CN1100728A - 具有2′位醚基的核苷和低聚核苷酸 - Google Patents
具有2′位醚基的核苷和低聚核苷酸 Download PDFInfo
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- CN1100728A CN1100728A CN94105715A CN94105715A CN1100728A CN 1100728 A CN1100728 A CN 1100728A CN 94105715 A CN94105715 A CN 94105715A CN 94105715 A CN94105715 A CN 94105715A CN 1100728 A CN1100728 A CN 1100728A
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- 108091034117 Oligonucleotide Proteins 0.000 title claims description 80
- 239000002777 nucleoside Substances 0.000 title claims description 31
- 125000003835 nucleoside group Chemical group 0.000 title claims description 28
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 title description 3
- 150000001875 compounds Chemical class 0.000 claims abstract description 118
- -1 4Be hydrogen Chemical class 0.000 claims description 140
- 229910052739 hydrogen Inorganic materials 0.000 claims description 103
- 239000001257 hydrogen Substances 0.000 claims description 96
- 150000002431 hydrogen Chemical class 0.000 claims description 47
- 238000002360 preparation method Methods 0.000 claims description 44
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 36
- 125000000217 alkyl group Chemical group 0.000 claims description 34
- 229910052799 carbon Inorganic materials 0.000 claims description 31
- 125000001997 phenyl group Chemical class [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 31
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 30
- 229910052794 bromium Inorganic materials 0.000 claims description 26
- 229910052801 chlorine Inorganic materials 0.000 claims description 26
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 26
- 230000000903 blocking effect Effects 0.000 claims description 25
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical group NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 229910052731 fluorine Inorganic materials 0.000 claims description 21
- 150000001721 carbon Chemical group 0.000 claims description 20
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 20
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 18
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 16
- 125000003729 nucleotide group Chemical group 0.000 claims description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims description 13
- 229910052740 iodine Inorganic materials 0.000 claims description 11
- 210000001541 thymus gland Anatomy 0.000 claims description 10
- 125000004103 aminoalkyl group Chemical group 0.000 claims description 9
- 238000005984 hydrogenation reaction Methods 0.000 claims description 9
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 9
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 claims description 9
- 239000011782 vitamin Substances 0.000 claims description 9
- 229940088594 vitamin Drugs 0.000 claims description 9
- 125000002768 hydroxyalkyl group Chemical group 0.000 claims description 8
- 125000000714 pyrimidinyl group Chemical group 0.000 claims description 8
- 125000006577 C1-C6 hydroxyalkyl group Chemical group 0.000 claims description 7
- 125000003545 alkoxy group Chemical group 0.000 claims description 7
- 125000004181 carboxyalkyl group Chemical group 0.000 claims description 7
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 7
- 229930003231 vitamin Natural products 0.000 claims description 7
- 235000013343 vitamin Nutrition 0.000 claims description 7
- 125000004642 (C1-C12) alkoxy group Chemical group 0.000 claims description 6
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 claims description 6
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 claims description 6
- 125000005078 alkoxycarbonylalkyl group Chemical group 0.000 claims description 6
- 125000004414 alkyl thio group Chemical group 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
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 229940104302 cytosine Drugs 0.000 claims description 6
- 125000004185 ester group Chemical group 0.000 claims description 6
- 239000011737 fluorine Substances 0.000 claims description 6
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 claims description 5
- 125000003277 amino group Chemical group 0.000 claims description 5
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 5
- 239000012442 inert solvent Substances 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 claims description 5
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 claims description 5
- OIVLITBTBDPEFK-UHFFFAOYSA-N 5,6-dihydrouracil Chemical compound O=C1CCNC(=O)N1 OIVLITBTBDPEFK-UHFFFAOYSA-N 0.000 claims description 4
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 claims description 4
- 125000002853 C1-C4 hydroxyalkyl group Chemical group 0.000 claims description 4
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 125000004177 diethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 4
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims description 4
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical class [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 claims description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 claims description 4
- 229910052701 rubidium Inorganic materials 0.000 claims description 4
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 4
- 125000006710 (C2-C12) alkenyl group Chemical group 0.000 claims description 3
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims description 3
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 claims description 3
- 208000026350 Inborn Genetic disease Diseases 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 125000002877 alkyl aryl group Chemical group 0.000 claims description 3
- 125000003710 aryl alkyl group Chemical group 0.000 claims description 3
- 125000003118 aryl group Chemical group 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 208000016361 genetic disease Diseases 0.000 claims description 3
- 125000002816 methylsulfanyl group Chemical group [H]C([H])([H])S[*] 0.000 claims description 3
- 125000001453 quaternary ammonium group Chemical group 0.000 claims description 3
- 239000003981 vehicle Substances 0.000 claims description 3
- 230000009385 viral infection Effects 0.000 claims description 3
- CFIBTBBTJWHPQV-UHFFFAOYSA-N 2-methyl-n-(6-oxo-3,7-dihydropurin-2-yl)propanamide Chemical compound N1C(NC(=O)C(C)C)=NC(=O)C2=C1N=CN2 CFIBTBBTJWHPQV-UHFFFAOYSA-N 0.000 claims description 2
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 claims description 2
- ZFTBZKVVGZNMJR-UHFFFAOYSA-N 5-chlorouracil Chemical compound ClC1=CNC(=O)NC1=O ZFTBZKVVGZNMJR-UHFFFAOYSA-N 0.000 claims description 2
- RYYIULNRIVUMTQ-UHFFFAOYSA-N 6-chloroguanine Chemical compound NC1=NC(Cl)=C2N=CNC2=N1 RYYIULNRIVUMTQ-UHFFFAOYSA-N 0.000 claims description 2
- 239000007818 Grignard reagent Substances 0.000 claims description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 2
- 239000005864 Sulphur Substances 0.000 claims description 2
- 125000004183 alkoxy alkyl group Chemical group 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 229960002949 fluorouracil Drugs 0.000 claims description 2
- 125000000524 functional group Chemical group 0.000 claims description 2
- 150000004795 grignard reagents Chemical class 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 150000002367 halogens Chemical class 0.000 claims description 2
- HZVOZRGWRWCICA-UHFFFAOYSA-N methanediyl Chemical compound [CH2] HZVOZRGWRWCICA-UHFFFAOYSA-N 0.000 claims description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 2
- 125000006501 nitrophenyl group Chemical group 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 125000004817 pentamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 2
- 150000003254 radicals Chemical class 0.000 claims description 2
- 150000002118 epoxides Chemical class 0.000 claims 4
- 125000002652 ribonucleotide group Chemical group 0.000 claims 2
- 230000002194 synthesizing effect Effects 0.000 claims 2
- 125000004890 (C1-C6) alkylamino group Chemical group 0.000 claims 1
- 125000006711 (C2-C12) alkynyl group Chemical group 0.000 claims 1
- CKOMXBHMKXXTNW-UHFFFAOYSA-N 6-methyladenine Chemical compound CNC1=NC=NC2=C1N=CN2 CKOMXBHMKXXTNW-UHFFFAOYSA-N 0.000 claims 1
- 125000003358 C2-C20 alkenyl group Chemical group 0.000 claims 1
- 150000007513 acids Chemical class 0.000 claims 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 1
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 claims 1
- 125000001153 fluoro group Chemical group F* 0.000 claims 1
- 125000006239 protecting group Chemical group 0.000 claims 1
- 210000002700 urine Anatomy 0.000 claims 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 69
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 67
- 239000011541 reaction mixture Substances 0.000 description 51
- 238000001704 evaporation Methods 0.000 description 46
- 230000008020 evaporation Effects 0.000 description 46
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Natural products CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 40
- 239000000243 solution Substances 0.000 description 39
- 239000000047 product Substances 0.000 description 37
- 239000000460 chlorine Substances 0.000 description 29
- 238000000605 extraction Methods 0.000 description 27
- 239000002585 base Substances 0.000 description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 25
- 238000003810 ethyl acetate extraction Methods 0.000 description 23
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- 239000007788 liquid Substances 0.000 description 20
- 238000005481 NMR spectroscopy Methods 0.000 description 19
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 19
- 238000004587 chromatography analysis Methods 0.000 description 19
- 239000000741 silica gel Substances 0.000 description 19
- 229910002027 silica gel Inorganic materials 0.000 description 19
- 229960001866 silicon dioxide Drugs 0.000 description 19
- 238000003756 stirring Methods 0.000 description 18
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Substances C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 18
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 15
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 14
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 12
- 239000000203 mixture Substances 0.000 description 11
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 9
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 101710163270 Nuclease Proteins 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 239000012074 organic phase Substances 0.000 description 7
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 description 6
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 238000009835 boiling Methods 0.000 description 5
- 229930182470 glycoside Natural products 0.000 description 5
- 150000002338 glycosides Chemical class 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical group [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 5
- 150000002924 oxiranes Chemical class 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 4
- JBWYRBLDOOOJEU-UHFFFAOYSA-N 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Cl)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 JBWYRBLDOOOJEU-UHFFFAOYSA-N 0.000 description 4
- QOPYYEHPKRREQO-UHFFFAOYSA-O CC(C)C1=[N+](C(C)C)NN=N1.N Chemical compound CC(C)C1=[N+](C(C)C)NN=N1.N QOPYYEHPKRREQO-UHFFFAOYSA-O 0.000 description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 4
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 4
- 125000003342 alkenyl group Chemical group 0.000 description 4
- 230000000692 anti-sense effect Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- LADPCMZCENPFGV-UHFFFAOYSA-N chloromethoxymethylbenzene Chemical compound ClCOCC1=CC=CC=C1 LADPCMZCENPFGV-UHFFFAOYSA-N 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- YDCHPLOFQATIDS-UHFFFAOYSA-N methyl 2-bromoacetate Chemical compound COC(=O)CBr YDCHPLOFQATIDS-UHFFFAOYSA-N 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N o-dimethylbenzene Natural products CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 238000010898 silica gel chromatography Methods 0.000 description 4
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 4
- XRRDFIPYLFCYLU-UHFFFAOYSA-N 1,3,5-trimethylbenzene Chemical group [CH2]C1=CC(C)=CC(C)=C1 XRRDFIPYLFCYLU-UHFFFAOYSA-N 0.000 description 3
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- BDAGIHXWWSANSR-UHFFFAOYSA-N Formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 3
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 3
- 229910002808 Si–O–Si Inorganic materials 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 3
- 125000000043 benzamido group Chemical group [H]N([*])C(=O)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
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- 235000017281 sodium acetate Nutrition 0.000 description 1
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- 238000010561 standard procedure Methods 0.000 description 1
- 125000004079 stearyl 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])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])[H] 0.000 description 1
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Classifications
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
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Abstract
本发明涉及I式化合物,式中各基团定义详见说明书。
Description
本发明涉及2′位羟基被多羟基衍生物醚化的核糖核苷类似物及其制备方法,还涉及含这些核苷的低聚核苷酸及核苷在制备分子内带有相同或不同核苷单元的低聚核苷酸时的应用。
核苷和低聚核苷酸均为抗病毒活性成分,并且由于其与核酸能相互作用(“反义”低聚核苷酸)和相应的生物活性而受到广泛的关注。例如见Uhlmann,E.Peyman,A,chemical Reviews 90:543-584(1990)。为制备具有新特点的核苷或改进天然核酸与反义低聚核苷酸的相互作用并提高它们对核酸酶的稳定性,已有许多方法来修饰核苷的糖基(或低聚核苷酸的核苷酸单位)或低聚核苷酸中的核苷酸间磷酸键;例如见:Marquez,V.Z.,Lim,M.I.,Medicinal Research Reviews 6:1-40(1986),Nelene,C,Tonlme,J.J.,Biochimica et Biophysica Acta 1049:99-125(1990),English,U.,Gams,D.H..Angewartte Chamie 103:629-646(1991),Natteutti,M.D.,Birchofberger,N.,Annual Reports in Medicinal Chemistry 26:87-296(1991).Cook,P.D.,Anti-Cancer Drug Design 6:585-60(1991)和WO91/06556描述了在糖的2′位羟基上已被修饰的核苷。所述修饰目的是提高对核酸酶的抵抗力;烷基越长,对核酸酶抵抗力就越强。人们发现,对于用短链的烷基如甲基、乙基、丙基的短烷基,键条和力略有提高,而对于较长链的烷基,键亲和力急剧下降。具有作为2′位羟基侧链的多羟基衍生物的核苷尚未见报道并从未与低聚核苷酸归如一类。令人惊奇的是,按本发明进行的修饰不仅对较短的链的情况下面且在较长链的情况下都提高了互补RNA的键亲和力。该结果无法用已知数据来预测。同样,对于2′位羟基修饰的低聚核苷酸,本发明的化合物由于其对核酸酶优越的抵抗力而与众不同。除此之外,包含本发明所述核苷的低聚核苷酸有增多的细胞吸收而且因此具有体内较好的生物利用度和活性。
本发明涉及式Ⅰ化合物
式中,R1和R2互相独立地为氢原子或保护基团或R1为哪些定义,
R2为形成一含磷核苷酸桥键的基团
B为嘌呤或嘧啶基团或其类似物;
R3为式Ⅰa、Ⅰb、Ⅰc基团
式中,R4为氢,C1-C21烷基,C2-C21链烯基,C2-C21炔基或-C(=O)-烷基;
R5为氢,C1-C10烷基,-CH2-O-R6或式Ⅰb基;
R6为氢,C1-C22烷基,C3-C21链烯基或部分或全部氟取代的C1-C10烷基或[(CH2)2-O]m-R7;
R7为氢或C1-C21烷基;
Z为-(CH2)p-或-(CH2-CH2-O)q-CH2-CH2-,
其中-CH2-不被取代或被一个或多个,相同或不同的C1-C10烷基;C5-C6环烷基,未取代或为C1-C4烷基取代的苯基取代。
n为1-12;
m为1-4;
p为1-10;
q为1-4;
当n=1,R5为氢时,R4不能是氢
R4优选为氢,C1-C21烷基,C2-C21链烯基或C2-C21炔基。R4更为优选为氢,C1-C8烷基,C2-C8链烯基或C2-C8炔基。R4特别优选为氢或C1-C4烷基,例如R4为甲基、乙基,正或异丙基,正、异和叔丁基,和戊基的异构体,己基,庚基,辛基,壬基,癸基,十一烷基,十二烷基,十三烷基,十四烷基、十五烷基,十六烷基,十七烷基,十八烷基,十九烷基,二十烷基,乙烯基,烯丙基,3-烯-1-丁基,2-烯-1-丁基,戊烯基,辛烯基,十二烯基,十八烯基,乙炔基,1-炔基-1-丙基,1-炔基-3-丙基,1-炔基-1-丁基或1-炔基-4-丁基。R4基特别优选为氢、甲基、乙基、正或异丙基、正丁基。n值优选1-8,更为优选1-6,特别优选1-3。
R5优选氢、C1-C5烷基或-CH2-O-R6,特别优选氢,甲基或-CH2-OH,-CH2-O-C1-C22烷基或-CH2-O-[(CH2)2-O]m-C1-C10烷基,其中m为1-4。
R1和R2优选为氢。
保护基团及衍生含有这些保护基团的羟基的方法已经在糖化学和核苷酸化学中公开并有所描述,例如Greene,B.T.,“在有机合成中的保护基团”,Wiley Interscience,New York(1991),Sonveawx,E,Bioorganic Chemistry 14:274-325(1986)或Beaacage,S.L.,Iyer,R.,Tetrahedron 48:2223-2311(1992),这些保护基团举例如下:苯甲基、甲基苯甲基、二甲基苯甲基、甲氧基苯甲基,二甲氧基苯甲基、溴代苯甲基、2,4-二氯苯甲基;二苯基甲基,二(甲基苯基)甲基,二(二甲基苯基)甲基,二(甲氧基苯基)甲基,二(二甲氧基苯基)甲基,三苯基甲基,三-4,4′,4″-叔-丁基苯基甲基,二-对-甲氧苯基苯基甲基,三(甲基苯基)甲基,三(二甲基苯基)甲基,甲氧基苯基(二苯基)甲基,二(甲氧基苯基)苯基甲基,三(甲氧基苯基)甲基,三(二甲氧基苯基)甲基;三苯基甲硅烷基,烷基二苯基甲硅烷基,二烷基苯基甲硅烷基和三烷基甲硅烷基,在其烷基基团中烷基为C1-C20,优选C1-C12特别优选C1-C8烷基,如三甲基甲硅烷基,三乙基甲硅烷基,三-正丙基甲硅烷基,异丙基-二甲基甲硅烷基,叔丁基-二甲基甲硅烷基,叔丁基-二苯基甲硅烷基,正-辛基-二甲基甲硅烷基,(1,1,2,2-四甲基乙基)-二甲基甲硅烷基;-(C1-C8烷基)2Si-O-Si(C1-C8烷基)2-,例如,式中烷基为甲基,乙基,正和异丙基或正、异或叔丁基;C2-C12酰基,特别是C2-C8酰基,如乙酰基,丙酰基,丁酰基,戊酰基,己酰基,苯甲酰基,甲基苯甲酰基,甲氧基苯甲酰基,氯苯甲酰基和溴苯甲酰基;Rs1-SO2-,式中Rs1为C1-C12烷基,特别为C1-C6烷基,C5或C6环烷基,苯基,苯甲基,C1-C12烷基苯基,特别为C1-C4烷基苯基,或C1-C12烷基苯甲基,特别为C1-C4烷基苯甲基,或卤代苯基或卤代苯甲基,例如:甲基-,乙基-,丙基-,丁基-,苯基-,苯甲基-,对-溴代-,对甲氧基-和对甲基苯基-磺酰基;C1-C12烷氧基羰基,优选为C1-C8烷氧基羰基,其中,其可未取代或被F,Cl,Br,C1-C4烷氧基,三(C1-C4烷基)-甲硅烷基或C1-C5烷基磺酰基取代,如甲氧基-,乙氧基-,正或异丙氧基-,正、异或叔丁氧羰基,2-三甲基甲硅烷基乙氧羰基,2-甲基磺酰基乙氧羰基,烯丙氧基羰基,或苯氧基羰基或苯甲氧基羰基,其中,烷氧羰基可未取代或被取代,如,甲基-,甲氧基-,氯苯氧基羰基或甲基-,甲氧基-,氯苯甲氧基羰基和9-芴基甲氧基羰基。如果R1和/或R2为烷基,它们可被氟,氯,溴,C1-C4烷氧基,苯氧基,氯苯氧基,甲氧苯氧基,苯甲氧基,甲氧苯甲氧基或氯苯氧基取代。式Ⅰ中R1和R2可为相同的或不同的保护基团。
R1和R2作为保护基团,优选苯甲基,甲基苯甲基,二甲基苯甲基,甲氧基苯甲基,二甲氧基苯甲基,卤代苯甲基,特别是溴苯甲基;二苯基甲基,二(甲基苯基)甲基,二(二甲基苯基)甲基,二(甲氧基苯基)甲基,二(甲氧基苯基)(苯基)甲基,三苯基甲基,三-4,4′,4″-叔-丁基苯基甲基,二-对-甲氧苯基苯基甲基,三(甲基苯基)甲基,三-(二甲基苯基)甲基,三(甲氧基苯基)甲基,三(三甲氧基苯基)甲基;三甲基甲硅烷基,三乙基甲硅烷基,三-正-丙基甲硅烷基,异丙基-二甲基甲硅烷基,叔丁基二甲基甲硅烷基,叔丁基二苯基甲硅烷基,正-辛基二甲基甲硅烷基,(1,1,2,2-四甲基乙基)-二甲基甲硅烷基,-(CH3)2Si-O-Si(CH3)2-,-(iso-C3H7)2Si-O-Si(iso-C3H7)2-;乙酰基,丙酰基,丁酰基,戊酰基,己酰基,苯甲酰基,甲基苯甲酰基,甲氧基苯甲酰基,氯苯甲酰基和溴苯甲酰基;甲基,乙基,丙基,丁基、苯基、苯甲基、对-溴代-,对-甲氧基-和对-甲基-苯基磺酰基;甲氧基-,乙氧基-,正或异丙氧基-,正、异或叔丁氧基羰基,或苯氧基羰基,苯甲氧基羰基,甲基或甲氧基或氯苯氧基羰基或甲基或甲氧基或氯苯甲氧基羰基或9-芴基甲氧基羰基,在这种条件下,R1和R2为相同的保护基团较好。
R2作为含磷的,构成核苷酸桥基的残基与式P1或P2相对应,
Xa为氧或硫;
Rb为氢,C1-C12烷基或C6-C12芳基;
在Ya、Ra和Rb中的烷基,芳基,芳烷基和烷芳基或未被取代,或被烷氧基,烷硫基,卤素,-CN,-NO2、苯基,硝基苯基或卤代苯基取代。
Ya包含的伯氨基,优选含1-12,特别优选含1-6个碳原子,其所包含的仲氨基,优选含2-12,特别优选含2-6个碳原子。
例如,伯氨基和仲氨基为式RcRdN残基,其中Rc为氢,或独立地具有Rd的定义,而Rd为C1-C20,优选C1-C12,特别优选C1-C6烷基,C1-C20,优选C1-C12-,特别优选C1-C6-氨基烷基或C1-C20-,优选C1-C12,特别优选C1-C6-羟基烷基;羧基烷基或烷氧羰基烷基,烷氧羰基基团含2-8个碳原子和烷基含1-6,优选1-4个碳原子;C2-C20,优选C2-C12-,特别优选C2-C6链烯基;苯基,单一或二-(C1-C4-烷基或C1-C4烷氧基)苯基、苯甲基,单或二-(C1-C4烷基或C1-C4烷氧基)苯甲基;或1,2-,1,3-或1,4-咪唑基-C1-C6烷基,或Rc和Rb同为四或五一亚甲基,3-恶-1,5-戊二烯亚戊基,-CH2-NRe-CH2CH2-或-CH2CH2-NR19-CH2CH2-,其中Re为氢或C1-C4烷基。氨基烷基中的氨基可被一或二个C1-C4烷基或C1-C4羟基烷基取代。羟基烷基中的羟基可被C1-C4烷基醚化。
Ya的伯、仲、叔和季铵,按M+的定义,可理解为它是式RfRgRhRiN
的离子,其中Rf为C1-C20、优选C1-C12、特别优选C1-C6-烷基,C1-C20,优选C1-C12,特别优选C1-C6-氨基烷基,C1-C20-、优选C1-C12-;特别优选C1-C6-羟基烷基;羧基烷基或烷氧羰基烷基,含2-8碳原子的烷氧羰基,和含1-6,优选1-4碳原子的烷基;C2-C20-、优选C2-C12-、特别优选C2-C6-链烯基;苯基;一或二(C1-C4烷基或C1-C4烷氧基)苯基,苯甲基、一或二-(C1-C4烷基或C1-C4烷氧基)苯甲基;或1,2-,1,3-或1,4-咪唑基-C1-C6-烷基,和Rg,Rh和Ri为相互独立的氢或有Rf的定义,或Rf和Rg均为四-或五-亚甲基,3-恶-1,5-戊二烯亚戊基,-CH2-NRe-CH2CH2-或-CH2CH2-NRe-CH2CH2-,其中Re为氢或C1-C4烷基;Rh和Ri相互独立,其中一个有Rf的定义。氨基烷基中的氨基可被一或二个C1-C4烷基或C1-C4羟基烷基取代。羟基烷基中的羟基可被C1-C4烷基醚化。
羧基烷基的实例为羰基甲基,羧基乙基,羧基丙基和羧基丁基,烷氧基羰基烷基的实例为被甲基或乙基酯化的羧基烷基基团。链烯基的实例为烯丙基,1-烯基-3-或4-丁基、3-或4-烯基-1或2-戊基,3-或4-或5-烯基-1-或2-己基,烷基-、烷氧-苯基、烷基-和烷氧-苯甲基的实例如甲基苯基,二甲基苯基,乙基苯基,二乙基苯基;甲基苯甲基,二甲基苯甲基,乙基苯甲基,二乙基苯甲基,甲氧基苯基,二甲氧基苯基,乙氧基苯基,二乙氧基苯基、甲氧苯甲基,二甲氧基苯甲基,乙氧基苯甲基和二乙氧苯甲基。烷基优选含2-4个碳原子的咪唑烷基的实例如1,2-,1,3-或1,4-咪唑-乙基或正-丙基或正-丁基,R19优选氢、甲基或乙基。
伯和仲氨基优选的实例为甲基-、乙基-、二甲基-、二乙基-、二异丙基-,一或二-(1-羟基-2-乙基)-,苯基-和苯甲基氨基,乙酰基氨基,苯酰基氨基,哌啶基,哌嗪基和吗啉基。
伯和仲铵的优选实例为甲基-,乙基-、二甲基、二乙基-,二异丙基-、一或二(1-羟基-2-乙基)-,苯基和苯甲基铵。
Ya、Ra和Rb为烷基的实例如甲基、乙基和丙基、丁基、戊基、己基、庚基、丁基的异构体;Ya、Ra和Rb为芳基的实例为苯基和萘基;Ra为链烯基的实例为烯丙基和(C1-C4烷基)CH=CH-CH2-.Ya为芳烷基的实例为苯基-CnH2n-,其中n为1-6,特别为苯甲基;Ya为烷芳基的实例为一、二和三(C1-C4烷基)苯基。取代基优选氯、溴,甲氧基,-NO2,-CN,2,4-二氯苯基和4-硝基苯基,Rb的实例为2,2,2-三氯乙基,4-氯苯基,2-氯苯基和2,4-二氯苯基;RbO为N-杂芳基的实例为吡咯N-基,三唑-N-基和苯并三唑-N-基。
Ra特别优选β-氰乙基和Ya特别优选二(异丙基氨基)。
如果B为嘌呤基团或其类似物,其可为式Ⅱ、Ⅱa、Ⅱb、Ⅱc、Ⅱd、Ⅱe或Ⅱf残基。
其中:Rb1为H、Cl、Br、OH或-O-C1-C12烷基,Rb2,Rb3和Rb5为相互独立的H、OH、SH、NH2、NHNH2,NHOH,NHO-C1-C12烷基,-N=CH-N(C1-C12烷基)2,-N=CH-N-环烷基,F,Cl,Br,C1-C12烷基,羟基-C1-C12烷基,氨基-C1-C12烷基,C1-C12烷氧基,苯甲氧基或C1-C12烷硫基-,未被取代或被保护基团取代的羟基和氨基,或苯基、苯甲基、C1-C20的伯基或C2-C30的仲氨基。
Rb4为氢、CN或-C=C-Rb7,Rb6和Rb7为氢或C1-C4烷基,
理想的保护基团如前所述,优选的保护基团为C1-C8酰基,如乙酰基,丙酰基、丁酰基和苯甲酰基。Rb6优选氢或甲基。
伯氨基优选含1-12个,特别优选1-6个碳原子,仲氨基优选含2-12个,特别优选2-6个碳原子。
一些优选含C1-C6的烷基,烷氧基、烷硫基,羟基烷基和氨基烷基的实例为甲基,乙基和丙基,丁基,戊基,己基,庚基,丁基,壬基,癸基,十一烷基,和十二烷基的异构体,还有其相应的烷氧基,烷硫基,羟基烷基和氨基烷基。烷基、烷氧基,烷硫基,羟基羰基和氨基烷基,其中烷基特别优选含1-4个碳原子。烷基、烷氧基、烷硫基、羟基烷基和氨基烷基优选为甲基、乙基、正和异丙基、正、异和叔丁基,甲氧基,乙氧基,甲硫基和乙硫基,氨基甲基,氨基乙基,羟甲基和羟乙基。
例如,伯氨基和仲氨基为式Ra1,Ra2N的残基,式中Ra1为氢,或独立地有Ra2的定义,Ra2为C1-C20-,优选C1-C12-;特别优选C1-C6烷基,C1-C20-,优选C1-C12-,特别优选C1-C6氨基烷基,C1-C20-,优选C1-C12-,特别优选C1-C6-羟烷基;羧烷基或烷氧基羰基烷基,含2-8个碳原子的烷氧基羰基和含1-6个碳原子,优选1-4个碳原子的烷基;C2-C20-,优选C2-C12-、特别优选C2-C6-链烯基;苯基,一或二(C1-C4-烷基或C1-C4烷氧基)苯基,苯甲基,一或二(C1-C4烷基或C1-C4烷氧基)苯甲基;或1,2-,1,3-,1,4-咪唑基-C1-C6烷基,或Ra1和Ra2一起为四或五亚甲基,3-恶-1,5-戊二烯亚戊基,-CH2-NRa3-CH2CH2-或-CH2CH2-NRa3-CH2CH2-,式中Ra3为氢或C1-C4烷基,氨基烷基中氨基可被一或二个C1-C4烷基或C1-C4羟烷基取代。羟烷基中的羟基可被C1-C4烷基醚化。
烷基的实例如前所述。氨基烷基的实例为氨甲基、氨乙基、1-氨-2-丙基或3-丙基,1-氨基-2-丁基、-3-丁基或-4-丁基,N-甲基或N,N-二甲基或N-乙基或N,N-二乙基或N-2-羟乙基或N,N-二-2-羟基-乙基-氨甲基、氨乙基、氨丙基或氨丁基。羟烷基的实例为羟甲基,1-羟基-2-乙基,1-羟基-2-丙基或-3-丙基和1-羟基-2-丁基-3-丁基、或-4-丁基。羧烷基的实例为羧甲基、羧乙基、羧丙基和羧丁基,烷氧基羰基烷基的实例为那些被甲基或乙基酯化的烷氧羰基。链烯基的实例为烯丙基、1-烯基-3-或-4-丁基,-3-或-4-烯基-1-或-2-戊基,-3-或-4-或-5-烯基-1-或-2-己基。烷基和烷氧基苯基与烷基和烷氧基苯甲基的实例为甲苯基、二甲苯基、乙苯基、二乙苯基、甲基苯甲基、二甲基苯甲基、乙基苯甲基、二乙基苯甲基、甲氧基苯基、二甲氧基苯基、乙氧基苯基、二乙氧基苯基,甲氧基苯甲基、二甲氧基苯甲基、乙氧基苯甲基和二乙氧基苯甲基。烷基优选含C2-C4的咪唑烷基的实例为1,2-,1,3-,1,4-咪唑基-乙基或正丙基或正丁基。Ra3优选为氢、甲基或乙基。
伯和仲氨基优选实例为甲基、乙基,二甲基、二乙基、烯丙基、一或二(1-羟基-2-乙基);苯基和苯甲基氨基,乙酰氨基,异丁酰基和苯甲酰氨基。
Rb1优选为氢,Rb5有时优选为氢,更为优选的是Rb2和Rb3为相互独立的H、F、Cl、Br、OH、SH、MH2、NHOH、NHNH2,甲氨基,二甲氨基、苯甲酰氨基、异丁酰氨基、甲氧基、乙氧基和甲硫基。
嘌呤类似物系列的一些实例,除嘌呤外,有黄嘌呤,次黄嘌呤,腺嘌呤,N-甲基-腺嘌呤,N-苯甲酰基-腺嘌呤,2-甲硫基腺嘌呤,2-氨基腺嘌呤,6-羟基嘌呤,2-氨基-6-氯嘌呤,2-氨基-6-甲硫基嘌呤,鸟嘌呤,N-异丁酰鸟嘌呤。特别优选腺嘌呤,2-氨基腺嘌呤,鸟嘌呤和其碱基保护的衍生物。
如果式Ⅰ中B为嘧啶基,则其优选为式Ⅲ、Ⅲa或Ⅲc的尿嘧啶,胸腺嘧啶或胞嘧啶或它们的二氢衍生物,
其中Rb6为氢或C1-C4烷基,Rb8为H,OH,SH,NH2,NHNH2,NHOH,NH-C1-C12烷基,-N=CH-N(C1-C12烷基)2,N=CH-N-环烷基,F,Cl,Br,C1-C12烷基,羟基-C1-C12烷基,氨基-C1-C12烷基,C1-C12烷氧基,苯甲氧基或C1-C12烷硫基,羟基和氨基可不被取代或被保护基团所取代,Rb8还可为苯基、苯甲基、含1-20个碳原子的伯氨基,含2-30个碳原子的仲氨基,C1-C12链烯基或C1-C12炔基,式Ⅲb中的NH2基可未被取代或被C1-C6烷基,苯甲酰基或保护基团所取代。式Ⅲ的Rb8优选为氢,C1-C6烷基或C1-C6羟烷基,C1-C6链烯基或C2-C6炔基,F,Cl,Br2,NH2,苯甲酰氨基,或一或二-C1-C6烷基氨基。式Ⅲb和Ⅲc的Rb8为优选为氢、C1-C6烷基或C1-C6烷氧基或C1-C6羟烷基,C1-C6链烯基或C1-C6炔基,F,Cl,Br,NH2,苯甲酰氨基或一或二-C1-C6烷基氨基。
Rb6优选为氢或甲基,式Ⅲ的Rb8优选H、F、Cl,Br,MH2,NHCH3,N(CH3)2,C1-C4烷基,C2-C4链烯基或C2-C4炔-1-基,式Ⅲb和Ⅲc的Rb8优选氢,C1-C4烷基,特别优选甲基,C2-C4链烯基,特别是乙烯基或C2-C4炔-1-基,特别是1-丙块-1-基,式NH2,NHCH3或(CH3)2N。
嘧啶类似物的实例为尿嘧啶,胸腺嘧啶、胞嘧啶,5-氟尿嘧啶,5-氯尿嘧啶,5-溴尿嘧啶,二氢尿嘧啶,5-甲基胞嘧啶,5-丙炔胸腺嘧啶和5-丙炔胞嘧啶。
本发明也涉及式Ⅰ化合物制备方法
其中,R1和R2为相互独立的氢或保护基团或R1有这些定义而R2为构成含磷核苷酸桥键的残基;B为嘌呤或嘧啶基或其类似物,和
(a)R3为式Ⅰa基团:
其中,R4为氢、C1-C21烷基,C2-C21链烯基,C2-C21炔基,或-C(=O)烷基;
R5为氢、C1-C10烷基、-CH2-O-R6或式Ⅰb残基;
R6为氢,C1-C22烷基,C3-C21链烯基,部分或全部被氟取代的C1-C10烷基或-[(CH2)2-O]m-R7;
R7为氢或C1-C21烷基;
n为1-12;
m为1-4;
当n=1和R5为氢时,R4不能是氢;
该方法包括
将式Ⅳa化合物
其中,R14和R15为相同或不同的保护基团,B为嘌呤或嘧啶或其类似物,碱基B中功能基团被保护基团保护起来,在惰性溶剂中,与式A化合物反应,
其中,R4、R5和n均如上所述定义,X为Cl,Br,I,甲苯磺酰基-O,甲磺酰基-O;
(b)R3为式Ⅰc基团
其制备方法为在惰性溶剂中将式Ⅳa化合物与式B化合物反应
其中X为Cl,Br,I,甲苯磺酰基-O或甲磺酰基-O;
(c)R3为式Ⅰb基团
其中,R5为氢,C1-C10烷基,-CH2-O-R6或式Ⅰb基团,R6为氢,C1-C22烷基,C3-C21链烯基,部分或全部被氟取代的C1-C10烷基或-[(CH2)2-O]m-R7;
R7为氢或C1-C21烷基;
Z为-(CH2)p-或-(CH2-CH-O)q-CH2CH2,Z中的-CH2-可不被取代或被一个或多个、相同或不同的取代基C1-C10烷基或C5-C6环烷基,和不被取代或被C1-C4烷基取代的苯基所取代。
m为1-4;
p为1-10;
q为1-4;
该方法为在H2O和BF3存在下,使从(b)获得的环氧化物开环并用式X′-(CH2)q-X′或X′-(CH2-CHO)rX′的化合物(X′为Cl,Br,I,甲苯磺酰基-O-或甲磺酰基-O)使之关环,形成一个新环;
(d)R3为式Ⅰa基团,
方法为在R6OH和BF3存在下,将(b)得到环氧化物反应,如需要使游离羟基转变为醚基或酯基,
(e)R3为式Ⅰa基团,
方法为在BF3和例如NaBH4的存在下,将(b)得到的环氧化物氢化,如需要使游离羟基转变为醚基或酯基;
(f)R3为式Ⅰa基团,
方法为将适当的Grignard试剂与(b)获得的环氧化物反应,如需要将游离羟基转变为醚基或酯基;或
(g)R3为式Ⅰa、Ⅰb、Ⅰc基团
方法如用(a)-(f)所述方法之一,在式Ⅳb化合物的2′-羟基上进行取代,
其中R14和R15如所述定义而A为离去基团,优选烷氧基、酰氧基、甲磺酰基-O,甲苯磺酰基-O,特别优选OCH3,OCOCH3和苯甲酰氧基,然后按照已知方法。通过取代引入碱基B[(Lukevics,E.,Zablorka,A.“核苷合成”,Ellis Howood,New York(1991)],如需要去掉保护基团R14和R15,引入构成合磷核苷酸桥键的基团。
式Ⅰ化合物(R5为氢,n=1)也可通过将式Ⅳa化合物与式X-CH2-CO-OR化合物反应,然后还原CO基团为CH2OH基团并对其进行烷基化而获得。
式Ⅳa和Ⅳb,A和B的化合物是已知的,其中有些是可市购的或可用已知或类似方法制备。
惰性溶剂举例如烃,卤代烃,烷基化羧酰胺和内酰胺,醚,腈,如乙腈,二烷基砜或二烷基亚砜或环状砜和亚砜。
在方法(a)-(g)中的反应温度为-50°-200℃,优选0-90℃。
本反应最好在碱存在下进行,例如碱金属氢化物,醇化物,氢氧化物,碳酸盐,三烷基胺或二氮杂二环十一碳烯。
例如,本反应可在催化下进行或在氢硼化合物存下进行。
式Ⅰ化合物可用已知方法分离和纯化,如沉淀或结晶,过滤和色谱法。
式Ⅰ化合物可用于合成由于核酸的相互作用而具有很好的生物活性的低聚核苷酸,还可用作药物活性成分或诊断试剂。
本发明还涉及式化合物在制备含式Ⅰ化合物的相同或不同的单体单元的低聚核苷酸中的应用,但是,至少一个式Ⅰ化合物的单体单元与其他天然或合成的核苷的单体单元结合,该低聚核苷酸含有2-200个单体单元,优选2-100个,特别优选2-50个,最特别优选4-30个单体单元。优选含式Ⅰ化合物的相同或不同单体单元的低聚核苷酸,优选也适于同时还含有从D-核糖成D-脱氧核糖衍生出的合成或天然核苷的单体单元的低聚核苷酸。
本发明还涉及式Ⅴ的低聚核苷酸
其中,X为0-200,Y为核苷酸桥键,U,V和W为相互独立的天然或合成的核苷的相同或不同残基,至少U.V和/或W残基之一为式Ⅵ的基团
;B和R3的优选和实例的定义如式Ⅰ化合物所设。
优选的桥基Y为存在天然低聚核苷酸之中的-P(O)O
基团。进一步讲,桥基的实例为-P(O)S
-,-P(S)S
-,-P(O)R16,P(O)N-R17R18,和-CH2-,其中R16为氢式C1-C6烷基,R17和R18相互独立的具有R16的定义,在式Ⅴ中,X优选0-100,特别优选1-50,更为优选3-29。式Ⅵ的基团可在未端或按核苷酸序列成链,全部或某些如,2-5个式Ⅵ基团可以相互连接,或Ⅵ基团可在天然或合成核苷的基团间成键,或在核苷酸序列中存在这些分布的混合形式。
一个特别优选的形式为式Ⅴ低聚核苷酸,其中X=2-50,优选2-30,Y为-P(O)O--基团,U,V和W为相互独立的相同或不同的天然核苷残基,和对应于式Ⅵ的至少U.V或W残基之一。适用的天然核苷如腺嘌呤核苷、胞嘧啶核苷、鸟嘌呤核苷,尿嘧啶核苷,2-氨基腺嘌呤,5-甲基胞嘧啶,2′-脱氧腺嘌呤核苷,2-′脱氧胞嘧啶核苷,2′-脱氧鸟嘌呤核苷和胸腺嘧啶核苷。还可能提及天然核苷碱基,特别优选腺嘌呤、胞嘧啶、鸟嘌呤,胸腺嘧啶和尿嘧啶。式Ⅵ残基可按未端或核苷酸序列成键,全部或某些,如2-5个相同或不同的式Ⅵ残基可相互连接,或式Ⅵ的相同或不同的残基在天然核苷残基之间成键,或在核苷酸序列中存在这些分布的混合形成。在式Ⅴ的低聚核苷酸的另一优选形式中,所有U.V和W残基为相同或不同的式Ⅵ残基。X优选3-29,式Ⅵ的残基总数优选为1-12。
本发明所述的低聚核苷酸可以使用市售的全自动DNA合成器和加工指令,用不同方法,按已知方式制备。对于桥基-P(O)O
-,例如,可用本领域专业人员所熟知的磷三酯法、亚磷酸三酯法,或H-磷酸盐法。例如,亚磷酸三酯法可按如下进行:R1和R2均为氢的式Ⅰ核苷与保护基团试剂反应,如,4,4′-2甲氧基三苯基甲基氯化物,得到式D的核苷
式D化合物在“连接剂”(如琥珀酸酐)作用下,附着在一个固体载体(如可控微孔玻璃(CPG)),并含有长链烷基氨基基团。在另一方法中,将式D化合物的羟基用R′OP[N(异丙基)2)]2派生,得到式E化合物,例如磷酰胺化物(phosphorus amidite)
例如其中R′为β-氰乙基
当保护基团;如附着在载体的物质的DMT基团,被除去后,随着-N(iso-C3H7)2的离去,式D化合物会发生偶联,任何游离的羟基会被阻断,生成的亚磷酸酯被氧化为磷酸酯。二聚物被去保护并反复进行与式E化合物循环反应,直至合成出含有所需数量的单体单元的低聚物,产物从载体上取出。低聚核苷酸可通过这个方法得到,其中与式V一致的所有U.V和W残基组成式Ⅵ残基。该方法也同样适于制备具有所需序列的所需单体单元的低聚核苷酸,其单元和序列决定于本发明合成和天然核苷结构单元,这些单元和它们序列用于个别反应循环中。
按本发明所述,R1和R2均为氢的式Ⅰ化合物具有抗病毒和抗生殖特性,因而可用作药物。除此之外,本发明所述的低聚核苷酸对核酸酶的降解有很高的稳定性。特别是它们可与互补核酸链,特别是RNA型,可极好地配对。除此之外,它们还具有相当高的细胞吸收。因此,本发明所述的低聚核苷酸特别适用于反义技术,即通过约束适当的mRNA的互补核苷序列而抑制不必要的蛋白质产物的表达(EP266099,WO87/07300和WO89/08146)。它们可用于感染和疾病的治疗,如通过在核酸(如致癌基因)水平上阻断生物活性蛋白质的表达。本发明所述的低聚核苷酸也适于用作诊断试剂并在控测病毒感染或遗传疾病上用作基因探针,通过在单或双链核酸水平上选择性地作用,特别是由于对核酸酶有较大的稳定性,它们不仅能用于体外诊断,还可用于体内诊断(如组织样品,血小板和血清)。例如在WO91/06556中对这些可能的应用有所描述。
本发明还涉及本发明所述的低聚核苷酸作为诊断试剂,在检测病毒感染或遗传疾病中的应用。
本发明还涉及本发明所述式Ⅰ核苷和式Ⅴ低聚核苷酸在通过纯化体内核苷酸序列从而治疗包括人类在内的热血动物疾病的治疗方法上的应用。例如体重约为70kg的热血动物的每月施药剂量为0.01-1000mg。给药优选以药物组合物的形式,非肠道给药,如静脉注射,腹腔注射给药。
本发明还涉及药物组合物,其中只含有效剂量的式Ⅰ核苷或式Ⅴ的低聚核苷酸或共同含这两种,另外还有其他活性成分、药物载体,优选以足够剂量,和适量赋形剂。
本发明所述的药理活性核苷和低聚核苷酸可以非肠道给药的组合物或滴注溶液的形式应用。该溶液优选等渗水溶液或悬浮液,例如含活性成分本身或加入载体(如甘露糖醇)的冷冻干燥组合物,这些溶液或悬浮液在使用之前制备。该药物组合物可以是灭菌的,和/或赋形剂,如防腐剂、稳定剂、湿润剂和/或乳化剂、助溶剂、调节渗透压的盐和/或缓冲剂。如有必要,可按已知方法制备含更多药理活性物质(如抗生素)的药物组合物,例如,按常规溶解或冷冻干燥方法,可含有约0.1%-90%,优选的0.5%-30%,如1%-5%的活性成分。
下面的实施例用来阐述本发明。该′H-NMR-波谱是基于下列环碳结构的碳原子的位次偏排:
初始化合物:
核苷(实例)
用于说明书和分子式中的缩写符号:
DMF N′N-二甲基甲酰胺
ClBnCl22,4-二氯苯甲基氯
Bn 苯甲基
Ac 乙酰基
φ 苯基
BSA N,N-双三甲基硅烷基乙酰氨
DBU 二氮杂双环[5,4,0]十一烷-7-烯
BOM-Cl 苯甲氧基甲基氯
DMTCl 4,4′二甲氧三苯甲基氯
THF 四氢呋喃
(A)核苷类似物的制备
实例A1:
在60℃,将28.0g的1-甲基核糖逐滴加到含13.5gNaH的130mlDMF中。当H2放完后,逐滴加入110.0g的ClBnCl2。将反应混合物在25℃搅拌16小时。为彻底除掉残留的NaH,逐滴小心加入甲醇,将反应混合物倒入冰水混合物中。滤去块状沉淀,用乙腈彻底洗涤而得到化合物(A1)
1H-NMR(250MHz,CDCl3):H-C(1)-质子出现在5.0ppm,呈单峰,MS:638(M+)
实例A2:
将65.9g由实例A1制备的产物溶于600ml二氯甲烷并冷却至0℃。逐滴加入800ml含121mlSnCl4的二氯甲烷,反应混合物在3℃放置。过26小时后再加2mlSnCl4。总共35小时后,将反应溶液小心倒入700ml的饱合NaHCO3溶液,用400ml二氯甲烷稀释后,滤掉含Sn沉淀,用MgSO4干燥滤液的有机相并蒸发浓缩而得到化合物(A2)。
1H-NMR(250MHz,CDCl3):H-C(1)质子出现在4.90ppm,呈双峰,J=5Hz。
实例A3:
将125.9g由实例A2获得的产物溶于1升吡啶。在20℃,加入25.5g乙酐和1g4-二甲基氨基吡啶。反应混和物搅拌17hr,在1升水中吸收,用浓盐酸酸化后,用乙酸乙酯萃取。用MgSO4干燥萃取液并蒸发浓缩。最后用己烷使残余物结晶而得到化合物(A3)
1H-NMR(250MHz,CDCl3):5.15[d,J=4.5Hz,H-C(1)];3.50(s,OCH3);2.17(s,OCOCH3);
MS:522(M+)
[α]Na(D)=87.4±1.0°,CHCl3(0.998%)
实例A4:
在100ml1,2-二氯乙烷中,将24g胸腺嘧啶制浆。加入116.4gBSA后,加热回流反应混合物直至溶液澄清。然后将反应混合物冷却至50℃,加入50g由实例A3制备产物和27.5g三氟甲基磺酸三甲基硅酯。在70℃搅拌反应混合物20小时,然后加入300mlNaHCO3溶液并搅拌,用二氯甲烷萃取后,用MgSO4干燥反应混合物并蒸发干燥。最后用甲醇使残余物结晶而得到化合物(A4)
1H-NMR(250MHz,CDCl3):8.25(s,NH);6.10[d,J=4.5Hz,H-C(1’)];2.13(s,OCOCH3);1.66(s,CH3);
MS:616(M+)
实例A5:
将85g由实例A4制备的产物悬浮于850ml乙腈中。在室温下,逐滴加入24.2gDBU和24.9g的BOM-Cl,搅拌反应混合物20小时,倒入水中并用乙酸乙酯萃取。用MgSO4干燥萃取液,蒸发浓缩后得到化合物(A5)
1H-NMR(250MHz,CDCl3):6.05[d,J=4.5Hz,H-C(1’)];5.5(AB,CH2);5.37[dd,H-C(2’)];2.13(s,OCOCH3);1.55(s,CH3);
MS:736(M+)
实例A6:
将106g由实例A5制备的产物悬浮于1升THF中,逐滴加入26g的30%NaOCH3/CH3OH溶液。搅拌2.5小时后,将反应溶液倒入水中,加入饱合氯化钠水溶液,用乙酸乙酯萃取反应溶液。用MgSO4干燥后,蒸发浓缩后得到化合物(A6)。
1H-NMR(250MHz,CDCl3):5.93[d,J=5Hz,H-C(1’)];5.5(AB,CH2);3.03(d,J=6.5Hz,OH);1.72(s,CH3)
MS:694(M+)
实例A7:
将20.3g由实例A6得到的产物溶于200mlTHF中。加入0.73gNaH,将反应混合物煮沸30分钟然后加入2.89g2-氯乙基甲基醚,将反应混合物再煮沸24小时。再加0.5gNaH和1.7g2-氯乙基甲基醚,继续沸腾。总共32小时后,反应混合物倒入水中并用乙酸乙酯萃取。用MgSO4干燥萃取液,蒸发浓缩。将残余用硅胶层析用甲苯/乙酸乙酯(4∶1)洗脱,得到化合物(A7)
1H-NMR(250MHz,CDCl3):7.65[s,H-C(6)];5.93[s,H-C(1’)];5.5(s,CH2);3.33(s,OCH3);1.6(s,CH3).
MS:752(M+).
实例A8:
将79.4g由实例A6获得的产物溶解于800mlTHF。加入3.3gNaH,使反应混合物沸腾一会儿,然后在40℃,滴加21g溴乙酸甲酯。反应混合物在60℃搅拌共27小时,在16小时后和20小时后分别加1gNaH和2ml溴乙酸甲酯。最后,将反应混合物倒入水中并用乙酸乙酯萃取。用MgSO4干燥萃取液,蒸发浓缩得到化合物(A8)
1H-NMR(250MHz,CDCl3):7.70[s,H-C(6)];5.92[s,H-C(1’)];5.48(AB,CH2);3.75(s,OCH3);1.58(s,CH3).
MS:766(M+).
实例A9:
a)将37g由实例A8得到的产物溶于400mlTHF。在20℃分批加入1.5g LiBH4,将反应混和物搅拌1小时。然后将反应混合物小心加入500ml水中,用32ml2N盐酸水溶液中和。用乙酸乙酯萃取并蒸发浓缩得到化合物(A9)。
1H-NMR(250MHz,CDCl3):7.65[s,H-C(6)];5.96[s,H-C(1’)];5.50(AB,CH2);2.57(broad s,OH);1.60(s,CH3).
MS:738(M+).
(b)在70℃,在含NaH的THF溶液中,用CH3I将化合物(A9)甲基化。8小时后,如实例A7所述进行反应而得到化合物(A7)。
实例A10:
将20.0g由实例A7所得产物溶于200ml THF。在常压(吸收H2102%)和25℃,在2g Pd/C(5%)上氢化4.5小时。过滤后通过蒸发浓缩滤液,残余液溶于170ml甲醇。用30%NaOCH3/CH3OH调PH于11。24小时后,反应混合物倒入250ml水,用2N盐酸水溶液酸化并用乙酸乙酯萃取。用MgSO4干燥萃取液并蒸发浓缩而得化合物(A10)。
1H-NMR(250MHz,CDCl3):8.75(s,NH);7.65[s,H-C(6)];5.97[s,H-C(1’)];3.33(s,OCH3);1.60(s,CH3).
MS:632(M+).
实例A11:
在常压50℃,在5g Pd/C(5%)上,在0.25g无水乙酸钠存在下,将15g由实例A10制备的产物在250ml甲醇中氢化。46小时后,过滤该氢化混合物并蒸发浓缩。为去掉盐,残余液在一小硅胶玻璃板上层析(乙酸乙酯/甲醇9∶1),从而获得化合物(A11)。
1H-NMR(250MHz,CDCl3):11.5(s,NH);7.95[s,H-C(6)];6.00[d,J=6Hz,H-C(1’)];5.33(broad s,OH);5.20(d,OH);3.32(s,OCH3);1.92(s,CH3).
MS:316(M+).
实例A12:
将0.45g由实例A11制备的产物分两次放入吡啶中并蒸发浓缩。再往18ml吡啶中依次加入:0.2g三乙胺,0.55g DMTCl和25mg 4-二甲基氨基吡啶。在室温搅拌该反应混合物16小时,然后用50ml乙酸乙酯稀释并倒入50ml水中。用MgSO4干燥有机相并浓缩。残余物在硅胶(己烷/乙酸乙酯/三乙胺86∶10∶4)上层析而得到化合物A12。
1H-NMR(250MHz,CDCl3):7.62[s,H-C(6)];6.02[d,J=4Hz,H-C(1’)];3.38(s,OCH3);1.36(s,CH3).
实例A13:
将300mg由实例A12制备的产物加入110mg二异丙基四唑铵,178mg2-氰乙基-N,N,N′,N′四异丙基磷酰胺和6ml二氯甲烷。在室温搅拌此反应混合物17小时,然后倒入饱合NaHCO3水溶液,用MgSO4干燥有机相并蒸发浓缩、残余物在硅胶(乙醇/乙酸乙酯4∶1),另外加入1%的三乙胺)上层析。将得到的泡沫溶于1ml甲基叔丁基醚并在0℃逐滴加入戊烷中而得到化合物(A13)(非对映异构体,1∶1)
1H-NMR(250MHz,CDCl3):7.70[s,H-C(6)] and 7.63[s,H-C(6)];6.08[d,J=4Hz,H-C(1’)];6.02[d,J=4Hz,H-C(1’)].
实例14:
将24.2g由实例A2得到的产物溶于250ml THF中。加入8.76g溴乙酸甲酯和1.38g NaH,然后在60℃搅拌此反应混合物3.5小时。在另加0.14g NaH和0.53ml溴乙酸甲酯后,在60℃再搅拌此反应混合物3小时,将悬浮液倒入300ml水中,用2N的盐酸水溶液中和并用乙酸乙酯萃取。用MgSO4干燥萃取液并蒸发浓缩而得到化合物(A14)
1H-NMR(250MHz,CDCl3):5.05[d,J=4Hz,H-C(1’)];3.50[s,OCH3] and 3.75[s,OCH3].
MS:552(M+).
实例A15:
将5.0g由实例14得到的产物溶于60ml乙腈中。加入4.88g二甲硅烷基化胸腺嘧啶,在60℃搅拌,滴加2.6g三氟甲基磺酸三甲基甲硅烷基酯。搅拌3.5小时后,冷却反应液,将其倒入饱合NaHCO3水溶液中搅拌。然后用乙酸乙酯萃取此反应液,用MgSO4干燥有机相并蒸发浓缩。残余物用硅胶(甲苯/乙酸乙酯1∶1)层析而得到化合物A15。
1H-NMR(250MHz,CDCl3):8.36(s,NH);7.70[s,H-C(6)];5.94[d,J=1.5Hz,H-C(1’)];3.73(s,OCH3);1.58[s,CH3].
MS:646(M+).
b)与苯甲氧基甲基氯反应,得到化合物(A8)。
实例16:
按照类似前述方法,可进行2′-OH修饰,见表1
表1 2′-OH修饰的实例
表1 2′-OH进一步修饰的实施例
实例A17:
将5.0g由实例A6制备的产物溶于50ml THF中。加入0.21g NaH后,在60℃加热反应混合物45分钟,然后加入1.57g二甘醇氯乙基甲基醚,最后在60℃总共持续搅拌54小时。在8小时和42小时,分别再加0.05g NaH和0.4g二甘醇氯乙基甲基醚。冷却混合物,倒入水中并用乙酸乙酯萃取,用MgSO4干燥并蒸发浓缩萃取液而得到化合物(A17),通过用己烷/乙酸乙酯1∶1进行硅胶层析而得。
1H-NMR(250MHz,CDCl3):1.60(s,CH3);3.33(s,OCH3);5.47(AB,CH2);5.93[d,H-C(1’)];7.65[s,H-C(6)].
MS:840(M-).
实例A18:
将3.2g由实例A17得到的产物,在常压和22℃,用0.6g Pd/C(5%)在THF中氢化。1小时后(吸收H2-96%),过滤氢化混合物至澄清并蒸发浓缩。残余物溶于30ml甲醇并加入3.5ml 30%的NaHCO3甲醇溶液。在20℃搅拌24小时后,将反应液倒入水中并用乙酸乙酯萃取。用MgSO4干燥并蒸发浓缩萃取液而得到化合物(A18)。
1H-NMR(250MHz,CDCl3):1.60(s,CH3);3.55(s,OCH3);5.96[d,J=2Hz,H-C(1’)];7.62[s,H-C(6)];8.96(s,NH).
MS:720/722/724(M+).
实例A19:
将2.3g由实例A18制备的产物溶于75ml甲醇并加入1.15g乙酸钠和1.0g Pd/C(5%)进行氢化反应。43小时后,滤掉催化剂,浓缩滤液。为分离出盐,用乙酸乙酯/甲醇(4∶1)在硅胶上过滤残余物而得到化合物(A19)。
1H-NMR[250MHz,methanol(D4)]:1.72(s,CH3);3.22(s,OCH3);5.80[d,J=5Hz,H-C(1-’)];7.78[s,H-C(6)].
MS:404(M).
实例A20:
将400mg由实例A19制备的产物溶于8ml无水吡啶中。在20℃加入0.34g DMTCl,搅拌反应混合物20小时,用30ml二氯甲烷稀释并倒入水中。用水再洗涤一次有机相,然后干燥(MgSO4)并蒸发浓缩。残余物用乙酸乙酯/三乙胺(97∶3)进行硅胶层析而得到化合物(A20)。
1H-NMR(250MHz,CDCl3;all signals are broad):1.40(s,CH3);3.35(s,OCH3);3.70(s,2x OCH3);6.02(s,H-C(1’);7.66[s,H-C(6)].
MS:705(M-H).
实例A21:
将0.39g由实例A20制备的产物的2.5ml二氯乙烷溶液滴加至含0.12g二异丙基四唑铵和0.2g氰乙基四异丙基磷酰胺(phosphous amidite)的2.5ml无水乙氯甲烷中。在20℃搅拌28小时,将反应液倒入饱合NaHCO3水溶液并用二氯乙烷萃取,蒸发浓缩并用乙酸乙酯/三乙胺(97∶3)进行硅胶层析而得到化合物(A21)。
1H-NMR(250MHz,CDCl3):The protons of H-C(1’)appear as doublets at 6.02 and 6.07 ppm.
31P-NMR(CDCl3):149.95 and 149.88 ppm
实例A22:
将21.4g尿嘧啶悬浮于250ml二氯乙烷中。加入116g BSA并将反应混合物加热到80℃。30分钟后形成溶液。将溶液冷却至40℃;加入50.0g溶于350ml二氯乙烷的由实例A3制备的产物和27.4g SnCl4。在80℃,放置溶液5小时,冷却后倒入饱合NaHCO3溶液。将得到的双相悬浮液过滤并将有机相分离出。用CH2Cl2萃取水相。用MgSO4干燥收集的萃取液并蒸发浓缩。在乙腈中浸煮残余物而得到化合物(A22)
NMR(250MHz,CDCl3):2.22(s,OAc),5.35(t,H-C(2’)),6.07(d,J=4Hz,H-C(1’)),5.53 and 7.23(each d,J=8Hz,H-C(5)and H-C(6)),8.57(s,NH).
MS:602
实例A23
在60℃,将溶于300ml THF的35.6g由实例A4制备的产物加到含1.47g NaH(100%)的60ml THF中,持续加热1小时。分批加入14.0g R-(-)缩水甘油甲苯磺酸酯。2.5小时后,再加0.1g NaH。加热此反应混合物4小时后冷却,倒在水中并用乙酸乙酯萃取。蒸发浓缩萃取液并进行层析(硅胶,甲苯/乙酸乙酯9∶1)而得到化合物(A23)。
NMR(250MHz,CDCl3):1.59(s,CH3),5.48(AB,CH2),5.92(s,1.5Hz,H-C(1’)),7.62(s,H-C(6)).
FAB-MS:751(M+H)
实例A24:
将5.1g由实例A23制备的产物和4.93g十六烷醇溶于50ml CH2Cl2。加入0.96g三氟化硼二乙基醚化物(d=1.13g/ml)。在室温搅拌该液19小时,然后倒入水中并用CH2Cl2萃取。蒸发浓缩萃取液并用硅胶(乙酸乙酯/己烷1∶1)层析而得到化合物(A24)。
NMR(250MHz,CDCl3):1.60(s,CH3),5.47(AB,CH2),5.93(d,J=1.5Hz,H-C(1’)),7.63(s,H-C(6)).
FAB-MS:1027(M+Cl)-
实例A25:
将3.45g由实例A24制备的产物加入含0.15g MaH(100%)的35ml THF中,在60℃加热45分钟。再加入0.63g MeI,再加热反应混合物1.5小时。冷却反应混合物,倒入水中并用乙酸乙酯萃取。蒸发浓缩残余物,然后在甲苯中通过短硅胶柱过滤而得到化合物(A25)。
NMR(250MHz,CDCl3):1.59(s,CH3),5.45(AB,CH2),5.93(d,J=1.5Hz,H-C(1′)),7.63(s,H-C(6)).
MS(DCl):1024(M+NH4)+
实例A26:
将3.3g由实例A25制备的产物在70ml THF中,在0.7g Pd/C(33小时,H2吸收104%)上氢化。将反应混和物过滤并蒸发浓缩。将残余物溶于10ml THF和10ml甲醇,加入NaCH3/HOCH3溶液(30%)。在室温搅拌反应混合物24小时,然后浓缩其体积至约一半,倒入水中并用乙酸乙酯萃取。蒸发浓缩该萃取液并用硅胶(乙酸乙酯/甲苯1∶2)层析而得到化合物(A26)。
NMR(250MHz,CDCl3):1.62(s,CH3),3.40(s,OCH3),5.98(d,J=2Hz,H-C(1′)),7.62(s,H-C(6)),8.57(s,NH).
MS(DCl):886
实例A27:
将1.21g由实例A26制备的产物在50ml甲醇和5ml甲苯中,用0.2g Pd/C(10%)和0.258g MgO(2小时,H2-吸收220ml)氢化。过滤该反应混和物,蒸发浓缩并用硅胶(去Mg盐)过滤而得到化合物(A27)。
NMR(250MHz,DMSO-D6):1.72(s,CH3),5.05(d,OH),5.18(t,OH),5.84(d,J=6Hz,H-C(1′)),7.77(s,H-C(6)).
FAB-MS:569(M-H).
实例A28:
将0.83g由实例A27制备的产物溶于50ml吡啶,蒸发浓缩溶液。向残余物加入8ml吡啶和0.6g DMT氯化物,在室温搅拌反应混合物44小时。将溶液倒入水中并用乙酸乙酯萃取。蒸发浓缩残余物并用硅胶(甲苯/乙酸乙酯1∶1)+1% NEt3)层析而得到化合物(A28)。
NMR(250MHz,CDCl3):1.35(s,CH3),3.47(s,OCH3),3.80(s,OCH3),6.03(d,J=4Hz,H-C(1′)),7.64(s,H-C(6)),8.20(s,NH).
FAB-MS:873(M+H).
实例A29:
将10ml CH2Cl2、0.245g二异丙基四唑铵和0.39g氰乙基四异丙基磷三酸胺加入0.95g由实例A28制备的产物。在室温下搅拌三天后,将反应混合物倒入饱和NaHCO3溶液并用CH2Cl2萃取。蒸发浓缩残余物并用硅胶(甲苯/乙酸乙酯2∶1+1%NEt3)层析而得到化合物(A29):
1H-NMR(250MHz,CDCl3:5.97 and 6.05(each d,J=4Hz,each H-C(1)),7.62 and 7.70(each s,each H-C(6)).
P-NMR(250MHz,CDCl3):150.012 and 150.102.
FAB-MS:1071(M-H).
实例A30:
将15.6g次黄嘌呤和46.6g BSA在300ml二氯乙烷加热回流。30分钟后形成溶液。将30.0g由实例A3制备的产物和16.5g的TMS三氟甲磺酸加入溶液中并再煮沸16小时。冷却反应混合物;倒入饱合NaHCO3溶液并过滤得到的悬浮液。滤液用CH2Cl2萃取。用MgSO4干燥有机相并蒸发浓缩。残余物经层析(甲醇/乙酸乙酯1∶1)而得到化合物(A30)。
1H-NMR(250MHz,CDCl3):6.22(d,3Hz,H-C(1′)),8.14 and 8.37(each s,H-C(2)and H-C(8)).
FAB-MS:625M-H
实例A31:
将3.8g由实例A30的产物加到150ml CH2Cl2中。煮沸时逐滴加入4.8ml SOCl2和2.4ml DMF混合物。再使反应混合物沸腾3小时,然后再加入1.2ml SOCl2和0.6ml DMF。共煮沸反应混合物5小时然后加到饱和NaHCO3溶液中,搅拌20分钟并用CH2Cl2萃取。用MgSO4干燥萃取液并蒸发浓缩而得到化合物(A31)。
NMR(250MHz,CDCl3):5.83(dd,H-C(2′)),6.33(d,J=3Hz,H-C(1′)),8.47 and 8.73(each s,H-C(2)and H-C(8)).
MS:664
实例A32:
将30.4g由实例A31制备的产物溶于200ml甲醇和100ml THF中,滴加16.9g 30% NaOMe/甲醇。搅拌反应混合物3小时;倒入水中并用乙酸乙酯萃取。用MgSO4干燥萃取液并蒸发浓缩而得到化合物(A32)。
NMR(250MHz,CDCl3):4.20(s,OCH3),6.10(d,J=6Hz,H-C(1′)),8.17 and 8.48(each s,H-C(2)and H-C(8)).
MS:598
实例A33:
将1.17g NaH(100%)加入到含25.4g由实例A32制备产物的250ml THF中。煮沸时,滴加6.76g 2-溴乙基甲基醚。再煮沸反应混合物6小时,再加入0.5g NaH和1.9ml溴化物。23小时后冷却反应混合物并倒入水中,用乙酸乙酯萃取并蒸发浓缩,残余物经层析(硅胶,己烷/丙酮2∶1)而得到化合物(A33)。
1H-NMR(250MHz,CDCl3):3.27(OCH3),4.20(OCH3),6.27(d,J=5Hz,H-C(1′)),8.24 and 8.52(each s,H-C(2)and H-C(8)).
MS(DCI):656
实例A34:
将2.2g由实例A33制备的产物在45ml含有0.66g Pd/C(10%)甲醇中,并加入402ml MgO的条件下氢化。13小时(H2-吸收485.5ml)后,过滤混合物至澄清并蒸发浓缩。为除去镁盐,残余物用硅胶(乙酸乙酯/甲醇9∶1)层析而得化合物(A34)。
NMR(250MHz,DMSO(-d6):4.4(t,OH),5.23(OH),5.94(d,J=6.5Hz,H-C(1′)),8.40 and 8.55(each s,H-C(2)and H-C(8)).
FAB MS:339(M-H)-.
实例A35:
将2.46g由实例A34制备的产物溶于80ml甲醇中并放入高压釜。加入20gNH3并在120℃保持12小时(内压:17bar)。蒸发浓缩反应混合物并用硅胶(乙酸乙酯/甲醇)层析而得到化合物(A35)。
NMR(250MHz,DMSO-D6):5.32(d,OH),5.60(t,OH),6.15(d,J=6Hz,H-C(1′)),8.33 and 8.57(each s,H-C(2)and H-C(8)).
MS(DCI):326(M+H).
实例A36:
将0.48g由实例A35得到的产物溶于5ml甲醇中并加入0.28gN-甲基-2,2-二甲氧基吡咯烷。搅拌反应混合物18小时后蒸发浓缩。加入两倍于残余物的吡啶,蒸发浓缩残余物。得到的残余物溶于10ml吡啶,加入0.5g DMT氯化物并在室温搅拌40小时。将得到的混合物倒入水中并用CH2Cl2萃取。蒸发浓缩萃取液并用硅胶(乙酸乙酯/甲醇9∶1)层析而得到化合物(A36)。
NMR(250MHz,CDCl3):3.09(s,NMe),3.72(s,OCH3),6.08(d,J=6Hz,H-C(1′)),7.98 and 8.43(each s,H-C(2)and H-C(8)).
FAB MS:709(M+H)
实例A37:
将6ml含0.63g由实例A36制备得到的三苯甲基衍生物的CH2Cl2溶液加入到6ml含0.38g氰乙基四异丙基磷二酸胺和0.24g二异丙基四唑铵的CH2Cl2溶液中。搅拌反应混合物40小时,倒入饱合的NaHCl3溶液中并用CH2Cl2萃取。蒸发浓缩萃取液并进行层析(乙酸乙酯+1% NEt3)而得到化合物(A37)。
31P=NMR(250MHz,CDCl3):149.8 and 150.4.
FAB-MS:909(M+H)
实例A38:
将7.1g由实例A23制备的环氧化物溶于70ml THF,加入0.43g NaBH4,在室温下搅拌反应混合物,加入0.5g BF3·OEt2。总共26小时后;将反应混合物倒入水中并用乙酸乙酯萃取。蒸发浓缩后得到化合物(A38)。
NMR(250MHz,CDCl3):1.16(d,J=7.5Hz,CH3),1.61(s,CH3),5.48(AB,CH2),5.92(d,J=1Hz,H-C(1′)),7.69(s,H-C(6)).
FAB-MS:787(M+Cl)-.
实例A39:
在70ml THF中,用1.56g NaH(100%)和156g MeI将6.9g由实例A38制备的醇甲基化(60℃,5小时)。通过常规反应得到化合物(A39)。
NMR(250MHz,CDCl3):1.13(d,J=7.5Hz,CH3),1.60(s,CH3),3.33(s,OCH3),5.49(AB,CH2),5.95(A,J=2Hz,H-C(1′)),7.64(s,H-C(6)).
MS(DCI):766(M-).
实例A40:
将14.4g由实例A4制备的衍生物,在60℃在加有0.65g NaH(100%)的150ml THF中加热30分钟。将反应混合物冷却至25℃并加入5.92g D-α,β-异亚丙基甘油-γ-甲苯磺酸酯。1.5小时后,在60℃再搅拌反应混合物3小时,冷却反应混合物并倒入水中,用乙酸乙酯萃取并蒸发浓缩。残余物用硅胶(甲苯/乙酸乙酯4∶1)层析而得到化合物(A40)。
NMR(250MHz,CDCl3):1.34 and 1.41(each s,each CH3),1.58(s,CH3),5.48(AB,CH2),5.92(d,J=1.5Hz,H-C(1′)),7.68(s,H-C(6)).
MS(DCI):843(M+Cl)-.
实例A41:
将3.31g由实例A13制备的磷酰胺化物溶于50ml乙腈中。加入9.4ml NEt3和6.17g1,2,4-三唑。在搅拌下,在温度控制在30℃以下的条件下滴加1.53gPOCl3。在室温再搅拌反应混合物4小时,倒入1N NaHCO3(150ml)并用乙酸乙酯萃取。蒸发浓缩萃取液,溶于少量CH2Cl2,搅拌加入100ml正戊烷中。沉淀出化合物(A41)。
1H-NMR(250MHz,CDCl3):6.02 and 6.06(each d,J=1.5Hz,H-C(1′)),8.44 and 8.47(each s,H-C(6)),8.07 and 9.35(each s,triazole H).
31P-NMR(250MHz,CDCl3):149.433 and 150.253
MS(DCI):869(M-)
实例A42:
将8.88g N2-异丁酰基-O6-苯甲基嘧啶(按Jenny,TF,Schneider,K.C.,Benner,S.A,Nucleosides and Nucleotide 11:1257-1261(1992)所述类似方法制备)和34.8gN,O-双(三甲基甲硅烷基)乙酰胺悬浮于130ml甲苯中,在100℃加热至溶液形成。在50℃,加入13.0g由实例A3制备的核糖衍生物和6.33g三氟甲基磺酸三甲基甲硅烷基酯。在100℃,搅拌该无色溶液4小时,冷却并倒入200ml饱合NaHCO3溶液。用乙酸乙酯萃取反应混合物,用MgSO4干燥并蒸发浓缩。残余物用1.2kg硅胶(甲苯/乙酸乙酯4∶1)层析而得到化合物(A42)。
1H-NMR(250MHz,CDCl3):1.26(d,J=7.5Hz,CH3),2.23(s,OAc),5.61(AB,OCH2),5.78(dd,H-C(2′)),6.19(d,J=3Hz,H-C(1′)),7.83(s,NH),8.00(s,H-C(8)).
MS(DCI):801(M-).
实例A43:
将0.83g 30%NaOMe/甲醇溶液加到80ml含7.4g由实例A42制备的衍生物的甲醇中,在室温搅拌反应溶液30分钟。将溶液倒入水中并用乙酸乙酯萃取。用MgSO4干燥萃取液,蒸发浓缩而得到化合物(A43)。
NMR(250MHz,CDCl3):1.29(d,J=7.5Hz,CH3),5.61(AB,OCH2),5.99(d,J=6Hz,H-C(1′)),7.97(s,NH),8.04(s,H-C(8)).
MS(DCI):760(M+H)+
实例44:
将630g NaH(100%)加到100ml含9.0g由实例A43制备的化合物的THF中,搅拌反应混合物15分钟。将1.97g α-溴乙基甲基醚喷撒在反应混合物中。25小时后,再加2.0g溴化物。共搅拌反应混合物48小时,然后倒入水中并用乙酸乙酯萃取。蒸发浓缩萃取液并用硅胶(甲苯/乙酸乙酯4∶1)层析向得到化合物(A44)。
NMR(250MHz,CDCl3):3.28(s,OCH3),5.56(AB,OCH2),6.10(d,J=5Hz,H-C(1′)),7.90(s,H-C(8)).
FAB-MS:748(M+H)+.
实例A45:
从化合物(A3)开始加入各种碱,产物列于表2。
表2:引入其它碱基的实施例
实例B:低聚核苷酸的制备
低聚核苷酸与固体载体(可控微孔玻璃,CPG)结合,用本发明所述的二甲氧基三苯甲基化和3′-活化的[3′-(β-氰乙氧基-二(异丙基氨基-磷酰胺化物)]核苷或类似活化的天然核苷结合,按照生产商品标准草案[参阅“低聚核苷酸合成,一种可行的方法“M.J.Gait;IRL Press 1984(Oxford-Washington DC)]在DNA合成器(应用生物系统,380B型,标准磷酰胺化物化学和iodoxidation)中合成,当最终核苷结构单元偶联后,通过用浓氨水处理过夜将5′位保护的低聚核苷酸从载体脱下,同时脱去所有保护基团,用50mM乙酸铵缓冲液(PH=7)/乙腈在反相HPLC上纯化。用80%乙酸处理20分钟除去5′-二甲氧基三苯甲基保护基团。用乙醇将低聚核苷酸沉淀出来并离心分离。低聚核苷酸的纯度用胶体电泳(聚乙酰胺)测试,其同一性用MALDI-TOFMS法测试。
实例C11:亲和力;低聚核苷酸(反义)和互补低聚核苷酸序列(有义)的相互作用。
低聚核苷酸与相应碱基互补的天然核糖核苷酸低聚物的相互作用可通过记录UV熔化曲线和确定的Tm值表征,例如Marky,L.A.Breslauer,K.J.,Biopolymers 26:1601-1620(1987)描述了该标准方法。
制备低聚核苷酸和相应碱基互补天然低聚核糖核苷酸的10mM磷酸盐缓冲液,100mM NaCl0.1mMEZDTA,PH=7.0(C=4×10-6M/低聚核苷酸),在260nm的消光变化记录为温度(15-95℃)的函数,从得到的熔化曲线确定其Tm值(表3)。
表3:亲合力
表3亲合力
(a)TTTTtCTCTCTCTCT(vs.RNA)
(b)CTCGTACCtTTCCGTCC(vs.RNA)
(c)CTCGTACttttCCGGTCC(vs.RNA)
实例D2:专一性,低聚核苷酸和己与异常核苷(Y)结合的碱基互补的低聚核糖核苷酸的相互作用。
制备低聚核苷酸和具有序列γ(GGA CCGGAA YGG TAC GAG)的相应碱基互补低聚核苷酸溶液的10mM磷酸盐缓冲液,100mM NaCl,0.1mMEDTA,PH=7,(C=4×10-6M/低聚核苷酸),测量在260nm的消光变化作为温度(15-95℃)的函数,Tm值从曲线确定。结果列于表4。
表4,专一性
有义:GAG CAU GGY AAG GCC AGG(RNA)
反义:CTC GTA CCt TTC CGG TCC(DNA)
Tm(℃)和△Tm
Y=A 63.3 65.0 64.1
Y=C 54.5 55.8 55.1
(-8.9) (-9.2) (-9.0)
Y=G 61.6 61.4 59.5
(-1.7 (-3.6) (-4.5)
Y=U 55.8 57.5 55.9
(-7.5) (-7.5) (-8.2)
Y=无 59.4 57.8 56.4
(-3.9) (-7.2) (-7.7)
实例C3:核酸酶稳定性;具有序列d(TCC AGG TGT CCG tttC)的不同低聚核苷酸的酶水解。
在37℃,在200ml 10%热灭活的小牛胚胎血清(C=70μg/ml)中培养14μg合成低聚核苷酸和14μg相应天然低聚物。在0.5,1,2,4,6,24和48小时后,加入25μl 9M尿素和三硼酸盐缓冲液(PH7)使15ml反应液骤冷,测量之前-20℃保存;通过聚乙酰胺凝胶电泳使骤冷的反应液分离出来并用含磷量(磷成像方法)测量断裂产物。完整低聚核苷酸浓度(Cnt),和由于从3′未端除去天然C结构单元而形成的片段浓度(Cn-1 t)之和与t=0时完整低聚核苷酸的初始浓度(Ch0)的比值R=(Cn(t)+Cn-1 t)/Cn°对时间作图,设立半衰期τ1/2,即R=0.5时的时间。
Claims (62)
1、式Ⅰ化合物
其中R1和R2互相独立为氢或保护基团,或R1为上述定义而R2为形成含磷核苷酸桥基的残基;
B为嘌呤或嘧啶残基或其类似物,
R3为式Ⅰa、Ⅰb或Ⅰc残基
其中,R4为氢,C1-C21烷基,C2-C21链烯基,C2-C21炔基或-C(=O)-烷基;
R5为氢,C1-C10烷基,-CH2-O-R6或式Ⅰb残基,
R6为氢,C1-C22烷基,C3-C21链烯基,部分或全部氟取代的C1-C10烷基或-[(OH2)-O]m-R7;
R7为氢或C1-C21烷基;
Z为-(CH2)p-或-(CH2-CH-O)q-CH2CH2-,其中-CH2-不取代或被一个或多个相同或不同的C1-C10烷基或C5-C6环烷基,和不取代或C1-C4烷基取代苯基所取代,
n为1-12;
m为1-4;
p为1-10;
q为1-4;
当n=1、R5为氢时,R4不能是氢。
2、权利要求1所述化合物的R1和R2均为氢。
3、权利要求1所述化合物的R1和R2为同一保护基团。
4、权利要求1所述化合物中作为嘌呤基或类似物的B为式Ⅱ、Ⅱa、Ⅱb、Ⅱc、Ⅱd、Ⅱe或Ⅱf残基,
其中,Rb1为H,Cl,Br,OH或-O-C1-C12烷基,
Rb2,Rb3和Rb5为互为独立的H、OH、SH,NH2,NHNH2,NHSH,NHO-C1-C12烷基,-N=CH-N(C1-C12烷基)2,-N=CH-N-环烷基,F,Cl,Br,C1-C12烷基,羟基-C1-C12烷基,氨基-C1-C12烷基,C1-C12烷氧基,苯甲氧基或C1-C12-烷硫基,未取代或被保护基团取代的羟基或氨基;或苯基,苯甲基,C1-C20伯氨基或C2-C30的仲氨基。
Rb4为氢;CH或-C=C-Rb7
Rb6和Rb7为氢或C1-C4烷基。
5、权利要求4所述化合物中,其中羟基和氨基的保护基团为C1-C8酰基。
6、权利要求4所述化合物中,伯氨基含1-12个碳原子和仲氨基含2-12个碳原子。
7、权利要求4所述化合物中,伯和仲氨基为式Ra1Ra2N残基,其中Ra1为氢或独立地有Ra2的定义,Ra2为C1-C20烷基,C1-C20氨基烷基或C1-C20羟基烷基;羧基烷基或烷氧基羰基烷基,其中羧基包含2-8个碳原子,烷基包含1-6,优选1-4个碳原子;C2-C20链烯基;苯基,一或二(C1-C4烷基或C1-C4烷氧基)苯基,苯甲基,一或二(C1-C4烷基或C1-C4烷氧基)苯甲基;或1,2-,1,3-,1,4-咪唑基-C1-C6烷基,或Ra1和Ra2均为四或五一亚甲基,3-恶-1,5-亚戊二烯亚戊基,-CH2-NRa3-CH2CH2-或-CH2CH2-NRa3-CH2CH2-,其中Ra3为氢或C1-4烷基,在氨基烷基中氨基未被取代或被一或二个C1-C4烷基或C1-C4羟烷基取代,羟基烷基中的羟基未取代或被C1-C4烷基醚化。
8、权利要求6所述化合物中的伯和仲氨基为甲基-,乙基-,二甲基-,二乙基-,烯丙基-,一或二(羟基-2-乙基)-,苯基-和苯甲基-,乙酰基-,异丁酰基-和苯甲酰基氨基。
9、权利要求4所述化合物中式Ⅱ、Ⅱb、Ⅱc、Ⅱd、Ⅱe中的Rb1为氢。
10、权利要求4所述化合物中式Ⅱd中的Rb5为氢。
11、权利要求4所述化合物中式Ⅱ,Ⅱa。Ⅱb、Ⅱc、Ⅱd、Ⅱf中的Rb2和Rb3为相互独立的H,F,Cl,Br,OH,SH,NH2,NHOH,NHNH2,甲氨基,二甲氨基,苯甲酰基氨基,甲氧基,乙氧基和甲硫基。
12、权利要求4所述化合物中,B为嘌呤或下列一组腺嘌呤类似物残基,N-甲基腺嘌呤,N-甲酰基腺嘌呤,2-甲硫基腺嘌呤,2-氨基腺嘌呤,6-羟基嘌呤,2-氨基-6-氯嘌呤,2-氨基-6-甲硫基嘌呤,鸟嘌呤和N-异丁酰鸟嘌呤。
13、权利要求1所述化合物中,式Ⅰ中的作为嘧啶的B为式Ⅲ、Ⅲa、Ⅲb、Ⅲc的尿嘧啶,胸腺嘧啶,胞嘧啶残基,
其中Rb6为氢或C1-C4烷基,Rb8为H,OH,SH,NH2,NHNH2,NHOH,NHO-C1-C12烷基,-N=CH-N(C1-C12烷基)2,-N=CH-N-环烷基,F,Cl,Br,C1-C12烷基,羟基-C1-C12烷基,氨基C1-C12烷基,C1-C12烷氧基,苯甲氧基或C1-C12烷硫基,未被取代或被保护基团取代的羟基和氨基,或为苯基,苯甲基,C1-C20的伯氨基,C2-C30的仲氨基,C1-C12链烯基或C1-C12炔基,式Ⅲb中的NH2未被取代或被C1-C6烷基,苯甲酰基,保护基团,式Ⅲ、Ⅲa、Ⅲb、和Ⅲc残基的二氢衍生物所取代。
14、权利要求13所述化合物中Rb8为氢,C1-C6烷基,或C1-C6羟烷基,C2-C6链烯基或C2-C6炔基,F,Cl,Br,NH2,苯甲酰基氨基或一或二C1-C6烷基氨基。
15、权利要求13所述化合物中Rb8为氢,C1-C6烷基或C1-C6烷氧基或C1-C6羟烷基,C2-C6链烯基或C2-C6炔基,F,Cl,Br,NH,苯甲酰基氨基或一或二-C1-C6烷基氨基。
16、权利要求14所述的化合物中Rb8为H,F,Cl,Br,NH2,NHCH3,N(CH3)2,C2-C4烷基,C2-C4链烯基-(1)或C2-C4炔基-(1)。
17、权利要求15所述化合物中Rb8为氢、C1-C4烷基,C2-C4链烯基-(1),C2-C4炔基-(1),NH2,NHCH3或(CH3)2N。
18、权利要求1所述化合物中作为嘧啶类似物残基的B可以下列化合物派生:尿嘧啶,胸腺嘧啶,胞嘧啶,5-氟尿嘧啶,5-氯尿嘧啶,5-溴尿嘧啶,二氢尿嘧啶,5-甲基胞嘧啶,5-丙炔胸腺嘧啶和5-丙炔胞嘧啶。
19、权利要求1所述化合物中R2为与式P1或P2相对应的含磷的构成核苷酸桥基的残基
其中,Ya为氢,C1-C12烷基,C6-C12芳基,C7-C20芳烷基,C7-C20烷基芳基,-ORb,-SRb,-NH2,伯氨基,仲氨基,O-M+或S-M+;
Xa为氧或硫;
Ra为氢,M+,C1-C12烷基,C2-C12链烯基或C6-C12芳基,或RaO-基团如有5元环和1-3个氮原子的N-氮杂芳基-N-基;
R6为氢,C1-C12烷基或C6-C12芳基;
M+为Na+,K+,Li+,NH4 +或伯、仲、叔、季铵;
Ya中的烷基,芳基,芳烷基和烷芳基及Ra和Rb未被取代或被烷氧基,烷硫基,卤素,-CN,-NO2,苯基,硝基苯基或卤代苯基取代。
20、权利要求19所述化合物中Ra为β-氰乙基,Ya为二(异丙基)氨基。
21、权利要求1所述化合物中R3为式Ⅰa残基。
22、权利要求21所述化合物中n为1-8。
23、权利要求22所述化合物中n为1-6。
24、权利要求21所述化合物中R4为氢,C1-C21烷基,C2-C21链烯基或C2-C21炔基。
25、权利要求24中所述化合物中R4为氢或C1-C4烷基。
26、权利要求21所述化合物中R5为氢,C1-C5烷基,-CH2-OH,-CH2-O-C1-C22烷基或-CH2-O-[(CH2)2-O]m-C1-C10烷基,式中m为1-4。
27、权利要求26所述化合物中R5为氢、甲基-CH2OH,-CH2-O-C1-C22烷基或-CH2-O-[(CH2)2-O]m-C1-C10烷基,式中m为1-4。
28、权利要求1所述化合物中,R3为式Ⅰb残基。
29、权利要求28所述化合物中,Z为-CH2-或取代-CH2-。
30、权利要求1所述化合物中,R3为式Ⅰc残基。
31、式Ⅰ化合物的制备方法
其中R1和R2为相互独立的氢或保护基团,或R1有此定义而R2为构成含烯核苷的残基,B为嘌呤基或嘧啶基或其类似物。
(a)R3为式Ⅰa残基
其中,R4为氢,C1-C21烷基,C2-C21链烯基,C2-C21炔基或-C(=O)烷基;
R5为氢,C1-C10烷基,-CH2-O-R6或式Ⅰb残基;R6为氢,C1-C22烷基,C2-C21链烯基,部分或全部氟取代的C1-C10烷基或-[(CH2)2-O]m-R7;
R7为氢或C1-C21烷基;
n为1-12;
m为1-4;
当n=1,R5=氢时,R4不能是氢,
该方法为:
在惰性溶剂中,将式Ⅳa化合物
其中R14和R15为相同的或不同的保护基团,B为嘌呤基或嘧啶基或其类似物,碱基B中功能基团被保护基团保护起来,与式A化合物反应,
其中R4,R5和n为前述定义,X为Cl,Br,I,甲苯磺酰基-O或甲磺酰基-O;
(b)R3为式Ⅰc残基
制备方法为在惰性溶剂中,将式Ⅳa化合物与式B化合物反应
其中X为Cl,Br,I,甲苯磺酰基-O或甲磺酰基-O;
(c)R3为式Ⅰb的残基
其中,R5为氢,C1-C10烷基,-CH2-O-R6或式Ⅰb残基;
R6为氢,C1-C22烷基,C3-C21链烯基,部分或全部氟代C1-C10烷基或-[(CH2)2-O]m-R7;
R7为氢或C1-C21烷基;
Z为-(OH2)p-或-(CH2-CH-O)q-CH2CH2-,Z中的-CH2-未被取代或被一个或多个、相同或不同的C1-C10烷基或C5-C6环烷基,未被取代或C1-C4烷基取代苯基取代;
m为1-4;
p为1-10;
q为1-4;
该方法为在H2O和BF3存在下打开由(b)得到的环氧化物并与式X′-(CH2)q-X′或
X′-(CH2-CH-O)r-X′化合物关环而形成一个新环,其中X′为Cl,Br,I,甲苯磺酸基-O或甲磺酰基-O,
(d)R3为式Ⅰa)残基
该方法为在R6OH和BF3存在下,使由(b)获得的环氧化物反应,将游离羟基转化为醚基或酯基;
(e)R3为式Ⅰa残基
该方法为在BF3和NaBH4存在下,将由(b)获得的环氧化物加氢,将游离羟基转化为醚基或酯基,
(f)R3为式Ⅰa残基,
该方法为将由(b)获得的环氧化物与适量的Grignard试剂反应,从而将游离羟基转化为醚基或酯基;
(g)R3为式Ⅰa、Ⅰb或Ⅰc残基
该方法包括用(a)-(f)所述方法之一取代式Ⅳb化合物的2′-OH
其中R14和R15如前述定义,A为离去基团,通过取代引入碱基B;进而去掉保护基R14和R15,引入构成含磷核苷酸桥基的残基。
32、权利要求1所述化合物在制备低聚核苷酸的应用,该低聚核苷酸含有式Ⅰ化合物相同或不同单体单元或含有式Ⅰ化合物至少一个单体单元与其他天然或合成核苷的单体单元结合,该低聚核苷酸含有2-200个单体单元。
33、权利要求32,其用途在于制备含有2-200单体单元的低聚核苷酸。
34、根据权利要求33,其用途在于制备含有2-50个单体单元的低聚核苷酸。
35、根据权利要求34,其用途在于制备含有4-30个单体单元的低聚核苷酸。
36、根据权利要求32,其用途在于制备含有式Ⅰ化合物相同或不同的单体单元的低聚核苷酸。
37、根据权利要求32,其用途在于制备含有式Ⅰ化合物相同的单体单元和天然或合成核苷的单体单元的低聚核苷酸。
38、式Ⅴ低聚核苷酸
其中X为0-200,Y为核苷酸桥基;U、V和W为相互独立的相同或不同的天然或合成核苷酸残基,至少U,V和/或W之一为式Ⅵ残基
其中B为嘌呤基或嘧啶基或其类似物;R3为式Ⅰa,Ⅰb,Ⅰc残基
其中,R4为氢,C1-C21烷基,C2-C21链烯基,C2-C21炔基,或-C(=O)烷基;
R5为氢,C1-C10烷基,-CH2-O-R6或式Ⅰb残基;
R6为氢,C1-C22烷基,C3-C21链烯基,部分或全部氟代的C1-C10烷基或[(CH2)2-O]m-R7;
R7为氢或C1-C21烷基
Z为-(CH2)p-或-(CH2-CH-O)q-CH2CH2-,Z中的-CH2-未取代或被一个或多个相同或不同的C1-C10烷基或C5-C6环烷基,未被取代或C1-C4烷基取代的苯基取代;
n为1-12;
m为1-4;
p为1-10;
q为1-4。
39、权利要求38所述低聚核苷酸中,桥基Y为-P(O)O-,-P(O)S-,-P(S)S-,-P(O)R16-,-P(O)NR17R18-或-CH2-,R16为氢或C1-C6烷基,R17和R18相互独立地具有R16的定义。
40、权利要求38所述低聚核苷酸中,桥基Y为-P(O)O-。
41、权利要求38所述低聚核苷酸中,X为0-100。
42、权利要求41所述低聚核苷酸中,X为1-50。
43、权利要求42所述低聚核苷酸,X为3-29。
44、权利要求38所述低聚核苷酸中,式Ⅵ残基在未端和/或在核苷酸序列中成键。
45、权利要求38所述低聚核苷酸中,式Ⅵ残基在天然或合成核苷酸残基之间成键。
46、权利要求44和45所述低聚核苷酸中,2-5个相同或不同式Ⅵ残基互相连接。
47、权利要求38所述低聚化合物中含有总共4-30核苷单元和1-12个式Ⅵ残基。
48、权利要求38所述低聚核苷酸中,作为嘌呤基或其类似物的B为式Ⅱ、Ⅱa、Ⅱb、Ⅱc、Ⅱd、Ⅱe或Ⅱf残基
其中,Rb1为H,Cl,Br,OH或-O-C1-C12烷基;
Rb2,Rb3和Rb5为相互独立的H,OH,SH,NH2,NHNH2,NHOH,NHO-C1-C12烷基,-N=CH-N(C1-C12烷基)2,-N=CH-N-环烷基,F,Cl,Br,C1-C12烷基,羟基-C1-C12烷基,氨基-C1-C12烷基,C1-C12烷氧基,苯甲氧基或C1-C12烷硫基;未取代或被保护基团取代的羟基和氨基;苯基,苯甲基,有C1-C20的伯氨基或C2-C30的仲氨基,
Rb4为氢,CN或-C=C-Rb7;
Rb6和Rb7为氢或C1-C4烷基
49、权利要求38所述的低聚核苷酸中,B为式Ⅲ、Ⅲa、II-Ib、Ⅲc的尿嘌嘧啶、胸腺嘧啶或胞嘌啶的残基,
其中Rb6为氢或C1-C4烷基,Rb8为H,OH,SH,NH2,NHNH2,NHOH,NH-C1-C12烷基,-N=CH-N(C1-C12烷基)2,N=CH-N-环烷基,F,Cl,Br,C1-C12烷基,羟基-C1-C12烷基,氨基-C1-C12烷基,C1-C12烷氧基,苯甲氧基或C1-C12烷硫基,未取代或被保护基团取代的羟基和氨基,或为苯基,苯甲基,有C1-C20的伯氨基,C2-C30的仲氨基,C2-C12链烯基或C2-C12炔基,或Ⅲb中的NH2基未被取代或被C1-C6烷基,苯甲酰基,保护基团,式Ⅲ、Ⅲa、Ⅲb、Ⅲc残基的二氢衍生物取代。
50、权利要求38所述的低聚核苷酸中,R3为式Ⅰa残基。
51、权利要求50所述的低聚核苷酸中,n为1-8。
52、权利要求51所述的低聚核苷酸中,n为1-6。
53、权利要求50所述的低聚核苷酸中,R4为氢,C1-C21烷基,C2-C21链烯基或C2-C21炔基。
54、权利要求53所述的低聚核苷酸中,R4为氢或C1-C4烷基。
55、权利要求50所述的低聚核苷酸中,R5为氢,C1-C5烷基或-CH2-OH,-CH2-O-C1-C22烷基或-CH2-O-[(CH2)2-O]m-C1-C10烷基,其中m为1-4。
56、权利要求55所述的低聚核苷酸中,R5为氢、甲基或-CH2-OH,-CH2-O-C1-C22烷基或-CH2-O-[(CH2)2-O]m-C1-C10烷基,其中m为1-4。
57、权利要求38所述的低聚核苷酸中,R3为式Ⅰb残基。
58、权利要求57所述的低聚核苷酸中,Z为-CH2或取代-CH2-。
59、权利要求38所述的低聚核苷酸中,R3为式Ⅰc残基。
60、式Ⅴ的低聚核苷酸作为诊断试剂在检测病毒感染或遗传疾病中的应用。
61、式Ⅴ低聚核苷酸在通过作用于体内核苷酸序列而治疗包括人类在内的热血动物疾病的治疗方法中的应用。
62、只含有有效量的式Ⅰ核苷或式Ⅴ低聚核苷酸或还含有其他活性成分,药物载体和适量赋形剂的药物组合物。
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CH146793 | 1993-05-12 |
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US (1) | US5969116A (zh) |
EP (1) | EP0626387B1 (zh) |
JP (1) | JP4012579B2 (zh) |
CN (1) | CN1100728A (zh) |
AT (1) | ATE177430T1 (zh) |
AU (1) | AU687796B2 (zh) |
CA (1) | CA2123241C (zh) |
DE (1) | DE59407895D1 (zh) |
DK (1) | DK0626387T3 (zh) |
ES (1) | ES2128535T3 (zh) |
IL (3) | IL109605A (zh) |
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CA2123241A1 (en) | 1994-11-13 |
DK0626387T3 (da) | 1999-09-27 |
AU6304494A (en) | 1994-11-17 |
ATE177430T1 (de) | 1999-03-15 |
ES2128535T3 (es) | 1999-05-16 |
IL123330A (en) | 1999-12-31 |
EP0626387B1 (de) | 1999-03-10 |
CA2123241C (en) | 2007-02-13 |
AU687796B2 (en) | 1998-03-05 |
IL109605A0 (en) | 1994-08-26 |
IL109605A (en) | 1999-12-31 |
DE59407895D1 (de) | 1999-04-15 |
JP4012579B2 (ja) | 2007-11-21 |
JPH072889A (ja) | 1995-01-06 |
IL123330A0 (en) | 1998-09-24 |
EP0626387A1 (de) | 1994-11-30 |
US5969116A (en) | 1999-10-19 |
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