CN114763324A - 一种脂质化合物及脂质体与药物组合物 - Google Patents
一种脂质化合物及脂质体与药物组合物 Download PDFInfo
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- CN114763324A CN114763324A CN202110057708.0A CN202110057708A CN114763324A CN 114763324 A CN114763324 A CN 114763324A CN 202110057708 A CN202110057708 A CN 202110057708A CN 114763324 A CN114763324 A CN 114763324A
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- -1 Lipid compound Chemical class 0.000 title claims abstract description 44
- 239000002502 liposome Substances 0.000 title claims abstract description 28
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 14
- 239000003814 drug Substances 0.000 claims abstract description 17
- 229940124597 therapeutic agent Drugs 0.000 claims abstract description 13
- 201000010099 disease Diseases 0.000 claims abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 3
- 125000003636 chemical group Chemical group 0.000 claims abstract description 3
- 108020004459 Small interfering RNA Proteins 0.000 claims description 22
- 229910052739 hydrogen Inorganic materials 0.000 claims description 10
- 239000001257 hydrogen Substances 0.000 claims description 10
- 125000002091 cationic group Chemical group 0.000 claims description 8
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 6
- 150000002632 lipids Chemical class 0.000 claims description 5
- 150000001841 cholesterols Chemical class 0.000 claims description 4
- 108020004999 messenger RNA Proteins 0.000 claims description 4
- 150000003904 phospholipids Chemical class 0.000 claims description 4
- 235000012000 cholesterol Nutrition 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 229910052717 sulfur Inorganic materials 0.000 claims description 3
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 claims description 2
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 claims description 2
- 108091034117 Oligonucleotide Proteins 0.000 claims description 2
- 239000000074 antisense oligonucleotide Substances 0.000 claims description 2
- 238000012230 antisense oligonucleotides Methods 0.000 claims description 2
- 150000001721 carbon Chemical group 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 claims description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 108091070501 miRNA Proteins 0.000 claims description 2
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 2
- 125000004434 sulfur atom Chemical group 0.000 claims description 2
- 230000005856 abnormality Effects 0.000 claims 1
- 230000002068 genetic effect Effects 0.000 claims 1
- 230000030279 gene silencing Effects 0.000 abstract description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 3
- 102000039446 nucleic acids Human genes 0.000 abstract description 3
- 150000007523 nucleic acids Chemical class 0.000 abstract description 3
- 238000012226 gene silencing method Methods 0.000 abstract description 2
- 125000004432 carbon atom Chemical group C* 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 112
- 230000015572 biosynthetic process Effects 0.000 description 90
- 238000003786 synthesis reaction Methods 0.000 description 90
- 239000007858 starting material Substances 0.000 description 76
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 69
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 65
- 238000004809 thin layer chromatography Methods 0.000 description 64
- 238000004440 column chromatography Methods 0.000 description 58
- 238000001514 detection method Methods 0.000 description 52
- 239000000047 product Substances 0.000 description 51
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 42
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 42
- 239000000243 solution Substances 0.000 description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 34
- 239000003921 oil Substances 0.000 description 32
- 239000002994 raw material Substances 0.000 description 32
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 27
- 239000012230 colorless oil Substances 0.000 description 26
- 235000019439 ethyl acetate Nutrition 0.000 description 26
- 239000012074 organic phase Substances 0.000 description 26
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 24
- 150000001875 compounds Chemical class 0.000 description 23
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 22
- 238000005160 1H NMR spectroscopy Methods 0.000 description 21
- 239000002904 solvent Substances 0.000 description 21
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 20
- 239000000203 mixture Substances 0.000 description 20
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 19
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 15
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 14
- 239000000741 silica gel Substances 0.000 description 14
- 229910002027 silica gel Inorganic materials 0.000 description 14
- HJUGFYREWKUQJT-UHFFFAOYSA-N tetrabromomethane Chemical compound BrC(Br)(Br)Br HJUGFYREWKUQJT-UHFFFAOYSA-N 0.000 description 13
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 13
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 12
- 229910052757 nitrogen Inorganic materials 0.000 description 12
- 239000007787 solid Substances 0.000 description 11
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 10
- 238000001914 filtration Methods 0.000 description 10
- 229910000104 sodium hydride Inorganic materials 0.000 description 10
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 10
- 238000003756 stirring Methods 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 239000012043 crude product Substances 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- ZWRUINPWMLAQRD-UHFFFAOYSA-N nonan-1-ol Chemical compound CCCCCCCCCO ZWRUINPWMLAQRD-UHFFFAOYSA-N 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 239000011541 reaction mixture Substances 0.000 description 8
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 7
- 239000011734 sodium Substances 0.000 description 7
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 6
- WTWWTKPAEZQYPW-UHFFFAOYSA-N heptadecan-9-ol Chemical compound CCCCCCCCC(O)CCCCCCCC WTWWTKPAEZQYPW-UHFFFAOYSA-N 0.000 description 6
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 description 6
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 5
- 239000008346 aqueous phase Substances 0.000 description 5
- 230000000717 retained effect Effects 0.000 description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 4
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 4
- AGEZXYOZHKGVCM-UHFFFAOYSA-N benzyl bromide Chemical compound BrCC1=CC=CC=C1 AGEZXYOZHKGVCM-UHFFFAOYSA-N 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000004821 distillation Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000010791 quenching Methods 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- SXCBDZAEHILGLM-UHFFFAOYSA-N heptane-1,7-diol Chemical compound OCCCCCCCO SXCBDZAEHILGLM-UHFFFAOYSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 108091081021 Sense strand Proteins 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- KBPLFHHGFOOTCA-UHFFFAOYSA-N caprylic alcohol Natural products CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- UBTQVPMVWAEGAC-UHFFFAOYSA-N ethyl 8-bromooctanoate Chemical compound CCOC(=O)CCCCCCCBr UBTQVPMVWAEGAC-UHFFFAOYSA-N 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- UKVIEHSSVKSQBA-UHFFFAOYSA-N methane;palladium Chemical compound C.[Pd] UKVIEHSSVKSQBA-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- QDRKDTQENPPHOJ-UHFFFAOYSA-N sodium ethoxide Chemical compound [Na+].CC[O-] QDRKDTQENPPHOJ-UHFFFAOYSA-N 0.000 description 2
- GOQZIPJCBUYLIR-UHFFFAOYSA-N tert-butyl n-[n-[(2-methylpropan-2-yl)oxycarbonyl]-n'-(trifluoromethylsulfonyl)carbamimidoyl]carbamate Chemical compound CC(C)(C)OC(=O)NC(=NS(=O)(=O)C(F)(F)F)NC(=O)OC(C)(C)C GOQZIPJCBUYLIR-UHFFFAOYSA-N 0.000 description 2
- AYMUQTNXKPEMLM-UHFFFAOYSA-N 1-bromononane Chemical compound CCCCCCCCCBr AYMUQTNXKPEMLM-UHFFFAOYSA-N 0.000 description 1
- LDLCZOVUSADOIV-UHFFFAOYSA-N 2-bromoethanol Chemical compound OCCBr LDLCZOVUSADOIV-UHFFFAOYSA-N 0.000 description 1
- FWOHDAGPWDEWIB-UHFFFAOYSA-N 2-bromoethoxymethylbenzene Chemical compound BrCCOCC1=CC=CC=C1 FWOHDAGPWDEWIB-UHFFFAOYSA-N 0.000 description 1
- CUZKCNWZBXLAJX-UHFFFAOYSA-N 2-phenylmethoxyethanol Chemical compound OCCOCC1=CC=CC=C1 CUZKCNWZBXLAJX-UHFFFAOYSA-N 0.000 description 1
- WCFJUSRQHZPVKY-UHFFFAOYSA-N 3-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)NCCC(O)=O WCFJUSRQHZPVKY-UHFFFAOYSA-N 0.000 description 1
- RDTALXUBMCLWBB-UHFFFAOYSA-N 4-(dimethylamino)butanoic acid;hydron;chloride Chemical compound Cl.CN(C)CCCC(O)=O RDTALXUBMCLWBB-UHFFFAOYSA-N 0.000 description 1
- JMLHQRQZCMCQNJ-UHFFFAOYSA-N 4-(methylamino)butanoic acid;hydrochloride Chemical compound Cl.CNCCCC(O)=O JMLHQRQZCMCQNJ-UHFFFAOYSA-N 0.000 description 1
- NVRVNSHHLPQGCU-UHFFFAOYSA-N 6-bromohexanoic acid Chemical compound OC(=O)CCCCCBr NVRVNSHHLPQGCU-UHFFFAOYSA-N 0.000 description 1
- DIUUESGNPCBJKC-UHFFFAOYSA-N 7-bromopentadecane Chemical compound CCCCCCCCC(Br)CCCCCC DIUUESGNPCBJKC-UHFFFAOYSA-N 0.000 description 1
- BKJFDZSBZWHRNH-UHFFFAOYSA-N 8-bromooctanoic acid Chemical compound OC(=O)CCCCCCCBr BKJFDZSBZWHRNH-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- ZSANYRMTSBBUCA-UHFFFAOYSA-N diethyl 3-oxopentanedioate Chemical compound CCOC(=O)CC(=O)CC(=O)OCC ZSANYRMTSBBUCA-UHFFFAOYSA-N 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- PQVSTLUFSYVLTO-UHFFFAOYSA-N ethyl n-ethoxycarbonylcarbamate Chemical compound CCOC(=O)NC(=O)OCC PQVSTLUFSYVLTO-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005861 gene abnormality Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- BNSOYWDFFBDEFB-UHFFFAOYSA-L hydroxy-(hydroxy(dioxo)chromio)oxy-dioxochromium;pyridine Chemical compound C1=CC=NC=C1.O[Cr](=O)(=O)O[Cr](O)(=O)=O BNSOYWDFFBDEFB-UHFFFAOYSA-L 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- GLXDVVHUTZTUQK-UHFFFAOYSA-M lithium hydroxide monohydrate Substances [Li+].O.[OH-] GLXDVVHUTZTUQK-UHFFFAOYSA-M 0.000 description 1
- 229940040692 lithium hydroxide monohydrate Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N n-Octanol Natural products CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- ZVEZMVFBMOOHAT-UHFFFAOYSA-N nonane-1-thiol Chemical compound CCCCCCCCCS ZVEZMVFBMOOHAT-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
- C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
-
- 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
- A61K31/13—Amines
- A61K31/133—Amines having hydroxy groups, e.g. sphingosine
-
- 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
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
- A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
本发明涉及一种脂质化合物,具体的提供了一种具有式(Ⅰ)所示结构的脂质化合物,式(Ⅰ)如下所示:
Description
技术领域
本发明涉及一种脂质化合物,具体的更涉及一种脂质化合物及脂质体与药物组合物。
背景技术
siRNA技术是最广泛应用的生物学技术之一,由于siRNA技术对特定基因mRNA的高度特异性和高效性表达调控,而被广泛的用于功能基因组学、遗传学、基因治疗、病毒性疾病治疗等许多领域。然而,无论在体内还是体外,siRNA都很容易被广泛存在的RNase降解,并且siRNA无法单独穿过细胞膜引发RNAi效应,因此,siRNA技术离不开高效、低毒的siRNA运载体的帮助。常用的siRNA转染运载体有病毒载体、脂质体、高聚物、无机纳米粒子、多肽生物载体等。
当前的siRNA转染运载体多存在包载量低、穿透能力差、毒性大、质量难以控制等缺陷,需要进一步改进并提高相关技术。
发明内容
在符合本领域常识的基础上,上述各优选条件,可任意组合,而不超出本发明的构思与保护范围。
为了解决上述的技术问题,本发明的第一个方面提供了一种具有式(Ⅰ)所示结构的脂质化合物,其特征在于,式(Ⅰ)如下所示:
其中,A为N或CH;X、Y、Z各自独立地选自以碳原子或碳链与A连接的化学基团。
作为一种优选的技术方案,所述的脂质化合物,当A为N时,X为且n1为1~4的整数;Y选自 且n2为2~8的整数,m1为4~12的整数,n3为2~8的整数,m2为4~12的整数,p1为1~5的整数,n4为2~6的整数,m3为4~10的整数,n5为2~6的整数,m4为1~5的整数,p2为3~8的整数,n6为4~8的整数,m5为7~9的整数,R1为氧原子或硫原子;
作为一种优选的技术方案,所述的脂质化合物,式(Ⅰ)所示结构选自:
编号式(Ⅰ)结构
作为一种优选的技术方案,其中所述脂质化合物呈化学上可接受的盐的形式。
作为一种优选的技术方案,其中所述脂质化合物呈阳离子脂质的形式。
本发明的第二个方面提供了一种脂质体,包含所述的脂质化合物。
作为一种优选的技术方案,所述脂质体还包含其它非阳离子形式的脂质化合物。
作为一种优选的技术方案,所述其它非阳离子形式的脂质化合物选自磷脂、胆固醇类衍生物。
作为一种优选的技术方案,所述磷脂选自二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二油酰磷脂酰乙醇胺。
作为一种优选的技术方案,所述胆固醇类衍生物为胆固醇。
作为一种优选的技术方案,所述脂质体还包含脂质缀合物。
作为一种优选的技术方案,所述脂质缀合物选自PEG-二酰基甘油缀合物、PEG-二烷氧基丙基缀合物。
作为一种优选的技术方案,所述脂质体的中值直径为30~150nm;优选的,所述脂质体的中值直径为50~100nm;更优选的,所述脂质体的中值直径为60~90nm。
本发明的第三个方面提供了一种药物组合物,包含所述的脂质化合物和治疗剂。
作为一种优选的技术方案,所述脂质化合物和治疗剂的摩尔比为20:1~200:1;优选的,所述脂质化合物和治疗剂的摩尔比为50:1~150:1;更优选的,所述脂质化合物和治疗剂的摩尔比为100:1。
本发明的第四个方面,提供了一种药物组合物,由所述的脂质体包裹治疗剂组成。
作为一种优选的技术方案,所述治疗剂分别独立的选自siRNA、反义寡核苷酸、微小RNA、mRNA、DNA。
作为一种优选的技术方案,所述药物组合物分别独立的用于治疗基因异常引发的疾病。
本发明相对于现有技术具有如下的显著优点及效果:
本发明提出了用于转染细胞的脂质化合物、脂质体及其在核酸等治疗剂运载和制备药物中的用途以及药物组合物,该脂质化合物可以高效递送核酸分子进入细胞,进入细胞后释放核酸分子,从而发挥核酸分子相应的功能,尤其适用于siRNA,通过RNA干扰达到基因沉默的目的,最终治疗疾病。并且,该脂质化合物毒性低,对细胞损伤小,应用前景好。
具体实施方式
下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。本发明所用试剂和原料均市售可得。下面结合实施例对本发明的技术方案进行详细描述,但并不因此将本发明限制在所述的实施例范围之中。
以下实施例中使用的各种简称的含义解释如下所示:
DCM:二氯甲烷
DCE:1,2-二氯乙烷
MeOH:甲醇
DMF:N,N-二甲基甲酰胺
EtOAc:乙酸乙酯
THF:四氢呋喃
EDCI:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐
DCC:二环己基碳二亚胺
PyBOP:1H-苯并***-1-基氧三吡咯烷基六氟磷酸盐
DMAP:4-二甲氨基吡啶
DIEA:N,N-二异丙基乙胺
TFA:三氟乙酸
PPh3:三苯基膦
CBr4:四溴化碳
NaH:氢化钠
NaOH:氢氧化钠
LiOH·H2O:氢氧化锂一水合物
PDC:重铬酸吡啶
TEA:三乙胺
Pd/C:钯炭
BnBr:溴化苄
Boc2O:二碳酸二叔丁酯
NaBH4:硼氢化钠
TLC:薄层层析色谱法
DOPE:1,2-二油酰-SN-甘油-3-磷酰乙醇胺
实施例1合成化合物LP-C1
(1)LP-Z1-01的合成
将原料8-溴辛酸(8.66g,39.0mmol)溶于DCM(100mL)中,搅拌下依次将9-十七醇(10.00g,39.0mmol),EDCI(11.20g,58.4mmol),DMAP(1.00g,8.2mmol)加入到反应瓶中,常温搅拌反应12h。TLC检测原料消失,有新点生成。将反应液用二氯甲烷和水洗涤2次,保留有机相,有机相用饱和食盐水洗涤一次,分液,保留有机相,用无水硫酸钠干燥有机相,过滤,保留有机相,减压蒸馏浓缩有机相,残留物通过柱层析提纯,得中间体LP-Z1-01(26.24g,浅黄色油状物,收率82%)。
(2)LP-Z1的合成
将中间体LP-Z1-01(26.00g,56.3mmol)溶于无水乙醇(20mL)中,加入2-羟基乙胺(103mL,1.77mol),加热到65℃搅拌反应18h。TLC检测有新点生成。将反应冷却至室温,用乙酸乙酯稀释,有机相用水洗涤2次,饱和食盐水洗涤1次,用无水硫酸钠将有机相干燥,减压蒸馏浓缩,残留物通过柱层析提纯,得中间体LP-Z1(13.0g,浅黄色油状物,收率52%)。MS(ESI):m/z(ESI):m/z[M+H]+822.97。
(3)LP-C1的合成
将中间体LP-Z1-01(26.00g,56.3mmol)溶于无水乙醇(20mL)中,加入2-羟基乙胺(103mL,1.77mol),加热到65℃搅拌反应18h。TLC检测有产物生成。反应完成后将反应冷却至室温,用乙酸乙酯稀释,有机相用水洗涤2次,饱和食盐水洗涤1次,用无水硫酸钠将有机相干燥,减压蒸馏浓缩,残留物通过柱层析提纯,得中间体LP-C1(140mg,浅黄色油状物,收率0.3%)。MS(ESI):m/z(ESI):m/z[M+H]+442.43。
1HNMR(500MHz,CDCl3)δ4.87(m,2H),3.72(br,2H),2.78-2.67(br,6H),2.28(m,4H),1.65-1.26(m,76H),0.88(m,12H)。
实施例2合成化合物LP-C2
LP-C2的合成
将中间体LP-Z1(250mg,0.56mmol)溶于无水乙醇(3mL)中,依次加入原料3-溴丙烷酸辛酯(174mg,0.62mmol)和DIEA(120mg,0.62mmol),加热到65℃反应16h。TLC检测原料剩余,也有新点生成。冷却到室温,减压浓缩除掉溶剂,残留物通过柱层析提纯,得到产物LP-C2(70mg,无色油状物,收率20%)。MS(ESI):m/z(ESI):m/z[M+H]+626.71。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.07(t,2H),3.56(br2H),2.82-2.47(br,8H),2.28(t,2H),1.32-1.26(m,50H),0.88(m,9H)。
实施例3合成化合物LP-C3
LP-C3的合成
将中间体LP-Z1(500mg,1.13mmol)溶于无水乙醇(5mL)中,依次加入原料3-溴丙烷十一烷基酯(382mg,1.24mmol)和DIEA(161mg,1.24mmol),加热到65℃反应16h。TLC检测少量原料剩余,有新点生成。冷却到室温,减压浓缩除掉溶剂,残留物通过柱层析提纯,得到产物LP-C3(410mg,无色油状物,收率54%)。MS(ESI):m/z[M+H]+668.64。
1HNMR(500MHz,CDCl3)δ4.87(m,1H),4.08(t,2H),3.58(br2H),2.84-2.47(br,8H),2.28(t,2H),1.62-1.29(m,56H),0.88(m,9H)。
实施例4合成化合物LP-C4
(1)LP-C4-01的合成
原料LP-Z1(500mg,1.1mmol,1.0eq)溶于无水乙醇(30mL)中,加入2-溴乙醇(560mg,4.4mmol,4.0eq)和DIEA(560mg,4.4mmol,4.0eq),氮气保护下升温到65℃反应16h。TLC检测发现有少量原料剩余,有新点产生。减压浓缩除去溶剂,剩余部分加硅胶拌样,通过柱层析纯化(DCM/MeOH=20:1,加5%氨水),浓缩后得到产品LP-C4-01(230mg,淡黄色油装物,收率42%),MS(ESI):m/z[M+H]+486.34。
(2)LP-C4的合成
原料LP-C4-01(100mg,0.2mmol,1.0eq)溶于DCM(20mL)中,加入正癸酸(35mg,0.2mmol,1.0eq),EDCI(40mg,0.22mmol,1.1eq),DMAP(5mg,0.04mmol,0.2eq)和DIEA(80mg,0.6mmol,3.0eq),氮气保护下室温反应16h。TLC检测原料消失,有新点产生。反应液加DCM(30mL)稀释,有机相用水(2x20mL)洗,饱和食盐水(20mL)洗,无水硫酸钠干燥,浓缩后通过柱层析纯化,得到产品LP-C4(41mg,黄色油状,收率32%),MS(ESI):m/z[M+H]+640.51。
1HNMR(500MHz,CDCl3)δ4.86(q,J=6.5Hz,1H),4.15(s,2H),3.55(s,2H),2.83–2.46(m,6H),2.33-2.26(m,4H),1.65-1.59(m,4H),1.55–1.41(m,6H),1.36–1.18(m,38H),0.88(t,J=6.9Hz,9H)。
实施例5合成化合物LP-C5
(1)LP-C5-01的合成
将原料6-(4-羟基丁氧基)己酸(0.25g,0.9mmol)溶于DCM(5mL)中,冰浴下,依次加入四溴化碳(0.34g,1.0mmol)和三苯基磷(0.36g,1.4mmol)。加完后升至室温反应2h。TLC检测原料消失,有新点生成。将反应液浓缩,残留物通过柱层析纯化,得到产品LP-C5-01(170mg,无色油状物,收率71%)。
(2)LP-C5-02的合成
将原料LP-C5-01(170mg,0.6mmol)溶于DCM(10mL)中,依次加入正辛醇(91mg,0.07mmol),TEA(83mg,0.8mmol))和PyBOP(364mg,0.7mmol,然后室温反应2h。TLC监控反应结束。将反应液减压浓缩,残留物通过柱层析提纯,得中间体LP-C5-02(70mg,无色油状物,收率31%)。
(3)LP-C5的合成
将原料LP-Z1(90mg,0.20mmol)溶于无水乙醇(5mL)中,依次加入中间体LP-C5-02(70mg,0.18mmol)和DIEA(40mg,0.21mmol),然后升温到65℃反应16h。TLC检测有原料剩余也有新点生成。冷却到室温,减压浓缩除掉溶剂,残留物通过柱层析提纯,得到产物LP-C5(37mg,无色油状物,收率27%)。MS(ESI):m/z[M+H]+740.92。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.06(dd,2H),3.90(br,2H),3.42(m,4H),3.01(br,6H),2.30(m,4H),1.6-1.26(m,60H),0.89(m,9H)。
实施例6合成化合物LP-C6
(1)LP-C6-01的合成
将原料1,4-丁二醇(5.0g,55.6mmol,1.0eq)溶于无水DMF(50mL),0℃下向体系中分批缓慢加入NaH(40%inoil)(2.2g,55.6mmol,1.0eq)并维持0℃下反应30min。然后在氮气保护下向体系中缓慢滴加BnBr(9.5g,55.6mmol,1.0eq)。加完后转室温反应16h。TLC检测有少量原料剩余,有新的点生成。加水淬灭反应,水相用EtOAc(200mL)萃取,有机相再用水(3x100mL)洗涤,饱和食盐水(100mL)洗涤,无水硫酸钠干燥,过滤浓缩,柱层析纯化(PE/EtOAc=3:1),得到产品LP-C6-01(7.4g,无色油状,收率74%)。MS(ESI):m/z[M+H]+181.21。
(2)LP-C6-02的合成
原料LP-C6-01(5.0g,27.8mmol,1.0eq)溶于DCM(80mL)中,加入CBr4(13.8g,41.7mmol,1.5eq),然后在0℃下缓慢加入PPh3(12.3g,47.26mmol,1.7eq)。加完后转室温反应1h。TLC检测原料消失,有新的点生成。反应液倒入MTBE中,有固体洗出,过滤除去固体,滤液浓缩后通过柱层析纯化(PE/EtOAc=20:1),得到产品LP-C6-02(6.1g,淡黄色油状,收率91%)。
(3)LP-C6-03的合成
0℃下向1,4-丁二醇(3.7g,41.32mmol,2.0eq)的DMF(70mL)溶液中缓慢加入NaH(1.2g,31mmol,1.5eq),并维持0℃反应30min。然后在氮气保护下,向体系中缓慢滴加LP-C6-02(5.0g,20.66mmol,1.0eq)溶于DMF(10mL)的溶液。加完后转室温反应16h。TLC检测原料消失,有新的点产生。加水淬灭反应,水相用乙酸乙酯(200mL)萃取,有机相再用水(3x100mL)洗涤,饱和食盐水(100mL)洗涤,无水硫酸钠干燥,过滤浓缩,柱层析纯化(PE/EtOAc=3:2),得到产品LP-C6-03(3.4g,淡黄色油状,收率65%),MS(ESI):m/z[M+H]+253.20。
(4)LP-C6-04的合成
原料LP-C6-03(3.4g,13.5mmol,1.0eq)溶于DMF(60mL)中,加入PDC(8.1g,21.6mmol,1.6eq),然后室温反应24h。TLC检测有少量原料剩余,有新点生成。加水淬灭反应,反应液用EtOAc(3x150mL)萃取,合并有机相,无水硫酸钠干燥,过滤浓缩,柱层析纯化(PE/EtOAc=1:1),得到产品LP-C6-04(900mg,淡黄色油状,收率25%)。MS(ESI):m/z[M-H]-265.26。
(5)LP-C6-05的合成
原料LP-C6-04(500mg,1.88mmol,1.0eq)和原料1-壬醇(540mg,3.76mmol,2.0eq)溶于DCM(20mL)中,依次加入EDCI(400mg,2.07mmol,1.1eq),DMAP(46mg,0.38mmol,0.2eq)和DIEA(730mg,5.64mmol,3.0eq),氮气保护下室温反应16h。TLC检测原料消失,有新点产生。反应液加水和DCM,分液,有机相用水洗,Brine洗,无水硫酸钠干燥,过滤浓缩,柱层析纯化(PE/EtOAc=15:1),得到产品LP-C6-05(430mg,无色油状,收率58%),MS(ESI):m/z[M+Na]+415.24。
(6)LP-C6-06的合成
原料LP-C6-05(300mg,0.77mmol,1.0eq)溶于乙酸乙酯(30mL),加入钯碳(60mg,20%wt),氢气置换三次,氢气球保护下室温反应2h。TLC检测原料消失,有新点产生。滤膜过滤除去不溶催化剂。滤液浓缩后得到产品LP-C6-06(210mg,淡灰色油状,收率91%),MS(ESI):m/z[M+Na]+325.14。
(7)LP-C6-07的合成
原料LP-C6-06(210mg,0.7mmol,1.0eq)溶于DCM(20mL),加入CBr4(350mg,1.05mmol,1.5eq),然后分批向体系中加入PPh3(310mg,1.19mmol,1.7eq),加完后室温反应1h。TLC检测原料消失,有新点产生。反应液直接加硅胶拌样,通过柱层析纯化(PE/EtOAc=20:1),得到产品LP-C6-07(245mg,无色油状,收率96%),MS(ESI):m/z[M+Na]+387.03。
(8)LP-C6的合成
原料LP-C6-07(190mg,0.52mmol)和原料LP-Z1(230mg,0.52mmol)溶于乙醇(20mL)中,加入DIEA(200mg,1.56mmol)升温到65℃反应16h。TLC检测有原料剩余,但是有主要新点产生。MS(ESI):m/z检测有产物,也有原料。减压浓缩除去溶剂,剩余部分用DCM稀释,有机相用水洗两次,饱和食盐水洗,无水硫酸钠干燥,过滤浓缩,通过柱层析纯化后得到产品LP-C6(65mg,黄色油状,收率16%)。MS(ESI):m/z[M+H]+726.66。
1HNMR(400MHz,CDCl3)δ4.85(d,J=8.5Hz,1H),4.04(t,J=7.5Hz,2H),3.65(br,1H),3.44–3.37(m,4H),2.68(br,4H),2.37(t,J=9.5Hz,2H),2.26(t,J=9.5Hz,2H),1.88(d,J=7.5Hz,2H),1.67–1.44(m,14H),1.30-1.24(m,46H),0.89-0.84(s,9H)。
实施例7合成化合物LP-C7
(1)LP-C7-01的合成
0℃下,原料6-溴己酸(2.0g,13.2mmol)溶于DMF(60mL)中,加入NaH(1.1g,27.7mmol)室温反应30min。然后向体系中加入2-苄氧基乙醇(2.6g,13.2mmol),氮气保护下室温反应过夜。在升温到50℃反应3h。TLC检测有新点产生。反应液用EtOAc稀释,加水淬灭,分液,水相用稀盐酸调节pH=2~3,水相用DCM萃取三次,合并有机相,无水硫酸钠干燥,过滤,浓缩,柱层析纯化后,得到产品LP-C7-01(840mg,黄色油状,收率24%)。
(2)LP-C7-02的合成
原料LP-C7-01(840mg,3.16mmol)和原料1-壬醇(500mg,3.48mmol)溶于DCM(30mL)中,依次加入EDCI(668mg,3.48mmol),DIEA(1.2g,9.48mmol)和DMAP(77mg,0.63mmol),氮气保护下室温反应16h。TLC检测有新点生成,反应液直接加入硅胶拌样,通过柱层析纯化,得到产品LP-C7-02(780mg,淡黄色油状,收率63%)。
(3)LP-C7-03的合成
原料LP-C7-02(370mg,0.94mmol)溶于乙酸乙酯(20mL)中,加入钯碳(74mg,20%wt),氢气置换三次,氢气球保护下室温反应3h。TLC检测原料消失,有新点产生。滤膜过滤,除去钯碳和不溶物,滤液浓缩后得到产品LP-C7-03(285mg,无色油状,定量),直接用于下步反应。
(4)LP-C7-04的合成
原料LP-C7-03(285mg,0.94mmol)和CBr4(1.09g,3.29mmol)溶于DCM中,0℃下,分批向体系中加入PPh3(616mg,2.35mmol),然后转室温反应1h。TLC检测原料消失,有新点产生。直接向反应液中加入硅胶拌样,通过柱层析纯化,得到产品LP-C7-04(310mg,无色油状,收率91%)。
(5)LP-C7的合成
原料LP-C7-04(150mg,0.41mmol)和原料LP-Z1(182mg,0.45mmol)溶于无水乙醇(10mL)中,加入DIEA(58mg,0.45mmol)升温到65℃反应16h。TLC检测少量原料剩余,主要是新点产生。反应液直接加入硅胶拌样,通过柱层析纯化,得到产品LP-C7(70mg,黄色油状,收率24%)。MS(ESI):m/z[M+H]+726.78。
1HNMR(500MHz,CDCl3)δ4.85(q,J=6.5Hz,1H),4.45(br,1H),4.05(t,J=6.5Hz,2H),4.03-3.95(m,4H),3.48(t,J=6.5Hz,2H),3.37-3.17(m,6H),2.29(dt,J=11.5,7.5Hz,4H),1.93-1.85(m,2H),1.72–1.45(m,14H),1.43–1.18(m,40H),0.90-0.87(m,9H)。
实施例8合成化合物LP-C8
(1)LP-C8-01的合成
将原料1,4-丁二醇(3.14g,34.9mmol)溶于DMF(50mL)中,冰水浴条件下,加入NaH(0.7g,17.5mmol),搅拌反应0.5h后,将((2-溴乙氧基)甲基)苯(2.5g,11.6mmol)加入到反应瓶中,升温至室温反应16h。TLC监控反应结束,加入水淬灭反应,并用100mL乙酸乙酯萃取反应液,有机相用饱和食盐水洗涤3次,保留有机相,无水硫酸钠干燥有机相,将有机相减压蒸馏浓缩,残留物通过柱层析提纯,得到产品LP-C8-01(1.9g,无色油状物,收率73%)。
(2)LP-C8-02的合成
将原料LP-C8-01(0.4g,1.8mmol)溶于DMF(15mL)中,搅拌条件下加入PDC(1.0g,2.6mmol),室温反应16h。TLC监控反应结束,加水淬灭反应,将反应液用乙酸乙酯萃取,合并有机相用饱和食盐水洗涤1次,无水硫酸钠干燥,减压蒸馏浓缩,残留物通过柱层析提纯,得到产品LP-C8-02(360mg,无色油状物,收率84%)。
(3)LP-C8-03的合成
将原料LP-C8-02(360mg,1.5mmol)溶于DCM(10mL)中,依次加入正壬醇(280mg,1.9mmol),TEA(197mg,2.0mmol)和PyBOP(858mg,1.6mmol)。然后室温反应2h。TLC监控反应结束。将反应液浓缩,残留物通过柱层析提纯,得到产品LP-C8-03(80mg,无色油状物,收率15%)。
(4)LP-C8-04的合成
将原料LP-C8-03(76mg,0.21mmol)溶于乙酸乙酯(10mL),加入Pd/C(40mg,20%wt),氢气置换三次,氢气球保护下室温反应2h。TLC检测反应结束。硅藻土过滤除去钯碳,滤液浓缩后得到产品LP-C8-04(24mg,浅灰色油状物,收率42%)。
(5)LP-C8-05的合成
将原料LP-C8-04(24mg,0.09mmol)溶于DCM(10mL)中,然后依次加入四溴化碳(44mg,0.13mmol)和三苯基磷(34mg,0.13mmol)。加完后室温反应2h。TLC检测反应结束。直接向反应液中加入硅胶拌样,柱层析纯化,得到产品LP-C8-05(29mg,无色油状物,收率99%)。
(6)LP-C8的合成
将原料LP-C8-05(29mg,0.09mmol)和LP-Z1(49mg,0.11mmol)溶于无水乙醇(5mL)中,然后加入DIEA(19mg,0.10mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,得到产品LP-C8(27mg,淡黄色油状物,收率42%)。MS(ESI):m/z[M+H]+698.61。
1HNMR(500MHz,CDCl3)δ4.84(m,1H),4.04(dd,2H),3.51(br,2H),3.45(t,2H),2.60(br,4H),2.37(t,4H),2.25(t,2H),1.88(m,2H),1.65-1.26(m,54H),0.86(m,9H)。
实施例9合成化合物LP-C9
(1)LP-C9的合成
将原料LP-C9-01(200mg,0.55mmol)和LP-Z1(266mg,0.60mmol)溶于无水乙醇(5mL)中,然后加入DIEA(116mg,0.60mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,得到产品LP-C9(47mg,淡黄色油状物,收率12%)。MS(ESI):m/z[M+H]+726.66。
1HNMR(500MHz,CDCl3)δ4.87(m,1H),4.05(dd,2H),3.65(br,2H),3.41(m,4H),2.63(br,6H),2.29(m,4H),1.66-1.29(m,58H),0.89(m,9H)。
实施例10合成化合物LP-C10
(1)LP-C10的合成
将原料LP-C10-01(154mg,0.50mmol)和LP-Z1(200mg,0.45mmol)溶于无水乙醇(5mL)中,然后加入DIEA(96mg,0.50mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,得到产品LP-C10(76mg,淡黄色油状物,收率24%)。MS(ESI):m/z[M+H]+698.83。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.09(t,2H),3.70(br,2H),3.41(m,4H),2.70(br,6H),2.30(m,4H),1.63-1.26(m,54H),0.89(m,9H)。
实施例11合成化合物LP-C11
(1)LP-C11的合成
将原料LP-C11-01(450mg,1.34mmol)和LP-Z1(531mg,1.20mmol)溶于无水乙醇(5mL)中,然后加入DIEA(282mg,1.47mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,得到产品LP-C11(340mg,淡黄色油状物,收率40%)。MS(ESI):m/z[M+H]+698.66。
1HNMR(500MHz,CDCl3)δ4.87(m,1H),4.10(t,2H),3.55(br,2H),3.41(m,4H),2.70(br,6H),2.30(m,4H),1.63-1.26(m,54H),0.89(m,9H)。
实施例12合成化合物LP-C12
(1)LP-C12的合成
将原料LP-C12-01(363mg,0.99mmol)和LP-Z1(400mg,0.90mmol))溶于无水乙醇(5mL)中,然后加入DIEA(191mg,1.00mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,得到产品LP-C12(253mg,淡黄色油状物,收率38%)。MS(ESI):m/z[M+H]+726.77。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.06(t,2H),3.97(br,2H),3.40(m,4H),3.07(br,6H),2.30(m,4H),1.63-1.26(m,58H),0.89(m,9H)。
实施例13合成化合物LP-C13
(1)LP-C13的合成
将原料LP-C13-01(273mg,0.75mmol)和LP-Z1(300mg,0.68mmol)溶于无水乙醇(5mL)中,然后加入DIEA(143mg,0.74mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,得到产品LP-C13(200mg,淡黄色油状物,收率40%)。MS(ESI):m/z[M+H]+726.77。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.22(m,2H),3.95(br,2H),3.62(m,4H),3.46(t,2H),3.04(br,6H),2.37(t,2H),2.28(t,2H),1.63-1.26(m,56H),0.88(m,9H)。
实施例14合成化合物LP-C14
(1)LP-C14的合成
将原料LP-C14-01(167mg,0.50mmol)和LP-Z1(200mg,0.45mmol)溶于无水乙醇(5mL)中,然后加入DIEA(96mg,0.50mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,得到产品LP-C14(100mg,淡黄色油状物,收率30%)。MS(ESI):m/z[M+H]+726.77。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.22(m,2H),3.91(br,2H),3.62(m,4H),3.46(t,2H),2.97(br,6H),2.34(t,2H),2.28(t,2H),1.70-1.26(m,54H),0.88(m,9H)。
实施例15合成化合物LP-C15
(1)LP-C15的合成
将原料LP-C15-01(660mg,1.81mmol)和LP-Z1(721mg,1.63mmol)溶于无水乙醇(5mL)中,然后加入DIEA(381mg,1.98mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,获得产品LP-C15(390mg,淡黄色油状物,收率33%)。MS(ESI):m/z[M+H]+726.75。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.09(t,2H),3.94(br,2H),3.41(m,4H),3.03(br,6H),2.30(dt,4H),1.72-1.26(m,54H),0.88(m,9H)。
实施例16合成化合物LP-C16
(1)LP-C16-01的合成
0℃下,原料1,7-庚二醇(3.8g,29.1mmol)溶于DMF(70mL)中,加入NaH(580mg,14.6mmol)反应30min。然后加入1-溴壬烷(2.0g,9.7mmol),转室温反应16h。TLC检测有新点产生。反应液用EtOAc稀释,加水淬灭,有机相用水洗三次,饱和食盐水洗一次,无水硫酸钠干燥,过滤浓缩,柱层析纯化,获得产品LP-C6-01(1.5g,淡黄色油状,收率60%)。
(2)LP-C16-02的合成
原料LP-C6-01(600mg,2.3mmol)和CBr4(2.7g,8.1mmol)溶于DCM(20mL)中,分批缓慢加入PPh3(1.5g,5.75mmol),加完后室温反应1h。TLC检测原料消失,有新点产生。反应液直接加入硅胶拌样,柱层析纯化,获得产品LP-C6-02(680mg,无色油状,收率91%)。
(3)LP-C16的合成
原料LP-C6-02(200mg,0.625mmol)和原料LP-Z1(304mg,0.69mmol)溶于乙醇(20mL)中,加入DIEA(89mg,0.69mmol),升温到65℃反应16h。TLC检测有少量原料剩余,也有新点产生。反应液直接加入硅胶拌样,柱层析纯化,得到产品LP-C6(40mg,黄色油状,收率10%)。MS(ESI):m/z[M+H]+682.68。
1HNMR(500MHz,CDCl3)δ4.87(t,J=6.5Hz,1H),3.71(s,2H),3.39(t,J=6.5Hz,4H),2.71(s,6H),2.28(t,J=7.5Hz,2H),1.71–1.46(m,14H),1.39–1.19(m,44H),0.88(t,J=7.0Hz,9H)。
实施例17合成化合物LP-C17
(1)LP-C17的合成
将原料LP-C17-01(218mg,0.75mmol)和LP-Z1(300mg,0.68mmol)溶于无水乙醇(5mL)中,然后加入DIEA(143mg,0.74mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,得到产品LP-C17(101mg,淡黄色油状物,收率23%)。MS(ESI):m/z[M+H]+655.15。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),3.88(br,2H),3.40(dt.,4H),2.92(br,6H),2.28(t,2H),1.60-1.20(m,58H),0.88(m,9H)。
实施例18合成化合物LP-C18
(1)LP-C18的合成
将原料LP-C18-01(28mg,0.08mmol)和LP-C19-03(30mg,0.07mmol)溶于无水乙醇(5mL)中,然后加入DIEA(143mg,0.74mmol)。加热到65℃反应16h。TLC检测反应结束。冷却到室温,减压浓缩除掉溶剂,剩余部分通过柱层析纯化,得到产品LP-C18(17mg,淡黄色油状物,收率34%)。MS(ESI):m/z[M+H]+682.75。
1HNMR(500MHz,CDCl3)δ4.85(m,1H),4.04(t,2H),3.45(br,5H),2.60(br,4H),2.37(t,2H),2.26(t,2H),1.88(m,2H),1.70-1.26(m,60H),0.88(m,9H)。
实施例19合成化合物LP-C19
(1)LP-C19-01的合成
将原料1,7-庚二醇(3.74g,28.3mmol)溶于DMF(50mL)中,冰水浴下加入NaH(0.95g,23.8mmol),搅拌反应1h。然后加入9-溴十七烷(890mg,2.8mmol),升至室温反应16h。TLC监控反应结束。加水淬灭反应,水相用乙酸乙酯萃取,合并有机相用饱和食盐水洗涤,无水硫酸钠干燥,过滤,浓缩,柱层析纯化,得到产品LP-C19-01(251mg,无色油状物,收率25%)。
(2)LP-C19-02的合成
将原料LP-C19-01(250mg,0.7mmol)溶于DCM(5mL)中,然后搅拌条件下依次加入四溴化碳(0.34g,1.0mmol)和三苯基磷(0.27g,1.0mmol),室温搅拌反应1h。TLC检测反应结束。将反应液浓缩,残留物通过柱层析提纯,得到产品LP-C19-02(31mg,无色油状物,收率11%)。
(3)LP-C19-03的合成
将原料LP-C19-02(31mg,0.07mmol)溶于无水乙醇(5mL)中,加入2-氨基乙醇(436mg,7.2mmol),然后加热至62℃搅拌反应16h。TLC监控反应结束。将反应液冷却至室温,加乙酸乙酯稀释,并用水洗涤3次,分液,保留有机相,用无水硫酸钠干燥,浓缩后得到产物LP-C19-03(31mg,淡黄色油状物,粗品)直接用于下步反应。
(4)LP-C19的合成
将原料LP-C19-03(31mg,0.08mmol)溶于无水乙醇(5mL)中,加入原料LP-C19-01(36mg,0.11mmol)和DIEA(17mg,0.09mmol),然后升温到65℃反应16h。TLC检测反应结束。冷却到室温,直接向反应液中加入硅胶拌样,然后通过柱层析纯化,得到产品LP-C19(20mg,棕黄色油状物,收率19%)。MS(ESI):m/z[M+H]+654.58。
1HNMR(500MHz,CDCl3)δ3.79(s,2H),3.39(t,2H),3.18(p,1H),2.84(d,6H),1.70-1.26(m,60H),0.88(m,9H)。
实施例20合成化合物LP-C20
(1)LP-C20-01的合成
原料1,7-庚二醇(2.0g,15.15mmol)溶于DMF(20mL)中,加入NaH(606mg,15.15mmol)室温反应20min。然后在氮气保护下向体系中缓慢加入BnBr(2.59g,15.15mmol)。加完后维持室温反应16h。TLC检测原料消失,有新点产生。反应液用EtOAc稀释,加水淬灭,有机相用水洗三次,饱和食盐水洗一次,无水硫酸钠干燥,过滤,浓缩后通过柱层析纯化,得到产品LP-C20-01(1.2g,无色油状,收率36%)。MS(ESI):m/z[M+H]+222.82。
(2)LP-C20-02的合成
原料LP-C20-01(1.2g,5.4mmol)和CBr4(2.7g,8.1mmol)溶于DCM(20mL)中,分批缓慢加入PPh3(2.4g,9.18mmol),加完后室温反应1h。TLC检测原料消失,有新点产生。反应液直接加入硅胶拌样,通过柱层析纯化,得到产品LP-C20-02(1.6g,无色油状,收率99%)。MS(ESI):m/z[M+K]+322.94。
(3)LP-C20-03的合成
原料1-壬硫醇(450mg,2.8mmol)溶于DMF(10mL)中,加入NaH(115mg,2.8mmol)室温反应30min。然后加入LP-C20-02(800mg,2.8mmol),氮气保护下升温到70℃反应16h。TLC显示显示原料基本消失,有新点产生。反应液用EtOAc稀释,加水淬灭,有机相用水洗三次,饱和食盐水洗一次,无水硫酸钠干燥,过滤浓缩,柱层析纯化,得到产品LP-C20-03(750mg,无色油状,收率73%)。MS(ESI):m/z[M+H]+365.40。
(4)LP-C20-04的合成
原料LP-C20-03(750mg,2.06mmol)溶于甲醇(20mL)中,加入钯碳(750mg,100%wt),氢气置换三次,氢气球保护下,加热到50℃反应16h。TLC检测有原料剩余,主要是新点产生。硅藻土过滤除去钯碳,滤液浓缩后通过柱层析纯化,得到产品LP-C20-04(170mg,白色固体,收率30%)。MS(ESI):m/z[M+H]+275.36。
(5)LP-C20-05的合成
原料LP-C20-04(170mg,0.62mmol)和CBr4(309mg,0.93mmol)溶于DCM(10mL),分批缓慢加入PPh3(276mg,1.05mmol),加完后室温反应1h。TLC检测原料消失,有新点产生。直接向反应液中加入硅胶拌样,通过柱层析纯化,得到产品LP-C20-05(190mg,无色油状,收率91%)。
(6)LP-C20的合成
原料LP-Z1(249mg,0.565mmol)和原料LP-C20-05(190mg,0.565mmol)溶于无水乙醇(10mL)中,加入DIEA(109mg,0.85mmol),升温到70℃反应过夜。TLC检测有部分原料剩余,也有新点产生。直接向反应液中加入硅胶拌样。通过柱层析纯化,得到产品LP-C20(79mg,黄色油状,收率20%)。MS(ESI):m/z[M+H]+698.60。
1HNMR(500MHz,CDCl3)δ4.86(t,J=6.5Hz,1H),3.74(s,2H),2.84(s,2H),2.72(s,4H)2.50(td,J=7.5,2.0Hz,4H),2.28(t,J=7.5Hz,2H),1.74–1.46(m,14H),1.43–1.18(m,44H),0.92–0.84(m,9H)。
实施例21合成化合物LP-C21
(1)LP-C21-01的合成
原料4-(甲基氨基)丁酸盐酸盐(2.0g,13mmol)溶于THF(30mL),加入NaOH(1.56g,39mmol)溶于水(10mL)的溶液和Boc2O(5.7g,26mmol),然后在氮气保护下加热到50℃反应16h。冷却到室温,减压浓缩除去THF,反应液用乙酸乙酯稀释,用稀盐酸调节pH=3-4,分液,有机相用水洗一次,饱和食盐水洗一次,无水硫酸钠干燥,过滤浓缩,通过柱层析纯化,得到产品LP-C21-01(1.8g,无色油状,收率64%)。
(2)LP-C21-02的合成
原料LP-C27-06(260mg,0.38mmol)和原料LP-C21-01(249mg,1.14mmol)溶于DCE(20mL)中,加入DCC(313mg,1.52mmol),TEA(192mg,1.9mmol)和DMAP(12mg,0.1mmol),然后升温到50℃反应16h。TLC检测有原料剩余,有新点产生,补加和延长时间没有进展。反应液直接拌样,通过柱层析纯化,得到产品LP-C21-02(135mg,无色油状,收率40%)。MS(ESI):m/z[M-Boc]+780.69和MS(ESI):m/z[M+NH4 +]+897.88(加氨)
(3)LP-C21-03的合成
原料LP-C21-02(120mg,0.14mmol)溶于DCM(10mL)中,加入TFA(2mL),室温反应2h。TLC检测反应结束。减压浓缩除去溶剂和TFA,油泵抽干,得到产品LP-C21-03(180mg,TFAsalt,定量),MS(ESI):m/z[M+H]+780.63。直接用于下步反应。
(4)LP-C21-04的合成
原料LP-C21-03(180mg,crudeTFAsalt)溶于DCM(20mL)中,加入TEA(282mg,2.8mmol)和1,3-二-Boc-2-(三氟甲基磺酰)胍(328mg,0.84mmol),室温反应48h。MS(ESI):m/z检测有少量原料剩余,主要是产物生成。反应液直接拌样,通过柱层析纯化,得到产品LP-C21-04(120mg,粗品,黄色油状,收率84%),MS(ESI):m/z[M+H]+1022.89。粗品直接用于下步反应。
(5)LP-C21的合成
原料LP-C21-04(120mg,粗品,0.12mmol)溶于DCM(10mL)中,加入TFA(5mL),室温反应16h。TLC检测有新点产生。减压浓缩除去溶剂和过量的TFA,然后通过柱层析纯化,得到产品LP-C21(13mg,黄色油状,收率14%)。MS(ESI):m/z[M+H]+822.75。
实施例22合成化合物LP-C22
(1)LP-C22-01的合成
原料LP-C27-06(300mg,0.44mmol)和原料Boc-β-丙氨酸(166mg,0.88mmol)溶于DCE(20mL)中,加入DCC(227mg,1.1mmol),TEA(222mg,2.2mmol)和DMAP(11mg,0.09mmol),氮气保护下升温到50℃反应16h。TLC检测有原料剩余,也有新点产生。冷却到室温后直接相反应液中加入硅胶拌样,通过柱层析纯化得LP-C22-01(185mg,粗品,无色油状,收率49%),MS(ESI):m/z[M-Boc]+752.66。直接用于下步反应。
(2)LP-C22-02的合成
原料LP-C22-01(185mg,0.22mmol)溶于DCM(10mL)中,加入TFA(3mL),室温反应1h。TLC检测反应结束。减压浓缩除去溶剂和TFA,剩余部分用DCM带两次,进一步除去TFA,油泵抽干得到产品LP-C22-02(260mg,crudeTFAsalt),直接用于下步反应。
(3)LP-C22-03的合成
原料LP-C22-02(260mg)溶于DCM(10mL)中,加入过量的TEA(1mL)和原料1,3-二-BOC-2-(三氟甲基磺酰)胍(95mg,0.242mmol),氮气保护下室温反应16h。MS(ESI):m/z检测有产物生成。反应液直接拌样,通过柱层析纯化,得到产品LP-C22-03(156mg,粗品,无色油状,收率72%)。MS(ESI):m/z[M+H]+752.66(原料)和MS(ESI):m/z[M+H]+995.14(产品),TLC显示一个点。粗品直接用于下步反应。
(4)LP-C22的合成
原料LP-C22-03(156mg,crude,0.16mmol)溶于DCM(10mL)中,加入TFA(4mL),室温反应16h。MS(ESI):m/z检测反应结束。减压浓缩除去溶剂和TFA,剩余部分用乙腈带两次,进一步去除TFA,然后通过柱层析纯化,得到产品LP-C22(40mg,无色油状,收率23%),MS(ESI):m/z[M+H]+794.71。
实施例23合成化合物LP-C27
(1)LP-C27-01的合成
向乙醇钠(35.7g,0.21mol)溶于无水乙醇(60mL)的溶液中加入原料1,3-丙酮二羧酸二乙酯(21.3g,0.105mol),升温到回流状态,缓慢加入8-溴辛酸乙酯(25g,0.105mol),回流反应2h。再次向反应体系中加入乙醇钠(35.7g,0.105mol)并再次缓慢加入8-溴辛酸乙酯(25g,0.105mol),加完后回流反应16h。减压浓缩除去溶剂,剩余部分加EtOAc稀释,有机相用饱和氯化铵溶液洗两次,饱和食盐水洗一次,无水硫酸钠干燥,过滤,浓缩得到产品LP-C27-01(58g,粗品,黄色油状,定量)。粗品直接用于下步反应。
(2)LP-C27-02的合成
原料LP-C27-01(58g,粗品,0.11mol)溶于浓盐酸/冰醋酸(110mL/60mL)中,加热至回流反应24h。MS(ESI):m/z检测有产物生成。冷却到室温,有固体析出,减压浓缩除去盐酸和醋酸,所得固体用水洗两次,然后减压浓缩除去多余水分,剩余部分用丙酮重结晶,得到产品LP-C27-02(8.3g,白色固体,收率24%),MS(ESI):m/z[M-H]-313.18。
(3)LP-C27-03的合成
原料LP-C27-02(10.2g,32.48mmol)和原料1-壬醇(10.3g,71.46mmol)溶于DCM(260mL)中,依次加入DCC(26.7g,129.92mmol)和DMAP(11.9g,97.44mmol),然后室温反应16h。TLC检测原料消失,有新点产生。过滤反应液除去不溶固体,滤液浓缩后,通过柱层析纯化,所得粗品再用PE/EtOAc打浆,得到产品LP-C27-03(9.4g,白色固体,收率51%),MS(ESI):m/z[M+Na]+589.36。
(4)LP-C27-04的合成
原料LP-C27-03(15.5g,27.39mmol)溶于THF(150mL)中,加入LiOH·H2O(1.38g,32.87mmol)溶于水(30mL)的溶液,然后室温反应过夜。TLC检测有原料剩余,有产物生成,也有双水解的产物。减压浓缩除去溶剂,剩余部分加水稀释,用稀盐酸调节pH=2-3,谁想用DCM萃取四次,合并有机相,无水硫酸钠干燥,过滤浓缩,柱层析纯化,回收原料再次水解,纯化,最终得到产品LP-C27-04(2.6g,白色固体,收率22%)。MS(ESI):m/z[M+Na]+463.22。
(5)LP-C27-05的合成
原料LP-C27-04(2.6g,5.9mmol)和原料9-十七醇(1.8g,7.08mmol)溶于DCM(100mL)中,加入DCC(2.43g,11.8mmol)和DMAP(1.1g,8.85mmol)。然后室温反应16h。TLC检测原料消失,有新点生成。过滤除去不溶固体,滤液浓缩后,通过柱层析纯化,得到产品LP-C27-05(4.47g,粗品,淡黄色固体,主要杂质是原料9-十七醇),粗品直接用于下步反应,MS(ESI):m/z[M+Na]+701.64。
(6)LP-C27-06的合成
原料LP-C27-05(4.47g,粗品,6.59mmol)溶于MeOH(40mL)中,分批缓慢加入NaBH4(376mg,9.9mmol),加完后室温反应30min。TLC检测原料消失,有新点产生。加少量水淬灭反应,反应液直接用无水硫酸钠干燥,过滤,滤液浓缩后,通过柱层析纯化,得到产品LP-C27-06(2.5g,浅黄色油状物,收率62%),MS(ESI):m/z[M+Na]+703.59。
(7)LP-C27的合成
原料LP-C27-06(100mg,0.15mmol)和4-二甲基氨基丁酸盐酸盐(50mg,0.3mmol)溶于DCM(15mL)中,依次加入DCC(68mg,0.33mmol),TEA(76mg,0.75mmol)和DMAP(4mg,0.03mmol),氮气保护下室温反应16h,在升温到40℃反应3h。TLC检测原料基本消失,有新点产生。直接向反应液中加入硅胶拌样,通过柱层析纯化,得到产品LP-C27(40mg,黄色油状,收率34%),MS(ESI):m/z[M+H]+794.60。
1HNMR(400MHz,CDCl3)δ4.84(t,J=6.4Hz,2H),4.08–4.00(m,2H),2.45–2.16(m,15H),1.90–1.73(m,4H),1.58(d,J=8.4Hz,7H),1.48(s,9H),1.25(d,J=9.1Hz,62H),0.87(d,J=6.0Hz,9H).
实施例23制备药物组合物:脂质体包载了siRNA治疗剂组成。
(1)溶液配制
以15%乙醇为介质,采用标准方法配制浓度为20mM、pH4.0的醋酸盐缓冲液;浓度为10mM的胆固醇乙醇溶液(A);浓度为10mM的DOPE乙醇溶液(B);浓度为10mM的DMG-PEG2000的乙醇溶液(C);配比为溶液(A)/溶液(B)/溶液(C)=38.5/10/1.5(v:v:v)的Mix溶液;浓度为10mM的阳离子脂质化合物溶液;浓度为1OD/100μL的siRNA溶液。
其中,1OD/100μL表示100μL溶液中含有1OD的siRNA,1OD的siRNA约为33μg。
(2)形成包载了siRNA治疗剂的脂质体
以阳离子脂质体包载siRNA遵循以下步骤,
步骤S1:15μL阳离子脂质化合物溶液与15μLmix溶液混合均匀;
步骤S2:将步骤S1混合物用乙醇稀释至1.12mg/mL;
步骤S3:取150μL步骤S2溶液加入到450μLpH4.0的醋酸盐缓冲液中,混合均匀;
步骤S4:将0.2ODsiRNA加入300μL步骤S3混合物中;
步骤S5:35度孵育30min;
步骤S6:加180μL1×PBS稀释至0.5mL。
其中制备药物组合物:所述脂质化合物选自式(Ⅰ)结构的LP-C2~LP-C20,分别由实施例1-实施例22制备得到。
实施例24脂质体制剂的体外基因沉默效力评价
本实施例比较了本发明的阳离子脂质体siRNA制剂在体外NSFB细胞(人皮肤成纤维细胞)模型中的有效性。
NSFB细胞模型在转染前24h用汇合度80%~90%的细胞铺板,铺板细胞数为100000cells/孔。转染时实验组用1×PBS稀释阳离子脂质体siRNA制剂至转染终浓度为10nM和15nM,同时设置siRNA为scramblesiRNA(正义链:5’-UUCUCCGAACGUGUCACGUTT-3’,反义链:5’-ACGUGACACGUUCGGAGAATT-3’)阴性对照组和不加脂质体siRNA制剂的空白对照组,实验组的siRNA(正义链:5’-GACAUCAAGAAGGUGGUGATT-3’,反义链:5’-UCACCACCUUCUUGAUGUCTT-3’)靶基因为GAPDH(甘油醛-3-磷酸脱氢酶)。24h后收细胞,Trizol法提取siRNA,反转录和PCR检测靶基因的mRNA表达水平,以人PPIBsiRNA为内参。
为了与阳性对照相比,将本发明的阳离子脂质与有效的阳离子脂质MC3(已知其能有效用于体内传送核酸,Angew.Chem.Int.Ed.2012,51,8529-8533)比较。MC3具有以下结构:
如表1所示,在剂量分别为10nM和50nM时,本发明部分阳离子脂质中,多数表现出比MC3更强的效力。
表1脂质体载siRNA治疗剂针对GAPDH的体外沉默效率
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (19)
5.如权利要求1-4任一项所述的脂质化合物,其特征在于,其中所述脂质化合物呈化学上可接受的盐的形式。
6.如权利要求1-4任一项所述的脂质化合物,其特征在于,其中所述脂质化合物呈阳离子脂质的形式。
7.一种脂质体,其特征在于,包含权利要求1-6任一项所述的脂质化合物。
8.如权利要求7所述的脂质体,其特征在于,所述脂质体还包含其它非阳离子形式的脂质化合物。
9.如权利要求8所述的脂质体,其特征在于,所述其它非阳离子形式的脂质化合物选自磷脂、胆固醇类衍生物。
10.如权利要求9所述的脂质体,其特征在于,所述磷脂选自二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二油酰磷脂酰乙醇胺。
11.如权利要求9所述的脂质体,其特征在于,所述胆固醇类衍生物为胆固醇。
12.如权利要求7-11任一项所述的脂质体,其特征在于,所述脂质体还包含脂质缀合物。
13.如权利要求12任一项所述的脂质体,其特征在于,所述脂质缀合物选自PEG-二酰基甘油缀合物、PEG-二烷氧基丙基缀合物。
14.如权利要求12所述的脂质体,其特征在于,所述脂质体的中值直径为30~150nm。
15.一种药物组合物,其特征在于,包含权利要求1-6任一项所述的脂质化合物和治疗剂。
16.一种药物组合物,其特征在于,由权利要求7-14任一项所述的脂质体包裹治疗剂组成。
17.如权利要求15或16所述药物组合物,其特征在于,所述治疗剂分别独立的选自siRNA、反义寡核苷酸、微小RNA、mRNA、DNA。
18.如权利要求15或16中所述药物组合物的应用,其特征在于,所述药物组合物分别独立的用于治疗基因异常引发的疾病。
19.如权利要求15所述的药物组合物,其特征在于,所述脂质化合物和治疗剂的摩尔比为20:1~200:1。
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