KR20240076588A - Amino acid-lipid binding compound for nucleic acid delivery and lipid nanoparticles comprising the same - Google Patents
Amino acid-lipid binding compound for nucleic acid delivery and lipid nanoparticles comprising the same Download PDFInfo
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- KR20240076588A KR20240076588A KR1020220157663A KR20220157663A KR20240076588A KR 20240076588 A KR20240076588 A KR 20240076588A KR 1020220157663 A KR1020220157663 A KR 1020220157663A KR 20220157663 A KR20220157663 A KR 20220157663A KR 20240076588 A KR20240076588 A KR 20240076588A
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- amino acid
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 138
- 150000002632 lipids Chemical class 0.000 title claims abstract description 114
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 title claims abstract description 74
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 70
- 108020004707 nucleic acids Proteins 0.000 title abstract description 30
- 102000039446 nucleic acids Human genes 0.000 title abstract description 30
- 150000007523 nucleic acids Chemical class 0.000 title abstract description 30
- -1 lipid compound Chemical class 0.000 claims abstract description 75
- 230000021615 conjugation Effects 0.000 claims abstract description 60
- 150000001413 amino acids Chemical class 0.000 claims abstract description 58
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 38
- 150000003839 salts Chemical class 0.000 claims abstract description 27
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 20
- 125000003277 amino group Chemical group 0.000 claims abstract description 17
- 150000002430 hydrocarbons Chemical class 0.000 claims abstract description 16
- 229930195733 hydrocarbon Natural products 0.000 claims abstract description 15
- 150000003862 amino acid derivatives Chemical class 0.000 claims abstract description 14
- 239000004215 Carbon black (E152) Substances 0.000 claims abstract description 13
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 6
- 229940024606 amino acid Drugs 0.000 claims description 55
- 235000001014 amino acid Nutrition 0.000 claims description 55
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- 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 44
- 150000003904 phospholipids Chemical class 0.000 claims description 24
- 235000012000 cholesterol Nutrition 0.000 claims description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical group CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- UAOUIVVJBYDFKD-XKCDOFEDSA-N (1R,9R,10S,11R,12R,15S,18S,21R)-10,11,21-trihydroxy-8,8-dimethyl-14-methylidene-4-(prop-2-enylamino)-20-oxa-5-thia-3-azahexacyclo[9.7.2.112,15.01,9.02,6.012,18]henicosa-2(6),3-dien-13-one Chemical compound C([C@@H]1[C@@H](O)[C@@]23C(C1=C)=O)C[C@H]2[C@]12C(N=C(NCC=C)S4)=C4CC(C)(C)[C@H]1[C@H](O)[C@]3(O)OC2 UAOUIVVJBYDFKD-XKCDOFEDSA-N 0.000 claims description 15
- 125000000524 functional group Chemical group 0.000 claims description 15
- 230000000379 polymerizing effect Effects 0.000 claims description 10
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 9
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 9
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 9
- 235000008206 alpha-amino acids Nutrition 0.000 claims description 9
- UDQTXCHQKHIQMH-KYGLGHNPSA-N (3ar,5s,6s,7r,7ar)-5-(difluoromethyl)-2-(ethylamino)-5,6,7,7a-tetrahydro-3ah-pyrano[3,2-d][1,3]thiazole-6,7-diol Chemical compound S1C(NCC)=N[C@H]2[C@@H]1O[C@H](C(F)F)[C@@H](O)[C@@H]2O UDQTXCHQKHIQMH-KYGLGHNPSA-N 0.000 claims description 8
- HUWSZNZAROKDRZ-RRLWZMAJSA-N (3r,4r)-3-azaniumyl-5-[[(2s,3r)-1-[(2s)-2,3-dicarboxypyrrolidin-1-yl]-3-methyl-1-oxopentan-2-yl]amino]-5-oxo-4-sulfanylpentane-1-sulfonate Chemical compound OS(=O)(=O)CC[C@@H](N)[C@@H](S)C(=O)N[C@@H]([C@H](C)CC)C(=O)N1CCC(C(O)=O)[C@H]1C(O)=O HUWSZNZAROKDRZ-RRLWZMAJSA-N 0.000 claims description 8
- YQOLEILXOBUDMU-KRWDZBQOSA-N (4R)-5-[(6-bromo-3-methyl-2-pyrrolidin-1-ylquinoline-4-carbonyl)amino]-4-(2-chlorophenyl)pentanoic acid Chemical compound CC1=C(C2=C(C=CC(=C2)Br)N=C1N3CCCC3)C(=O)NC[C@H](CCC(=O)O)C4=CC=CC=C4Cl YQOLEILXOBUDMU-KRWDZBQOSA-N 0.000 claims description 8
- HCCNBKFJYUWLEX-UHFFFAOYSA-N 7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)-3-(pyrazin-2-ylmethylamino)pyrido[3,4-b]pyrazin-2-one Chemical compound O=C1N(CCOCCC)C2=CC(C=3C=NC(OC)=CC=3)=NC=C2N=C1NCC1=CN=CC=N1 HCCNBKFJYUWLEX-UHFFFAOYSA-N 0.000 claims description 8
- 239000004472 Lysine Substances 0.000 claims description 8
- LJOOWESTVASNOG-UFJKPHDISA-N [(1s,3r,4ar,7s,8s,8as)-3-hydroxy-8-[2-[(4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-7-methyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-yl] (2s)-2-methylbutanoate Chemical compound C([C@H]1[C@@H](C)C=C[C@H]2C[C@@H](O)C[C@@H]([C@H]12)OC(=O)[C@@H](C)CC)CC1C[C@@H](O)CC(=O)O1 LJOOWESTVASNOG-UFJKPHDISA-N 0.000 claims description 8
- PSLUFJFHTBIXMW-WYEYVKMPSA-N [(3r,4ar,5s,6s,6as,10s,10ar,10bs)-3-ethenyl-10,10b-dihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-6-(2-pyridin-2-ylethylcarbamoyloxy)-5,6,6a,8,9,10-hexahydro-2h-benzo[f]chromen-5-yl] acetate Chemical compound O([C@@H]1[C@@H]([C@]2(O[C@](C)(CC(=O)[C@]2(O)[C@@]2(C)[C@@H](O)CCC(C)(C)[C@@H]21)C=C)C)OC(=O)C)C(=O)NCCC1=CC=CC=N1 PSLUFJFHTBIXMW-WYEYVKMPSA-N 0.000 claims description 8
- 229940125773 compound 10 Drugs 0.000 claims description 8
- 229940127204 compound 29 Drugs 0.000 claims description 8
- 229940125936 compound 42 Drugs 0.000 claims description 8
- 229940125844 compound 46 Drugs 0.000 claims description 8
- 229940126545 compound 53 Drugs 0.000 claims description 8
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 claims description 8
- IOMMMLWIABWRKL-WUTDNEBXSA-N nazartinib Chemical compound C1N(C(=O)/C=C/CN(C)C)CCCC[C@H]1N1C2=C(Cl)C=CC=C2N=C1NC(=O)C1=CC=NC(C)=C1 IOMMMLWIABWRKL-WUTDNEBXSA-N 0.000 claims description 8
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 claims description 7
- ASGMFNBUXDJWJJ-JLCFBVMHSA-N (1R,3R)-3-[[3-bromo-1-[4-(5-methyl-1,3,4-thiadiazol-2-yl)phenyl]pyrazolo[3,4-d]pyrimidin-6-yl]amino]-N,1-dimethylcyclopentane-1-carboxamide Chemical compound BrC1=NN(C2=NC(=NC=C21)N[C@H]1C[C@@](CC1)(C(=O)NC)C)C1=CC=C(C=C1)C=1SC(=NN=1)C ASGMFNBUXDJWJJ-JLCFBVMHSA-N 0.000 claims description 6
- AOSZTAHDEDLTLQ-AZKQZHLXSA-N (1S,2S,4R,8S,9S,11S,12R,13S,19S)-6-[(3-chlorophenyl)methyl]-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-azapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one Chemical compound C([C@@H]1C[C@H]2[C@H]3[C@]([C@]4(C=CC(=O)C=C4[C@@H](F)C3)C)(F)[C@@H](O)C[C@@]2([C@@]1(C1)C(=O)CO)C)N1CC1=CC=CC(Cl)=C1 AOSZTAHDEDLTLQ-AZKQZHLXSA-N 0.000 claims description 6
- ABJSOROVZZKJGI-OCYUSGCXSA-N (1r,2r,4r)-2-(4-bromophenyl)-n-[(4-chlorophenyl)-(2-fluoropyridin-4-yl)methyl]-4-morpholin-4-ylcyclohexane-1-carboxamide Chemical compound C1=NC(F)=CC(C(NC(=O)[C@H]2[C@@H](C[C@@H](CC2)N2CCOCC2)C=2C=CC(Br)=CC=2)C=2C=CC(Cl)=CC=2)=C1 ABJSOROVZZKJGI-OCYUSGCXSA-N 0.000 claims description 6
- GLGNXYJARSMNGJ-VKTIVEEGSA-N (1s,2s,3r,4r)-3-[[5-chloro-2-[(1-ethyl-6-methoxy-2-oxo-4,5-dihydro-3h-1-benzazepin-7-yl)amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide Chemical compound CCN1C(=O)CCCC2=C(OC)C(NC=3N=C(C(=CN=3)Cl)N[C@H]3[C@H]([C@@]4([H])C[C@@]3(C=C4)[H])C(N)=O)=CC=C21 GLGNXYJARSMNGJ-VKTIVEEGSA-N 0.000 claims description 6
- 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 claims description 6
- GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 claims description 6
- IUSARDYWEPUTPN-OZBXUNDUSA-N (2r)-n-[(2s,3r)-4-[[(4s)-6-(2,2-dimethylpropyl)spiro[3,4-dihydropyrano[2,3-b]pyridine-2,1'-cyclobutane]-4-yl]amino]-3-hydroxy-1-[3-(1,3-thiazol-2-yl)phenyl]butan-2-yl]-2-methoxypropanamide Chemical compound C([C@H](NC(=O)[C@@H](C)OC)[C@H](O)CN[C@@H]1C2=CC(CC(C)(C)C)=CN=C2OC2(CCC2)C1)C(C=1)=CC=CC=1C1=NC=CS1 IUSARDYWEPUTPN-OZBXUNDUSA-N 0.000 claims description 6
- YJLIKUSWRSEPSM-WGQQHEPDSA-N (2r,3r,4s,5r)-2-[6-amino-8-[(4-phenylphenyl)methylamino]purin-9-yl]-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound C=1C=C(C=2C=CC=CC=2)C=CC=1CNC1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O YJLIKUSWRSEPSM-WGQQHEPDSA-N 0.000 claims description 6
- VIJSPAIQWVPKQZ-BLECARSGSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-acetamido-5-(diaminomethylideneamino)pentanoyl]amino]-4-methylpentanoyl]amino]-4,4-dimethylpentanoyl]amino]-4-methylpentanoyl]amino]propanoyl]amino]-5-(diaminomethylideneamino)pentanoic acid Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(C)=O VIJSPAIQWVPKQZ-BLECARSGSA-N 0.000 claims description 6
- WWTBZEKOSBFBEM-SPWPXUSOSA-N (2s)-2-[[2-benzyl-3-[hydroxy-[(1r)-2-phenyl-1-(phenylmethoxycarbonylamino)ethyl]phosphoryl]propanoyl]amino]-3-(1h-indol-3-yl)propanoic acid Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)O)C(=O)C(CP(O)(=O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1C=CC=CC=1)CC1=CC=CC=C1 WWTBZEKOSBFBEM-SPWPXUSOSA-N 0.000 claims description 6
- STBLNCCBQMHSRC-BATDWUPUSA-N (2s)-n-[(3s,4s)-5-acetyl-7-cyano-4-methyl-1-[(2-methylnaphthalen-1-yl)methyl]-2-oxo-3,4-dihydro-1,5-benzodiazepin-3-yl]-2-(methylamino)propanamide Chemical compound O=C1[C@@H](NC(=O)[C@H](C)NC)[C@H](C)N(C(C)=O)C2=CC(C#N)=CC=C2N1CC1=C(C)C=CC2=CC=CC=C12 STBLNCCBQMHSRC-BATDWUPUSA-N 0.000 claims description 6
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 claims description 6
- IWZSHWBGHQBIML-ZGGLMWTQSA-N (3S,8S,10R,13S,14S,17S)-17-isoquinolin-7-yl-N,N,10,13-tetramethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-amine Chemical compound CN(C)[C@H]1CC[C@]2(C)C3CC[C@@]4(C)[C@@H](CC[C@@H]4c4ccc5ccncc5c4)[C@@H]3CC=C2C1 IWZSHWBGHQBIML-ZGGLMWTQSA-N 0.000 claims description 6
- MPDDTAJMJCESGV-CTUHWIOQSA-M (3r,5r)-7-[2-(4-fluorophenyl)-5-[methyl-[(1r)-1-phenylethyl]carbamoyl]-4-propan-2-ylpyrazol-3-yl]-3,5-dihydroxyheptanoate Chemical compound C1([C@@H](C)N(C)C(=O)C2=NN(C(CC[C@@H](O)C[C@@H](O)CC([O-])=O)=C2C(C)C)C=2C=CC(F)=CC=2)=CC=CC=C1 MPDDTAJMJCESGV-CTUHWIOQSA-M 0.000 claims description 6
- KQZLRWGGWXJPOS-NLFPWZOASA-N 1-[(1R)-1-(2,4-dichlorophenyl)ethyl]-6-[(4S,5R)-4-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-5-methylcyclohexen-1-yl]pyrazolo[3,4-b]pyrazine-3-carbonitrile Chemical compound ClC1=C(C=CC(=C1)Cl)[C@@H](C)N1N=C(C=2C1=NC(=CN=2)C1=CC[C@@H]([C@@H](C1)C)N1[C@@H](CCC1)CO)C#N KQZLRWGGWXJPOS-NLFPWZOASA-N 0.000 claims description 6
- WZZBNLYBHUDSHF-DHLKQENFSA-N 1-[(3s,4s)-4-[8-(2-chloro-4-pyrimidin-2-yloxyphenyl)-7-fluoro-2-methylimidazo[4,5-c]quinolin-1-yl]-3-fluoropiperidin-1-yl]-2-hydroxyethanone Chemical compound CC1=NC2=CN=C3C=C(F)C(C=4C(=CC(OC=5N=CC=CN=5)=CC=4)Cl)=CC3=C2N1[C@H]1CCN(C(=O)CO)C[C@@H]1F WZZBNLYBHUDSHF-DHLKQENFSA-N 0.000 claims description 6
- ONBQEOIKXPHGMB-VBSBHUPXSA-N 1-[2-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 ONBQEOIKXPHGMB-VBSBHUPXSA-N 0.000 claims description 6
- UNILWMWFPHPYOR-KXEYIPSPSA-M 1-[6-[2-[3-[3-[3-[2-[2-[3-[[2-[2-[[(2r)-1-[[2-[[(2r)-1-[3-[2-[2-[3-[[2-(2-amino-2-oxoethoxy)acetyl]amino]propoxy]ethoxy]ethoxy]propylamino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-1-oxopropan-2-yl Chemical compound O=C1C(SCCC(=O)NCCCOCCOCCOCCCNC(=O)COCC(=O)N[C@@H](CSC[C@@H](COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC)C(=O)NCC(=O)N[C@H](CO)C(=O)NCCCOCCOCCOCCCNC(=O)COCC(N)=O)CC(=O)N1CCNC(=O)CCCCCN\1C2=CC=C(S([O-])(=O)=O)C=C2CC/1=C/C=C/C=C/C1=[N+](CC)C2=CC=C(S([O-])(=O)=O)C=C2C1 UNILWMWFPHPYOR-KXEYIPSPSA-M 0.000 claims description 6
- FQMZXMVHHKXGTM-UHFFFAOYSA-N 2-(1-adamantyl)-n-[2-[2-(2-hydroxyethylamino)ethylamino]quinolin-5-yl]acetamide Chemical compound C1C(C2)CC(C3)CC2CC13CC(=O)NC1=CC=CC2=NC(NCCNCCO)=CC=C21 FQMZXMVHHKXGTM-UHFFFAOYSA-N 0.000 claims description 6
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 6
- PYRKKGOKRMZEIT-UHFFFAOYSA-N 2-[6-(2-cyclopropylethoxy)-9-(2-hydroxy-2-methylpropyl)-1h-phenanthro[9,10-d]imidazol-2-yl]-5-fluorobenzene-1,3-dicarbonitrile Chemical compound C1=C2C3=CC(CC(C)(O)C)=CC=C3C=3NC(C=4C(=CC(F)=CC=4C#N)C#N)=NC=3C2=CC=C1OCCC1CC1 PYRKKGOKRMZEIT-UHFFFAOYSA-N 0.000 claims description 6
- YSUIQYOGTINQIN-UZFYAQMZSA-N 2-amino-9-[(1S,6R,8R,9S,10R,15R,17R,18R)-8-(6-aminopurin-9-yl)-9,18-difluoro-3,12-dihydroxy-3,12-bis(sulfanylidene)-2,4,7,11,13,16-hexaoxa-3lambda5,12lambda5-diphosphatricyclo[13.2.1.06,10]octadecan-17-yl]-1H-purin-6-one Chemical compound NC1=NC2=C(N=CN2[C@@H]2O[C@@H]3COP(S)(=O)O[C@@H]4[C@@H](COP(S)(=O)O[C@@H]2[C@@H]3F)O[C@H]([C@H]4F)N2C=NC3=C2N=CN=C3N)C(=O)N1 YSUIQYOGTINQIN-UZFYAQMZSA-N 0.000 claims description 6
- TVTJUIAKQFIXCE-HUKYDQBMSA-N 2-amino-9-[(2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-7-prop-2-ynyl-1H-purine-6,8-dione Chemical compound NC=1NC(C=2N(C(N(C=2N=1)[C@@H]1O[C@@H]([C@H]([C@H]1O)F)CO)=O)CC#C)=O TVTJUIAKQFIXCE-HUKYDQBMSA-N 0.000 claims description 6
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Classifications
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- 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
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- 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
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- A—HUMAN NECESSITIES
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- 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
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
본 발명은 아미노산 유래 중합 단위 및 지질 유래 중합 단위를 포함하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염을 제공한다. 상기 아미노산 유래 중합 단위는 아미노산-지질 접합 화합물의 일 부분이고, 일측단에 카르복실기를 가지고, 타측단에 아미노기를 가지는 아미노산, 또는 아미노산 유도체로부터 중합에 의해 얻어진다. 상기 지질 유래 중합 단위는 아미노산-지질 접합 화합물의 일 부분이고, 탄소 수가 10 내지 20인 탄화수소를 포함하는 지질 화합물로부터 중합에 의해 얻어진다. 본 발명에 따른 아미노산-지질 접합 화합물은 핵산을 전달하기 위한 지질 나노입자의 일 구성인 이온화성 지질로서 이용된다.The present invention provides an amino acid-lipid conjugation compound comprising a polymerized unit derived from an amino acid and a polymerized unit derived from a lipid, or a pharmaceutically acceptable salt thereof. The amino acid-derived polymerized unit is a part of an amino acid-lipid conjugation compound, and is obtained by polymerization from an amino acid or amino acid derivative having a carboxyl group at one end and an amino group at the other end. The lipid-derived polymerized unit is a part of an amino acid-lipid conjugation compound and is obtained by polymerization from a lipid compound containing a hydrocarbon having 10 to 20 carbon atoms. The amino acid-lipid conjugation compound according to the present invention is used as an ionizable lipid, which is a component of lipid nanoparticles for delivering nucleic acids.
Description
본 발명은 핵산 전달용 아미노산-지질 접합 화합물 및 이를 포함하는 지질 나노입자에 관한 것이다.The present invention relates to amino acid-lipid conjugation compounds for nucleic acid delivery and lipid nanoparticles containing the same.
약물 치료법에 있어서, 약물의 부작용을 줄이고 효능 및 효과를 극대화시켜 필요한 양의 약물을 효과적으로 전달할 수 있도록 설계한 약물전달시스템(Drug Delivery System, DDS)은 중요하다. 상기 약물전달시스템은 신약개발과 맞먹는 경제적 이익을 창출할 수 있으며, 성공 가능성이 큰 고부가가치 핵심 기술로서 약물투여를 효율화함으로써 환자 치료의 질을 높이는데 기여할 수 있다.In drug therapy, a drug delivery system (DDS) designed to effectively deliver the required amount of drug by reducing the side effects of the drug and maximizing its efficacy and effectiveness is important. The drug delivery system can generate economic benefits equivalent to the development of new drugs, and as a high-value core technology with a high probability of success, it can contribute to improving the quality of patient treatment by streamlining drug administration.
특히, 생물학적 약물전달시스템에서 원하는 반응에 영향을 주기 위해 핵산의 전달과 관련된 다양한 연구가 진행되어 왔다. Antisense RNA, siRNA, mRNA 등의 핵산은 생체 내에서 특정 단백질의 발현을 억제할 수 있는 물질로, 암, 유전병, 감염질병, 자가면역 질환 등의 치료에 중요한 도구로 각광받고 있다. 일부 핵산, 예를 들어, mRNA 또는 플라스미드는 예를 들어 단백질 또는 효소의 결핍과 관련된 질환 치료에 유용하도록 특정 세포 산물의 발현을 달성하기 위해 사용될 수 있다. 핵산의 발현 산물은 기존의 단백질 레벨을 증대시킬 수 있고 단백질의 결핍 또는 비기능적 버전을 치환할 수 있고, 또한 세포 또는 생물에 새로운 단백질 및 관련된 기능성을 도입할 수 있다. 핵산 기반 치료제는 엄청난 가능성을 가지지만 이러한 가능성을 실현하기 위해 세포 또는 유기체 내의 적절한 부위에 핵산을 보다 효과적으로 전달할 필요가 있다.In particular, various studies related to the delivery of nucleic acids have been conducted to influence the desired response in biological drug delivery systems. Nucleic acids such as antisense RNA, siRNA, and mRNA are substances that can inhibit the expression of specific proteins in vivo, and are attracting attention as important tools in the treatment of cancer, genetic diseases, infectious diseases, and autoimmune diseases. Some nucleic acids, such as mRNA or plasmids, can be used to achieve expression of specific cellular products, such as to be useful in treating diseases associated with deficiencies of proteins or enzymes. Expression products of nucleic acids can augment existing protein levels, replace deficient or non-functional versions of proteins, and also introduce new proteins and associated functionality into cells or organisms. Nucleic acid-based therapeutics have tremendous potential, but to realize this potential, there is a need to more effectively deliver nucleic acids to the appropriate site within a cell or organism.
유전정보를 가진 핵산을 전달함에 있어서, 바이러스 전달체가 효과적일 수 있으나, 면역원성, 주입된 DNA 크기의 한계, 대량 생산의 어려움과 같은 몇 가지 결점으로 인해 바이러스 전달체에 대한 이용이 제한되고, 이에 대한 대체수단으로 양이온성 리포좀 및 중합체와 같은 비 바이러스성 전달체가 주목을 받고 있다. 그러나, 이러한 비 바이러스성 전달체는 열악한 생체적합성 및 비 생분해성으로 인해 현저하게 높은 세포독성을 나타낼 수 있으며, 트랜스픽션 효율이 낮은 문제점이 존재한다.Although viral carriers can be effective in delivering nucleic acids containing genetic information, several drawbacks, such as immunogenicity, limitations in the size of the injected DNA, and difficulties in mass production, limit the use of viral carriers. As an alternative, non-viral delivery vehicles such as cationic liposomes and polymers are attracting attention. However, these non-viral carriers can exhibit significantly high cytotoxicity due to poor biocompatibility and non-biodegradability, and have low transfection efficiency.
이에 본 발명자는 비 바이러스성 전달체로 지질 나노입자에 대한 지속적인 연구 끝에 지질 나노입자의 구성성분으로 적합한 신규한 이온화성 지질 화합물을 합성함으로써 발명을 완성하였다.Accordingly, after continuous research on lipid nanoparticles as non-viral carriers, the present inventor completed the invention by synthesizing a novel ionizable lipid compound suitable as a component of lipid nanoparticles.
본 발명은 핵산 전달을 위한 지질 나노입자의 핵심 구성 요소인 이온화 지질로서, 지질 나노입자에서 세포 및 동물에 효율적으로 핵산을 전달하여 관련 질병 예방 또는 치료를 가능하게 하는 아미노산-지질 접합 화합물을 제공하고자 한다.The present invention is an ionized lipid, which is a core component of lipid nanoparticles for nucleic acid delivery, and aims to provide an amino acid-lipid conjugation compound that enables the prevention or treatment of related diseases by efficiently delivering nucleic acids from lipid nanoparticles to cells and animals. do.
본 발명의 제1 측면에 따르면,According to the first aspect of the present invention,
본 발명은 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염을 제공한다.The present invention provides an amino acid-lipid conjugation compound, or a pharmaceutically acceptable salt thereof.
본 발명의 일 구체예에 따르면, 상기 아미노산-지질 접합 화합물은 아미노산 유래 중합 단위 및 지질 유래 중합 단위를 포함한다.According to one embodiment of the present invention, the amino acid-lipid conjugation compound includes a polymerized unit derived from an amino acid and a polymerized unit derived from a lipid.
본 발명의 일 구체예에 따르면, 상기 아미노산 유래 중합 단위는 아미노산-지질 접합 화합물의 일 부분이고, 일측단에 카르복실기를 가지고, 타측단에 아미노기를 가지는 아미노산, 또는 아미노산 유도체로부터 중합에 의해 얻어진다.According to one embodiment of the present invention, the amino acid-derived polymerized unit is a part of an amino acid-lipid conjugation compound and is obtained by polymerization from an amino acid or amino acid derivative having a carboxyl group at one end and an amino group at the other end.
본 발명의 일 구체예에 따르면, 상기 지질 유래 중합 단위는 아미노산-지질 접합 화합물의 일 부분이고, 탄소 수가 10 내지 20인 탄화수소를 포함하는 지질 화합물로부터 중합에 의해 얻어진다.According to one embodiment of the present invention, the lipid-derived polymerized unit is a part of an amino acid-lipid conjugation compound and is obtained by polymerization from a lipid compound containing a hydrocarbon having 10 to 20 carbon atoms.
본 발명의 일 구체예에 따르면, 상기 아미노산은 알파 아미노산을 포함한다.According to one embodiment of the present invention, the amino acid includes an alpha amino acid.
본 발명의 일 구체예에 따르면, 상기 알파 아미노산은 이소류신, 류신, 리신, 발린, 메티오닌, 페닐알라닌, 트레오닌, 트립토판, 히스티딘, 아르기닌, 알라닌, 아스파라긴, 아스파르트산, 시스테인, 글루탐산, 글루타민, 글리신, 프롤린, 세린, 티로신 및 이의 조합으로 이루어진 군으로부터 선택되는 아미노산을 포함한다.According to one embodiment of the present invention, the alpha amino acids are isoleucine, leucine, lysine, valine, methionine, phenylalanine, threonine, tryptophan, histidine, arginine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, It contains an amino acid selected from the group consisting of serine, tyrosine, and combinations thereof.
본 발명의 일 구체예에 따르면, 상기 아미노산 유도체는 아미노산의 카르복실기가 알코올 화합물과 중합 반응하여 형성된 에스테르 화합물이거나 아미노산의 카르복실기가 아민 화합물과 중합 반응하여 형성된 아마이드 화합물을 포함한다.According to one embodiment of the present invention, the amino acid derivative includes an ester compound formed by polymerizing the carboxyl group of an amino acid with an alcohol compound or an amide compound formed by polymerizing the carboxyl group of an amino acid with an amine compound.
본 발명의 일 구체예에 따르면, 상기 알코올 화합물은 메탄올, 에탄올, 프로판올, 부탄올 또는 이의 조합을 포함한다.According to one embodiment of the present invention, the alcohol compound includes methanol, ethanol, propanol, butanol, or a combination thereof.
본 발명의 일 구체예에 따르면, 상기 아민 화합물은 아미노 메탄올, 아미노 에탄올, 아미노 프로판올, 아미노 부탄올, 메틸아미노 메탄올, 메틸아미노 에탄올, 메틸아미노 프로판올, 메틸아미노 부탄올, 에틸아미노 메탄올, 에틸아미노 에탄올, 에틸아미노 프로판올, 에틸아미노 부탄올, 아미노메틸 이미다졸, 아미노에틸 이미다졸, 아미노프로필 이미다졸, 아미노부틸 이미다졸 또는 이의 조합을 포함한다.According to one embodiment of the present invention, the amine compound is amino methanol, amino ethanol, amino propanol, amino butanol, methylamino methanol, methylamino ethanol, methylamino propanol, methylamino butanol, ethylamino methanol, ethylamino ethanol, ethyl Includes amino propanol, ethylamino butanol, aminomethyl imidazole, aminoethyl imidazole, aminopropyl imidazole, aminobutyl imidazole, or combinations thereof.
본 발명의 일 구체예에 따르면, 상기 지질 화합물은 탄화수소의 일측단에 아미노산의 아미노기와 중합할 수 있는 작용기를 갖는다.According to one embodiment of the present invention, the lipid compound has a functional group capable of polymerizing with the amino group of an amino acid at one end of the hydrocarbon.
본 발명의 일 구체예에 따르면, 상기 지질 화합물의 작용기는 카르복실기, 알데히드기 또는 아세테이트기이다.According to one embodiment of the present invention, the functional group of the lipid compound is a carboxyl group, an aldehyde group, or an acetate group.
본 발명의 일 구체예에 따르면, 상기 아미노산-지질 접합 화합물은 1개 내지 10개의 아미노산 유래 중합 단위를 포함한다.According to one embodiment of the present invention, the amino acid-lipid conjugation compound includes 1 to 10 amino acid-derived polymerization units.
본 발명의 일 구체예에 따르면, 상기 아미노산-지질 접합 화합물이 2개 이상의 아미노산 유래 중합 단위를 포함하는 경우, 각각의 아미노산 유래 중합 단위는 동일하거나 상이하다.According to one embodiment of the present invention, when the amino acid-lipid conjugation compound includes two or more amino acid-derived polymerized units, each amino acid-derived polymerized unit is the same or different.
본 발명의 일 구체예에 따르면, 상기 아미노산-지질 접합 화합물은 하기 화합물 1 내지 53이다.According to one embodiment of the present invention, the amino acid-lipid conjugation compound is the following compounds 1 to 53.
본 발명의 제2 측면에 따르면,According to the second aspect of the present invention,
본 발명은 상술한 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체를 포함하는 지질 나노입자를 제공한다.The present invention provides lipid nanoparticles comprising the above-described amino acid-lipid conjugate compound, phospholipid, cholesterol, and PEG-lipid conjugate.
본 발명의 일 구체예에 따르면, 상기 지질 나노입자는 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 30몰% 내지 60몰%의 아미노산-지질 접합 화합물을 포함한다.According to one embodiment of the present invention, the lipid nanoparticles include 30 mol% to 60 mol% of the amino acid-lipid conjugate compound based on the total number of moles of the amino acid-lipid conjugate compound, phospholipid, cholesterol, and PEG-lipid conjugate. do.
핵산 전달을 위해 사용되는 지질 나노입자의 경우, 조성물 중에서 이온화 지질이 효능에 가장 큰 영향을 미치는 것으로 알려져 있다. 구체적으로, 세포 내에서 핵산의 엔도솜(endosome) 탈출을 가능하게 함으로써, 다른 조성물보다 핵산 전달 효율에 직접적인 영향을 미친다. 따라서, 보다 효능이 증가된 핵산 기반 유전자 치료제를 개발하기 위해서는 전달 효율이 증대된 신규한 이온화 지질이 필요하다. 본 발명은 아미노산을 포함하는 지질 접합 화합물을 합성하고, 핵산 전달 능력을 세포 및 동물 모델에서 평가하여, 핵산 치료제 개발에 필요한 지질 나노입자 조성물로서의 유용성을 확인하였다.In the case of lipid nanoparticles used for nucleic acid delivery, ionized lipids are known to have the greatest effect on efficacy among the compositions. Specifically, it has a more direct effect on nucleic acid delivery efficiency than other compositions by enabling the escape of nucleic acids from endosomes within cells. Therefore, in order to develop nucleic acid-based gene therapy with increased efficacy, novel ionized lipids with increased delivery efficiency are needed. The present invention synthesized a lipid conjugation compound containing amino acids, evaluated its nucleic acid delivery ability in cell and animal models, and confirmed its usefulness as a lipid nanoparticle composition required for the development of nucleic acid therapeutics.
도 1은 본 발명의 실험예 4에 따라 지질 나노입자(화합물 28, 29 및 40)에 대한 HEK293 세포주에서의 단백질 발현을 평가한 luminescence 측정 결과를 나타낸 그래프이다.
도 2는 본 발명의 실험예 5에 따라 지질 나노입자(화합물 28, 29, 40 및 42)에 대한 마우스에서의 단백질 발현 분포를 나타낸 이미지이다.
도 3은 본 발명의 실험예 5에 따라 지질 나노입자(화합물 45, 46, 47 및 53)에 대한 마우스에서의 단백질 발현 분포를 나타낸 이미지이다.
도 4는 본 발명의 실험예 5에 따라 지질 나노입자(화합물 28, 29, 40, 42, 45, 46, 47 및 53)에 대한 마우스 간 부위의 luminescence 측정 결과를 나타낸 그래프이다.Figure 1 is a graph showing the results of luminescence measurement evaluating protein expression in HEK293 cell line for lipid nanoparticles (compounds 28, 29, and 40) according to Experimental Example 4 of the present invention.
Figure 2 is an image showing the protein expression distribution in mice for lipid nanoparticles (compounds 28, 29, 40, and 42) according to Experimental Example 5 of the present invention.
Figure 3 is an image showing the protein expression distribution in mice for lipid nanoparticles (compounds 45, 46, 47, and 53) according to Experimental Example 5 of the present invention.
Figure 4 is a graph showing the luminescence measurement results of mouse liver for lipid nanoparticles (compounds 28, 29, 40, 42, 45, 46, 47, and 53) according to Experimental Example 5 of the present invention.
본 발명에 따라 제공되는 구체예는 하기의 설명에 의하여 모두 달성될 수 있다. 하기의 설명은 본 발명의 바람직한 구체예를 기술하는 것으로 이해되어야 하며, 본 발명이 반드시 이에 한정되는 것은 아님을 이해해야 한다.The embodiments provided according to the present invention can all be achieved by the following description. It should be understood that the following description describes preferred embodiments of the present invention, and that the present invention is not necessarily limited thereto.
본 발명은 핵산 전달용으로 이용되는 신규한 아미노산-지질 접합 화합물 및 이를 포함하는 지질 나노입자를 제공한다. 본 발명에 따른 아미노산-지질 접합 화합물은 화합물 그 자체에 국한되지 않으며, 토토머(tautomer) 또는 입체 이성질체(stereoisomer)와 같은 이성질체까지 확장되며, 이의 약학적으로 허용가능한 염을 포함한다.The present invention provides novel amino acid-lipid conjugation compounds used for nucleic acid delivery and lipid nanoparticles containing the same. The amino acid-lipid conjugation compound according to the present invention is not limited to the compound itself, but extends to isomers such as tautomers or stereoisomers, and includes pharmaceutically acceptable salts thereof.
상기 “약학적으로 허용가능한 염”은 산 부가 염과 염기 부가 염의 양쪽 모두가 포함된다. 상기 산 부가 염은 예를 들어, 염산, 트리플루오로아세트산, 포름산, 시트르산, 푸마르산, 푸마레이트 모노-나트륨, p-톨루엔설폰산, 스테아르산, 시트레이트 다이-나트륨, 타르타르산, 말산, 락트산, 석신산, 및 살리실산 등이 부가된 염일 수 있지만, 해당 기술분야에서 일반적으로 사용되는 산 부가 염이면 특별히 제한되지 않는다. 상기 염기 부가 염은 예를 들어, 암모늄, 나트륨, 칼륨, 칼슘, 마그네슘, 이소프로필 아민, 디에틸아민, 에탄올아민, 트리메틸아민, 디사이클로헥실아민, 콜린 및 카페인 등이 부가된 염일 수 있지만, 해당 기술분야에서 일반적으로 사용되는 염기 부가 염이면 특별히 제한되지 않는다.The “pharmaceutically acceptable salt” includes both acid addition salts and base addition salts. The acid addition salts include, for example, hydrochloric acid, trifluoroacetic acid, formic acid, citric acid, fumaric acid, mono-sodium fumarate, p-toluenesulfonic acid, stearic acid, di-sodium citrate, tartaric acid, malic acid, lactic acid, sulfuric acid. It may be a salt to which acidic acid, salicylic acid, etc. have been added, but there is no particular limitation as long as it is an acid addition salt commonly used in the relevant technical field. The base addition salt may be, for example, a salt to which ammonium, sodium, potassium, calcium, magnesium, isopropyl amine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, caffeine, etc. are added. There is no particular limitation as long as it is a base addition salt commonly used in the technical field.
또한, 본 발명에 따른 아미노산-지질 접합 화합물은 단순히 아미노산과 지질이 중합에 의해 접합된 화합물을 의미할 뿐만 아니라, 나아가 아미노산 유도체와 지질 유도체가 중합에 의해 접합된 화합물을 의미할 수도 있으며, 아미노산과 지질이 중합된 후 산화나 환원에 의해 수소가 제거되거나 첨가되는 화합물을 의미할 수도 있다. 구체적으로, 산화에 의해 이웃하는 원자에 연결된 2개의 수소가 제거되면 단일결합이 이중결합으로 바뀔 수 있으며, 환원에 의해 이중결합으로 연결된 이웃하는 원자에 2개의 수소가 첨가되면 이중결합이 단일결합으로 바뀔 수 있다. 예를 들어, 아미노산-지질 접합 화합물에 존재하는 카보닐기는 환원되어 알코올이 될 수 있다. 상기 아미노산-지질 접합 화합물은 일부 또는 전부가 산화되거나 환원된 형태일 수 있다.In addition, the amino acid-lipid conjugation compound according to the present invention not only refers to a compound in which an amino acid and a lipid are conjugated by polymerization, but may also refer to a compound in which an amino acid derivative and a lipid derivative are conjugated by polymerization. It may also refer to a compound in which hydrogen is removed or added by oxidation or reduction after lipid polymerization. Specifically, when two hydrogens connected to neighboring atoms are removed by oxidation, a single bond can be changed to a double bond, and when two hydrogens are added to neighboring atoms connected to a double bond by reduction, a double bond can be changed to a single bond. It can change. For example, carbonyl groups present in amino acid-lipid conjugation compounds can be reduced to alcohols. The amino acid-lipid conjugation compound may be partially or entirely oxidized or reduced.
본 발명에 따른 아미노산-지질 접합 화합물은 이온화성 지질로서 주변 pH에 따라 전하상태가 변하는 지질일 수 있다. 이러한 이온화성 지질은 지질과 유사한 특성을 가지며, 약물(예를 들면, 음이온성 약물 및/또는 핵산)과의 정전기적 상호작용을 통하여 상기 약물이 지질 나노입자 내에 높은 효율로 봉입되도록 하는 역할을 수행할 수 있다.The amino acid-lipid conjugation compound according to the present invention may be an ionizable lipid whose charge state changes depending on the surrounding pH. These ionizable lipids have properties similar to lipids and play a role in ensuring that the drug is encapsulated within lipid nanoparticles with high efficiency through electrostatic interaction with drugs (e.g., anionic drugs and/or nucleic acids). can do.
이하에서, 본 발명에 따른 아미노산-지질 접합 화합물의 구체예를 상세히 설명한다.Hereinafter, specific examples of the amino acid-lipid conjugation compound according to the present invention will be described in detail.
본 발명의 일 구체예는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염을 제공한다. 여기서, “약학적으로 허용가능한 염”은 상술한 내용에 따른 산 부가 염 또는 염기 부가 염일 수 있다.One embodiment of the present invention provides an amino acid-lipid conjugation compound, or a pharmaceutically acceptable salt thereof. Here, the “pharmaceutically acceptable salt” may be an acid addition salt or a base addition salt according to the above description.
상기 아미노산-지질 접합 화합물은 아미노산 단량체와 지질 단량체가 중합된 화합물로서, 아미노산 유래 중합 단위 및 지질 유래 중합 단위를 포함한다. 상기 아미노산-지질 접합 화합물은 아미노산 또는 아미노산 유도체와 지질 화합물의 공중합체로도 표현될 수 있다. 여기서, 상기 아미노산 유래 중합 단위는 아미노산-지질 접합 화합물의 일 부분이고, 일측단에 카르복실기를 가지고, 타측단에 아미노기를 가지는 아미노산 또는 아미노산 유도체로부터 중합에 의해 얻어질 수 있다. 또한, 상기 지질 유래 중합 단위는 아미노산-지질 접합 화합물의 일 부분이고, 탄소 수가 10 내지 20인 탄화수소를 포함하는 지질 화합물로부터 중합에 의해 얻어질 수 있다. 상기 탄화수소는 탄소와 수소로 이루어진 포화 또는 불포화 작용기를 의미하고, 본 발명의 일 구체예에 따르면, 상기 탄화수소는 알킬기 또는 알케닐기를 포함한다. 본 발명의 일 구체예에 따르면, 지질 유래 중합 단위에서 포함되는 탄화수소는 10 이상, 11 이상, 12 이상, 13 이상, 14 이상, 15 이상, 16 이상이고, 20 이하, 19 이하, 18 이하일 수 있다.The amino acid-lipid conjugation compound is a compound in which an amino acid monomer and a lipid monomer are polymerized, and includes a polymerized unit derived from an amino acid and a polymerized unit derived from a lipid. The amino acid-lipid conjugation compound may also be expressed as a copolymer of an amino acid or an amino acid derivative and a lipid compound. Here, the amino acid-derived polymerized unit is a part of an amino acid-lipid conjugation compound, and can be obtained by polymerization from an amino acid or amino acid derivative having a carboxyl group at one end and an amino group at the other end. Additionally, the lipid-derived polymerized unit is a part of an amino acid-lipid conjugation compound and can be obtained by polymerization from a lipid compound containing a hydrocarbon having 10 to 20 carbon atoms. The hydrocarbon refers to a saturated or unsaturated functional group consisting of carbon and hydrogen, and according to one embodiment of the present invention, the hydrocarbon includes an alkyl group or an alkenyl group. According to one embodiment of the present invention, the number of hydrocarbons included in the lipid-derived polymerization unit may be 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 20 or less, 19 or less, and 18 or less. .
상기 아미노산은 양측단에 중합 반응이 가능한 작용기인 카르복실기 또는 아미노기를 가지고 있기 때문에, 아미노산은 서로 중합되어 펩타이드와 같은 이량체 또는 다량체를 형성할 수 있다. 이와 달리, 상기 지질 화합물은 일측단이 알킬기로 중합 반응이 가능한 작용기를 포함하지 않기 때문에, 중합 반응이 가능한 작용기를 포함하는 지질 화합물의 타측단에서 아미노산과 중합되는 경우, 아미노산-지질 접합 화합물은 지질 화합물의 일측단 방향으로 더 이상 중합 반응이 진행될 수 없다. 본 발명의 일 구체예에 따른 지질 화합물은 아미노산의 아미노기와 중합할 수 있는 작용기를 포함하며, 이 경우, 본 발명의 일 구체예에 따른 아미노산-지질 접합 화합물은 '(아미노산 유래 중합 단위)n-(지질 유래 중합 단위)'와 같은 형태의 화학식을 가진다.Since the amino acid has a carboxyl group or amino group at both ends, which are functional groups capable of polymerization, the amino acids can be polymerized with each other to form a dimer or multimer such as a peptide. In contrast, since the lipid compound does not contain a functional group capable of polymerization with an alkyl group at one end, when the lipid compound containing a functional group capable of polymerization is polymerized with an amino acid at the other end, the amino acid-lipid conjugate compound is a lipid compound. The polymerization reaction can no longer proceed in the direction of one side of the compound. The lipid compound according to an embodiment of the present invention includes a functional group capable of polymerizing with the amino group of an amino acid. In this case, the amino acid-lipid conjugation compound according to an embodiment of the present invention is '(amino acid-derived polymerization unit) n - It has a chemical formula of the form ‘(lipid-derived polymerization unit)’.
본 발명의 일 구체예에 따르면, 상기 아미노산은 알파 아미노산을 포함한다. 상기 알파 아미노산은 카르복실기와 아미노기가 하나의 탄소인 메틸렌으로 연결된 구조의 화합물을 의미하며, 상기 메틸렌의 하나 이상의 수소는 다른 작용기로 치환될 수 있다.According to one embodiment of the present invention, the amino acid includes an alpha amino acid. The alpha amino acid refers to a compound in which a carboxyl group and an amino group are linked to one carbon methylene, and one or more hydrogens of the methylene may be substituted with other functional groups.
본 발명의 일 구체예에 따르면, 상기 알파 아미노산은 대표적인 20종의 아미노산을 포함하며, 구체적으로, 이소류신(isoleucine, ile), 류신(leucine, leu), 리신(lysine, lys), 발린(valine, val), 메티오닌(methionine, met), 페닐알라닌(phenylalanine, phe), 트레오닌(threonine, thr), 트립토판(tryptophan, trp), 히스티딘(histidine, his), 아르기닌(arginine, arg), 알라닌(alanine, ala), 아스파라긴(asparagine, asn), 아스파르트산(aspartic acid, asp), 시스테인(cysteine, cys), 글루탐산(glutamic acid, glu), 글루타민(glutamine, gln), 글리신(glycine, gly), 프롤린(proline, pro), 세린(serine, ser), 티로신(tyrosine, tyr) 및 이의 조합으로 이루어진 군으로부터 선택되는 아미노산을 포함한다. 보다 구체적으로, 상기 알파 아미노산은 이소류신, 류신, 리신, 발린, 메티오닌, 페닐알라닌, 트레오닌, 트립토판, 히스티딘, 아르기닌, 알라닌, 시스테인, 글리신, 프롤린, 세린, 티로신 및 이의 조합으로 이루어진 군으로부터 선택될 수 있다. 본 발명의 일 구체예에 따르면, 상기 알파 아미노산은 히스티딘을 포함할 수 있다.According to one embodiment of the present invention, the alpha amino acid includes 20 representative amino acids, specifically, isoleucine (ile), leucine (leu), lysine (lys), and valine (valine). val), methionine (met), phenylalanine (phe), threonine (thro), tryptophan (trp), histidine (his), arginine (arg), alanine (ala) ), asparagine (asn), aspartic acid (asp), cysteine (cys), glutamic acid (glu), glutamine (gln), glycine (gly), proline (proline) , pro), serine (ser), tyrosine (tyrosine, tyr), and combinations thereof. More specifically, the alpha amino acid may be selected from the group consisting of isoleucine, leucine, lysine, valine, methionine, phenylalanine, threonine, tryptophan, histidine, arginine, alanine, cysteine, glycine, proline, serine, tyrosine, and combinations thereof. . According to one embodiment of the present invention, the alpha amino acid may include histidine.
본 발명의 일 구체예에 따르면, 상기 아미노산 유도체는 아미노산의 카르복실기가 알코올 화합물과 중합 반응하여 형성된 에스테르 화합물이거나 아미노산의 카르복실기가 아민 화합물과 중합 반응하여 형성된 아마이드 화합물을 포함한다.According to one embodiment of the present invention, the amino acid derivative includes an ester compound formed by polymerizing the carboxyl group of an amino acid with an alcohol compound or an amide compound formed by polymerizing the carboxyl group of an amino acid with an amine compound.
상기 알코올 화합물은 탄소 수가 7 이하, 구체적으로는 4 이하의 탄화수소를 가진 화합물 일 수 있다. 본 발명의 일 구체예에 따르면, 상기 알코올 화합물은 메탄올, 에탄올, 프로판올, 부탄올 또는 이의 조합을 포함한다.The alcohol compound may be a hydrocarbon compound having a carbon number of 7 or less, specifically, a hydrocarbon number of 4 or less. According to one embodiment of the present invention, the alcohol compound includes methanol, ethanol, propanol, butanol, or a combination thereof.
상기 아민 화합물은 1차 또는 2차 아민 화합물일 수 있으며, 탄소 수가 7 이하, 구체적으로는 4 이하의 탄화수소를 가진 화합물 일 수 있다. 상기 탄화수소의 타측단에는 아릴기 또는 헤테로아릴기가 치환될 수 있다. 본 발명의 일 구체예에 따르면, 상기 아민 화합물은 아미노 메탄올, 아미노 에탄올, 아미노 프로판올, 아미노 부탄올, 메틸아미노 메탄올, 메틸아미노 에탄올, 메틸아미노 프로판올, 메틸아미노 부탄올, 에틸아미노 메탄올, 에틸아미노 에탄올, 에틸아미노 프로판올, 에틸아미노 부탄올, 아미노메틸 이미다졸, 아미노에틸 이미다졸, 아미노프로필 이미다졸, 아미노부틸 이미다졸 또는 이의 조합을 포함한다.The amine compound may be a primary or secondary amine compound, and may be a compound with hydrocarbons having 7 or less carbon atoms, specifically 4 or less carbon atoms. The other end of the hydrocarbon may be substituted with an aryl group or heteroaryl group. According to one embodiment of the present invention, the amine compound is amino methanol, amino ethanol, amino propanol, amino butanol, methylamino methanol, methylamino ethanol, methylamino propanol, methylamino butanol, ethylamino methanol, ethylamino ethanol, ethyl Includes amino propanol, ethylamino butanol, aminomethyl imidazole, aminoethyl imidazole, aminopropyl imidazole, aminobutyl imidazole, or combinations thereof.
상기 지질 화합물은 탄화수소의 일측단에 아미노산의 아미노기와 중합할 수 있는 작용기를 가질 수 있다. 이에 따라, 본 발명의 일 구체예에 따른 아미노산-지질 접합 화합물은 '(아미노산 유래 중합 단위)n-(지질 유래 중합 단위)'와 같은 형태의 화학식을 가질 수 있으며, 상기 화학식에서 아미노산 유래 중합 단위측 말단에는 카르복실기가 존재하고, 지질 유래 중합 단위측 말단에는 알킬기, 알케닐기와 같은 탄화수소가 존재할 수 있다. 말단의 아미노산 유래 중합 단위가 아미노산 유도체로부터 중합에 의해 얻어지는 경우, 예를 들어, 카르복실기와 중합 반응하는 알코올 화합물 또는 아민 화합물에 의해 말단이 형성될 수 있다.The lipid compound may have a functional group capable of polymerizing with the amino group of an amino acid at one end of the hydrocarbon. Accordingly, the amino acid-lipid conjugation compound according to one embodiment of the present invention may have a chemical formula of the form '(amino acid-derived polymerized unit) n -(lipid-derived polymerized unit)', and in the formula, the amino acid-derived polymerized unit A carboxyl group may be present at the side terminal, and a hydrocarbon such as an alkyl group or alkenyl group may be present at the end of the lipid-derived polymer unit. When the terminal amino acid-derived polymerized unit is obtained by polymerization from an amino acid derivative, the terminal may be formed, for example, by an alcohol compound or amine compound that polymerizes with a carboxyl group.
본 발명의 일 구체예에 따르면, 상기 지질 화합물의 작용기는 카르복실기, 알데히드기 또는 아세테이트기이다. 예를 들어, 상기 카르복실기는 아미노산의 아미노기와 중합 반응하여 아마이드 작용기를 형성한다. 상기 알데히드기는 아미노산의 아미노기와 중합 반응하여 아미노기의 수소에 지질 화합물의 탄화수소가 치환된다. 상기 아세테이트기는 아미노산의 아미노기와 중합 반응하여 아세테이트기의 탄소와 탄소 사이의 이중 결합이 끊어지면서 아미노기의 질소와 연결된다.According to one embodiment of the present invention, the functional group of the lipid compound is a carboxyl group, an aldehyde group, or an acetate group. For example, the carboxyl group polymerizes with the amino group of an amino acid to form an amide functional group. The aldehyde group undergoes a polymerization reaction with the amino group of the amino acid, and the hydrogen of the amino group is replaced with the hydrocarbon of the lipid compound. The acetate group undergoes a polymerization reaction with the amino group of the amino acid, thereby breaking the double bond between the carbons of the acetate group and connecting it to the nitrogen of the amino group.
상기 아미노산은 양측단에 중합 반응을 할 수 있는 작용기를 갖기 때문에, 양측단에서 아미노산, 아미노산 유도체 또는 지질 화합물 등과 중합 반응을 할 수 있다. 이와 달리, 상기 아미노산 유도체 또는 지질 화합물은 일측단에 중합 반응을 할 수 있는 작용기를 갖기 때문에, 일측단에서 아미노산, 아미노산 유도체 또는 지질 화합물 등과 중합 반응을 할 수 있다. 상기 아미노산 유도체 또는 지질 화합물은 중합 반응을 할 수 있는 작용기를 포함하고 있지 않은 타측단은 더 이상 중합 반응이 일어날 수 없기 때문에, 아미노산-지질 접합 화합물의 말단을 형성할 수 있다. 이러한 특징을 고려할 때, 본 발명의 일 구체예에 따른 아미노산-지질 접합 화합물은 '(아미노산 유래 중합 단위)n-(지질 유래 중합 단위)'와 같은 형태의 화학식을 가질 수 있다.Since the amino acid has functional groups capable of polymerization at both ends, it can undergo polymerization with amino acids, amino acid derivatives, or lipid compounds at both ends. In contrast, since the amino acid derivative or lipid compound has a functional group capable of polymerization at one end, it can undergo a polymerization reaction with the amino acid, amino acid derivative, or lipid compound at one end. Since the other end of the amino acid derivative or lipid compound does not contain a functional group capable of polymerization and no further polymerization reaction can occur, it can form the terminal of an amino acid-lipid conjugation compound. Considering these characteristics, the amino acid-lipid conjugation compound according to one embodiment of the present invention may have a chemical formula such as '(amino acid-derived polymerization unit) n -(lipid-derived polymerization unit)'.
본 발명의 일 구체예에 따르면, 상기 아미노산-지질 접합 화합물은 1개 내지 10개의 아미노산 유래 중합 단위를 포함한다. 구체적으로, 상기 아미노산-지질 접합 화합물에서 아미노산 유래 중합 단위는 1개 이상이고, 10개 이하, 9개 이하, 8개 이하, 7개 이하, 6개 이하, 5개 이하, 4개 이하, 3개 이하, 2개 이하이며, 1개 내지 10개, 1개 내지 5개, 1개 내지 3개, 1개 내지 2개일 수 있다. 상기 아미노산-지질 접합 화합물이 2개 이상의 아미노산 유래 중합 단위를 포함하는 경우, 각각의 아미노산 유래 중합 단위는 동일하거나 상이할 수 있다.According to one embodiment of the present invention, the amino acid-lipid conjugation compound includes 1 to 10 amino acid-derived polymerization units. Specifically, in the amino acid-lipid conjugation compound, the amino acid-derived polymerization unit is one or more, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, and 3 or less. Hereinafter, it may be 2 or less, and may be 1 to 10, 1 to 5, 1 to 3, or 1 to 2. When the amino acid-lipid conjugation compound includes two or more amino acid-derived polymerized units, each amino acid-derived polymerized unit may be the same or different.
본 발명의 일 구체예에 따르면, 상기 아미노산-지질 접합 화합물은 하기 표 1의 화합물 1 내지 53이다.According to one embodiment of the present invention, the amino acid-lipid conjugation compound is compounds 1 to 53 in Table 1 below.
상기 표 1에서 화합물 10은 이중결합으로 연결된 이웃하는 원자 모두에 수소가 첨가되어 환원된 형태이며, 화합물 10 또한 본 발명에 따른 아미노산-지질 접합 화합물에 포함된다.In Table 1, Compound 10 is in a reduced form by adding hydrogen to all neighboring atoms connected by a double bond, and Compound 10 is also included in the amino acid-lipid conjugation compound according to the present invention.
이하에서, 상술한 아미노산-지질 접합 화합물을 포함하는 지질 나노입자의 구체예를 상세히 설명한다.Hereinafter, specific examples of lipid nanoparticles containing the above-described amino acid-lipid conjugation compound will be described in detail.
본 발명의 일 구체예는 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체를 포함하는 지질 나노입자를 제공한다. 상기 아미노산-지질 접합 화합물은 상술한 내용에 따른다.One embodiment of the present invention provides lipid nanoparticles containing amino acid-lipid conjugation compounds, phospholipids, cholesterol, and PEG-lipid conjugates. The amino acid-lipid conjugation compound is as described above.
상기 인지질은 지질 나노입자 내에서 아미노산-지질 접합 화합물 및 약물과 상호작용하여 형성된 코어를 감싸서 보호하는 역할을 한다. 또한, 상기 인지질은 타겟 세포의 인지질 이중층과 결합하여 약물의 세포 내 전달시 세포막 통과 및 엔도좀 탈출(endosomal escape)을 용이하게 한다. 상기 인지질은 상술한 기능성을 가지고, 해당 기술분야에서 일반적으로 사용되는 물질이면 특별히 제한되지 않는다. 본 발명의 일 구체예에 따르면, 상기 인지질은 디스테아로일포스파티딜콜린(distearoylphosphatidylcholine, DSPC), 팔미토일올레오일포스파티딜콜린(palmitoyloleoylphosphatidylcholine, POPC), 에그 포스파티딜콜린(egg phosphatidylcholine, EPC), 디올레오일포스파티딜콜린(dioleoylphosphatidylcholine, DOPC), 디팔미토일포스파티딜콜린(dipalmitoylphosphatidylcholine, DPPC), 디올레오일포스파티딜글리세롤(dioleoylphosphatidylglycerol, DOPG), 디팔미토일포스파티딜글리세롤(dipalmitoylphosphatidylglycerol, DPPG), 디스테아로일포스파티딜에탄올아민(distearoylphosphatidylethanolamine, DSPE), 포스파티딜에탄올아민(Phosphatidylethanolamine, PE), 디팔미토일포스파티딜에탄올아민(dipalmitoylphosphatidylethanolamine), 1,2-디올레일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1-팔미토일-2-올레일-sn-글리세로-3-포스포에탄올아민(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE), 1-팔미토일-2-올레일-sn-글리세로-3-포스포콜린(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC), 1,2-디올레일-sn-글리세로-3-[포스포-L-세린](1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], DOPS) 및 이의 조합으로 이루어진 군으로부터 선택된다. 상기 인지질은 전달하고자 하는 핵산의 종류에 따라 효과가 우수한 종을 선택할 수 있다.The phospholipid serves to surround and protect the core formed by interacting with the amino acid-lipid conjugation compound and drug within the lipid nanoparticle. In addition, the phospholipid binds to the phospholipid bilayer of the target cell, facilitating passage through the cell membrane and endosomal escape during intracellular delivery of the drug. The phospholipid is not particularly limited as long as it has the above-described functionality and is a material commonly used in the relevant technical field. According to one embodiment of the present invention, the phospholipids include distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), and dioleoylphosphatidylcholine (dioleoylphosphatidylcholine, DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DS) PE), phosphatidylethanol Amine (Phosphatidylethanolamine, PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE), 1-palmitoyl-2 -Oleyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC), 1,2-dioleyl-sn-glycero-3-[phos phospho-L-serine](1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], DOPS) and combinations thereof. The phospholipid may be selected from a species with excellent effectiveness depending on the type of nucleic acid to be delivered.
상기 콜레스테롤은 지질 나노입자 내에서 지질 충전에 형태적 측면에서 견고성을 부여하여, 나노입자의 코어 및 표면에 분산되어 나노입자의 안정성을 향상시키는 역할을 한다.The cholesterol provides morphological rigidity to the lipid filling within the lipid nanoparticles and is dispersed on the core and surface of the nanoparticles to improve the stability of the nanoparticles.
상기 PEG-지질 접합체는 지질의 일측 단부에 친수성 중합체인 PEG(polyethyleneglycol) 중합체가 결합된 지질을 의미한다. 상기 PEG-지질 접합체는 지질 나노입자 내에서 나노입자의 혈청 내 입자 안정성에 기여하며, 나노입자 간 응집을 막는 역할을 할 뿐만 아니라, 핵산의 생체 내 전달시 분해효소로부터 핵산을 보호하여 핵산의 체내 안정성을 강화시키며, 나노입자 내 봉입된 약물의 반감기를 증가시킬 수 있다. 상기 PEG-지질 접합체에서 PEG와 지질은 해당 기술분야에서 일반적으로 사용되는 방법에 따라 접합될 수 있으며, PEG는 경우에 따라 연결기에 의해 지질과 접합될 수 있다. 본 발명의 일 구체예에 따르면, 상기 PEG-지질 접합체에서 지질은 세라마이드(ceramide), 디미리스톨글리세롤(dimyristoylglycerol, DMG), 석시노일 디아글리세롤(succinoyl-diacylglycerol, s-DAG), 디스테아로일포스파티딜콜린(distearoylphosphatidylcholine, DSPC), 디스테아로일포스파티딜에탄올아민(distearoylphosphatidylethanolamine, DSPE), 콜레스테롤 및 이의 조합으로 이루어진 군으로부터 선택된다.The PEG-lipid conjugate refers to a lipid in which PEG (polyethyleneglycol) polymer, a hydrophilic polymer, is bound to one end of the lipid. The PEG-lipid conjugate not only contributes to the stability of nanoparticles in serum within lipid nanoparticles and prevents aggregation between nanoparticles, but also protects nucleic acids from degrading enzymes during in vivo delivery of nucleic acids, thereby preserving the nucleic acids in the body. It enhances stability and can increase the half-life of drugs encapsulated in nanoparticles. In the PEG-lipid conjugate, PEG and lipids may be conjugated according to methods commonly used in the relevant technical field, and PEG may be conjugated to lipids by a linking group in some cases. According to one embodiment of the present invention, the lipid in the PEG-lipid conjugate is ceramide, dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s-DAG), and distearo. It is selected from the group consisting of distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), cholesterol, and combinations thereof.
상기 지질 나노입자는 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체를 혼합하여 지질 혼합물을 형성한 후, Antisense RNA, siRNA, mRNA 등의 핵산을 혼합함으로써, 제조된다. 따라서, 상기 지질 나노입자는 핵산을 더 포함할 수 있다. 구체적인 제조방법은 해당 기술분야에서 일반적인 방법을 사용하면 특별히 제한되지 않는다.The lipid nanoparticles are manufactured by mixing amino acid-lipid conjugation compounds, phospholipids, cholesterol, and PEG-lipid conjugates to form a lipid mixture, and then mixing nucleic acids such as antisense RNA, siRNA, and mRNA. Therefore, the lipid nanoparticles may further include nucleic acids. The specific manufacturing method is not particularly limited as long as general methods in the relevant technical field are used.
상기 지질 혼합물에서 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체는 각 성분의 기능성을 고려하여 적절한 비율로 혼합된다.In the lipid mixture, the amino acid-lipid conjugate compound, phospholipid, cholesterol, and PEG-lipid conjugate are mixed in an appropriate ratio considering the functionality of each component.
본 발명의 일 구체예에 따르면, 상기 아미노산-지질 접합 화합물은 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 30몰% 내지 60몰%가 지질 혼합물에 포함된다. 구체적으로, 상기 아미노산-지질 접합 화합물은 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 30몰% 이상, 31몰% 이상, 32몰% 이상, 33몰% 이상, 34몰% 이상, 35몰% 이상이 지질 혼합물에 포함되고, 60몰% 이하, 59몰% 이하, 58몰% 이하, 57몰% 이하, 56몰% 이하, 55몰% 이하, 54몰% 이하, 53몰% 이하, 52몰% 이하, 51몰% 이하, 50몰% 이하가 지질 혼합물에 포함될 수 있다.According to one embodiment of the present invention, the amino acid-lipid conjugation compound is included in the lipid mixture in an amount of 30 mol% to 60 mol% based on the total number of moles of the amino acid-lipid conjugation compound, phospholipid, cholesterol, and PEG-lipid conjugate. . Specifically, the amino acid-lipid conjugation compound is 30 mol% or more, 31 mol% or more, 32 mol% or more, 33 mol% or more based on the total number of moles of amino acid-lipid conjugation compound, phospholipid, cholesterol and PEG-lipid conjugate. , 34 mol% or more, 35 mol% or more is contained in the lipid mixture, 60 mol% or less, 59 mol% or less, 58 mol% or less, 57 mol% or less, 56 mol% or less, 55 mol% or less, 54 mol% Hereinafter, 53 mol% or less, 52 mol% or less, 51 mol% or less, and 50 mol% or less may be included in the lipid mixture.
본 발명의 일 구체예에 따르면, 상기 인지질은 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 5몰% 내지 50몰%가 지질 혼합물에 포함된다. 구체적으로, 상기 인지질은 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 5몰% 이상, 6몰% 이상, 7몰% 이상, 8몰% 이상, 9몰% 이상, 10몰% 이상이 지질 혼합물에 포함되고, 50몰% 이하, 49몰% 이하, 48몰% 이하, 47몰% 이하, 46.5몰% 이하가 지질 혼합물에 포함될 수 있다.According to one embodiment of the present invention, the phospholipid is included in the lipid mixture in an amount of 5 mol% to 50 mol% based on the total number of moles of the amino acid-lipid conjugate compound, phospholipid, cholesterol, and PEG-lipid conjugate. Specifically, the phospholipid is 5 mol% or more, 6 mol% or more, 7 mol% or more, 8 mol% or more, 9 mol% based on the total number of moles of amino acid-lipid conjugation compound, phospholipid, cholesterol and PEG-lipid conjugate. Above, 10 mol% or more may be included in the lipid mixture, and 50 mol% or less, 49 mol% or less, 48 mol% or less, 47 mol% or less, and 46.5 mol% or less may be included in the lipid mixture.
본 발명의 일 구체예에 따르면, 상기 콜레스테롤은 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 10몰% 내지 45몰%가 지질 혼합물에 포함된다. 구체적으로, 상기 콜레스테롤은 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 10몰% 이상, 11몰% 이상, 12몰% 이상, 13몰% 이상, 14몰% 이상, 15몰% 이상, 16몰% 이상이 지질 혼합물에 포함되고, 45몰% 이하, 44몰% 이하, 43몰% 이하, 42몰% 이하, 41몰% 이하, 40몰% 이하, 39몰% 이하, 38.5몰% 이하가 지질 혼합물에 포함될 수 있다.According to one embodiment of the present invention, the cholesterol is included in the lipid mixture in an amount of 10 mol% to 45 mol% based on the total number of moles of the amino acid-lipid conjugate compound, phospholipid, cholesterol, and PEG-lipid conjugate. Specifically, the cholesterol is 10 mol% or more, 11 mol% or more, 12 mol% or more, 13 mol% or more, 14 mol% based on the total number of moles of amino acid-lipid conjugation compound, phospholipid, cholesterol and PEG-lipid conjugate. 15 mol% or more, 16 mol% or more is included in the lipid mixture, 45 mol% or less, 44 mol% or less, 43 mol% or less, 42 mol% or less, 41 mol% or less, 40 mol% or less, 39 moles. % or less, 38.5 mol% or less may be included in the lipid mixture.
본 발명의 일 구체예에 따르면, 상기 PEG-지질 접합체는 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 0.5몰% 내지 3.0몰%가 지질 혼합물에 포함된다. 구체적으로, 상기 PEG-지질 접합체는 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 0.5몰% 이상, 0.6몰% 이상, 0.7몰% 이상, 0.8몰% 이상, 0.9몰% 이상, 1.0몰% 이상, 1.1몰% 이상, 1.2몰% 이상, 1.3몰% 이상, 1.4몰% 이상, 1.5몰% 이상이 지질 혼합물에 포함되고, 3.0몰% 이하, 2.9몰% 이하, 2.8몰% 이하, 2.7몰% 이하, 2.6몰% 이하, 2.5몰% 이하가 지질 혼합물에 포함될 수 있다.According to one embodiment of the present invention, the PEG-lipid conjugate is included in the lipid mixture in an amount of 0.5 mol% to 3.0 mol% based on the total number of moles of the amino acid-lipid conjugate compound, phospholipid, cholesterol, and PEG-lipid conjugate. Specifically, the PEG-lipid conjugate is 0.5 mol% or more, 0.6 mol% or more, 0.7 mol% or more, 0.8 mol% or more, based on the total number of moles of amino acid-lipid conjugate compound, phospholipid, cholesterol and PEG-lipid conjugate. 0.9 mol% or more, 1.0 mol% or more, 1.1 mol% or more, 1.2 mol% or more, 1.3 mol% or more, 1.4 mol% or more, 1.5 mol% or more are included in the lipid mixture, 3.0 mol% or less, 2.9 mol% or less , 2.8 mol% or less, 2.7 mol% or less, 2.6 mol% or less, and 2.5 mol% or less may be included in the lipid mixture.
본 발명의 일 구체예에 따르면, 상기 지질 나노입자에 포함되는 핵산은 소간섭리보핵산(siRNA), 리보좀 리보핵산(rRNA), 리보핵산(RNA), 디옥시리보핵산(DNA), 상보성 디옥시리보핵산(cDNA), 앱타머(aptamer), 전령 리보핵산(mRNA), 운반 리보핵산(tRNA), 안티센스 올리고뉴클레오티드, shRNA, miRNA, 리보자임(ribozyme), PNA, DNAzyme, 및 유전자교정을 위한 sgRNA 및 이의 조합으로 이루어진 군으로부터 선택된다.According to one embodiment of the present invention, the nucleic acids contained in the lipid nanoparticles include small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and complementary deoxyribonucleic acid (cDNA). ), aptamer, messenger ribonucleic acid (mRNA), transport ribonucleic acid (tRNA), antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, DNAzyme, and sgRNA for gene editing and combinations thereof. is selected from the group consisting of
실시예Example
화합물의 제조:Preparation of compounds:
이하에서는 상기 표 1의 화합물 1 내지 53에 대한 구체적인 제조방법을 설명한다. 하기 실시예 1 내지 21에서 A-B-C와 같이 단량체의 중합 형태로 표시된 펩타이드는 A, B 및 C 단량체의 중합으로 형성된 펩타이드를 의미한다. 여기서, 단량체가 아미노산인 경우, 좌측 말단에는 아미노기가 위치하고, 우측 말단에는 카르복실기가 위치하도록 중합된다. 예를 들면, A, B 및 C가 아미노산인 A-B-C 중합 펩타이드는 A 측에 아미노기를 가지고, C 측에 카르복실기를 가지는 펩타이드이다.Below, specific preparation methods for compounds 1 to 53 in Table 1 will be described. In Examples 1 to 21 below, peptides expressed in the form of polymerization of monomers such as A-B-C refer to peptides formed by polymerization of A, B, and C monomers. Here, when the monomer is an amino acid, it is polymerized so that an amino group is located at the left end and a carboxyl group is located at the right end. For example, an A-B-C polymerized peptide in which A, B, and C are amino acids is a peptide that has an amino group on the A side and a carboxyl group on the C side.
실시예 1: 화합물 1의 제조Example 1: Preparation of Compound 1
[화합물 1][Compound 1]
His-His-His-His-His-His-His-His-His-His 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 스테아르산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고, C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 1을 얻었다.His-His-His-His-His-His-His-His-His-His polymerized peptide was synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of stearic acid were dissolved in dimethylformamide and put into a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. Then it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed phase HPLC using a C18 reversed phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 1.
수득한 화합물 1을 HPLC 및 LC-MS로 확인하였다.The obtained compound 1 was confirmed by HPLC and LC-MS.
HPLC purity: 99%, LC-MS found: 1655HPLC purity: 99%, LC-MS found: 1655
실시예 2: 화합물 2의 제조Example 2: Preparation of Compound 2
[화합물 2][Compound 2]
His-His-His-His-His 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고, C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 2를 얻었다.The His-His-His-His-His polymerized peptide was synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed phase HPLC using a C18 reversed phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 2.
수득한 화합물 2를 HPLC 및 LC-MS로 확인하였다.The obtained compound 2 was confirmed by HPLC and LC-MS.
HPLC purity: 95%, LC-MS found: 941HPLC purity: 95%, LC-MS found: 941
실시예 3: 화합물 3의 제조Example 3: Preparation of Compound 3
[화합물 3][Compound 3]
His-His-Lys-His-His 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고, C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 3을 얻었다.The His-His-Lys-His-His polymerized peptide was synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed phase HPLC using a C18 reversed phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 3.
수득한 화합물 3을 HPLC 및 LC-MS로 확인하였다.The obtained compound 3 was confirmed by HPLC and LC-MS.
HPLC purity: 99%, LC-MS found: 1171HPLC purity: 99%, LC-MS found: 1171
실시예 4: 화합물 4의 제조Example 4: Preparation of Compound 4
[화합물 4][Compound 4]
His-His 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 스테아르산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 4를 얻었다.His-His polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of stearic acid were dissolved in dimethylformamide and put into a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. Then it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 4.
수득한 화합물 4를 HPLC 및 LC-MS로 확인하였다.The obtained compound 4 was confirmed by HPLC and LC-MS.
HPLC purity: 95%, LC-MS found: 558HPLC purity: 95%, LC-MS found: 558
실시예 5: 화합물 5의 제조Example 5: Preparation of Compound 5
[화합물 5][Compound 5]
His-His-Lys 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 스테아르산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 5를 얻었다.His-His-Lys polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of stearic acid were dissolved in dimethylformamide and put into a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. Then it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 5.
수득한 화합물 5를 HPLC 및 LC-MS로 확인하였다.The obtained compound 5 was confirmed by HPLC and LC-MS.
HPLC purity: 89%, LC-MS found: 952HPLC purity: 89%, LC-MS found: 952
실시예 6: 화합물 6의 제조Example 6: Preparation of Compound 6
[화합물 6][Compound 6]
Tyr-Tyr-His-His-His 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 미리스트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 6을 얻었다.Tyr-Tyr-His-His-His polymerized peptide was synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of myristic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 6.
수득한 화합물 6을 HPLC 및 LC-MS로 확인하였다.The obtained compound 6 was confirmed by HPLC and LC-MS.
HPLC purity: 97%, LC-MS found: 965HPLC purity: 97%, LC-MS found: 965
실시예 7: 화합물 7의 제조Example 7: Preparation of Compound 7
[화합물 7][Compound 7]
Thr-Thr-His-His-His 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 미리스트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 7을 얻었다.Thr-Thr-His-His-His polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of myristic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 7.
수득한 화합물 7을 HPLC 및 LC-MS로 확인하였다.The obtained compound 7 was confirmed by HPLC and LC-MS.
HPLC purity: 99%, LC-MS found: 841HPLC purity: 99%, LC-MS found: 841
실시예 8: 화합물 8의 제조Example 8: Preparation of Compound 8
[화합물 8][Compound 8]
Lys-His-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 8을 얻었다.Lys-His-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 8.
수득한 화합물 8을 HPLC 및 LC-MS로 확인하였다.The obtained compound 8 was confirmed by HPLC and LC-MS.
HPLC purity: 92%, LC-MS found: 774HPLC purity: 92%, LC-MS found: 774
실시예 9: 화합물 9의 제조Example 9: Preparation of Compound 9
[화합물 9][Compound 9]
Lys-Gly-His-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 9를 얻었다.Lys-Gly-His-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 9.
수득한 화합물 9를 HPLC 및 LC-MS로 확인하였다.The obtained compound 9 was confirmed by HPLC and LC-MS.
HPLC purity: 85%, LC-MS found: 831HPLC purity: 85%, LC-MS found: 831
실시예 10: 화합물 10의 제조Example 10: Preparation of Compound 10
[화합물 10][Compound 10]
Gly-Lys-His-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 10을 얻었다.Gly-Lys-His-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 10.
수득한 화합물 10을 HPLC 및 LC-MS로 확인하였다.The obtained compound 10 was confirmed by HPLC and LC-MS.
HPLC purity: 90%, LC-MS found: 831HPLC purity: 90%, LC-MS found: 831
실시예 11: 화합물 11의 제조Example 11: Preparation of Compound 11
[화합물 11][Compound 11]
Lys-Ser-His-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 11을 얻었다.Lys-Ser-His-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 11.
수득한 화합물 11을 HPLC 및 LC-MS로 확인하였다.The obtained compound 11 was confirmed by HPLC and LC-MS.
HPLC purity: 85%, LC-MS found: 860HPLC purity: 85%, LC-MS found: 860
실시예 12: 화합물 12의 제조Example 12: Preparation of Compound 12
[화합물 12][Compound 12]
Lys-Thr-His-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 12를 얻었다.Lys-Thr-His-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 12.
수득한 화합물 12를 HPLC 및 LC-MS로 확인하였다.The obtained compound 12 was confirmed by HPLC and LC-MS.
HPLC purity: 85%, LC-MS found: 875HPLC purity: 85%, LC-MS found: 875
실시예 13: 화합물 13의 제조Example 13: Preparation of Compound 13
[화합물 13][Compound 13]
Lys-Arg-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 13을 얻었다.Lys-Arg-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 13.
수득한 화합물 13을 HPLC 및 LC-MS로 확인하였다.The obtained compound 13 was confirmed by HPLC and LC-MS.
HPLC purity: 85%, LC-MS found: 793HPLC purity: 85%, LC-MS found: 793
실시예 14: 화합물 14의 제조Example 14: Preparation of Compound 14
[화합물 14][Compound 14]
Lys-Lys-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 14를 얻었다.Lys-Lys-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 14.
수득한 화합물 14를 HPLC 및 LC-MS로 확인하였다.The obtained compound 14 was confirmed by HPLC and LC-MS.
HPLC purity: 85%, LC-MS found: 765HPLC purity: 85%, LC-MS found: 765
실시예 15: 화합물 15의 제조Example 15: Preparation of Compound 15
[화합물 15][Compound 15]
Pro-Lys-Lys-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 15를 얻었다.Pro-Lys-Lys-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 15.
수득한 화합물 15를 HPLC 및 LC-MS로 확인하였다.The obtained compound 15 was confirmed by HPLC and LC-MS.
HPLC purity: 85%, LC-MS found: 862HPLC purity: 85%, LC-MS found: 862
실시예 16: 화합물 16의 제조Example 16: Preparation of Compound 16
[화합물 16][Compound 16]
Arg-Arg 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 16을 얻었다.Arg-Arg polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 16.
수득한 화합물 16을 HPLC 및 LC-MS로 확인하였다.The obtained compound 16 was confirmed by HPLC and LC-MS.
HPLC purity: 88%, LC-MS found: 568HPLC purity: 88%, LC-MS found: 568
실시예 17: 화합물 17의 제조Example 17: Preparation of Compound 17
[화합물 17][Compound 17]
Lys-Lys 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 17을 얻었다.Lys-Lys polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 17.
수득한 화합물 17을 HPLC 및 LC-MS로 확인하였다.The obtained compound 17 was confirmed by HPLC and LC-MS.
HPLC purity: 89%, LC-MS found: 512HPLC purity: 89%, LC-MS found: 512
실시예 18: 화합물 18의 제조Example 18: Preparation of Compound 18
[화합물 18][Compound 18]
Pro-Lys-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 18을 얻었다.Pro-Lys-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 18.
수득한 화합물 18을 HPLC 및 LC-MS로 확인하였다.The obtained compound 18 was confirmed by HPLC and LC-MS.
HPLC purity: 85%, LC-MS found: 495HPLC purity: 85%, LC-MS found: 495
실시예 19: 화합물 19의 제조Example 19: Preparation of Compound 19
[화합물 19][Compound 19]
Lys-His-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 미리스트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 19를 얻었다.Lys-His-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of myristic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 19.
수득한 화합물 19를 HPLC 및 LC-MS로 확인하였다.The obtained compound 19 was confirmed by HPLC and LC-MS.
HPLC purity: 88%, LC-MS found: 718HPLC purity: 88%, LC-MS found: 718
실시예 20: 화합물 20의 제조Example 20: Preparation of Compound 20
[화합물 20][Compound 20]
Lys-Arg-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 20을 얻었다.Lys-Arg-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 20.
수득한 화합물 20을 HPLC 및 LC-MS로 확인하였다.The obtained compound 20 was confirmed by HPLC and LC-MS.
HPLC purity: 85%, LC-MS found: 793HPLC purity: 85%, LC-MS found: 793
실시예 21: 화합물 21의 제조Example 21: Preparation of Compound 21
[화합물 21][Compound 21]
Lys-Lys-MeOH 중합 펩타이드는 표준 고체상 펩타이드 합성 프로토콜을 사용하여 합성하였다. 이와 팔미트산 10 당량을 다이메틸폼아마이드에 용해하여 레진이 있는 반응기에 투입하고, 2M 하이드록시벤조트리아졸 10 당량, 2M 1,3-디이소프로필 카보디아미드 10 당량을 다이메틸폼아마이드에 넣은 후 4 시간 동안 교반하였다. 2% 트리플루오로아세트산/디클로로메탄을 레진이 있는 반응기에 투입하고, 2분 동안 교반시킨 후, 여액을 수득 및 건조하여 조 펩타이드를 얻었다. 조 펩타이드를 DW에 용해시키고 C18 역상 컬럼을 사용하는 역상 HPLC로 정제하였다. 용출은 0.1%(v/v) 트리플루오로아세트산을 함유하는 물-아세토니트릴 선형 구배(아세토니트릴의 10~75%(v/v))로 수행되었다. 펩타이드의 순수한 부분을 수집하고 동결건조하여 화합물 21을 얻었다.Lys-Lys-MeOH polymerized peptides were synthesized using standard solid-phase peptide synthesis protocols. 10 equivalents of palmitic acid were dissolved in dimethylformamide and added to a reactor containing resin, and 10 equivalents of 2M hydroxybenzotriazole and 10 equivalents of 2M 1,3-diisopropyl carbodiamide were added to dimethylformamide. After addition, it was stirred for 4 hours. 2% trifluoroacetic acid/dichloromethane was added to the reactor containing the resin, stirred for 2 minutes, and the filtrate was collected and dried to obtain a crude peptide. The crude peptide was dissolved in DW and purified by reversed-phase HPLC using a C18 reversed-phase column. Elution was performed with a water-acetonitrile linear gradient (10–75% (v/v) of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The pure portion of the peptide was collected and lyophilized to obtain compound 21.
수득한 화합물 21을 HPLC 및 LC-MS로 확인하였다.The obtained compound 21 was confirmed by HPLC and LC-MS.
HPLC purity: 85%, LC-MS found: 765HPLC purity: 85%, LC-MS found: 765
실시예 22: 화합물 22의 제조Example 22: Preparation of Compound 22
[화합물 22][Compound 22]
디클로로메탄(DCM)(1mL) 중 글리신 메틸 에스테르 하이드로클로라이드(25mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 92% 도데칸알(100mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc /헥산)로 정제하여 화합물 22를 얻었다.A solution of glycine methyl ester hydrochloride (25 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in dichloromethane (DCM) (1 mL) was stirred at room temperature for 30 min. A solution of 92% dodecaneal (100 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 22.
수득한 화합물 22를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 22 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 3.24 (s, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s, 36H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 426.42 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 3.24 (s, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s, 36H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 426.42 (M+H).
실시예 23: 화합물 23의 제조Example 23: Preparation of Compound 23
[화합물 23][Compound 23]
DCM(1mL) 중 알라닌 메틸 에스테르 하이드로클로라이드(28mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 92% 도데칸알(100mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 23을 얻었다.A solution of alanine methyl ester hydrochloride (28 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of 92% dodecaneal (100 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 23.
수득한 화합물 23을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 23 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 3.46 (q, 1H), 2.5 (m, 2H), 2.4 (m, 2H), 1.36 (br s, 4H), 1.18 (br s, 36H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 440.45 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 3.46 (q, 1H), 2.5 (m, 2H), 2.4 (m, 2H), 1.36 (br s, 4H), 1.18 ( br s, 36H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 440.45 (M+H).
실시예 24: 화합물 24의 제조Example 24: Preparation of Compound 24
[화합물 24][Compound 24]
DCM(1mL) 중 발린 메틸 에스테르 하이드로클로라이드(34mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 92% 도데칸알(100mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 24를 얻었다.A solution of valine methyl ester hydrochloride (34 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of 92% dodecaneal (100 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 24.
수득한 화합물 24를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 24 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 2.8 (t, 1H), 2.6 (t, 2H), 2.2 (t, 2H), 2.0 (q, 1h) 1.36 (br s, 4H), 1.18 (br s, 36H), 0.89 (t, 6H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 482.48 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 2.8 (t, 1H), 2.6 (t, 2H), 2.2 (t, 2H), 2.0 (q, 1h) 1.36 (br s , 4H), 1.18 (br s, 36H), 0.89 (t, 6H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 482.48 (M+H).
실시예 25: 화합물 25의 제조Example 25: Preparation of Compound 25
[화합물 25][Compound 25]
DCM(1mL) 중 시스테인 메틸 에스테르 하이드로클로라이드(34 mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 92% 도데칸알(100mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 25를 얻었다.A solution of cysteine methyl ester hydrochloride (34 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of 92% dodecaneal (100 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 25.
수득한 화합물 25를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 25 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.7 (t, 1H) 3.67 (s, 3H), 3.5 (t, 2H), 2.9 (br s, 1H), 2.5 (m, 4H), 2.4 (m, 2H), 1.4 (m, 4H), 1.18 (br s, 36H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 456.44 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.7 (t, 1H) 3.67 (s, 3H), 3.5 (t, 2H), 2.9 (br s, 1H), 2.5 (m, 4H), 2.4 (m , 2H), 1.4 (m, 4H), 1.18 (br s, 36H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 456.44 (M+H).
실시예 26: 화합물 26의 제조Example 26: Preparation of Compound 26
[화합물 26][Compound 26]
DCM(1mL) 중 메티오닌 메틸 에스테르 하이드로클로라이드(40mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 92% 도데칸알(100mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 26을 얻었다.A solution of methionine methyl ester hydrochloride (40 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of 92% dodecaneal (100 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 26.
수득한 화합물 26을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 26 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 3.46 (q, 1H), 2.5 (m, 4H), 2.4 (m, 2H), 2.1 (s, 3H), 1.93 (m, 1H), 1.9 (m, 1H), 1.36 (br s, 4H), 1.18 (br s, 36H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 500.45 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 3.46 (q, 1H), 2.5 (m, 4H), 2.4 (m, 2H), 2.1 (s, 3H), 1.93 (m) , 1H), 1.9 (m, 1H), 1.36 (br s, 4H), 1.18 (br s, 36H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 500.45 (M+H).
실시예 27: 화합물 27의 제조Example 27: Preparation of Compound 27
[화합물 27][Compound 27]
트리플루오로에탄올(TFE)(1mL) 중 히스티딘 메틸 에스테르 디하이드로클로라이드(48mg, 0.2mmol) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데실 아크릴레이트(148mg, 0.5mmol) 용액을 첨가하고, 반응 용액을 90℃에서 24시간 동안 교반하였다. 반응 용액을 진공에서 농축하였다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 50% EtOAc/헥산)로 정제하여 화합물 27을 얻었다.A solution of histidine methyl ester dihydrochloride (48 mg, 0.2 mmol) and triethylamine (84 μL, 0.6 mmol) in trifluoroethanol (TFE) (1 mL) was stirred at room temperature for 30 min. Hexadecyl acrylate (148mg, 0.5mmol) solution was added, and the reaction solution was stirred at 90°C for 24 hours. The reaction solution was concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 50% EtOAc/hexane) to obtain compound 27.
수득한 화합물 27을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 27 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.35 (s, 1H), 6.7 (s, 1H), 4.05 (t, 4H), 3.67 (s, 3H), 3.6 (t, 1H), 2.8 (m, 3H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 762.6 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.35 (s, 1H), 6.7 (s, 1H), 4.05 (t, 4H), 3.67 (s, 3H), 3.6 (t, 1H), 2.8 (m , 3H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 762.6 (M+H).
실시예 28: 화합물 28의 제조Example 28: Preparation of Compound 28
[화합물 28][Compound 28]
DCM(1mL) 중 히스티딘 메틸 에스테르 디하이드로클로라이드(48mg, 0.2mmol) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 33% EtOAc/헥산)로 정제하여 화합물 28을 얻었다.A solution of histidine methyl ester dihydrochloride (48 mg, 0.2 mmol) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 33% EtOAc/hexane) to obtain compound 28.
수득한 화합물 28을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 28 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 618.59 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q , 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 618.59 (M+H).
실시예 29: 화합물 29의 제조Example 29: Preparation of Compound 29
[화합물 29][Compound 29]
DCM(1mL) 중 히스티딘 메틸 에스테르 디하이드로클로라이드(48mg, 0.2mmol) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. 시스-11-헥사데센알(119mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 33% EtOAc/헥산)로 정제하여 화합물 29를 얻었다.A solution of histidine methyl ester dihydrochloride (48 mg, 0.2 mmol) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of cis-11-hexadecenal (119 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 33% EtOAc/hexane) to obtain compound 29.
수득한 화합물 29를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 29 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.5 (s, 1H), 6.7 (s, 1H), 5.3 (m, 4H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 614.55 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.5 (s, 1H), 6.7 (s, 1H), 5.3 (m, 4H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q , 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 614.55 (M+H).
실시예 30: 화합물 30의 제조Example 30: Preparation of Compound 30
[화합물 30][Compound 30]
[환원적 아민화] DCM(1mL) 중 리신(Z) 메틸 에스테르 하이드로클로라이드(66.2mg, 0.2mmol, Z=카보벤질옥시(carbobenzyloxy, CBz)) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 24시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 50% EtOAc/헥산)로 정제하여 디헥사데실 리신(Z) 메틸 에스테르를 얻었다. [CBz-탈보호] EtOH(1.0mL) 중 디헥사데실 리신(Z) 메틸 에스테르(71mg, 0.098mmol)을 10% Pd/C(7.1mg)로 처리하고, 생성된 혼합물을 H2로 수소화하기 전에 N2-가스로 탈기시켰다. 30분 동안 교반한 후, 생성된 혼합물을 셀라이트 패드를 통해 여과하고, 진공에서 농축시켰다. 잔류물을 컬럼 크로마토그래피(2% Et3N, 7% MeOH/CH2Cl2)로 정제하여 화합물 30을 얻었다. [Reductive amination] A solution of lysine (Z) methyl ester hydrochloride (66.2 mg, 0.2 mmol, Z = carbobenzyloxy (CBz)) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) Stirred at room temperature for 30 minutes. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 24 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 50% EtOAc/hexane) to obtain dihexadecyl lysine (Z) methyl ester. [CBz-deprotection] Dihexadecyl lysine (Z) methyl ester (71 mg, 0.098 mmol) in EtOH (1.0 mL) was treated with 10% Pd/C (7.1 mg) and the resulting mixture was hydrogenated with H 2 It was previously degassed with N 2 -gas. After stirring for 30 minutes, the resulting mixture was filtered through a pad of Celite and concentrated in vacuo. The residue was purified by column chromatography (2% Et 3 N, 7% MeOH/CH 2 Cl 2 ) to obtain compound 30.
수득한 화합물 30을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 30 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 3.6 (t, 1H), 2.75 (m, 2H), 2.6 (q, 1H), 2.4 (t, 2H), 1.6 (m, 2H), 1.5 (m, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 609.62 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 3.6 (t, 1H), 2.75 (m, 2H), 2.6 (q, 1H), 2.4 (t, 2H), 1.6 (m , 2H), 1.5 (m, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 609.62 (M+H).
실시예 31: 화합물 31의 제조Example 31: Preparation of Compound 31
[화합물 31][Compound 31]
[환원적 아민화] DCM(1mL) 중 리신(Z) 메틸 에스테르 하이드로클로라이드(66.2mg, 0.2mmol, Z=카보벤질옥시(carbobenzyloxy, CBz)) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. 시스-11-헥사데센알(119mg, 0.5mmol)의 용액을 첨가하고 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 24시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 50% EtOAc/헥산)로 정제하여 디헥사데세닐 리신(Z) 메틸 에스테르을 얻었다. [CBz-탈보호] CH2Cl2(3.0mL) 중 냉각(0℃)된 디헥사데세닐 리신(Z) 메틸 에스테르(37.9mg, 0.064mmol)을 BF3OEt2(20.0μL, 0.127mmol, 2당량) 및 Me2S(20.0μL, 0.253mmol, 3 당량)로 처리하였다. 25℃에서 7시간 동안 교반한 후, 생성된 혼합물을 0℃에서 포화 중탄산나트륨을 천천히 첨가하여 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 7% MeOH/CH2Cl2)로 정제하여 화합물 31을 얻었다. [Reductive amination] A solution of lysine (Z) methyl ester hydrochloride (66.2 mg, 0.2 mmol, Z = carbobenzyloxy (CBz)) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) Stirred at room temperature for 30 minutes. A solution of cis-11-hexadecenal (119 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 24 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 50% EtOAc/hexane) to obtain dihexadecenyl lysine (Z) methyl ester. [CBz-deprotection] Dihexadecenyl lysine (Z) methyl ester (37.9 mg, 0.064 mmol) cooled (0°C) in CH 2 Cl 2 (3.0 mL) was added to BF 3 OEt 2 (20.0 μL, 0.127 mmol, 2 equivalents) and Me 2 S (20.0 μL, 0.253 mmol, 3 equivalents). After stirring at 25°C for 7 hours, the resulting mixture was quenched by slowly adding saturated sodium bicarbonate at 0°C and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 7% MeOH/CH 2 Cl 2 ) to obtain compound 31.
수득한 화합물 31을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 31 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 5.3 (m, 4H), 3.67 (s, 3H), 3.3 (t, 1H), 2.8 (t, 1H), 2.5 (m, 2H), 2.4 (m, 2H), 2.2 (m, 1H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 605.59 (M+H). 1H NMR (300 MHz, CDCl 3 ) δ: 5.3 (m, 4H), 3.67 (s, 3H), 3.3 (t, 1H), 2.8 (t, 1H), 2.5 (m, 2H), 2.4 (m) , 2H), 2.2 (m, 1H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 605.59 (M+H).
실시예 32: 화합물 32의 제조Example 32: Preparation of Compound 32
[화합물 32][Compound 32]
DCM(1mL) 중 트립토판 메틸 에스테르 하이드로클로라이드(51mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(20% EtOAc/헥산)로 정제하여 화합물 32를 얻었다.A solution of tryptophan methyl ester hydrochloride (51 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (20% EtOAc/hexane) to obtain compound 32.
수득한 화합물 32를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 32 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 8.0 (s, 1H), 7.6 (s, 1H), 7.5-7.0 (m, 4H), 3.75 (t, 1H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 667.60 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 8.0 (s, 1H), 7.6 (s, 1H), 7.5-7.0 (m, 4H), 3.75 (t, 1H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 667.60 (M+H).
실시예 33: 화합물 33의 제조Example 33: Preparation of Compound 33
[화합물 33][Compound 33]
DCM(1mL) 중 메티오닌 메틸 에스테르 하이드로클로라이드(40mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 33을 얻었다.A solution of methionine methyl ester hydrochloride (40 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 33.
수득한 화합물 33을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 33 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 3.53 (t, 1H), 2.55 (m, 4H), 2.45 (m, 2H), 2.1 (s, 3H), 1.8 (m, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 612.57 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 3.53 (t, 1H), 2.55 (m, 4H), 2.45 (m, 2H), 2.1 (s, 3H), 1.8 (m) , 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 612.57 (M+H).
실시예 34: 화합물 34의 제조Example 34: Preparation of Compound 34
[화합물 34][Compound 34]
DCM(1mL) 중 발린 메틸 에스테르 하이드로클로라이드(34mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 34를 얻었다.A solution of valine methyl ester hydrochloride (34 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 34.
수득한 화합물 34를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 34 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 2.8 (t, 1H), 2.6 (t, 2H), 2.25 (t, 2H), 2.0 (m, 1H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.92 (d, 6H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 594.61 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 2.8 (t, 1H), 2.6 (t, 2H), 2.25 (t, 2H), 2.0 (m, 1H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.92 (d, 6H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 594.61 (M+H).
실시예 35: 화합물 35의 제조Example 35: Preparation of Compound 35
[화합물 35][Compound 35]
DCM(1mL) 중 알라닌 메틸 에스테르 하이드로클로라이드(28 mg, 0.2 mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 35를 얻었다.A solution of alanine methyl ester hydrochloride (28 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 35.
수득한 화합물 35를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 35 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 3.6 (t, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.95 (d, 3H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 552.56 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 3.6 (t, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 ( br s,52H), 0.95 (d, 3H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 552.56 (M+H).
실시예 36: 화합물 36의 제조Example 36: Preparation of Compound 36
[화합물 36][Compound 36]
DCM(1mL) 중 페닐알라닌 메틸 에스테르 하이드로클로라이드(43mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 36을 얻었다.A solution of phenylalanine methyl ester hydrochloride (43 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 36.
수득한 화합물 36을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 36 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.4-7.2 (aromatic, 5H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 628.59 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.4-7.2 (aromatic, 5H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 628.59 (M+H).
실시예 37: 화합물 37의 제조Example 37: Preparation of Compound 37
[화합물 37][Compound 37]
DCM(1mL) 중 글리신 메틸 에스테르 하이드로클로라이드(25mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 37을 얻었다.A solution of glycine methyl ester hydrochloride (25 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 37.
수득한 화합물 37을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 37 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 3.3 (s, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 538.55 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 3.3 (s, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s, 52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 538.55 (M+H).
실시예 38: 화합물 38의 제조Example 38: Preparation of Compound 38
[화합물 38][Compound 38]
DCM(1mL) 중 류신 메틸 에스테르 하이드로클로라이드(36mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 38을 얻었다.A solution of leucine methyl ester hydrochloride (36 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 38.
수득한 화합물 38을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 38 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 3.4 (t, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.7 (m, 1H), 1.5 (q, 2H) 1.36 (br s, 4H), 1.18 (br s,52H), 0.92 (m, 6H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 594.61 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 3.4 (t, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.7 (m, 1H), 1.5 (q , 2H) 1.36 (br s, 4H), 1.18 (br s,52H), 0.92 (m, 6H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 594.61 (M+H).
실시예 39: 화합물 39의 제조Example 39: Preparation of Compound 39
[화합물 39][Compound 39]
DCM(1mL) 중 이소류신 메틸 에스테르 하이드로클로라이드(36mg, 0.2mmol) 및 트리에틸아민(42μL, 0.3mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(10% EtOAc/헥산)로 정제하여 화합물 39를 얻었다.A solution of isoleucine methyl ester hydrochloride (36 mg, 0.2 mmol) and triethylamine (42 μL, 0.3 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% EtOAc/hexane) to obtain compound 39.
수득한 화합물 39를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 39 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 3.67 (s, 3H), 3.0 (d, 1H), 2.6 (m, 2H), 2.4 (m, 2H), 1.75 (m, 2H), 1.6 (m, 1H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 594.61 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 3.67 (s, 3H), 3.0 (d, 1H), 2.6 (m, 2H), 2.4 (m, 2H), 1.75 (m, 2H), 1.6 (m) , 1H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 594.61 (M+H).
실시예 40: 화합물 40의 제조Example 40: Preparation of Compound 40
[화합물 40][Compound 40]
DCM(1mL) 중 히스티딘 메틸 에스테르 디하이드로클로라이드(48mg, 0.2mmol) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. 옥타데칸알(134mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 33% EtOAc/헥산)로 정제하여 화합물 40을 얻었다.A solution of histidine methyl ester dihydrochloride (48 mg, 0.2 mmol) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of octadecanal (134 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 33% EtOAc/hexane) to obtain compound 40.
수득한 화합물 40을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 40 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.67 (s, 3H), 3.6 (t, 1H), 3.1 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,60H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 674.65 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.67 (s, 3H), 3.6 (t, 1H), 3.1 (q, 1H), 2.8 (q , 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,60H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 674.65 (M+H).
실시예 41: 화합물 41의 제조Example 41: Preparation of Compound 41
[화합물 41][Compound 41]
DCM(1mL) 중 히스티딘 에틸 에스테르 디하이드로클로라이드(51mg, 0.2mmol) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 33% EtOAc/헥산)로 정제하여 화합물 41을 얻었다.A solution of histidine ethyl ester dihydrochloride (51 mg, 0.2 mmol) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 33% EtOAc/hexane) to obtain compound 41.
수득한 화합물 41을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 41 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.5 (s, 1H), 6.7 (s, 1H), 4.15 (m, 2H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 9H). UPLC-MS (ESI) m/z 632.60 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.5 (s, 1H), 6.7 (s, 1H), 4.15 (m, 2H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q , 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 9H). UPLC-MS (ESI) m/z 632.60 (M+H).
실시예 42: 화합물 42의 제조Example 42: Preparation of Compound 42
[화합물 42][Compound 42]
DCM(1mL) 중 글리실-L-히스티딘 메틸 에스테르 디하이드로클로라이드(60mg, 0.2mmol) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 50% EtOAc/헥산)로 정제하여 화합물 42를 얻었다.A solution of glycyl-L-histidine methyl ester dihydrochloride (60 mg, 0.2 mmol) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 50% EtOAc/hexane) to obtain compound 42.
수득한 화합물 42를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 42 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 8.25 (d, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 4.8 (m, 1H), 3.67 (s, 3H), 3.2 (m, 2H), 3.0 (s, 2H), 2.45 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 676.10 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 8.25 (d, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 4.8 (m, 1H), 3.67 (s, 3H), 3.2 (m) , 2H), 3.0 (s, 2H), 2.45 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 676.10 (M+H).
실시예 43: 화합물 43의 제조Example 43: Preparation of Compound 43
[화합물 43][Compound 43]
DCM(1mL) 중 히스티딜-L-글리신 메틸 에스테르 디하이드로클로라이드(60mg, 0.2mmol) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 50% EtOAc/헥산)로 정제하여 화합물 43을 얻었다.A solution of histidyl-L-glycine methyl ester dihydrochloride (60 mg, 0.2 mmol) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 50% EtOAc/hexane) to obtain compound 43.
수득한 화합물 43을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 43 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 8.25 (d, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 4.1 (m, 2H), 4.0 (m, 2H), 3.67 (s, 3H), 2.9 (d, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 676.10 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 8.25 (d, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 4.1 (m, 2H), 4.0 (m, 2H), 3.67 (s) , 3H), 2.9 (d, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 676.10 (M+H).
실시예 44: 화합물 44의 제조Example 44: Preparation of Compound 44
[화합물 44][Compound 44]
DCM(1mL) 중 히스티딜-L-히스티딘 메틸 에스테르 트리하이드로클로라이드(83mg, 0.2mmol) 및 트리에틸아민(126μL, 0.9mmol)의 용액을 실온에서 30분 동안 교반하였다. 헥사데칸알(120mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 50% EtOAc/헥산)로 정제하여 화합물 44를 얻었다.A solution of histidyl-L-histidine methyl ester trihydrochloride (83 mg, 0.2 mmol) and triethylamine (126 μL, 0.9 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of hexadecaneal (120 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 50% EtOAc/hexane) to obtain compound 44.
수득한 화합물 44를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 44 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 8.25 (d, 1H), 7.5 (s, 2H), 6.7 (s, 2H), 4.8 (m, 1H), 3.67 (s, 3H), 3.4 (d, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 756.19 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 8.25 (d, 1H), 7.5 (s, 2H), 6.7 (s, 2H), 4.8 (m, 1H), 3.67 (s, 3H), 3.4 (d) , 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 756.19 (M+H).
실시예 45: 화합물 45의 제조Example 45: Preparation of Compound 45
[화합물 45][Compound 45]
DCM(1mL) 중 히스티딘 메틸 에스테르 디하이드로클로라이드(48mg, 0.2mmol) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. (Z)-9-헥사데센알(119mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고, 반응을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(0-50% EtOAc/헥산)로 정제하여 화합물 45를 얻었다.A solution of histidine methyl ester dihydrochloride (48 mg, 0.2 mmol) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of (Z)-9-hexadecenal (119 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added, the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-50% EtOAc/hexane) to obtain compound 45.
수득한 화합물 45를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 45 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.5 (s, 1H), 6.7 (s, 1H), 5.3 (m, 4H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,48H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 614.55 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.5 (s, 1H), 6.7 (s, 1H), 5.3 (m, 4H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q , 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,48H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 614.55 (M+H).
실시예 46: 화합물 46의 제조Example 46: Preparation of Compound 46
[화합물 46][Compound 46]
TFE(0.6mL, 0.1M) 중 화합물 28(37.3mg, 0.060mmol)의 용액을 AcOH(액적, 10mol%) 및 아미노 에탄올(18.0μL, 0.300mmol, 5 당량)로 처리하였다. 생성된 혼합물을 100℃로 가열하였다. 24시간 동안 교반한 후, 생성된 혼합물을 실온으로 냉각시키고, Et3N으로 염기성화한 후 진공에서 농축시켰다. 잔류물을 컬럼 크로마토그래피(2% Et3N, 3% MeOH/CH2Cl2)로 정제하여 화합물 46을 얻었다.A solution of compound 28 (37.3 mg, 0.060 mmol) in TFE (0.6 mL, 0.1 M) was treated with AcOH (droplets, 10 mol %) and amino ethanol (18.0 μL, 0.300 mmol, 5 equiv). The resulting mixture was heated to 100°C. After stirring for 24 hours, the resulting mixture was cooled to room temperature, basified with Et 3 N and concentrated in vacuo. The residue was purified by column chromatography (2% Et 3 N, 3% MeOH/CH 2 Cl 2 ) to obtain compound 46.
수득한 화합물 46을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 46 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.9 (t, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 647.61 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.9 (t, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m , 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 647.61 (M+H).
실시예 47: 화합물 47의 제조Example 47: Preparation of Compound 47
[화합물 47][Compound 47]
TFE(0.3mL, 0.1M) 중 화합물 28(20.2mg, 0.033mmol)의 용액을 AcOH (방울, 10mol%) 및 3-아미노-1-프로판올(13.0μL, 0.165mmol, 5 당량)로 처리하였다. 생성된 혼합물을 100℃로 가열하였다. 24시간 동안 교반한 후, 생성된 혼합물을 실온으로 냉각시키고, Et3N으로 염기성화한 후 진공에서 농축시켰다. 잔류물을 컬럼 크로마토그래피(2% Et3N, 3% MeOH/CH2Cl2)로 정제하여 화합물 47을 얻었다.A solution of compound 28 (20.2 mg, 0.033 mmol) in TFE (0.3 mL, 0.1 M) was treated with AcOH (drops, 10 mol %) and 3-amino-1-propanol (13.0 μL, 0.165 mmol, 5 equiv). The resulting mixture was heated to 100°C. After stirring for 24 hours, the resulting mixture was cooled to room temperature, basified with Et 3 N and concentrated in vacuo. The residue was purified by column chromatography (2% Et 3 N, 3% MeOH/CH 2 Cl 2 ) to obtain compound 47.
수득한 화합물 47을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 47 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.9 (t, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 661.63 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.9 (t, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m , 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 661.63 (M+H).
실시예 48: 화합물 48의 제조Example 48: Preparation of Compound 48
[화합물 48][Compound 48]
TFE(0.7mL, 0.1M) 중 화합물 28(42.5mg, 0.069mmol)의 용액을 AcOH(액적, 10mol%) 및 4-아미노-1-부탄올(32.0μL, 0.345mmol, 5 당량)로 처리하였다. 생성된 혼합물을 100℃로 가열하였다. 24시간 동안 교반한 후, 생성된 혼합물을 실온으로 냉각시키고, Et3N으로 염기성화한 후 진공에서 농축시켰다. 잔류물을 컬럼 크로마토그래피(2% Et3N, 3% MeOH/CH2Cl2)로 정제하여 화합물 48을 얻었다.A solution of compound 28 (42.5 mg, 0.069 mmol) in TFE (0.7 mL, 0.1 M) was treated with AcOH (droplets, 10 mol %) and 4-amino-1-butanol (32.0 μL, 0.345 mmol, 5 equiv). The resulting mixture was heated to 100°C. After stirring for 24 hours, the resulting mixture was cooled to room temperature, basified with Et 3 N and concentrated in vacuo. The residue was purified by column chromatography (2% Et 3 N, 3% MeOH/CH 2 Cl 2 ) to obtain compound 48.
수득한 화합물 48을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 48 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.9 (t, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 675.64 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.9 (t, 1H), 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m , 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 675.64 (M+H).
실시예 49: 화합물 49의 제조Example 49: Preparation of Compound 49
[화합물 49][Compound 49]
TFE(0.6mL, 0.1M) 중 화합물 28(36.7mg, 0.059mmol)의 용액을 AcOH (액적, 10mol%) 및 2-(메틸아미노)에탄올(24.0μL, 0.295mmol, 5 당량)로 처리하였다. 생성된 혼합물을 100℃로 가열하였다. 24시간 동안 교반한 후, 생성된 혼합물을 실온으로 냉각시키고, Et3N으로 염기성화한 후 진공에서 농축시켰다. 잔류물을 컬럼 크로마토그래피(2% Et3N, 10% MeOH/CH2Cl2)로 정제하여 화합물 49를 얻었다.A solution of compound 28 (36.7 mg, 0.059 mmol) in TFE (0.6 mL, 0.1 M) was treated with AcOH (droplet, 10 mol %) and 2-(methylamino)ethanol (24.0 μL, 0.295 mmol, 5 equiv). The resulting mixture was heated to 100°C. After stirring for 24 hours, the resulting mixture was cooled to room temperature, basified with Et 3 N and concentrated in vacuo. The residue was purified by column chromatography (2% Et 3 N, 10% MeOH/CH 2 Cl 2 ) to obtain compound 49.
수득한 화합물 49를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 49 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m, 2H), 2.9 (s, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 661.63 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m, 2H), 2.9 (s) , 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 661.63 (M+H).
실시예 50: 화합물 50의 제조Example 50: Preparation of Compound 50
[화합물 50][Compound 50]
TFE(0.6mL, 0.1M) 중 화합물 28(36.7mg, 0.059mmol)의 용액을 AcOH(액적, 10mol%) 및 3-메틸아미노-1-프로판올(28.0μL, 0.295mmol, 5 당량)로 처리하였다. 생성된 혼합물을 100℃로 가열하였다. 24시간 동안 교반한 후, 생성된 혼합물을 실온으로 냉각시키고, Et3N으로 염기성화한 후 진공에서 농축시켰다. 잔류물을 컬럼 크로마토그래피(2% Et3N, 10% MeOH/CH2Cl2)로 정제하여 화합물 50을 얻었다.A solution of compound 28 (36.7 mg, 0.059 mmol) in TFE (0.6 mL, 0.1 M) was treated with AcOH (droplets, 10 mol %) and 3-methylamino-1-propanol (28.0 μL, 0.295 mmol, 5 equiv). . The resulting mixture was heated to 100°C. After stirring for 24 hours, the resulting mixture was cooled to room temperature, basified with Et 3 N and concentrated in vacuo. The residue was purified by column chromatography (2% Et 3 N, 10% MeOH/CH 2 Cl 2 ) to obtain compound 50.
수득한 화합물 50을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 50 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m, 2H), 2.9 (s, 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 675.64 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.7 (t, 2H), 3.45 (m, 3H), 2.95 (m, 2H), 2.9 (s) , 2H), 2.5 (t, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 675.64 (M+H).
실시예 51: 화합물 51의 제조Example 51: Preparation of Compound 51
[화합물 51][Compound 51]
MeOH:H2O=10:1(총 1.1mL) 중 화합물 28(52.8mg, 0.085mmol)의 용액을 LiOH (10.3mg, 0.429mmol, 5 당량)로 처리하였다. 실온에서 24시간 동안 교반한 후, 생성된 혼합물을 진공에서 농축하였다. 잔류물을 컬럼 크로마토그래피(2% Et3N, 25% MeOH/CH2Cl2) 로 정제하여 화합물 51을 얻었다.A solution of compound 28 (52.8 mg, 0.085 mmol) in MeOH:H 2 O=10:1 (1.1 mL total) was treated with LiOH (10.3 mg, 0.429 mmol, 5 equiv). After stirring at room temperature for 24 hours, the resulting mixture was concentrated in vacuo. The residue was purified by column chromatography (2% Et 3 N, 25% MeOH/CH 2 Cl 2 ) to obtain compound 51.
수득한 화합물 51을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 51 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 604.57 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.5 (s, 1H), 6.7 (s, 1H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t , 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 604.57 (M+H).
실시예 52: 화합물 52의 제조Example 52: Preparation of Compound 52
[화합물 52][Compound 52]
TFE(0.4mL, 0.1M) 중 화합물 28(22.9mg, 0.037mmol)의 용액을 AcOH(액적, 10mol%) 및 1-(3-아미노프로필)이미다졸(41.0μL, 0.370mmol, 10 당량)로 처리하였다. 생성된 혼합물을 100℃로 가열하였다. 24시간 동안 교반한 후, 생성된 혼합물을 실온으로 냉각시키고, Et3N으로 염기성화한 후 진공에서 농축시켰다. 잔류물을 컬럼 크로마토그래피(5% Et3N, 10% MeOH/CH2Cl2)로 정제하여 화합물 52를 얻었다.A solution of compound 28 (22.9 mg, 0.037 mmol) in TFE (0.4 mL, 0.1 M) was diluted with AcOH (droplets, 10 mol %) and 1-(3-aminopropyl)imidazole (41.0 μL, 0.370 mmol, 10 equiv). Processed. The resulting mixture was heated to 100°C. After stirring for 24 hours, the resulting mixture was cooled to room temperature, basified with Et 3 N and concentrated in vacuo. The residue was purified by column chromatography (5% Et 3 N, 10% MeOH/CH 2 Cl 2 ) to obtain compound 52.
수득한 화합물 52를 1H NMR 및 LC-MS로 확인하였다.The obtained compound 52 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.7 (s, 1H), 7.6 (t, 1H), 7.5 (s, 1H), 7.2 (m, 3H), 6.7 (s, 1H), 4.0 (m, 2H), 3.4 (m, 2H), 2.4 (t, 4H), 2.0 (m, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 697.64 (M+H). 1 H NMR (300 MHz, CDCl 3 ) δ: 7.7 (s, 1H), 7.6 (t, 1H), 7.5 (s, 1H), 7.2 (m, 3H), 6.7 (s, 1H), 4.0 (m , 2H), 3.4 (m, 2H), 2.4 (t, 4H), 2.0 (m, 4H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 697.64 (M+H).
실시예 53: 화합물 53의 제조Example 53: Preparation of Compound 53
[화합물 53][Compound 53]
DCM(1mL) 중 히스티딘 메틸 에스테르 디하이드로클로라이드(48 mg, 0.2 mmol) 및 트리에틸아민(84μL, 0.6mmol)의 용액을 실온에서 30분 동안 교반하였다. (9Z,12Z)-옥타데카-9,12-디엔알(132mg, 0.5mmol)의 용액을 첨가하고, 혼합물을 0℃로 냉각시켰다. 나트륨 트리아세톡시보로하이드라이드(105mg, 0.5mmol)를 첨가하고 반응물을 실온으로 되돌리고 16시간 동안 교반하였다. 포화 중탄산나트륨을 천천히 첨가하여 반응을 ?칭하고 DCM으로 추출하였다. 합한 추출물을 염수로 세척하고, 무수 Na2SO4 상에서 건조시키고, 여과하고, 진공에서 농축시켰다. 잔류물을 실리카겔 컬럼 크로마토그래피(2% Et3N, 50% EtOAc/헥산)로 정제하여 화합물 53을 얻었다.A solution of histidine methyl ester dihydrochloride (48 mg, 0.2 mmol) and triethylamine (84 μL, 0.6 mmol) in DCM (1 mL) was stirred at room temperature for 30 min. A solution of (9Z,12Z)-octadeca-9,12-dienal (132 mg, 0.5 mmol) was added and the mixture was cooled to 0°C. Sodium triacetoxyborohydride (105 mg, 0.5 mmol) was added and the reaction was returned to room temperature and stirred for 16 hours. The reaction was quenched by slow addition of saturated sodium bicarbonate and extracted with DCM. The combined extracts were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2% Et 3 N, 50% EtOAc/hexane) to obtain compound 53.
수득한 화합물 53을 1H NMR 및 LC-MS로 확인하였다.The obtained compound 53 was confirmed by 1 H NMR and LC-MS.
1H NMR (300 MHz, CDCl3) δ: 7.75 (s, 1H), 6.7 (s, 1H), 5.4 (m, 8H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q, 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 666.59 (M+H). 1H NMR (300 MHz, CDCl 3 ) δ: 7.75 (s, 1H), 6.7 (s, 1H), 5.4 (m, 8H), 3.67 (s, 3H), 3.6 (t, 1H), 3.0 (q , 1H), 2.8 (q, 1H), 2.6 (t, 2H), 2.4 (t, 2H), 1.36 (br s, 4H), 1.18 (br s,52H), 0.84 (t, 6H). UPLC-MS (ESI) m/z 666.59 (M+H).
실험예Experiment example
실험예 1: 지질 나노입자의 제조Experimental Example 1: Preparation of lipid nanoparticles
이온화성 지질, 인지질, 콜레스테롤, PEG-지질 접합체를 에탄올에 50:10:38.5:1.5의 몰 비율로 용해시킨 후, mRNA가 용해된 citrate buffer(pH 4, 50 mM)와 1:3의 부피비로 혼합하였다. 상기 이온화성 지질로 상기 실시예에서 수득한 일부 화합물(화합물 28, 29, 40, 42, 45, 46, 47 및 53)을 사용하였고, 상기 인지질로 DSPC(Avanti Polar Lipids, USA) 및 DOPE(Avanti Polar Lipids, USA)를 사용하였다. 또한, 상기 콜레스테롤로 cholesterol(Sigma Aldrich, USA)를 사용하였고, 상기 PEG-지질 접합체로 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(DMG-PEG2000)(Avanti Polar Lipids, USA)를 사용하였다. 상기 mRNA로 CleanCap® Firefly Luciferase mRNA(TriLink, USA)를 사용하였다.Ionizable lipids, phospholipids, cholesterol, and PEG-lipid conjugates were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5, and then mixed with citrate buffer (pH 4, 50 mM) in which mRNA was dissolved at a volume ratio of 1:3. Mixed. Some of the compounds obtained in the above examples (compounds 28, 29, 40, 42, 45, 46, 47, and 53) were used as the ionizable lipids, and DSPC (Avanti Polar Lipids, USA) and DOPE (Avanti Polar Lipids, USA) was used. In addition, cholesterol (Sigma Aldrich, USA) was used as the cholesterol, and 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (Avanti Polar Lipids, USA) was used as the PEG-lipid conjugate. was used. CleanCap® Firefly Luciferase mRNA (TriLink, USA) was used as the mRNA.
지질 나노입자 생성을 위해서 NanoAssemblr IgniteTM(Precision Nanosystems, Inc., Canada)를 사용하여 총 유속(total flow rate; TFR)이 12 mL/min 조건 하에 이온화성 지질과 mRNA 간의 무게 비가 10:1이 되도록 혼합하였다. 제조된 지질 나노입자는 에탄올 제거, 버퍼 교환, 및 농축을 위하여 Amicon Ultra Centrifugal Filter, MWCO 10 kDa(Millipore, USA)를 사용하였고, 희석, 농축, 및 교환을 위하여 1X DPBS (Thermo Scientific, USA)를 사용하였다.To generate lipid nanoparticles, NanoAssembly Ignite TM (Precision Nanosystems, Inc., Canada) was used to achieve a weight ratio between ionizable lipids and mRNA of 10:1 under the condition of a total flow rate (TFR) of 12 mL/min. Mixed. The prepared lipid nanoparticles were filtered using an Amicon Ultra Centrifugal Filter, MWCO 10 kDa (Millipore, USA) for ethanol removal, buffer exchange, and concentration, and 1X DPBS (Thermo Scientific, USA) for dilution, concentration, and exchange. used.
실험예 2: 지질 나노입자의 물성 평가Experimental Example 2: Evaluation of physical properties of lipid nanoparticles
실험예 1에서 제조된 지질 나노입자의 물성 분석을 위하여, Zetasizer Pro(Malvern Instruments, United Kingdom)를 사용하여 지질 나노입자의 입자 크기(Z-average)을 측정하였다. 입자 크기를 측정하기 위해서, 1X DPBS를 희석액으로 사용하였고, 이에 따른 입자 크기 결과를 하기 표 2에 나타내었다.To analyze the physical properties of the lipid nanoparticles prepared in Experimental Example 1, the particle size (Z-average) of the lipid nanoparticles was measured using Zetasizer Pro (Malvern Instruments, United Kingdom). To measure the particle size, 1X DPBS was used as a diluent, and the particle size results are shown in Table 2 below.
표 2에 따르면, 화합물 28, 29, 40, 42, 45, 46, 47 또는 53을 포함하는 지질 나노입자는 모두 200nm 미만의 z-average 값을 가져 지질 나노입자로서 적정 수준의 크기를 갖는 것을 확인할 수 있다.According to Table 2, lipid nanoparticles containing compounds 28, 29, 40, 42, 45, 46, 47 or 53 all have z-average values of less than 200 nm, confirming that they have an appropriate size as lipid nanoparticles. You can.
실험예 3: 지질 나노입자의 mRNA 함량 및 봉입 효율 평가Experimental Example 3: Evaluation of mRNA content and encapsulation efficiency of lipid nanoparticles
실험예 1에서 제조된 mRNA가 봉입된 지질 나노입자의 mRNA 함량 및 봉입 효율(encapsulation efficiency) 측정을 위하여, Ribogreen RNA assay kit (Invitrogen, USA)를 사용하였다. 구체적으로, Ribosomal RNA를 사용하여 0-1000ng/mL 범위의 표준 용액을 제조하고, 범위 내에 들어오도록 샘플을 희석하였다. 96-well microplate에 샘플 또는 표준 용액을 50 μL씩 로딩하고, 1X TE buffer나 1% TritonX-100 용액을 50 μL씩 로딩하였다. 상온에서 10분간 정치 후, 1/200 비율로 희석된 RiboGreen reagent 용액을 100 μL씩 로딩한 후 Microplate reader기 (Enspire 2300, PerkinElmer, USA)를 사용하여 형광을 측정하였고(Excitation 485 nm, Emission 530 nm), 이에 따른 봉입 효율 결과를 하기 표 3에 나타내었다.To measure the mRNA content and encapsulation efficiency of the mRNA-encapsulated lipid nanoparticles prepared in Experimental Example 1, the Ribogreen RNA assay kit (Invitrogen, USA) was used. Specifically, a standard solution in the range of 0-1000ng/mL was prepared using ribosomal RNA, and the sample was diluted to fall within the range. 50 μL of sample or standard solution was loaded into a 96-well microplate, and 50 μL of 1X TE buffer or 1% TritonX-100 solution was loaded. After standing at room temperature for 10 minutes, 100 μL of RiboGreen reagent solution diluted at a ratio of 1/200 was loaded, and fluorescence was measured using a microplate reader (Enspire 2300, PerkinElmer, USA) (Excitation 485 nm, Emission 530 nm) ), and the resulting encapsulation efficiency results are shown in Table 3 below.
표 3에 따르면, 화합물 28, 29, 40, 42, 45, 46, 47 또는 53을 포함하는 지질 나노입자는 모두 79% 이상의 mRNA 봉입 효율을 가져 지질 나노입자로서 적정 수준으로 mRNA를 봉입할 수 있는 것을 확인할 수 있다.According to Table 3, lipid nanoparticles containing compounds 28, 29, 40, 42, 45, 46, 47 or 53 all have an mRNA encapsulation efficiency of 79% or more, showing that lipid nanoparticles can encapsulate mRNA at an appropriate level. You can check that.
실험예 4: 지질 나노입자의 세포 내 핵산 트랜스펙션 효율 평가Experimental Example 4: Evaluation of intracellular nucleic acid transfection efficiency of lipid nanoparticles
실험예 1에서 제조된 mRNA가 봉입된 지질 나노입자(화합물 28, 29 및 40)의 세포 내 핵산 트랜스펙션 효율(In vitro transfection efficiency) 측정을 위하여, HEK293 세포주를 96-well microplate에 DMEM 배지(media)(10% FBS, 1% penicillin/streptomycin 첨가)를 사용하여 15,000 cells/well 수로 시딩(seeding)한 후 하룻밤 동안 배양(overnight incubation)하였다(5% CO2 incubator). Well 당 25 ng mRNA에 해당하는 샘플을 처리 후 24시간 배양하였다(최종 농도 0.25 μg/mL). 트랜스펙션 후 배지 부피와 동일 부피(100 μL)의 Bright-GloTM Luciferase Assay System(Promega, USA) 용액을 처리한 후 GloMax Navigator Luminator(Promega, USA)를 사용하여 luciferase 활성(activity)을 측정하였고(integration time: 0.3 sec), 이에 따른 Luminescence 측정 결과를 하기 도 1에 나타내었다.To measure the in vitro transfection efficiency of the mRNA-encapsulated lipid nanoparticles (compounds 28, 29, and 40) prepared in Experimental Example 1, the HEK293 cell line was cultured in DMEM medium (DMEM medium) in a 96-well microplate. Media) (10% FBS, 1% penicillin/streptomycin added) was used to seed the cells at 15,000 cells/well and then incubated overnight (5% CO 2 incubator). Samples corresponding to 25 ng mRNA per well were cultured for 24 hours after treatment (final concentration 0.25 μg/mL). After transfection, the Bright-GloTM Luciferase Assay System (Promega, USA) solution was treated with the same volume (100 μL) as the medium volume, and then the luciferase activity was measured using GloMax Navigator Luminator (Promega, USA) ( integration time: 0.3 sec), and the resulting Luminescence measurement results are shown in Figure 1 below.
도 1에 따르면, 봉입되지 않은 mRNA를 처리하는 것과 비교하여, 화합물 28, 29 또는 40을 포함하는 지질 나노입자 모두 mRNA를 봉입하여 세포 내에 효율적으로 약물을 전달하는 것을 확인할 수 있다.According to Figure 1, it can be seen that compared to processing unencapsulated mRNA, lipid nanoparticles containing compounds 28, 29, or 40 all encapsulate mRNA and efficiently deliver the drug into cells.
실험예 5: 지질 나노입자의 생체 내 핵산 트랜스펙션 효율 평가Experimental Example 5: Evaluation of in vivo nucleic acid transfection efficiency of lipid nanoparticles
실험예 1에서 제조된 mRNA가 봉입된 지질 나노입자를 전신 투여 후, 약물 전달 분포 및 생체 내 핵산 트랜스펙션 효율(in vivo transfection efficiency) 측정을 위하여, 0.5 mg/kg mRNA에 해당하는 지질나노입자를 Balb/c 마우스(male, 5주령)에 꼬리 혈관 인젝션(tail vein injection)을 통하여 주입하였다(투여 부피: 200 μL). 주사 후 6시간에 D-Luciferin (Perkin Elmer, USA) 150 mg/kg 복강 투여 15분 후 IVIS Luminar XR (Perkin Elmer, USA) 장비를 사용하여 생체 발광을 측정하였다. 이에 따른 마우스에서의 단백질 발현 분포를 하기 도 2(화합물 28, 29, 40 및 42) 및 도 3(화합물 45, 46, 47 및 53)에 나타내고, 마우스 간 부위의 luminescence 측정 결과를 하기 도 4에 나타내었다.After systemic administration of the lipid nanoparticles encapsulated with mRNA prepared in Experimental Example 1, lipid nanoparticles corresponding to 0.5 mg/kg mRNA were used to measure drug delivery distribution and in vivo nucleic acid transfection efficiency. was injected into Balb/c mice (male, 5 weeks old) via tail vein injection (administration volume: 200 μL). At 6 hours after injection, bioluminescence was measured using an IVIS Luminar XR (Perkin Elmer, USA) device 15 minutes after intraperitoneal administration of 150 mg/kg D-Luciferin (Perkin Elmer, USA). Accordingly, the protein expression distribution in mice is shown in Figure 2 (compounds 28, 29, 40, and 42) and Figure 3 (compounds 45, 46, 47, and 53), and the luminescence measurement results in the mouse liver region are shown in Figure 4 below. indicated.
도 2 내지 도 4에 따르면, 봉입되지 않은 mRNA를 처리하는 것과 비교하여, 화합물 28, 29, 40, 42, 45, 46, 47 또는 53을 포함하는 지질 나노입자 모두 mRNA를 봉입하여 생체 내에 효율적으로 약물을 전달할 수 있으며, 특히 간으로의 약물을 전달하는 효과가 우수한 것을 확인할 수 있다.According to Figures 2 to 4, compared to processing unencapsulated mRNA, lipid nanoparticles containing compounds 28, 29, 40, 42, 45, 46, 47, or 53 all encapsulate mRNA efficiently in vivo. It can be confirmed that drugs can be delivered, and in particular, the effect of delivering drugs to the liver is excellent.
상술한 특정 구체예를 중심으로 하여 본 발명을 설명하였으나, 해당 기술 분야의 통상의 기술자는 본 발명을 다양하게 변형 및 변경할 수 있으며, 이 또한 이하에 첨부한 청구범위에 의해 정의되는 본 발명의 범주 내에 든다는 사실을 이해하여야 한다.Although the present invention has been described focusing on the specific embodiments described above, those skilled in the art may make various modifications and changes to the present invention, and this also falls within the scope of the present invention as defined by the claims appended below. You must understand that it is within the scope.
Claims (12)
상기 아미노산-지질 접합 화합물은 아미노산 유래 중합 단위 및 지질 유래 중합 단위를 포함하고,
상기 아미노산 유래 중합 단위는 아미노산-지질 접합 화합물의 일 부분이고, 일측단에 카르복실기를 가지고, 타측단에 아미노기를 가지는 아미노산, 또는 아미노산 유도체로부터 중합에 의해 얻어지며,
상기 지질 유래 중합 단위는 아미노산-지질 접합 화합물의 일 부분이고, 탄소 수가 10 내지 20인 탄화수소를 포함하는 지질 화합물로부터 중합에 의해 얻어지는 것인 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.An amino acid-lipid conjugation compound, or a pharmaceutically acceptable salt thereof,
The amino acid-lipid conjugation compound includes an amino acid-derived polymerized unit and a lipid-derived polymerized unit,
The amino acid-derived polymerized unit is a part of an amino acid-lipid conjugation compound and is obtained by polymerization from an amino acid or amino acid derivative having a carboxyl group at one end and an amino group at the other end,
The lipid-derived polymerization unit is a part of an amino acid-lipid conjugation compound, and is obtained by polymerization from a lipid compound containing a hydrocarbon having 10 to 20 carbon atoms, or a pharmaceutically acceptable salt thereof.
상기 아미노산은 알파 아미노산을 포함하는 것을 특징으로 하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.In claim 1,
An amino acid-lipid conjugation compound, or a pharmaceutically acceptable salt thereof, wherein the amino acid includes an alpha amino acid.
상기 알파 아미노산은 이소류신, 류신, 리신, 발린, 메티오닌, 페닐알라닌, 트레오닌, 트립토판, 히스티딘, 아르기닌, 알라닌, 아스파라긴, 아스파르트산, 시스테인, 글루탐산, 글루타민, 글리신, 프롤린, 세린, 티로신 및 이의 조합으로 이루어진 군으로부터 선택되는 아미노산을 포함하는 것을 특징으로 하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.In claim 2,
The alpha amino acids are isoleucine, leucine, lysine, valine, methionine, phenylalanine, threonine, tryptophan, histidine, arginine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, and combinations thereof. An amino acid-lipid conjugation compound comprising an amino acid selected from, or a pharmaceutically acceptable salt thereof.
상기 아미노산 유도체는 아미노산의 카르복실기가 알코올 화합물과 중합 반응하여 형성된 에스테르 화합물이거나 아미노산의 카르복실기가 아민 화합물과 중합 반응하여 형성된 아마이드 화합물을 포함하는 것을 특징으로 하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.In claim 1,
The amino acid derivative is an amino acid-lipid conjugate compound, which is characterized in that it contains an ester compound formed by polymerizing the carboxyl group of an amino acid with an alcohol compound or an amide compound formed by polymerizing the carboxyl group of an amino acid with an amine compound, or a pharmaceutically acceptable compound thereof. Possible salt.
상기 알코올 화합물은 메탄올, 에탄올, 프로판올, 부탄올 또는 이의 조합을 포함하는 것을 특징으로 하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.In claim 4,
The alcohol compound is an amino acid-lipid conjugation compound, or a pharmaceutically acceptable salt thereof, wherein the alcohol compound includes methanol, ethanol, propanol, butanol, or a combination thereof.
상기 아민 화합물은 아미노 메탄올, 아미노 에탄올, 아미노 프로판올, 아미노 부탄올, 메틸아미노 메탄올, 메틸아미노 에탄올, 메틸아미노 프로판올, 메틸아미노 부탄올, 에틸아미노 메탄올, 에틸아미노 에탄올, 에틸아미노 프로판올, 에틸아미노 부탄올, 아미노메틸 이미다졸, 아미노에틸 이미다졸, 아미노프로필 이미다졸, 아미노부틸 이미다졸 또는 이의 조합을 포함하는 것을 특징으로 하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.In claim 4,
The amine compounds include amino methanol, amino ethanol, amino propanol, amino butanol, methylamino methanol, methylamino ethanol, methylamino propanol, methylamino butanol, ethylamino methanol, ethylamino ethanol, ethylamino propanol, ethylamino butanol, aminomethyl An amino acid-lipid conjugation compound comprising imidazole, aminoethyl imidazole, aminopropyl imidazole, aminobutyl imidazole, or a combination thereof, or a pharmaceutically acceptable salt thereof.
상기 지질 화합물은 탄화수소의 일측단에 아미노산의 아미노기와 중합할 수 있는 작용기를 갖는 것을 특징으로 하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.In claim 1,
The lipid compound is an amino acid-lipid conjugation compound, or a pharmaceutically acceptable salt thereof, characterized in that it has a functional group capable of polymerizing with the amino group of an amino acid at one end of the hydrocarbon.
상기 지질 화합물의 작용기는 카르복실기, 알데히드기 또는 아세테이트기인 것을 특징으로 하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.In claim 7,
An amino acid-lipid conjugation compound, or a pharmaceutically acceptable salt thereof, wherein the functional group of the lipid compound is a carboxyl group, an aldehyde group, or an acetate group.
상기 아미노산-지질 접합 화합물은 1개 내지 10개의 아미노산 유래 중합 단위를 포함하고,
상기 아미노산-지질 접합 화합물이 2개 이상의 아미노산 유래 중합 단위를 포함하는 경우, 각각의 아미노산 유래 중합 단위는 동일하거나 상이한 것을 특징으로 하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.In claim 1,
The amino acid-lipid conjugation compound contains 1 to 10 amino acid-derived polymerization units,
When the amino acid-lipid conjugation compound includes two or more amino acid-derived polymerization units, each amino acid-derived polymerization unit is the same or different, or a pharmaceutically acceptable salt thereof.
상기 아미노산-지질 접합 화합물은 하기 화합물 1 내지 53인 것을 특징으로 하는 아미노산-지질 접합 화합물, 또는 이의 약학적으로 허용가능한 염.
화합물 1;
화합물 2;
화합물 3;
화합물 4;
화합물 5;
화합물 6;
화합물 7;
화합물 8;
화합물 9;
화합물 10;
화합물 11;
화합물 12;
화합물 13;
화합물 14;
화합물 15;
화합물 16;
화합물 17;
화합물 18;
화합물 19;
화합물 20;
화합물 21;
화합물 22;
화합물 23;
화합물 24;
화합물 25;
화합물 26;
화합물 27;
화합물 28;
화합물 29;
화합물 30;
화합물 31;
화합물 32;
화합물 33;
화합물 34;
화합물 35;
화합물 36;
화합물 37;
화합물 38;
화합물 39;
화합물 40;
화합물 41;
화합물 42;
화합물 43;
화합물 44;
화합물 45;
화합물 46;
화합물 47;
화합물 48;
화합물 49;
화합물 50;
화합물 51;
화합물 52;
화합물 53;
.In claim 1,
The amino acid-lipid conjugation compound is an amino acid-lipid conjugation compound, or a pharmaceutically acceptable salt thereof, characterized in that the compounds 1 to 53 below.
Compound 1;
Compound 2;
Compound 3;
Compound 4;
Compound 5;
Compound 6;
Compound 7;
Compound 8;
Compound 9;
Compound 10;
Compound 11;
Compound 12;
Compound 13;
Compound 14;
Compound 15;
Compound 16;
Compound 17;
Compound 18;
Compound 19;
Compound 20;
Compound 21;
Compound 22;
Compound 23;
Compound 24;
Compound 25;
Compound 26;
Compound 27;
Compound 28;
Compound 29;
Compound 30;
Compound 31;
Compound 32;
Compound 33;
Compound 34;
Compound 35;
Compound 36;
Compound 37;
Compound 38;
Compound 39;
Compound 40;
Compound 41;
compound 42;
Compound 43;
Compound 44;
Compound 45;
Compound 46;
Compound 47;
Compound 48;
Compound 49;
Compound 50;
Compound 51;
Compound 52;
Compound 53;
.
상기 지질 나노입자는 아미노산-지질 접합 화합물, 인지질, 콜레스테롤 및 PEG-지질 접합체의 총 몰 수를 기준으로 30 몰% 내지 60 몰%의 아미노산-지질 접합 화합물을 포함하는 것을 특징으로 하는 지질 나노입자.
In claim 11,
The lipid nanoparticle is a lipid nanoparticle characterized in that it contains 30 mol% to 60 mol% of an amino acid-lipid conjugate compound based on the total number of moles of the amino acid-lipid conjugate compound, phospholipid, cholesterol, and PEG-lipid conjugate.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20240076588A true KR20240076588A (en) | 2024-05-30 |
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