR(400MHz,CDCl3)δ3.89(dd,J=4.0,5.6Hz,1H),3.76-3.81(m,1H),3.60-3.70(m,5H),3.39-3.56(m,3H),3.29(q,J=8.0Hz,2H),2.33(t,J=8.0Hz,2H),1.52-1.72(m,12H),1.27-1.38(m,50H),0.87-0.93(m,6H)
5化合物7的制备
将化合物6(510mg,0.748mmol)溶入THF(6.0mL)和H2O(3.0mL)中,加入LiOH(62.7mg,1.49mmol),反应在25摄氏度下反应16小时。TLC(PE/EA=3/1)显示有新点生成。反应液旋干,加入10.0mL水,水相用1M HCl调节pH至~4,水相用二甲基四氢呋喃(10.0mL x 2)萃取,合并的有机相用饱和食盐水洗涤(10.0mL x 1),无水硫酸钠干燥,过滤,旋干得到白色固体粗产品7(460mg,0.55mmol,73.6%)。1H NMR(400MHz,CD3OD)δ3.83-3.90(m,1H),3.36-3.65(m,6H),3.19-3.27(m,2H),2.29(t,J=8.0Hz,2H),1.49-1.68(m,8H),1.23-1.37(m,54H),0.86-0.94(m,6H)
6化合物9的制备
将化合物7(326mg,0.489mmol)溶入DCM(5.0mL)中,加入HATU(219mg,0.57mmol)和DIEA(0.22mL,1.33mmol),反应液在25摄氏度下反应30分钟,再加入化合物8(200mg,0.44mmol),反应液在25摄氏度下反应16小时。TLC(DCM/MeOH=10/1)显示有新点生成。向反应液中加入10.0mL H2O,反应液用DCM(10.0mL x 2)萃取,向反应液中加入碳酸氢钠溶液(10.0mL x 1),饱和食盐水洗涤(10.0mL x 1)洗涤,无水硫酸钠干燥,过滤,旋干得到粗产品,粗产品通过柱层析纯化(DCM/MeOH=1/0-5/1),得到白色固体粗品产物9(194mg,0.17mmol,39.7%)。1H NMR(400MHz,CDCl3)δ7.38-7.46(m,1H),7.27-7.35(m,7H),7.18(d,J=8.0Hz,1H),6.84(d,J=8.0Hz,4H),4.75-4.80(m,1H),3.87(s,2H),3.77-3.85(m,6H),3.69-3.76(m,3H),3.57-3.68(m,4H),3.37-3.56(m,5H),3.11-3.36(m,3H),2.72-2.81(m,1H),2.81(s,1H),2.18-2.45(m,2H),1.66-1.75(m,4H),1.43-1.51(m,4H),1.26(s,54H),0.86-0.92(m,6H).
7 DL0142的制备
将化合物9(390mg,0.48mmol)溶在DCM(7.00mL)中,加入DIEA(0.16mL,0.98mmol),DMAP(9.91mg,0.08mmol)和化合物10(98.28mg,0.982mmol),反应在25摄氏度下反应3小时。LCMS显示有所需产物的MS。反应液旋干,粗产品用prep-HPLC纯化(柱:Waters Xbridge BEH C18 19*150mm;条件:TEAA-ACN;Begin B 65-95;梯度时间:15分钟;100%B保持时间:2分钟;流速:15ml/分钟),得到白色固体产物DL0142(106mg,0.08mmol,53.9%)。1H NMR(400MHz,CDCl3)δ7.31-7.39(m,2H),7.21-7.27(m,5H),7.12-7.17(m,1H),6.89-6.96(m,1H),6.73-6.79(m,4H),4.29-4.40(m,1H),3.94-4.04(m,1H),3.77-3.92(m,3H),3.70-3.76(m,6H),3.67(d,J=4.0Hz,1H),3.29-3.51(m,6H),3.04-3.28(m,5H),2.94(q,J=8.0Hz,3H),2.47-2.59(m,4H),2.17-2.27(m,2H),1.43-1.61(m,8H),1.18-1.26(m,54H),0.78-0.85(m,6H)
实施例1.15 DL0143的制备
1.化合物3的制备
在25℃下将化合物2(2.00g,11.0mmol)溶于THF(60.0mL)中,依次将TBAB(0.71g,2.20mmol)、化合物1(10.5mL,43.9mmol)和KOH(3.08g,54.9mmol)加入到上述混合液体中。反应液体在25℃下搅拌12小时。薄层色谱(PE/EA=10/1)显示原料反应完全,有新点生成。将反应液用EA(50.0mL)稀释,依次用水洗(30.0mL X 3)、饱和柠檬酸溶液洗(30.0mL X 3)、水洗(30.0mL X 3)和饱和NaCl溶液(30.0mL X 3)洗涤,有机相经无水硫酸钠干燥后减压浓缩得到粗品,粗品经柱层析(PE/EA=97/3~96/4)纯化,得到无色油状化合物3(5.21g,收率91.48%)。1H NMR(400MHz,CD3OD)δ7.21-7.38(m,5H),4.53(s,2H),3.43-3.65(m,9H),1.49-1.62(m,4H),1.25-1.37(m,36H),0.87-0.93(m,6H)
2.化合物4的制备
将化合物3(2.77g,5.34mmol)加入到EtOH(30.0mL)中,加入Pd(OH)2(0.30g,0.214mmol),用氢气(14.696psi)置换体系气体3次,油浴加热50℃下反应12小时。薄层色谱(PE/EA=10/1)显示原料消失,有新点生成。反应液用硅藻土过滤,有机相减压浓缩得粗品。粗品经柱层析(PE/EA=95/5~94/6)纯化,得到无色油状粗品化合物4(2.20g)。1H NMR(400MHz,CD3OD)δ3.42-3.62(m,9H),1.50-1.63(m,4H),1.27-1.39(m,36H),0.84-0.97(m,6H)
3.化合物5的制备
在25℃下将化合物4(700mg,1.63mmol)溶于DCM(10.0mL)和DMF(5.00mL)中,将PDC(2.15g,5.71mmol)加入到其中。混合液体在25℃下搅拌12小时。薄层色谱(PE/EA=3/1)显示原料消失,新点生成。向反应液体中加入水(50.0mL),混合液体用乙酸乙酯萃取(100mL X 3),有机相在减压条件下浓缩悬干得到粗产品。粗产品经柱层析纯化(PE/EA=5/1~3/1),得到白色固体化合物5(320mg,收率44.27%)。1H NMR(400MHz,CDCl3)δ4.05(dd,J=3.2,5.2Hz,1H),3.78-3.83(m,1H),3.69-3.75(m,1H),3.64(t,J=6.4Hz,2H),3.42-3.56(m,2H),1.52-1.70(m,4H),1.23-1.37(m,36H),0.89(t,J=6.4Hz,6H)
4.化合物7的制备
在25℃下将化合物5(320mg,0.723mmol)加入到DCM(5.00mL)中,依次加入HATU(412mg,1.08mmol),DIEA(0.358mL,2.17mmol)。混合液体在25℃下搅拌0.5小时后将化合物6(158mg,0.867mmol)加入到其中。反应液体在25℃下搅拌12小时。TLC(PE/EA=3/1,PMA)显示原料消耗完全,有新点生成。向反应液体中加入二氯甲烷(100mL),依次用饱和碳酸氢钠溶液(15.0mL X 3)和饱和食盐水(15.0mL X 3)洗涤三次。有机相用无水硫酸钠干燥后,在减压条件下浓缩后得到粗产品化合物。粗产品经柱层析纯化(PE/EA=3/1~1/1,PMA),得到无色液体化合物7(200mg,收率48.55%)。1H NMR(400MHz,CD3OD)δ3.86(dd,J=3.2,5.2Hz,1H),3.64-3.70(m,4H),3.40-3.63(m,5H),3.20-3.24(m,2H),2.33(t,J=7.6Hz,2H),1.47-1.69(m,8H),1.22-1.43(m,38H),0.86-0.94(m,6H)
5.化合物8的制备
在25℃下将化合物7(200mg,0.351mmol)溶于THF(4.00mL)和H2O(2.00mL)中,将LiOH(44.2mg,1.05mmol)加入到其中。混合液体在25℃下搅拌12小时。薄层色谱(PE/EA=3/1,PMA)显示原料消耗完全,有新点生成。用1M HCl将反应液体调pH至5,用二甲基四氢呋喃将产物萃取(30.0mL X 3),有机相在减压条件下悬干,得到无色油状粗品化合物8(200mg)。1H NMR(400MHz,CD3OD)δ3.83-3.89(m,1H),3.64-3.71(m,1H),3.39-3.63(m,5H),3.18-3.28(m,2H),2.29(t,J=7.6Hz,2H),1.50-1.68(m,8H),1.24-1.40(m,38H),0.86-0.94(m,6H)
6.化合物10的制备
在25℃下将化合物8(193mg,0.347mmol)加入到DCM(5.00mL)中,依次将HATU(165mg,0.433mmol),DIEA(0.143mL,0.867mmol)加入到其中。反应液在25℃下搅拌0.5小时后将化合物9(130mg,0.289mmol)加入到其中,继续搅拌12小时。LCMS显示产物的MS生成。TLC(DCM/MeOH=1/1)显示新点生成。向反应液体中加入二氯甲烷(100mL),依次用饱和碳酸氢钠溶液(15.0mL X 3)和饱和食盐水(15.0mL X 3)洗涤三次。有机相用无水硫酸钠干燥后,在减压条件下浓缩后得到粗产品
化合物。粗产品经柱层析纯化(DCM/MeOH=10/1至8/1,PMA),得到无色液体化合物10(180mg,收率63.04%)。1H NMR(400MHz,CD3OD)δ7.69-7.87(m,1H),7.43(d,J=8.0Hz,1H),7.17-7.33(m,6H),7.07-7.15(m,1H),6.79-6.91(m,4H),3.83-4.05(m,2H),3.74-3.81(m,6H),3.63-3.73(m,3H),3.56-3.63(m,3H),3.40-3.56(m,6H),3.32-3.40(m,1H),3.15-3.28(m,4H),2.14-2.46(m,2H),1.46-1.68(m,8H),1.28(s,34H),0.84-0.94(m,6H)
7.化合物DL0143的制备
在25℃下将化合物10(180mg,0.182mmol)溶于DCM(5.00mL)中,依次加入化合物11(109mg,1.09mmol),DIEA(0.181mL,1.09mmol),DMAP(5.57mg,0.046mmol)。反应液在25℃下搅拌12小时。LCMS显示产物生成。反应液体直接在减压条件下悬干得到粗品,粗品通过反相柱(色谱柱:01-Waters Xbridge BEH C18 19*150mm;流动相:TEAA-ACN;梯度:75%-95%/18分钟;流速:15ml/分钟)制备分离,得到无色油状化合物DL0143(110mg,收率55.49%,纯度99.49%)。1H NMR(400MHz,CD3OD)δ7.43(d,J=7.6Hz,2H),7.18-7.33(m,7H),6.84-6.87(m,4H),4.09-4.28(m,2H),3.83-4.03(m,4H),3.78(d,J=2.0Hz,7H),3.61-3.70(m,2H),3.41-3.61(m,7H),3.33-3.41(m,1H),3.10-3.26(m,4H),2.51-2.66(m,4H),2.18-2.44(m,2H),1.45-1.69(m,8H),1.20-1.43(m,41H),0.85-0.93(m,6H)
实施例1.16 DL0144的制备
1.化合物3的制备
在25℃下将化合物1(1.00g,2.59mmol)溶于DCM(16.0mL),加入吡啶(Py)(0.418mL,5.17mmol),降温至0℃,然后加入化合物2(0.630g,3.10mmol),反应液在25℃下搅拌2小时。薄层色谱(PE/EA=3/1,PE/EA=10/1)显示原料没反应完全,有新点生成。将反应液用DCM(50.0mL)稀释,用饱和NaHCO3水溶液(20.0mL x 3)洗涤,有机相用饱和食盐水溶液(20.0mL x 3)洗涤,然后将有机相用Na2SO4干燥,有机相旋干得到粗品。粗品经柱层析(PE/EA=1/0-10/1)纯化,得到白色固体化合物3(800mg,55.94%)。1H NMR(400MHz,CDCl3)δ8.22-8.34(m,2H),7.36-7.44(m,2H),5.44(d,J=4.8Hz,1H),4.56-4.68(m,1H),2.43-2.55(m,2H),1.68-2.10(m,7H),1.44-1.54(m,4H),1.24-1.40
(m,4H),0.95-1.24(m,14H),0.92(d,J=6.4Hz,3H),0.87(dd,J=6.57,1.6Hz,6H),0.69(s,3H).
2.化合物5的制备
在25℃下将化合物4(200mg,1.10mmol)溶于DCM(10.0mL),依次加入TEA(0.656mL,4.72mmol)和化合物3(434mg,0.787mmol),反应液在25℃下搅拌16小时。薄层色谱(PE/EA=10/1,PE/EA=3/1)显示原料消耗完全且有新点生成。将反应液用DCM(50.0mL)稀释,用饱和NaHCO3水溶液(20.0mL X 3)洗涤,有机相再用饱和食盐水溶液(20.0mL X 3)洗涤,然后将有机相用Na2SO4干燥,有机相旋干得到粗品。粗品进行柱层析(PE/EA=1/0-5/1)纯化,得到白色固体粗品化合物5(380mg)。1H NMR(400MHz,CDCl3)δ5.35-5.41(m,1H),4.42-4.54(m,1H),3.68(s,3H),3.17(q,J=6.4Hz,2H),2.24-2.40(m,4H),1.92-2.05(m,2H),1.78-1.90(m,3H),1.65(dt,J=15.2,7.2Hz,3H),1.46-1.54(m,6H),1.23-1.45(m,8H),0.97-1.22(m,13H),0.92(d,J=6.4Hz,3H),0.87(dd,J=6.4,1.6Hz,6H),0.68(s,3H).
3.化合物6的制备
在25℃下将化合物5(380mg,0.681mmol)溶于THF(5.00mL)和H2O(2.50mL)的混合溶剂中,加入LiOH(32.6mg,1.36mmol),反应液在25℃下反应18小时。薄层色谱(PE/EA=3/1)显示原料消耗完全且有新点生成。向反应液中加入1M HCl(10.0mL)溶液调酸性,然后加入DCM(20.0mL),萃取收集有机相,并用Na2SO4干燥、旋干得到白色固体化合物6(230mg,收率62.09%)。1H NMR(400MHz,CDCl3)δ5.36-5.40(m,1H),4.45-4.56(m,1H),3.18(q,J=6.4Hz,2H),2.27-2.41(m,4H),1.96-2.05(m,2H),1.67(dt,J=15.2,7.2Hz,3H),1.46-1.59(m,9H),1.29-1.44(m,8H),0.97-1.18(m,13H),0.92(d,J=6.4Hz,3H),0.87(dd,J=6.4,1.6Hz,6H),0.68(s,3H).
4.化合物8的制备
在25℃下将化合物6(232mg,0.427mmol)溶于DCM(10.0mL),依次加入HATU(203mg,0.534mmol)和DIEA(0.353mL,2.14mmol),反应液在25℃下搅拌0.5小时,然后加入化合物7(160mg,0.356mmol),反应液在25℃下搅拌16小时。LCMS显示有产物的mass值。薄层色谱(DCM/MeOH=10/1)显示有新点生成。将反应液用DCM(20.0mL)稀释,先后用饱和柠檬酸水溶液(5.00mL X 3)、饱和NaHCO3水溶液(5.00mL X 3)和饱和食盐水溶液(5.00mL X 3)洗涤,然后将有机相用无水Na2SO4干燥,有机相旋干得到粗品。粗品进行柱层析(DCM/MeOH=1/0-10/1)纯化,得到无色油状液体粗品
化合物8(500mg)。1H NMR(400MHz,CDCl3)δ7.27-7.34(m,5H),7.15-7.21(m,4H),6.81-6.87(m,4H),5.35-5.41(m,1H),4.63-4.80(m,1H),4.31-4.52(m,1H),3.86-3.94(m,2H),3.79-3.83(m,6H),3.57-3.74(m,7H),3.47-3.55(m,1H),3.12-3.22(m,2H),2.24-2.41(m,4H),1.81-2.05(m,6H),1.45-1.59(m,11H),1.21-1.31(m,4H),1.08-1.17(m,6H),0.95(d,J=5.2Hz,6H),0.90-0.94(m,3H),0.86-0.89(m,6H),0.66-0.71(m,3H).
5.DL0144的制备
在25℃下将化合物8(500mg,0.308mmol)溶于DCM(10.0mL),依次加入DIEA(0.305mL,1.85mmol)、DMAP(15.0mg,0.123mmol)和化合物9(185mg,1.85mmol),反应液在25℃下搅拌2小时。LCMS显示有产物的mass值。将反应液旋干得到粗品。粗品通过反相柱(色谱柱:01-Waters Xbridge BEH C18 19*150mm;流动相:TEAA-ACN;B%:75%-95%,15min;流速:15ml/min)制备分离,得到白色固体化合物DL0144(218mg,收率65.91%,纯度91.17%)。1H NMR(400MHz,CDCl3)δ7.37-7.46(m,2H),7.27-7.35(m,7H),6.83(d,J=8.8Hz,4H),5.38(s,1H),4.33-4.72(m,2H),3.83-4.04(m,3H),3.79-3.81(m,6H),3.05-3.58(m,8H),2.96(q,J=7.2Hz,5H),2.49-2.69(m,5H),2.23-2.40(m,5H),1.79-2.05(m,6H),1.42-1.62(m,11H),1.31-1.39(m,4H),1.26(t,J=7.2Hz,7H),1.08-1.18(m,6H),0.97-1.07(m,6H),0.92(d,J=6.4Hz,3H),0.87(dd,J=6.4,1.6Hz,6H),0.68(s,3H).
实施例1.17 DL0145的制备
1化合物3的制备
在25℃下将化合物2(134mg,0.667mmol)溶于DCM(10.0mL)中,依次加入HATU(380mg,1.00mmol)和DIEA(0.331mL,2.00mmol),混合液体在25℃下搅拌0.5小时后将化合物1(300mg,0.667mmol)加入到其中。反应液体在25℃下搅拌12小时。薄层色谱(DCM/MeOH=10/1,PMA)和(DCM/MeOH=10/1,PMA)显示两种原料反应完全,有新点生成。向其中加入乙酸乙酯(10.0mL),混合液体用盐水洗涤三次(10mL X 3),有机相经减压浓缩后得到粗产品化合物。粗品经柱层析纯化(DCM/MeOH=93/7~90/10),得到无色油状化合物3(412mg,收率97.71%)。1H NMR(400MHz,METHANOL-d4)δ7.39-7.51(m,2H),7.16-7.35(m,7H),6.86(dt,J=2.89,5.83Hz,4H),3.79-4.04(m,2H),3.78(d,J=1.25Hz,6H),3.41-3.76(m,5H),3.32-3.38(m,1H),3.18(br d,J=6.27Hz,1H),2.94-3.14(m,1H),2.16-2.42(m,2H),1.52(br d,J=6.02Hz,2H),1.19-1.39(m,18H),0.87-0.92(m,3H)
2 DL0145的制备
在25℃将化合物3(200mg,0.317mmol)溶于DCM(3.00mL)中,依次将DIEA(0.314mL,1.90mmol),DMAP(9.67mg,0.079mmol),化合物4(31.7mg,0.317mmol)加入到上述混合液体中。反应液体在25℃下搅拌3小时。薄层色谱(DCM/MeOH=10/1)显示原料反应完全,有新点生成。液质(LCMS)显示原料反应完全。将反应液用二氯甲烷稀释(10.0mL),依次用饱和NaHCO3溶液(10.0mL X 3)和饱和NaCl溶液(10.0mL X 3)洗涤3遍,无水硫酸钠干燥后减压浓缩,经柱层析(DCM/MeOH=95/5~90/10)纯化得粗品。粗品通过Prep-HPLC(色谱柱:Waters Xbridge BEH C18 100*25mm*5um;流动相:TEAA-ACN;梯度:45%-95%/16min;流速:15ml/min)纯化,得到无色油状产物DL0145(84.0mg,收率36.26%,纯度98.70%)。1H NMR(400MHz,METHANOL-d4)δ7.43(br d,J=7.53Hz,2H),7.18-7.36(m,7H),6.82-6.93(m,4H),4.16-4.27(m,1H),4.11(d,J=5.52Hz,1H),3.97(br d,J=11.29Hz,1H),3.85(br s,1H),3.78(s,6H),3.43-3.75(m,3H),3.34-3.43(m,1H),3.10-3.28(m,5H),2.55-2.64(m,2H),2.52(d,J=6.02Hz,2H),2.33(br d,J=12.30Hz,2H),1.52(br s,2H),1.18-1.38(m,21H),0.83-0.95(m,3H)
实施例2:siRNA的合成
使用本领域熟知的固相亚磷酰胺法制备本发明的siRNA。具体方法可参考例如PCT公开号WO2016081444和WO2019105419,并简述如下。
1.正义链3’端未连接配体的siRNA的制备
1.1正义链(SS链)的合成
通过固相亚磷酰胺合成法,利用空白的CPG固相载体做为起始循环,按照正义链核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包含了脱保护、偶联、盖帽、氧化或硫代四步反应,合成规模为5umol的寡核酸。合成条件如下:
核苷单体以0.05mol/L的乙腈溶液提供,每一步反应的条件相同,即温度为25度,脱保护使用3%的三氯乙酸-二氯甲烷溶液,脱保护3次;偶联反应使用的活化剂为0.25mol/L的5-乙硫基四氮唑(ETT)-乙腈溶液,偶联2次;盖帽使用10%醋酐-乙腈和吡啶/N-甲基咪唑/乙腈(10:14:76,v/v/v),盖帽2次;氧化使用0.05mol/L碘的四氢呋喃/吡啶/水(70/20/10,v/v/v),氧化2次;硫代使用0.2mol/L苯乙酰二硫化物(PADS)的乙腈/3-甲基吡啶(1/1,v/v),硫代2次。
1.2反义链(AS链)的合成
通过固相亚磷酰胺合成法,利用空白的CPG固相载体做为起始循环,按照反义链核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包含了脱保护、偶联、盖帽、氧化或硫代四步反应,反义链的5umol的寡核酸合成条件和正义链的相同。
1.3寡核苷酸的纯化与退火
1.3.1氨解
将合成好的固相载体(正义链或者反义链)加入到5mL的离心管中,加入3%的二乙胺/氨水(v/v),35度(或者55度)恒温水浴下反应16小时(或者8小时),过滤,固相载体用乙醇/水洗涤三次,每次1mL,滤液离心浓缩后粗品进行纯化。
1.3.2纯化
纯化和脱盐的方法是本领域人员所熟知的。例如,可采用强阴离子填料装柱,氯化钠-氢氧化钠体系进行洗脱纯化,产品收集并管,可采用凝胶填料纯化柱进行脱盐,洗脱体系是纯水。
1.3.3退火
根据说明书将正义链(SS链)与反义链(AS链)以摩尔比(SS链/AS链=1/1.05)混合,水浴锅加热至70-95度,保持3-5min,自然冷却至室温,将体系冻干得到产品。
测试实施例
实施例3:体内活性检测(CNS递送)
1.单链序列信息
本文中,各缩写的意义如下:
A、U、G和C分别表示天然的腺嘌呤核糖核苷酸、尿嘧啶核糖核苷酸、鸟嘌呤核糖核苷酸和胞嘧啶核糖核苷酸。
d表示其右侧相邻的核苷酸是脱氧核糖核苷酸。例如dA、dT、dG和dC分别表示腺嘌呤脱氧核糖核苷酸、胸腺嘧啶脱氧核糖核苷酸、鸟嘌呤脱氧核糖核苷酸和胞嘧啶脱氧核糖核苷酸。
i表示肌苷核糖核苷酸。
m表示其左侧相邻的核苷酸是2’-OCH3修饰的核苷酸。例如,Am、Um、Gm和Cm表示2’-OCH3修饰的A、U、G和C。
f表示其左侧相邻的核苷酸是2’-F修饰的核苷酸。例如,Af、Uf、Gf和Cf分别表示2’-F修饰的A、U、G和C。
“s”表示其左右相邻的两个核苷酸和/或递送载体通过硫代磷酸酯连接。
VP表示其右侧相邻的核苷酸是乙烯基磷酸酯修饰的核苷酸。
Ib表示反向无碱基脱氧核糖核苷酸,根据其在siRNA中所在位置/连接方式的不同可包括以下三种结构。
2.使用的双链序列
3.实验方法
实验动物:SD大鼠,雄性,6-8周,每组2-3只;
实验操作:
·动物经异氟烷气体麻醉后,放置与恒温电热毯,臀背部备皮并用碘伏及75%酒精消毒,涂眼膏;
·准确定位L3-L5水平,并切开此段皮肤暴露脊柱,经椎间孔穿刺,见到明显的甩尾动物(做为穿刺成功标志)后,缓慢推注30uL(0.9mg,30mg/mL)相应化合物,注射完毕后留针15-30s,继而缝合皮肤;
·动物术后护理:经皮下注射美洛昔康和抗生素;
·与注射后14天,将所有动物安乐死处理,取颈段和胸段脊髓,取脑组织并分离小脑,脑干,海马,额叶皮层,置于RNAlater妥善保存,用于后续SOD1mRNA抽提和QPCR检测。
mRNA抽提和QPCR检测的方法是本领域熟知的,使用的引物信息如下:
通过以下公式计算剩余抑制率:
计算2-△△Ct值并换算成百分比以得到剩余抑制率。
△△Ct=[(Ct实验组目的基因-Ct实验组内参)-(Ct对照组目的基因-Ct对照组内参)]。
△△Ct=[(Ct实验组目的基因-Ct实验组内参)-(Ct对照组目的基因-Ct对照组内参)]。
目的基因为SOD1,内参为GAPDH,对照组注射人工脑脊液(aCSF)。
4.实验结果
剩余抑制率数据如下表所示(也可见图1),DR005713、DR005714、DR005715、DR005717、DR005718、DR005716、DR005735在中枢神经系统内均降低了SOD1的表达量。其中DR005716和
DR005735的敲减效果优于其他序列。
实施例4:体内活性检测(眼部递送)
本实施例中所用序列信息如下:
给药及分离眼部组织
将C57BL/6小鼠(雄性,6~8周)进行随机分组,采用双侧眼玻璃体内注射方式单次给药,每只眼给药剂量为2μg,siRNA缀合物以5mg/mL的溶液(磷酸盐缓冲溶液作为溶剂)给药;具体地,在实验前,用磷酸盐缓冲溶液将siRNA缀合物溶解且定容至所需溶液浓度和体积,磷酸盐缓冲溶液和siRNA缀合物的给药体积为1.5μL/眼。
给药后第14天摘取眼球,并分离为三部分:①视网膜色素上皮细胞(RPE)+脉络膜+巩膜;②视网膜;③角膜+虹膜+睫状体;分离样本立即放于液氮中急冻,之后储存于-80℃用于检测mTTR mRNA。
RNA提取与检测
按照高通量组织RNA提取试剂盒(凡知医疗,FG0412)的操作protocol使用核酸提取仪(杭州奥盛,Auto-pure96)进行细胞RNA提取;参考PrimeScriptTMII 1st Strand cDNA Synthesis Kit(Takara,6210B)反转录;参考TaqManTMFast Advanced Master Mix(ABI,4444965)20μL体系进行荧光定量PCR反应(ABI,QuantStudio3)检测,引物见下表。
数据统计
计算2-△△Ct值并换算成百分比以得到剩余抑制率;
△△Ct=[(Ct实验组目的基因-Ct实验组内参)-(Ct对照组目的基因-Ct对照组内参)]。
目的基因为mTTR,内参为mGAPDH。
实验结果见图2。结果显示,DR005938、DR007050和DR007871均可在眼部降低TTR基因的表达量,降低效果与阳性序列DR005933相当或更优。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
Claims (33)
- 式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,L1和L2独立地选自H、反应性磷基团、羟基保护基或固相载体;Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基,其任选地被氘代,直至完全氘代;m=0、1、2、3、4、5或6;R为-C(O)-C0-10亚烷基-L-R1、-C(O)-C2-10亚烯基-L-R1或-C(O)-C2-10亚炔基-L-R1;L为化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)-或-NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-;R1独立地为C1-30烷基、C2-30烯基或C2-30炔基,其中所述基团中不相邻的1、2、3、4、5、6、7、8、9、10个碳原子可以被选自O、S和N的杂原子替换,或者-CH2CH2-基团可以被-OC(O)-、-C(O)O-、-NHC(O)-或-C(O)NH-替换,或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环;其中C0-10亚烷基、C2-10亚烯基、C2-10亚炔基、C1-30烷基、C2-30烯基和C2-30炔基中的氢原子可以任选地被1、2、3、4、5、6、7、8、9、10或更多个卤素、C1-6烷基或C1-6卤代烷基替换,而且其任选地被氘代,直至完全氘代。 - 权利要求1的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,R为-C(O)-C0-10亚烷基-L-R1,优选-C(O)-L-R1,优选-C(O)-C2-8亚烷基-L-R1,更优选-C(O)-C3-7亚烷基-L-R1,更优选-C(O)-C4-6亚烷基-L-R1,更优选-C(O)-C1-3亚烷基-L-R1。
- 权利要求1或2的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,L为化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-O-CH(CH2OH)CH(OH)-、-NH-CH(CH2OH)CH(OH)-、-O-CH2CH(OH)CH(OH)-、-O-CH2CH(NH2)CH(OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-、-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-、-NHC(O)-CH2-O-CH2CH(OH)CH(OH)-或-NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-,优选化学键、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S-S-、-NHC(O)O-、-NHC(O)NH-、-OC(O)O-、-OC(O)NH-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-或-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-,优选化学键、-NHC(O)-、-S-S-、-NHC(O)O-、-O-CH(CH(OH)CH2OH)-、-O-CH(CH(NH2)CH2OH)-、-NHC(O)-CH2-O-CH(CH(OH)CH2OH)-或-NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-,更优选化学键、-NHC(O)-、-S-S-或-NHC(O)O-,更优选-NHC(O)-。
- 权利要求1或2的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,L为-NHC(O)-CH(OR1)CH2O-、-C(O)NH-CH(OR1)CH2O-、-OC(O)-CH(OR1)CH2O-、-C(O)O-CH(OR1)CH2O-、-NHC(O)-CH(R1)-、-C(O)NH-CH(R1)-、-OC(O)-CH(R1)-、-C(O)O-CH(R1)-、-CH(OR1)CH2O-、-O-CH(R1)CH2O-、-O-CH2CH(R1)O-,优选-NHC(O)-CH(OR1)CH2O-、-NHC(O)-CH(R1)-或-CH(OR1)CH2O-,更优选-NHC(O)-CH(OR1)CH2O-。
- 权利要求1-4中任一项的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,R1独立地为C1-30烷基或C2-30烯基,其中所述基团中不相邻的1、2、3、4、5、6、7或8个碳原子可以被选自O、S和N的杂原子替换,或者-CH2CH2-基团可以被-NHC(O)-或-C(O)NH-替换,或者一个或多个碳原子上的取代基可以连接成饱和或不饱和环;优选地,R1独立地为C5-25烷基、含有1、2、3、4、5或6个双键的C10-25烯基、其中1、2、3、4或5个碳原子被N杂原子替换和/或1、2或3个-CH2CH2-基团被-C(O)NH-替换的C5-25烷基、或者一个或多个碳原子上的取代基连接成甾体环的C5- 25烷基;优选地,R1选自以下基团:C6烷基、C8烷基、C11烷基、C12烷基、C13烷基、C15烷基、C16烷基、C17烷基、C21烷基、
- 权利要求1-5中任一项的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,L1和L2之一为反应性磷基团,优选亚磷酰胺、H-膦酸酯、烷基-膦酸酯、磷酸酯或磷酸酯模拟物,例如天然磷酸酯、硫代磷酸酯、二硫代磷酸酯、硼烷磷酸酯、硼烷硫代磷酸酯、膦酸酯、卤素取代的膦酸酯和磷酸酯、氨基磷酸酯、磷酸二酯、磷酸三酯、硫代磷酸二酯、硫代磷酸三酯、二磷酸酯或三磷酸酯,优选-P(OCH2CH2CN)(N(iPr)2)。
- 权利要求1-5中任一项的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,L1和L2选自保护基,优选羟基保护基,例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基,优选-C(O)CH2CH2C(O)OH或4,4'-二甲氧基三苯甲基,更优选-C(O)CH2CH2C(O)OH。
- 权利要求1-7中任一项的式(I)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其选自以下通式:
其中各基团如权利要求1-7所定义。 - 权利要求1-8中任一项的化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,所述化合物选自以下:
- 寡核苷酸,其包含一个或多个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,表示H或羟基保护基,或表示与相邻核苷酸连接的位置;L2表示H或固相载体,或表示与相邻核苷酸连接的位置;Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基,其任选地被氘代,直至完全氘代;m=0、1、2、3、4、5或6;R为疏水性基团;优选地,表示H或羟基保护基,或表示与相邻核苷酸连接的位置;L2表示H或固相载体,或表示与相邻核苷酸连接的位置;Rs、m和R如权利要求1-5中任一项所定义。 - 权利要求10的寡核苷酸,其中所述式(I’)化合物选自以下通式化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,表示H或羟基保护基,或表示与相邻核苷酸连接的位置;L2表示H或固相载体,或表示与相邻核苷酸连接的位置;其他各基团如权利要求1-5所定义。 - 权利要求10的寡核苷酸,其中所述式(I’)化合物选自以下化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,所述化合物选自以下:
其中之一表示H或羟基保护基,或表示与相邻核苷酸连接的位置,并且另一个表示H或固相载体,或表示与相邻核苷酸连接的位置。 - 权利要求10-12中任一项的寡核苷酸,其具有14至30个核苷酸。
- 权利要求10-13中任一项的寡核苷酸,其在5’端包含一个权利要求10-12中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
- 权利要求10-14中任一项的寡核苷酸,其在3’端包含一个权利要求10-12中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
- 权利要求10-15中任一项的寡核苷酸,其在5’端和3’端分别包含一个权利要求10-12中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
- 寡核苷酸,其在寡核苷酸内部、5’端和/或3’端包含两个或多个疏水性基团;优选地,所述疏水性基团如式(I)化合物中R基团所定义;优选地,所述疏水性基团通过接头,例如可生物降解的接头,连接至寡核苷酸上。
- 双链RNA,其具有正义链和反义链,各链具有14至30个核苷酸,所述反义链包含与所述正义链和靶标mRNA充分互补的序列,其中所述正义链和/或反义链包含一个或多个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,表示H,或表示与相邻核苷酸连接的位置;L2表示H或羟基保护基,或表示与相邻核苷酸连接的位置;Rs选自H、D、卤素、C1-6烷基或C1-6卤代烷基,其任选地被氘代,直至完全氘代;m=0、1、2、3、4、5或6;R为疏水性基团;优选地,表示H,或表示与相邻核苷酸连接的位置;L2表示H或羟基保护基,或表示与相邻核苷酸连接的位置;Rs、m和R如权利要求1-5中任一项所定义。 - 权利要求18的双链RNA,其中所述式(I’)化合物选自以下通式化合物,或其药学上可接受的盐、互变异构体或立体异构体:
其中,表示H,或表示与相邻核苷酸连接的位置;L2表示H或羟基保护基,或表示与相邻核苷酸连接的位置;Rs、m和R如权利要求1-5所定义。 - 权利要求18的双链RNA,其中所述式(I’)化合物选自以下化合物,或其药学上可接受的盐、互变异构体或立体异构体,其中,所述化合物选自以下:
其中之一表示H,或表示与相邻核苷酸连接的位置,并且另一个表示H或羟基保护基,或与相邻核苷酸连接的位置。 - 权利要求18-20中任一项的双链RNA,其中所述正义链在5’端包含一个权利要求10-12中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
- 权利要求18-21中任一项的双链RNA,其中所述正义链在3’端包含一个权利要求10-12中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
- 权利要求18-22中任一项的双链RNA,其中所述正义链在5’端和3’端分别包含一个权利要求10-12中任一项的式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体。
- 权利要求18-23中任一项的双链RNA,其中所述正义链和/或反义链上的两个或多个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体之间间隔至少5-30个核苷酸。
- 权利要求18-24中任一项的双链RNA,其中所述双链RNA包含两个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其位于以下位点中的任两个:正义链的5’端、正义链的3’ 端、反义链的5’端和反义链的3’端;优选位于正义链的5’端和正义链的3’端。
- 权利要求18-25中任一项的双链RNA,其中所述双链RNA包含三个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其位于以下位点中的任三个:正义链的5’端、正义链的3’端、反义链的5’端和反义链的3’端;优选位于正义链的5’端、正义链的3’端和反义链的3’端。
- 权利要求18-26中任一项的双链RNA,其中所述双链RNA包含四个式(I’)化合物,或其药学上可接受的盐、互变异构体或立体异构体,其位于以下位点:正义链的5’端、正义链的3’端、反义链的5’端和反义链的3’端。
- 权利要求18-26中任一项的双链RNA,其进一步在所述反义链的5’端偶联末端磷酸保护基团或前药保护基团,优选乙烯基磷酸酯基或式(X)所示的前药保护基团:
其中,X1选自OH或Ra选自H、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;Rb和Rc独立地选自H、C1-6烷基或C1-6卤代烷基,所述Rb和Rc可任选地被D、C6-10芳基或5-10元杂芳基取代,直至完全氘代;X2是与所述反义链的5’端的第一个核苷酸连接的化学键,优选通过羟基连接;X3独立地选自O或S;T选自每个RT1独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C1-6卤代烷基、C2-6烯基、C2- 6炔基或包含GalNAc的链,其任选地被氘代,直至完全氘代;每个RT2独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;每个RT3独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;每个RT4独立地选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;m为0、1、2、3、4或5;n为0、1、2、3、4或5;p为0、1、2、3、4或5;X选自化学键、-O-、-S-、-C(O)-、-C(O)O-、-OC(O)-、-OC(O)NRX1-、-NRX1C(O)O-、-NRX1C(O)-或-C(O)NRX1-;RX1选自H、C1-6烷基或C1-6卤代烷基,其任选地被氘代,直至完全氘代;L为-Ar-(CH2)1-6-O-,其中每一个CH2可任选地被R#取代,R#选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;L中的Ar与X相连,氧原子与磷原子相连;Ar选自C3-10环烷基、3-10元杂环基、C6-10芳基或5-14元杂芳基,所述C3-10环烷基、3-10元杂环基、C6-10芳基或5-10元杂芳基可任选被1个、2个、3个、4个或5个R*取代;R*选自H、D、卤素、CN、C1-6烷基、C1-6卤代烷基、C2-6烯基或C2-6炔基,其任选地被氘代,直至完全氘代;其中P1选自保护基,优选羟基保护基,例如三甲基硅基(TMS)、三乙基硅基(TES)、二甲基异丙基硅基(DMIPS)、二乙基异丙基硅基(DEIPS)、叔丁基二甲基硅基(TBDMS)、叔丁基二苯基硅基(TBDPS)、三异丙基硅基(TIPS)、乙酰基(Ac)、氯乙酰基、二氯乙酰基、三氯乙酰基、三氟乙酰基(TFA)、苯甲酰基、对甲氧基苯甲酰基、9-芴基甲氧基羰基(Fmoc)、烯丙氧羰基(Alloc)、2,2,2-三氯乙氧羰基(Troc)、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、苯甲基(Bn)、对甲氧基苄基(PMB)、烯丙基、三苯基甲基(Tr)、双对甲氧基三苯甲基(DMTr)、甲氧基甲基(MOM)、苯氧基甲基(BOM)、2,2,2-三氯乙氧基甲基、2-甲氧基乙氧基甲基(MEM)、甲硫基甲基(MTM)、对甲氧基苄氧基甲基(PMBM)、4,4'-二甲氧基三苯甲基、-P(OCH2CH2CN)(N(iPr)2)或-C(O)CH2CH2C(O)OH,优选-P(OCH2CH2CN)(N(iPr)2)或-C(O)CH2CH2C(O)OH。 - 权利要求18-28中任一项的双链RNA,其选自小干扰RNA(siRNA)和短发夹RNA(shRNA),优选用于抑制在眼部表达的基因。
- 双链RNA,其具有正义链和反义链,各链具有14至30个核苷酸,所述反义链包含与所述正义链和靶标mRNA充分互补的序列,其中所述正义链和/或反义链在内部、5’端和/或3’端包含两个或多个疏水性基团;优选地,所述疏水性基团如式(I)化合物中R基团所定义;优选地,所述疏水性基团通过接头,例如可生物降解的接头,连接至正义链和/或反义链上。
- 细胞,其含有如权利要求18-30中任一项所述的双链RNA。
- 药物组合物,其包含如权利要求18-30中任一项所述的双链RNA、或如权利要求31所述的细胞,以及任选的药学上可接受的载剂或赋形剂。
- 试剂盒,其包含如权利要求18-30中任一项所述的双链RNA、或如权利要求31所述的细胞。
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