CN116354836A - 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 - Google Patents
阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 Download PDFInfo
- Publication number
- CN116354836A CN116354836A CN202310189716.XA CN202310189716A CN116354836A CN 116354836 A CN116354836 A CN 116354836A CN 202310189716 A CN202310189716 A CN 202310189716A CN 116354836 A CN116354836 A CN 116354836A
- Authority
- CN
- China
- Prior art keywords
- compound
- cationic lipid
- reaction
- added
- lipid compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- -1 Cationic lipid compound Chemical class 0.000 title claims abstract description 70
- 238000002360 preparation method Methods 0.000 title claims abstract description 38
- 108020004999 messenger RNA Proteins 0.000 title claims abstract 10
- 239000003814 drug Substances 0.000 claims abstract description 19
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 14
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 14
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 14
- 125000001924 fatty-acyl group Chemical group 0.000 claims abstract description 8
- 150000003512 tertiary amines Chemical class 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 99
- 150000001875 compounds Chemical class 0.000 claims description 87
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 36
- 239000000203 mixture Substances 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 19
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 18
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 15
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 14
- 229940125904 compound 1 Drugs 0.000 claims description 11
- 229940126214 compound 3 Drugs 0.000 claims description 10
- 229940125898 compound 5 Drugs 0.000 claims description 10
- 229940125782 compound 2 Drugs 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 150000001263 acyl chlorides Chemical class 0.000 claims description 4
- MZSAMHOCTRNOIZ-UHFFFAOYSA-N 3-[4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy-N-phenylaniline Chemical compound NCC1=CC(=NC(=C1)C(F)(F)F)OC=1C=C(NC2=CC=CC=C2)C=CC=1 MZSAMHOCTRNOIZ-UHFFFAOYSA-N 0.000 claims description 2
- 239000011261 inert gas Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 4
- 210000000056 organ Anatomy 0.000 abstract description 5
- 125000002091 cationic group Chemical group 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 165
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 56
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 51
- 239000000243 solution Substances 0.000 description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 31
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 30
- 239000012074 organic phase Substances 0.000 description 28
- 239000003208 petroleum Substances 0.000 description 28
- 239000002105 nanoparticle Substances 0.000 description 25
- 150000002632 lipids Chemical class 0.000 description 22
- 239000007858 starting material Substances 0.000 description 21
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 20
- 239000000741 silica gel Substances 0.000 description 20
- 229910002027 silica gel Inorganic materials 0.000 description 20
- 238000001035 drying Methods 0.000 description 19
- 238000004440 column chromatography Methods 0.000 description 17
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 15
- 229910052740 iodine Inorganic materials 0.000 description 15
- 239000011630 iodine Substances 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 238000000605 extraction Methods 0.000 description 14
- 239000012043 crude product Substances 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- 238000002156 mixing Methods 0.000 description 11
- 230000014509 gene expression Effects 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 201000010099 disease Diseases 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 238000009472 formulation Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 229910052938 sodium sulfate Inorganic materials 0.000 description 8
- 235000011152 sodium sulphate Nutrition 0.000 description 8
- 238000000926 separation method Methods 0.000 description 7
- 210000000952 spleen Anatomy 0.000 description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 238000010992 reflux Methods 0.000 description 5
- 238000002390 rotary evaporation Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- ABRVLXLNVJHDRQ-UHFFFAOYSA-N [2-pyridin-3-yl-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound FC(C1=CC(=CC(=N1)C=1C=NC=CC=1)CN)(F)F ABRVLXLNVJHDRQ-UHFFFAOYSA-N 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 description 3
- 239000012263 liquid product Substances 0.000 description 3
- 239000012312 sodium hydride Substances 0.000 description 3
- 229910000104 sodium hydride Inorganic materials 0.000 description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 206010013801 Duchenne Muscular Dystrophy Diseases 0.000 description 2
- 102000003951 Erythropoietin Human genes 0.000 description 2
- 108090000394 Erythropoietin Proteins 0.000 description 2
- 208000031220 Hemophilia Diseases 0.000 description 2
- 208000009292 Hemophilia A Diseases 0.000 description 2
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 108091027974 Mature messenger RNA Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- REAYFGLASQTHKB-UHFFFAOYSA-N [2-[3-(1H-pyrazol-4-yl)phenoxy]-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound N1N=CC(=C1)C=1C=C(OC2=NC(=CC(=C2)CN)C(F)(F)F)C=CC=1 REAYFGLASQTHKB-UHFFFAOYSA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 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 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 229940105423 erythropoietin Drugs 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 102000044890 human EPO Human genes 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000005311 nuclear magnetism Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002342 ribonucleoside Substances 0.000 description 2
- 229920002477 rna polymer Polymers 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- SZHOJFHSIKHZHA-UHFFFAOYSA-N tridecanoic acid Chemical compound CCCCCCCCCCCCC(O)=O SZHOJFHSIKHZHA-UHFFFAOYSA-N 0.000 description 2
- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 description 1
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 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 description 1
- VMKOFRJSULQZRM-UHFFFAOYSA-N 1-bromooctane Chemical compound CCCCCCCCBr VMKOFRJSULQZRM-UHFFFAOYSA-N 0.000 description 1
- IKPSIIAXIDAQLG-UHFFFAOYSA-N 1-bromoundecane Chemical compound CCCCCCCCCCCBr IKPSIIAXIDAQLG-UHFFFAOYSA-N 0.000 description 1
- ASZMYJSJEOGSBR-UHFFFAOYSA-N 1-chlorotridecane Chemical compound CCCCCCCCCCCCCCl ASZMYJSJEOGSBR-UHFFFAOYSA-N 0.000 description 1
- JYWKEVKEKOTYEX-UHFFFAOYSA-N 2,6-dibromo-4-chloroiminocyclohexa-2,5-dien-1-one Chemical compound ClN=C1C=C(Br)C(=O)C(Br)=C1 JYWKEVKEKOTYEX-UHFFFAOYSA-N 0.000 description 1
- PYSGFFTXMUWEOT-UHFFFAOYSA-N 3-(dimethylamino)propan-1-ol Chemical compound CN(C)CCCO PYSGFFTXMUWEOT-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 description 1
- 208000024556 Mendelian disease Diseases 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- ZEEBGORNQSEQBE-UHFFFAOYSA-N [2-(3-phenylphenoxy)-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound C1(=CC(=CC=C1)OC1=NC(=CC(=C1)CN)C(F)(F)F)C1=CC=CC=C1 ZEEBGORNQSEQBE-UHFFFAOYSA-N 0.000 description 1
- 229940127024 acid based drug Drugs 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 229940126543 compound 14 Drugs 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- HVTICUPFWKNHNG-UHFFFAOYSA-N iodoethane Chemical compound CCI HVTICUPFWKNHNG-UHFFFAOYSA-N 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical class [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C201/00—Preparation of esters of nitric or nitrous acid or of compounds containing nitro or nitroso groups bound to a carbon skeleton
- C07C201/06—Preparation of nitro compounds
- C07C201/14—Preparation of nitro compounds by formation of nitro groups together with reactions not involving the formation of nitro groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
- C07C205/13—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups
- C07C205/20—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups having nitro groups and hydroxy groups bound to carbon atoms of six-membered aromatic rings
- C07C205/21—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups having nitro groups and hydroxy groups bound to carbon atoms of six-membered aromatic rings having nitro groups and hydroxy groups bound to carbon atoms of the same non-condensed six-membered aromatic ring
- C07C205/22—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups having nitro groups and hydroxy groups bound to carbon atoms of six-membered aromatic rings having nitro groups and hydroxy groups bound to carbon atoms of the same non-condensed six-membered aromatic ring having one nitro groups bound to the ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C213/00—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C213/06—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton from hydroxy amines by reactions involving the etherification or esterification of hydroxy groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C219/16—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by an inorganic acid or a derivative thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
-
- 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
-
- 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
- C07C229/12—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 acyclic carbon skeletons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C269/04—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups from amines with formation of carbamate groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C329/00—Thiocarbonic acids; Halides, esters or anhydrides thereof
- C07C329/02—Monothiocarbonic acids; Derivatives thereof
- C07C329/04—Esters of monothiocarbonic acids
- C07C329/06—Esters of monothiocarbonic acids having sulfur atoms of thiocarbonic groups bound to acyclic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C333/00—Derivatives of thiocarbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C333/02—Monothiocarbamic acids; Derivatives thereof
- C07C333/04—Monothiocarbamic acids; Derivatives thereof having nitrogen atoms of thiocarbamic groups bound to hydrogen atoms or to acyclic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/28—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group
- C07C67/29—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group by introduction of oxygen-containing functional groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
- C07C69/22—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety
- C07C69/30—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety esterified with trihydroxylic compounds
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Optics & Photonics (AREA)
- Nanotechnology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Dispersion Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
本申请要求于2022年03月25日提交中国专利局、申请号为2022103011731、发明名称为“阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统”的中国专利申请的优先权。其中,本申请的化合物1与优先权说明书中的化合物14的结构一致。
技术领域
本发明涉及一种医药生物技术领域,特别是涉及一种阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统。
背景技术
mRNA(信使核糖核酸)是由DNA (脱氧核糖核酸)双链中的一条链为模板(template),在RNA聚合酶(RNA polymerase)的催化作用下,以4种三磷酸核糖核苷(A、U、G、C)为底物,通过磷酸二酯键聚合而成的一类单链核糖核酸。
mRNA能够携带并传递细胞核内DNA所存储的遗传信息,在遗传信息向功能性蛋白的转换过程中发挥着关键作用。在细胞质中,未成熟的mRNA通过加帽,加尾,以及内含子剪切等步骤完成加工修饰变成成熟的mRNA,成熟的mRNA能够精确指导细胞质中蛋白质的合成过程。相对来说,由于mRNA比DNA的分子量小得多,易于转染,并且不会有整合到宿主DNA而引发插入突变的致癌风险。因此,以mRNA为预防性和治疗性药物,在多种疾病的防治方面有着巨大的优势和潜力。
mRNA核酸类药物是利用分子生物学的方法将目的功能基因或者目的基因的功能亚单位通过信使核糖核酸的形式导入患者体内,经靶向性胞内递送,晚期内含体逃逸,细胞内翻译及翻译后加工修饰,表达出具有特定功能的蛋白质,用以预防(功能性蛋白或者亚单位激活宿主免疫系统产生相应体液免疫或细胞免疫反应)或治疗疾病(表达出的蛋白或者亚单位具有治疗疾病的功能或调节其他基因表达的作用)的一种防治策略。与其他方法相比,其优势在于它可以直接在分子水平上激活机体产生针对特定病原体的功能性抗体或细胞免疫反应,或者有针对性地修复致病基因或纠正异常基因的表达,从而达到多种疾病预防和治疗的作用。
mRNA核酸类药物可以达到传统药物无法替代的效果,例如单克隆抗体药物只能作用于细胞表面,而mRNA核酸类药物不仅可以针对细胞膜外蛋白发挥作用,也可以对细胞内蛋白发挥作用,甚至可以在细胞核内发挥作用,并具有精确的靶向性。在人类面临的7000多种疾病中,约1/3的疾病是由于功能性基因的表达出现了问题(缺失、降低或过表达),如血友病(Hemophilia)、杜氏肌营养不良(DMD)、囊性纤维化(Cysticfibrosis)和严重的免疫缺陷综合征(SCID)等,在临床上几乎是无药可治,而mRNA核酸类药物对这种单基因疾病非常有优势。在个性化医疗以及精准医疗普及的时代背景下。理论上,由患者基因差异或基因表达异常引起的疾病,都可利用mRNA核酸药物对其进行精准有效的治疗。
mRNA核酸类药物虽然在调控基因表达及恶性疾病的预防和治疗中有巨大的优势和潜力。但此类药物的研发、制备以及后续系统给药中均面临着诸多困难。首先,mRNA是以单链的形式存在,导致mRNA在体外及生理条件下极不稳定,不仅易被空气中或血液中的RNA核酸酶(RNAase))所降解,还易被肝脏、脾脏等组织器官中单核巨噬细胞清除;其次,由于mRNA带负电性,使其难以穿过细胞膜进入到细胞内部;再次,mRNA难以从内涵体逃逸并进入细胞质中发挥作用。此外,mRNA的尿嘧啶核糖核苷(U)易产生免疫原性,在某些情况下,免疫原性的产生有可能会增加mRNA药物潜在的毒副作用。最后,易产生脱靶效应也是mRNA核酸类药物在制备以及给药过程中面临的重要挑战。因此,mRNA核酸类药物胞内递送系统的开发,是其能够大规模临床应用的关键所在。在此,阳离子脂质是mRNA 递送系统(LNP)的重要组成部分,所以,亟需开发递送效率高、生物安全性好的阳离子脂质。
发明内容
有鉴于此,本发明提供一种阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统,主要目的在于开发一种递送效率高、生物安全性好的阳离子脂质化合物。
为达到上述目的,本发明主要提供如下技术方案:
一方面,本发明的实施例提供一种阳离子脂质化合物,其中,所述阳离子脂质化合物的结构如下述式(I)所示:
其中,在式(I)中,R1为包含三级胺的链状结构;R2为链状脂肪酰基;R3为具有支链的链状脂肪酰基。
优选的,所述R1为以下基团中的任一种:
优选的,所述R2为以下基团中的任一种:
优选的,所述R3为以下基团中的任一种:
优选的,所述阳离子脂质化合物为以下化合物中的任一种:
另一方面,本发明实施例还提供了上述任一项所述的阳离子脂质化合物的制备方法,其中,所述阳离子脂质化合物的制备方程式如下:
优选的,当所述阳离子脂质化合物为化合物1、化合物2、化合物3时:
其中,在化合物1-5的无水四氢呋喃溶液中,加入N,N-二异丙基乙胺,惰性气体保护,在0-5℃的温度下,再向其中滴加酰氯,然后在70-75℃的温度下反应;反应结束后,进行后处理,得到油状物为化合物1-6。
优选的,当所述阳离子脂质化合物为化合物4时:
其中,在反应容器中加入化合物4-10,并使化合物4-10溶于四氢呋喃,再于室温下向其中加入化合物酰氯,室温搅拌至清,然后在室温下,再向其中再加入N,N-二异丙基乙胺,室温搅拌后盖好盖子,在70-75℃的温度下进行反应,反应结束后进行后处理,得到黄色液体化合物4-11。
优选的,当所述阳离子脂质化合物为化合物5时:
其中,在反应容器中加入化合物5-7,并使化合物5-7溶于四氢呋喃中,再于室温下向其中加入酰氯,室温搅拌至清,然后在室温下向其中再加入三乙胺,室温搅拌后盖好盖子,在70-75℃的温度下,进行搅拌反应,反应结束后进行后处理,得到黄色液体化合物5-8。
再一方面,本发明实施例还提供了上述任一项所述的阳离子脂质化合物在制备mRNA递送系统中的应用。
优选的,所述mRNA递送系统为LNP组合物。
再一方面,本发明实施例还提供了上述任一项所述的阳离子脂质化合物在制备核酸类药物中的应用。
再一方面,本发明实施例还提供一种mRNA递送系统,其中,所述mRNA递送系统包括上述任一项所述的阳离脂质化合物。
优选的,所述mRNA递送系统为LNP组合物。
优选的,所述mRNA递送系统在制备核酸类药物中的应用。
与现有技术相比,本发明的阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统至少具有下列有益效果:
一方面,本发明实施例开发一种新的阳离子脂质化合物,其结构如通式(I)所示,该阳离子脂质化合物用于制备mRNA递送系统,具有递送效率高、器官靶向递送、生物安全性好的特点。
另一方面,本发明实施例提供一种mRNA 递送系统,由于该mRNA 递送系统包括了上述的阳离子脂质化合物,因此,该mRNA递送系统具有送效率高、器官靶向递送、生物安全性好的特点。
另外,上述的阳离子脂质化合物、mRNA递送系统主要用于制备核酸类药物。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是由本发明实施例的化合物制备的脂质纳米颗粒制剂的蛋白表达水平对比图;
图2是由本发明实施例的化合物制备的脂质纳米颗粒制剂、MC3脂质纳米颗粒制剂的脾脏蛋白表达水平对比图;
图3是由本发明实施例的化合物制备的脂质纳米颗粒制剂、MC3脂质纳米颗粒制剂的生物安全测试对比图。
具体实施方式
为更进一步阐述本发明为达成预定发明目的所采取的技术手段及功效,以下结合附图及较佳实施例,对依据本发明申请的具体实施方式、结构、特征及其功效,详细说明如后。在下述说明中,不同的“一实施例”或“实施例”指的不一定是同一实施例。此外,一或多个实施例中的特定特征、结构、或特点可由任何合适形式组合。
本发明实施例开发一种递送效率高、器官靶向递送、生物安全性好的阳离子脂质化合物。其中,所述阳离子脂质化合物的结构如下述式(I)所示:
其中,在式(I)中,R1为包含三级胺的链状结构;R2为链状脂肪酰基;R3为具有支链的链状脂肪酰基。
较佳地,本发明的阳离子脂质化合物为化合物1-5中的任一种,但不限于此,下面详细说明化合物1-5的制备方法:
1.化合物1
其中,化合物1的制备步骤如下:
在化合物1-1(15.0g,103mmol)的无水四氢呋喃(150mL)溶液中,加入N,N-二异丙基乙胺(26.5g,205mmol),在冰浴的条件下,再向其中缓慢滴加正十三酸酰氯(35.8g,154mmol),氮气保护,零度下反应8小时,TLC检测反应完全。然后向反应体系中加水(500mL),再加乙酸乙酯萃取三次(200mL×3)。采用有机相分离,合并,用饱和食盐水洗,Na2SO4干燥,旋蒸除去溶剂得到粗品。粗品过硅胶柱(洗脱:石油醚/乙酸乙酯=20/1)得到化合物1-2(26.0g),为油状物。其中,化合物1-2的核磁数据如下:
1H NMR(400MHz,CDCl3)δ4.15(dd,J=7.0,2.2Hz,2H),4.00~3.95(m,2H),3.75~3.70(m,2H),2.30(t,J=7.6Hz,2H),2.01~1.98(m,1H),1.65~1.58(m,2H),1.43(s,3H),1.40(s,3H),1.29~1.25(m,18H),0.89~0.85(m,3H)。
将化合物1-2(26.0g,75.9mmol)溶解在甲醇(100mL)中,并向其中加入盐酸(3mol/L,10mL)。在25℃的温度下,搅拌反应30分钟。TLC检测反应完全,并向其中加入水(500mL),乙酸乙酯萃取三次(200mL×3)。用有机相分离、合并、用饱和食盐水洗,Na2SO4干燥,旋蒸除去溶剂得到粗品。粗品过硅胶柱(洗脱:二氯甲烷/甲醇=20/1)得到白色固体为化合物1-3(18.0g)。其中,化合物1-3的核磁数据如下:
1H NMR(400MHz,DMSO-d6)δ4.48(t,J=5.2Hz,2H),4.01(d,J=6.4Hz,2H),3.46~3.36(m,4H),2.27(t,J=7.4Hz,2H),1.86~1.77(m,1H),1.54~1.47(m,2H),1.28~1.24(m,18H),0.87~0.83(m,3H)。
在化合物1-3(6.00g,19.8mmol)的无水四氢呋喃(50mL)溶液中,加入N,N-二异丙基乙胺(5.12g,39.6mmol,2.0eq.),氮气保护,在0℃的温度下缓慢滴加酰氯(10.7g,29.7mmol,1.5eq.),在0℃的温度下反应8小时。TLC检测反应完全,加入水(200mL),用乙酸乙酯萃取三次(100mL×3)。有机相分离,合并,用饱和食盐水洗,Na2SO4干燥,旋蒸除去溶剂得到粗品。粗品过硅胶柱(洗脱:石油醚/乙酸乙酯=10/1)得到油状物为化合物1-5(4.50g)。其中,化合物1-5的核磁数据如下:
1H NMR(400MHz,CDCl3)δ4.22~4.10(m,4H),3.60(d,J=5.6Hz,2H),2.35~2.29(m,2H),2.22~2.16(m,1H),1.63~1.55(m,4H),1.47~1.39(m,4H),1.32~1.18(m,47H),0.87(t,J=6.4Hz,9H)。
在化合物1-5(4.50g,7.36mmol)的无水四氢呋喃(50mL)溶液中,加入N,N-二异丙基乙胺(1.90g,14.7mmol),氮气保护,在0℃的温度下向其中缓慢滴加酰氯(2.22g,11.0mmol),在70℃的温度下反应2小时。TLC检测反应完全,加入水(100mL),用乙酸乙酯萃取三次(100mL×3)。有机相分离,合并,用饱和食盐水洗,Na2SO4干燥,旋蒸除去溶剂得到粗品。粗品过硅胶柱(洗脱:石油醚/乙酸乙酯=20/1)得到油状物为化合物1-6(3.40g)。其中,化合物1-6的核磁数据如下:
1H NMR(400MHz,CDCl3)δ8.28(d,J=8.8Hz,2H),7.38(dd,J=9.2,2.8Hz,2H),4.36(d,J=5.6Hz,2H),4.21(d,J=6.0Hz,4H),2.56~2.47(m,1H),2.39~2.30(m,2H),1.65~1.42(m,8H),1.32~1.23(m,47H),0.89~0.85(m,9H)。
在25mL的史莱克管中,加入化合物1-6(3.40g,4.38mmol),在室温下再向其中缓慢滴加3-(二乙胺基)丙基-1-醇(10mL),所得溶液在氮气保护下室温反应8小时。TLC检测反应完全,加入水(500mL),乙酸乙酯萃取三次(50mL×3)。有机相分离,合并,用饱和食盐水洗,Na2SO4干燥,旋蒸除去溶剂得到粗品。粗品过硅胶柱(洗脱:石油醚/乙酸乙酯=1/1)得到浅黄色油状物为化合物1(1.40g)。其中,化合物1的核磁数据如下:
1H NMR(400MHz,CDCl3)δ4.19~4.13(m,8H),2.50(q,J=6.8Hz,6H),2.44~2.38(m,1H),2.34~2.27(m,3H),1.83~1.76(m,2H),1.63~1.52(m,4H),
1.46~1.37(m,2H),1.28~1.23(m,50H),1.00(t,J=7.2Hz,6H),0.87(t,J=6.4Hz,9H)。
2.化合物2
其中,化合物2的制备步骤如下:
在室温条件,于500mL的茄型瓶中,加入化合物2-1(10g,66.8mmol)、硫代乙酸钾(9.9g,86.9mmol))和N,N-二甲基甲酰胺(50mL)。再加入碳酸钾(44g,133.6mmol),室温反应2h。TLC点板,显示原料反应完全,加水淬灭反应。然后加入乙酸乙酯(500mL)与水(500mL)进行萃取分液,然后有机相在用饱和氯化钠溶液(300mL×3)洗涤2-3次,然后有机相干燥旋干,加入硅胶进行拌样,通过柱层析(二氯甲烷/甲醇=200/1到20/1)得到无色的液体状产品2-2(6.5g)。
在室温条件,于250mL的茄型瓶中,加入化合物2-2(6.5g,34.3mmol),氢氧化钠溶液(4.1g,103mmol,溶于10mL水)和甲醇20mL。室温搅拌反应2h。TLC点板,显示原料反应完全,加3mol/L的盐酸淬灭反应。然后挥走甲醇,加入乙酸乙酯(200mL)与水(200mL)进行萃取分液,然后有机相在用饱和氯化钠溶液(300mL×3)洗涤2-3次,然后有机相干燥旋干,加入硅胶进行拌样,通过柱层析(二氯甲烷/甲醇=200/1到10/1)得到无色的液体状产品2-3(2.6g)。
在室温条件下,于100mL的茄型瓶中,加入化合物1-6(3g,3.5mmol)。逐渐缓慢加入化合物2-3(2.6g,17.7mmol,3eq),搅拌反应50分钟,TLC点板,显示原料反应完全,加水淬灭反应。然后加入乙酸乙酯(200mL)与水(200mL)进行萃取分液,然后有机相在用饱和氯化钠溶液(200mL×3)洗涤2-3次,然后有机相干燥旋干,加入硅胶进行拌样,通过柱层析(二氯甲烷/甲醇=300/1到60/1)得到粗品,再经制备纯化,得到淡黄色的油状液体产品化合物2(603mg)。其中,化合物2的核磁数据如下:
1H NMR(400MHz,CDCl3)δ4.28(d,J=5.9Hz,2H),4.13(d,J=6.0Hz,4H),2.88(t,J=7.2Hz,2H),2.50(q,J=7.2Hz,6H),2.42(p,J=5.9Hz,1H),2.37–2.27(m,3H),1.79(q,J=7.2Hz,4H),1.59(m,4H),1.44(m,3H),1.34–1.18(m,51H),1.01(t,J=7.1Hz,6H),0.88(t,J=6.7Hz,9H).C47H91NO6SExact Mass:797.66,found[M+H]+:798.73.
3.化合物3
其中,化合物3的制备步骤如下:
在室温条件下,于50mL的茄型瓶中,先加入化合物1-6(3g,3.5mmol),然后再逐渐缓慢加入二甲胺基丙醇(1.8g,17.7mmol),搅拌反应50分钟,TLC点板,显示原料反应完全,加水淬灭反应。然后加入乙酸乙酯(200mL)与水(200mL)进行萃取分液,然后有机相在用饱和氯化钠溶液(200mL×3)洗涤2-3次,然后用有机相干燥旋干,加入硅胶进行拌样,通过柱层析(二氯甲烷/甲醇=300/1到50/1)得到粗品,再经进一步纯化,得到黄色的油体状产品化合物3(517mg)。其中,化合物3的核磁数据如下:
1H NMR(400MHz,CDCl3)δ4.26–4.12(m,8H),2.49–2.38(m,3H),2.38–2.29(m,3H),2.27(s,6H),1.88(p,J=6.9Hz,2H),1.61(m,4H),1.46(m,3H),1.37–1.17(m,50H),0.90(t,J=6.7Hz,9H).C45H87NO7 Exact Mass:753.65,found[M+H]+:754.64.
4.化合物4
其中,化合物4的制备步骤如下:
在2000mL的三口瓶中加入化合物4-1(30.0g,187mmol),将其溶于1,4-二氧六环(600mL),在水浴的条件下,再缓慢加入氢化钠(6.74g,280mmol),搅拌5min,加入化合物1-溴正十一烷(48.46g,206mmol),在100℃进行反应,回流搅拌过夜,TLC监测反应。TLC(石油醚/乙酸乙酯=20/1)碘显色显示原料已反应完全,有一新的主点产生,停止反应。加入水溶液洗涤,加乙酸乙酯萃取三次,收集有机相,硫酸钠干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(石油醚/乙酸乙酯=20/1)得到无色油状液体化合物4-2(15g)。
在1000mL的三口瓶中加入化合物4-2(15g,47.7mmol),溶于1,4-二氧六环(300.0mL),水浴缓慢加入氢化钠(4.58g,190.8mmol),搅拌5min,加入化合物碘乙烷(22.32g,143.1mmol),在100℃下反应,回流搅拌过夜,TLC监测反应。TLC(石油醚/乙酸乙酯=20/1)碘显色显示原料已反应完全,有一新的主点产生,停止反应。用乙酸乙酯和水萃取,分液,收集有机相,干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(石油醚:乙酸乙酯=30/1)得到无色油状液体4-3(10.1g)。
在250mL的单口瓶中加入化合物4-4(10.1g,29.49mmol)),并使其溶于无水乙醇(10.0mL)。将氢氧化钾(16.55g,294.87mmol)溶于H2O(28.0mL),搅拌至清,然后加入到含化合物4-4的反应液中,在95℃的温度下搅拌反应过夜,TLC监测反应。TLC(石油醚/乙酸乙酯=20/1)碘显色显示原料已反应完全,有一新的主点产生,停止反应。减压浓缩,旋掉大部分乙醇,6mol/L的盐酸溶液调至PH=2-3。用乙酸乙酯和水萃取,分液,收集有机相,干燥,减压浓缩,得到白色固体化合物4-5(7.5g)。
在100mL的单口瓶中加入化合物4-5(7.5g,26.18mmol),接上冷凝管,插上气球,升温至170℃,回流搅拌16h,观察气球,气球不再膨胀,气体释放停止,停止反应,核磁监测反应。得到褐色液体化合物4-6(6.1g)。不做纯化直接用于下一步。
在100mL的单口瓶中加入化合物4-6(6.1g,25.17mmol),用无水二氯甲烷DCM(6.0mL)稀释,再加入草酰氯(6.39mL,75.50mmol),室温搅拌反应1h,插上气球,观察气球,气球不再膨胀,TLC监测反应。取样加1滴甲醇进行TLC,TLC(石油醚/乙酸乙酯=20/1)显示原料已反应完全,有一新的主点产生,停止反应。旋干后直接用于下一步,得到褐色油状中间体4-7(6.3g)。
在500mL的单口瓶中加入化合物1-1(8.0g,23.49mmol),并将其溶于四氢呋喃(160.0mL)中,然后向其中加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(6.75g,35.23mmol),室温搅拌0.5h,再向其中加入4-二甲氨基吡啶(5.74g,46.98mmol),室温搅拌15min,再向其中加入羧酸类化合物(4.46g,30.54mmol),室温搅拌反应过夜,TLC监测反应。TLC(石油醚/乙酸乙酯=20/1)碘显色显示原料已反应完全,有一新的主点产生,停止反应。用乙酸乙酯和水萃取,分液,收集有机相,干燥,旋干,通过柱层析(石油醚/乙酸乙酯=20/1)得到无色油状液体化合物4-8(7.0g)。
在250mL的单口瓶中加入化合物4-8(7.0g,14.93mmol),溶于乙醇(30.0mL),向其中加入3mol/L稀盐酸溶液(3.48mL,10.45mmol),室温搅拌10分钟。TLC监测反应。碘显色显示原料已反应完全,有一新的主点产生,停止反应,用碳酸氢钠溶液调节pH=8,减压浓缩,旋掉大部分甲醇,用乙酸乙酯和水萃取,分液,收集有机相,干燥,减压浓缩,得到白色固体化合物4-9(6.2g)。
在500mL的三口瓶中加入化合物4-9(6.2g,14.46mmol),将其溶于四氢呋喃(120.0mL)中,再加入三乙胺TEA(4.39g,43.39mmol),冰浴下滴加4-7(5.66g,21.69mmol),冰浴搅拌15分钟,撤冰浴,室温搅拌反应5h,TLC监测反应。TLC(石油醚/乙酸乙酯=5/1)碘显色显示有一新的主点产生,原料反应完全,停止反应。加入饱和氯化铵溶液洗涤,加乙酸乙酯萃取三次,收集有机相,硫酸钠干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(石油醚/乙酸乙酯=10/1)得到淡黄透明液体化合物4-10(3.8g)。
在250mL的封管中加入化合物4-10(3.38g,5.18mmol),并将其溶于四氢呋喃(25.0mL)中,向其中再加入化合物酰氯(1.56g,7.76mmol),室温搅拌5分钟,搅拌至清,在室温条件下,向其中再加入N,N-二异丙基乙胺DIPEA(1.05g,10.35mmol),室温搅拌5分钟,盖好盖子,在70℃的温度下进行反应,搅拌过夜,TLC监测反应。TLC(石油醚/乙酸乙酯=10/1)碘显色显示原料已反应完全,有一新的主点产生,有紫外,停止反应。依次加入水溶液,乙酸乙酯和饱和氯化铵溶液洗涤,用乙酸乙酯萃取2遍,收集有机相,硫酸钠干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(石油醚/乙酸乙酯=40/1)得到黄色液体化合物4-11(2.89g)。
在50mL的单口瓶中加入化合物4-11(2.89g,3.53mmol)),再加入化合物3-(二乙胺基)丙基-1-醇(4.63g,35.32mmol),室温搅拌过夜,TLC和LCMS监测反应。TLC(二氯甲烷/甲醇=10/1)碘显色显示原料已反应完全,有一新的主点产生,停止反应。LCMS有检测到目标分子量,加入饱和氯化铵溶液洗涤,用乙酸乙酯萃取3遍,收集有机相,硫酸钠干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(二氯甲烷/甲醇=70/1)得到黄色液体1.85克,核磁不纯,用3mol/L盐酸进行洗涤,乙酸乙酯萃取,再用饱和碳酸钾溶液进行洗涤,用纯净水进行洗涤,减压浓缩,得到黄色油状液体化合物4(536mg)。其中,化合物4的核磁数据如下:
1H NMR(400MHz,CDCl3)δ4.25–4.13(m,8H),2.57(q,J=7.1Hz,6H),2.45(p,J=6.0Hz,1H),2.39–2.24(m,2H),1.85(p,J=6.7Hz,2H),1.69–1.51(m,5H),1.51–1.40(m,3H),1.26(s,50H),1.05(t,J=7.2Hz,6H),0.89(t,J=6.8Hz,12H).C49H95NO7 Exact Mass:809.71,found[M+H]+:810.74.
5.化合物5
其中,化合物5的制备步骤如下:
在500mL的三口瓶中加入化合物4-1(5.0g,31.22mmol),并将其溶于四氢呋喃(100.0mL)中,在水浴条件下,向其中缓慢加入氢化钠(60%)(4.99g,124.87mmol),搅拌5min,加入化合物1-溴正辛烷(24.12g,124.87mmol),在80℃的温度下反应,冷凝回流搅拌过夜,TLC监测反应。TLC(石油醚/乙酸乙酯=20/1)碘显色显示原料已反应完全,有一新的主点产生,停止反应。加入水溶液洗涤,加乙酸乙酯萃取三次,收集有机相,硫酸钠干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(石油醚/乙酸乙酯=20/1)得到无色油状液体化合物5-1(3.7g)。
在100mL的单口瓶中加入化合物5-1(2.0g,5.20mmol),将其溶于无水乙醇(2.0mL)中。将氢氧化钾(2.92g,52.00mmol)溶于H2O(5.6mL),搅拌至清,加入到含化合物5-1的反应液,在95℃的温度下,搅拌反应过夜,TLC监测反应。TLC(石油醚/乙酸乙酯=20/1)碘显色显示原料已反应完全,有一新的主点产生,停止反应。减压浓缩,旋掉大部分乙醇,6mol/L的盐酸溶液调至pH=2-3。用乙酸乙酯和水萃取,分液,收集有机相,干燥,减压浓缩,得到白色固体5-2(1.2g)。
在500mL的单口瓶中加入化合物5-2(41.0g,124.81mmol),接上冷凝管,插上气球,升温至170℃,回流搅拌5h,观察气球,气球不再膨胀,气体释放停止,停止反应,核磁监测反应。得到褐色液体化合物5-3,减压浓缩,然后加入硅胶进行拌样,通过柱层析(石油醚/乙酸乙酯=10/1)得到黄色粘稠固体5-3(27.0g)。
在100mL的单口瓶中加入化合物5-3(5.7g,20.04mmol)),用无水二氯甲烷(6.0mL)稀释,再加入草酰氯(6.0mL,70.91mmol,3.54eq),室温搅拌反应1h,插上气球,观察气球,气球不再膨胀,TLC监测反应。取样加1滴甲醇进行TLC,TLC(石油醚/乙酸乙酯=20/1)显示原料已反应完全,有一新的主点产生,停止反应。旋干后直接用于下一步,得到褐色油状中间体5-4。
在500mL的单口瓶中加入化合物1-1(19.11g,102.61mmol),溶于四氢呋喃(150.0mL),加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(29.50g,153.9mmol),室温搅拌0.5h,加入4-二甲氨基吡啶(25.07g,205.2mmol),室温搅拌15min,再加入正十三酸(15.0g,102.6mmol),室温搅拌反应过夜,TLC监测反应。TLC(石油醚/乙酸乙酯=20/1)碘显色显示原料已反应完全,有一新的主点产生,停止反应。用乙酸乙酯和水萃取,分液,收集有机相,干燥,旋干,通过柱层析(石油醚/乙酸乙酯=20/1)得到无色油状液体化合物5-5(26.0g)。
在500mL的单口瓶中加入化合物5-5(26.0g,82.68mmol),溶于甲醇(130.0mL),加入3mol/L稀盐酸溶液(19.92mL,57.88mmol),室温搅拌1h。TLC监测反应。碘显色显示原料已反应完全,有一新的主点产生,停止反应,用碳酸钠溶液调节pH=8,减压浓缩,旋掉大部分甲醇,用乙酸乙酯和水萃取,分液,收集有机相,干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(石油醚/乙酸乙酯=100/1)得到白色固体化合物5-6(5.42g)。
在500mL的三口瓶中加入化合物5-6(5.42g,19.75mmol),并将其溶于四氢呋喃(60.0mL)中,再向其中加入三乙胺(6.0g,26mmol,3.0eq),在冰浴条件下,向其中滴加5-4(5.98g,19.75mmol),搅拌15分钟,撤除冰浴,室温搅拌反应5h,TLC监测反应。TLC(石油醚/乙酸乙酯=5/1)碘显色显示有一新的主点产生,原料反应完全,停止反应。加入饱和氯化铵溶液洗涤,加乙酸乙酯萃取三次,收集有机相,硫酸钠干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(石油醚/乙酸乙酯=10/1)得到淡黄透明液体化合物5-7(4.04g)。
在250mL的封管中加入化合物5-7(4.04g,7.47mmol),并将其溶于四氢呋喃(35.0mL),再向其中加入酰氯(2.26g,11.20mmol),室温搅拌5分钟,搅拌至清,在室温条件下,向其中加入DIPEA(1.51g,14.94mmol),室温搅拌5分钟,盖好盖子,在70℃下反应搅拌过夜,TLC监测反应。TLC(石油醚/乙酸乙酯=10/1)碘显色显示原料已反应完全,有一新的主点产生,有紫外,停止反应。依次加入水溶液,乙酸乙酯和饱和氯化铵溶液洗涤,用乙酸乙酯萃取2遍,收集有机相,硫酸钠干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(石油醚/乙酸乙酯=40/1)得到黄色液体化合物5-8(4.3g)。
在50mL的单口瓶中加入化合物5-8(4.30g,6.09mmol),再加入化合物3-(二乙胺基)丙基-1-醇(7.99g,60.91mmol),室温搅拌过夜,TLC和LCMS监测反应。TLC(二氯甲烷/甲醇=10/1)碘显色显示原料已反应完全,有一新的主点产生,停止反应。LCMS有检测到目标分子量,加入饱和氯化铵溶液洗涤,用乙酸乙酯萃取3遍,收集有机相,硫酸钠干燥,减压浓缩,然后加入硅胶进行拌样,通过柱层析(二氯甲烷/甲醇=70/1)得到黄色液体1.7g,进一步纯化得到黄色油状液体化合物5(710mg)。其中,化合物5的核磁数据如下:
1H NMR(400MHz,CDCl3)δ4.25–4.11(m,8H),2.54(q,J=6.9Hz,6H),2.43(p,J=6.0Hz,1H),2.38–2.27(m,3H),1.83(p,J=6.8Hz,2H),1.60(m,4H),1.51–1.38(m,2H),1.36–1.16(m,38H),1.03(t,J=7.1Hz,6H),0.93–0.83(m,9H).C41H79NO7 Exact Mass:697.59,found[M+H]+:698.55.
下面说明本发明的阳离子脂质化合物应用在mRNA递送系统LNP中的技术效果。
实施例1
脂质纳米颗粒制剂的制备及检测。
1.制备步骤:
将本发明的上述化合物分别与DSPC、胆固醇和DMG-PEG2000以50:10:38.5:1.5的摩尔比溶于乙醇制备乙醇脂质溶液。另外将荧光素酶(Firefly luciferase,Fluc)mRNA或促红细胞生成素(Human Erythropoietin,hEPO)mRNA用50mM柠檬酸盐缓冲液(pH=4)稀释得到mRNA水溶液。
通过微流控装置以1:3的体积比混合乙醇脂质溶液和mRNA水溶液制备脂质纳米颗粒,经过1x PBS透析18小时以除去乙醇和完成柠檬酸盐缓冲液换液过程。最后,脂质纳米颗粒溶液通过0.2μm的无菌过滤和超滤浓缩过程,最终得到包封荧光素酶或促红细胞生成素mRNA的脂质纳米颗粒制剂。
另外,用相同方法制备MC3脂质纳米颗粒制剂作为对照。
2.检测步骤:
利用LitesizerTM500(Anton Paar,Austrian)测定脂质纳米颗粒粒径(Size),多分散指数(PDI)和电位(Zeta)。其中,粒径及电位均在0.1% PBS中测定。通过RiboGreen法检测脂质纳米颗粒制剂包封效率。以包封Fluc mRNA为例,测试结果如表1所示:
表1
制剂名称 | Size(nm) | PDI | Zeta(mV) | 包封效率 |
MC3 | 93.55 | 0.156 | 27.1 | 89.6% |
化合物1 | 100.79 | 0.184 | 29.4 | 90.78% |
化合物2 | 94.66 | 0.157 | 22.1 | 93.9% |
化合物3 | 86.74 | 0.103 | 29.5 | 95.5% |
化合物4 | 83.59 | 0.197 | 18.8 | 95.4% |
化合物5 | 90.54 | 0.159 | 24.9 | 89.6% |
从表1可以看出:本发明的化合物均可作为可离子化脂质形成脂质纳米颗粒。
实施例2
(1)脂质纳米颗粒制剂蛋白表达水平有效性验证:
以0.3mg/kg剂量对8-9周龄雌性Balb/c小鼠通过尾静脉注射实施例1制备的包封hEPO mRNA的脂质纳米颗粒,并在给药后6小时采集小鼠颌下血液,室温静置1.5小时。通过在4℃的温度下,以3000g离心15分钟分离出血清,使用市售试剂盒(Human EPO ELISA Kit)测得hEPO浓度。
验证结果参见图1所示,从图1可以看出:由本发明的化合物制备的脂质纳米颗粒制剂均具有较好的hEPO蛋白表达,则转染效果优异。
(2)脂质纳米颗粒制剂脾脏蛋白表达水平验证:
以0.3mg/kg的剂量对8-9周龄雌性Balb/c小鼠通过尾静脉注射MC3或实施例1制备的包封Fluc mRNA脂质纳米颗粒,并在给药后6小时腹腔注射荧光素酶底物,等待10分钟后取出小鼠脾脏进行成像及定量。
验证结果参见图2所示,从图2可以看出:由本发明化合物1、化合物3、化合物5制备的脂质纳米颗粒制剂在脾脏的Fluc表达量优于MC3脂质纳米颗粒制剂,表明其具有优异的脾脏靶向性。
(3)脂质纳米颗粒制剂生物安全性验证:
为考察实施例1中制备的脂质纳米颗粒制剂的生物安全性,以0.3mg/kg的剂量对8-9周龄雌性Balb/c小鼠通过尾静脉注射包封hEPO mRNA的MC3或实施例1制备的脂质纳米颗粒,并分别在给药前,给药后1天、2天、3天及4天记录小鼠体重变化。
验证结果参见图3所示,从图3可以看出:由本发明的化合物制备的脂质纳米颗粒均未引起体重的显著降低,说明其生物安全性良好。
综上所述,本发明的阳离子脂质化合物在体外mRNA递送上,具有递送效率高、脾脏靶向性好、生物安全性好的技术效果。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (15)
10.权利要求1-5任一项所述的阳离子脂质化合物在制备mRNA递送系统中的应用。
11.根据权利要求10所述的阳离子脂质化合物在制备mRNA递送系统中的应用,其特征在于,所述mRNA递送系统为LNP组合物。
12.权利要求1-5任一项所述的阳离子脂质化合物在制备核酸类药物中的应用。
13.一种mRNA递送系统,其特征在于,所述mRNA递送系统包括权利要求1-5任一项所述的阳离脂质化合物。
14.根据权利要求13所述的mRNA递送系统,其特征在于,所述mRNA递送系统为LNP组合物。
15.权利要求13或14所述的mRNA递送系统在制备核酸类药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CN2023/082056 WO2023179463A1 (zh) | 2022-03-25 | 2023-03-17 | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210301173 | 2022-03-25 | ||
CN2022103011731 | 2022-03-25 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116354836A true CN116354836A (zh) | 2023-06-30 |
Family
ID=86793615
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310189716.XA Pending CN116354836A (zh) | 2022-03-25 | 2023-03-02 | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 |
CN202310203981.9A Active CN116283624B (zh) | 2022-03-25 | 2023-03-06 | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310203981.9A Active CN116283624B (zh) | 2022-03-25 | 2023-03-06 | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 |
Country Status (2)
Country | Link |
---|---|
CN (2) | CN116354836A (zh) |
WO (2) | WO2023179463A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117466777A (zh) * | 2023-12-27 | 2024-01-30 | 北京新合睿恩生物医疗科技有限公司 | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 |
Family Cites Families (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2156289C (en) * | 1993-02-19 | 2006-01-03 | Junichi Yano | Drug composition containing nucleic acid copolymer |
AU2855099A (en) * | 1998-03-24 | 1999-10-18 | Nippon Shinyaku Co. Ltd. | Remedies for hepatitis |
CN1981044B (zh) * | 2004-06-07 | 2010-05-05 | 普洛体维生物治疗公司 | 包封干扰rna的脂质 |
CN103096875B (zh) * | 2010-06-03 | 2016-08-17 | 阿尔尼拉姆医药品有限公司 | 用于递送活性剂的生物可降解脂质 |
AU2012347637B2 (en) * | 2011-12-07 | 2017-09-14 | Alnylam Pharmaceuticals, Inc. | Biodegradable lipids for the delivery of active agents |
CN110003066B (zh) * | 2013-11-18 | 2021-09-03 | 阿克丘勒斯治疗公司 | 用于rna递送的可电离的阳离子脂质 |
EP3872066A1 (en) * | 2013-12-19 | 2021-09-01 | Novartis AG | Lipids and lipid compositions for the delivery of active agents |
EP3083579B1 (en) * | 2013-12-19 | 2022-01-26 | Novartis AG | Lipids and lipid compositions for the delivery of active agents |
WO2017117528A1 (en) * | 2015-12-30 | 2017-07-06 | Acuitas Therapeutics, Inc. | Lipids and lipid nanoparticle formulations for delivery of nucleic acids |
CN107281103A (zh) * | 2016-04-11 | 2017-10-24 | 百奥迈科生物技术有限公司 | 具有高含量阳性脂质化合物的脂质体制剂及其应用 |
US10383952B2 (en) * | 2016-12-21 | 2019-08-20 | Arcturus Therapeutics, Inc. | Ionizable cationic lipid for RNA delivery |
KR102591743B1 (ko) * | 2018-06-08 | 2023-10-19 | 후지필름 가부시키가이샤 | 화합물 또는 그 염 및 지질 입자 |
MA54385A (fr) * | 2018-12-05 | 2021-10-13 | Intellia Therapeutics Inc | Lipides aminés modifiés |
MX2021012934A (es) * | 2019-04-25 | 2022-04-06 | Intellia Therapeutics Inc | Lipidos de amina ionizables y nanoparticulas lipidicas. |
CA3143865A1 (en) * | 2019-06-07 | 2020-12-10 | Fujifilm Corporation | Lipid composition |
JP2023549011A (ja) * | 2020-09-15 | 2023-11-22 | ヴァーヴ・セラピューティクス,インコーポレーテッド | 遺伝子編集のための脂質製剤 |
CN112979483B (zh) * | 2021-05-14 | 2021-08-06 | 苏州艾博生物科技有限公司 | 一种阳离子脂质化合物、包含其的组合物及应用 |
-
2023
- 2023-03-02 CN CN202310189716.XA patent/CN116354836A/zh active Pending
- 2023-03-06 CN CN202310203981.9A patent/CN116283624B/zh active Active
- 2023-03-17 WO PCT/CN2023/082056 patent/WO2023179463A1/zh unknown
- 2023-03-17 WO PCT/CN2023/082055 patent/WO2023179462A1/zh unknown
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117466777A (zh) * | 2023-12-27 | 2024-01-30 | 北京新合睿恩生物医疗科技有限公司 | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 |
CN117466777B (zh) * | 2023-12-27 | 2024-03-19 | 北京新合睿恩生物医疗科技有限公司 | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 |
Also Published As
Publication number | Publication date |
---|---|
CN116283624A (zh) | 2023-06-23 |
WO2023179462A1 (zh) | 2023-09-28 |
CN116283624B (zh) | 2023-10-20 |
WO2023179463A1 (zh) | 2023-09-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104910025B (zh) | 氨基醇类脂质和其用途 | |
JP5600846B2 (ja) | キトサン基材高分子接合体及びその製造方法 | |
US20140350233A1 (en) | Sirna conjugate and preparation method thereof | |
JP2024507482A (ja) | イオン化可能な脂質分子、その製造方法及び脂質ナノ粒子の製造における応用 | |
CN114805113B (zh) | 一种安全高效的可降解脂质纳米颗粒及其制备方法和应用 | |
CN113461577B (zh) | 一种氨基脂质及其应用 | |
CN116354836A (zh) | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 | |
CN114105799B (zh) | 一种氨基脂质及其制备方法和应用 | |
CN116655486A (zh) | 长链烷基酯胺类化合物及其制备方法和在核酸递送方面的应用 | |
CN114874107A (zh) | 一种氨基脂质及其制备方法和应用 | |
CN117466768B (zh) | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 | |
CN117466777B (zh) | 阳离子脂质化合物及其制备方法和应用、以及mRNA递送系统 | |
CN117886711A (zh) | 一种阳离子脂质化合物及其制备方法和应用、及lnp组合物 | |
CN114853828A (zh) | 化合物、缀合物及其用途 | |
WO2024140546A1 (zh) | 一种阳离子脂质化合物及制备方法和应用、mRNA递送系统 | |
WO2023241577A1 (zh) | 阳离子脂质化合物及其制备方法和应用 | |
CN113292616B (zh) | 一种单糖配体功能化的阳离子脂质类化合物及其制备方法与应用 | |
CN114957364B (zh) | 一种碘苷碱基及其制备方法和构建的两亲核酸 | |
CN116947669B (zh) | 一种转染效率高的可电离脂质化合物及其应用 | |
WO2024067639A1 (zh) | 一种转染效率高的可电离脂质化合物及其应用 | |
CN114805410B (zh) | 一类两亲性树形分子、合成及其在核酸递送方面的应用 | |
CN115869262A (zh) | 新型peg脂质化合物、其制备方法、组合物及应用 | |
CN118084699A (zh) | 一种可电离脂质化合物及其制备方法与医学上的应用 | |
CN114349811A (zh) | 阳离子胆固醇衍生物、纳米复合物及其制备方法和应用 | |
CN117658840A (zh) | 一种可电离阳离子脂质化合物、制备方法及应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |