AMINO LIPID COMPOUND, PREPARATION METHOD THEREFOR, AND USE THEREOF

Abstract

The present invention relates to an amino lipid compound, namely a compound represented by chemical formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof. Moreover, also disclosed are a preparation method for the compound and the use of the compound in delivering a genetic material and preparing a drug.

##STR00001##

Claims

1. An amino lipid compound represented by chemical formula I, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, a prodrug thereof, or a solvate thereof: ##STR00084## wherein: R.sup.1 and R.sup.2 are independently to each other selected from the following structures: a linear or branched, substituted or unsubstituted alkyl structure containing 6 to 24 carbon atoms, wherein in the substituted alkyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; a linear or branched, substituted or unsubstituted alkenyl structure containing 6 to 24 carbon atoms, wherein in the substituted alkenyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; a linear or branched, substituted or unsubstituted alkynyl structure containing 6 to 24 carbon atoms, wherein in the substituted alkynyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; a linear or branched, saturated or unsaturated, substituted or unsubstituted acyl structure containing 4 to 24 carbon atoms, wherein in the substituted acyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; R.sup.3 and R.sup.4 are independently to each other selected from the following structures: a linear or branched, substituted or unsubstituted alkyl structure containing 1 to 12 carbon atoms, wherein in the substituted alkyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; a linear or branched, substituted or unsubstituted alkenyl structure containing 2 to 12 carbon atoms, wherein in the substituted alkenyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; a linear or branched, substituted or unsubstituted alkynyl structure containing 2 to 12 carbon atoms, wherein in the substituted alkynyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; R.sup.3 and R.sup.4 combine with each other to form a 4 to 10-membered heterocycle structure, wherein the heteroatom is one or more heteroatoms of nitrogen, sulfur, and oxygen, and the heterocycle optionally comprises 1 to 6 heteroatoms; L is selected from the following structures: a linear or branched, substituted or unsubstituted alkylene structure containing 1 to 12 carbon atoms, wherein said substituent group is one or more of a hydrocarbyl group, a carboxyl group, an acyl group, and an alkoxy group; a linear or branched, substituted or unsubstituted alkenylene structure containing 2 to 12 carbon atoms, wherein said substituent group is one or more of a hydrocarbyl group, a carboxyl group, an acyl group, and an alkoxy group; a linear or branched, substituted or unsubstituted alkynylene structure containing 2 to 12 carbon atoms, wherein said substituent group is one or more of a hydrocarbyl group, a carboxyl group, an acyl group, and an alkoxy group; a 4 to 10-membered heterocycle structure, the heteroatom is one or more heteroatoms of nitrogen, sulfur, and oxygen, and the heterocycle optionally comprises 1 to 6 heteroatoms; n=1 or 2; X is one selected from CH.sub.2, NH, O, S, S(?O), S(?O).sub.2 and (SS).

2. The amino lipid compound according to claim 1, which is characterized in that said R.sup.1 is one selected from the following N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N18, N19, and N20: N6: CH.sub.3(CH.sub.2).sub.5; N7: CH.sub.3(CH.sub.2).sub.6; N8: CH.sub.3(CH.sub.2).sub.7; N9: CH.sub.3(CH.sub.2).sub.8; N10: CH.sub.3(CH.sub.2).sub.9; N11: CH.sub.3(CH.sub.2).sub.10; N12: CH.sub.3(CH.sub.2).sub.11; N13: CH.sub.3(CH.sub.2).sub.12; N14: CH.sub.3(CH.sub.2).sub.13; N15: CH.sub.3(CH.sub.2).sub.14; N16: CH.sub.3(CH.sub.2).sub.15; N18: CH.sub.3(CH.sub.2).sub.17; ##STR00085## said R.sup.2 is one selected from the following A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A18, A19, and A20: A6: CH.sub.3(CH.sub.2).sub.4; A7: CH.sub.3(CH.sub.2).sub.5; A8: CH.sub.3(CH.sub.2).sub.6; A9: CH.sub.3(CH.sub.2).sub.7; A10: CH.sub.3(CH.sub.2).sub.8; A11: CH.sub.3(CH.sub.2).sub.9; A12: CH.sub.3(CH.sub.2).sub.10; A13: CH.sub.3(CH.sub.2).sub.11; A14: CH.sub.3(CH.sub.2).sub.12; A15: CH.sub.3(CH.sub.2).sub.13; A16: CH.sub.3(CH.sub.2).sub.14; A18: CH.sub.3(CH.sub.2).sub.16; ##STR00086## X-L-N(R.sup.3)(R.sup.4) is any one selected from the following O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, D1, D2, D3, D4, D5, D6, D7, D8, D9, and D10: ##STR00087## ##STR00088##

3. The amino lipid compound according to claim 1, which is characterized in that R.sup.1 and R.sup.2 are independently to each other selected from the following structures: a linear or branched, substituted or unsubstituted alkyl structure containing 6 to 24 carbon atoms, wherein in the substituted alkyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; a linear or branched, substituted or unsubstituted alkenyl structure containing 6 to 24 carbon atoms, wherein in the substituted alkenyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; R.sup.3 and R.sup.4 are independently to each other selected from the following structures: a linear or branched, substituted or unsubstituted alkyl structure containing 1 to 12 carbon atoms, wherein in the substituted alkyl structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms; R.sup.3 and R.sup.4 combine with each other to form a 4 to 10-membered heterocycle structure, wherein the heteroatom is one or more heteroatoms of nitrogen, sulfur, and oxygen, and the heterocycle optionally comprises 1 to 6 heteroatoms; X is NH or O.

4. The amino lipid compound according to claim 1, which is characterized in that said L is a linear or branched, substituted or unsubstituted alkylene structure containing 1 to 4 carbon atoms, wherein in the substituted alkylene structure, said substituent group is a hydrocarbyl group containing 1 to 6 carbon atoms.

5. The amino lipid compound according to claim 1, wherein said X is O.

6. The amino lipid compound according to claim 1, wherein said R.sup.1 is one selected from the following N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N18, N19, and N20: N6: CH.sub.3(CH.sub.2).sub.5; N7: CH.sub.3(CH.sub.2).sub.6; N8: CH.sub.3(CH.sub.2).sub.7; N9: CH.sub.3(CH.sub.2).sub.8; N10: CH.sub.3(CH.sub.2).sub.9; N11: CH.sub.3(CH.sub.2).sub.19; N12: CH.sub.3(CH.sub.2).sub.11; N13: CH.sub.3(CH.sub.2).sub.12; N14: CH.sub.3(CH.sub.2).sub.13; N15: CH.sub.3(CH.sub.2).sub.14; N16: CH.sub.3(CH.sub.2).sub.15; N18: CH.sub.3(CH.sub.2).sub.17; ##STR00089## said R.sup.2 is one selected from the following A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A18, A19, and A20: A6: CH.sub.3(CH.sub.2).sub.4; A7: CH.sub.3(CH.sub.2).sub.5; A8: CH.sub.3(CH.sub.2).sub.6; A9: CH.sub.3(CH.sub.2).sub.7; A10: CH.sub.3(CH.sub.2).sub.8; A11: CH.sub.3(CH.sub.2).sub.9; A12: CH.sub.3(CH.sub.2).sub.10; A13: CH.sub.3(CH.sub.2).sub.11; A14: CH.sub.3(CH.sub.2).sub.12; A15: CH.sub.3(CH.sub.2).sub.13; A16: CH.sub.3(CH.sub.2).sub.14; A18: CH.sub.3(CH.sub.2).sub.16; ##STR00090## and any one of N19, N20, A19 and A20 is at least contained in the molecule; O-L-N(R.sup.3)(R.sup.4) is any one selected from the following O2, O4, O5, O8, O9, and O10; ##STR00091##

7. The amino lipid compound according to claim 1, wherein when said R.sup.1 is N19 or N20, said R.sup.2 is any one of A10, A11, A12, A14, A15, A16, and A18; or when said R.sup.2 is A19 or A20, said R.sup.1 is any one of N10, N11, N12, N14, N15, N16, and N18.

8. The amino lipid compound according to claim 1, wherein said amino lipid compound is: ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096##

9. A process for preparing the amino lipid compound according to claim 1, which comprises the following steps: S1, stirring compounds NH.sub.2R.sup.1 and R.sup.2CHO in a solvent to react, removing the solvent by distillation, adding a cyclic acid anhydride, warming up and reacting, and purifying to obtain a compound (II) having the following structural formula, ##STR00097## S2, reacting compound (II) with an alcohol or amine represented by ##STR00098## in the presence of a condensation agent to obtain a compound (I) having the following structural formula, ##STR00099## wherein: wherein each variable such as R.sup.1, R.sup.2, R.sup.3, R.sup.4, n, X, and L is defined as in claim 1.

10. A composition for delivering genetic substances (for example nucleic acids, such as mRNA) into cells, wherein the composition comprises the amino lipid compound according to claim 1, preferably said composition further comprises one or more substances of a helper lipid (e.g. non-cationic lipid), a sterol (e.g. cholesterol), a polyethylene glycol lipid (e.g. PEG2000-DMG) and a bioactivator.

11. The composition according to claim 10, wherein the composition is present in the form of lipid particles.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG. 1 shows the humoral antibody titers produced by the delivery of OVA mRNA via the subcutaneous administration of LNPs prepared with the representative compounds N12A12C4O2, N16A10C5O10, N12A12C4D1, and N12A20C4O10 of the Examples of the present invention; and

[0070] FIG. 2 shows the humoral antibody titers produced by the delivery of OVA mRNA via the subcutaneous administration of LNPs prepared with the representative compounds Lipid 1 to Lipid 10 of the Examples of the present invention.

DETAILED DESCRIPTION

[0071] As used herein, the following compound represented by chemical formula I exists in the form of the racemate, and the stereochemistry information shown in chemical formula I is schematic.

##STR00041##

[0072] As used herein, the term substituted means optionally substituted, i.e., one or more hydrogen atoms attached to an atom or group are independently unsubstituted, or are substituted by one or more substituents, e.g. one, two, three or four substituents, the substituents are independently selected from: deuterium (D), halogen, OH, mercapto, cyano, CD.sub.3, C.sub.1-C.sub.6 alkyl (preferably C.sub.1-C.sub.3 alkyl), C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, cycloalkyl (preferably C.sub.3-C.sub.8 cycloalkyl), aryl, heterocyclyl (preferably 3-8-membered heterocyclyl), heteroaryl, arylC.sub.1-C.sub.6 alkyl-, heteroarylC.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, OC.sub.1-C.sub.6 alkyl (preferably OC.sub.1-C.sub.3 alkyl), OC.sub.2-C.sub.6 alkenyl, OC.sub.1-C.sub.6 alkylphenyl, C.sub.1-C.sub.6 alkyl-OH (preferably C.sub.1-C.sub.4 alkyl-OH), C.sub.1-C.sub.6 alkyl-SH, C.sub.1-C.sub.6 alkyl-OC.sub.1-C.sub.6 alkyl, OC.sub.1-C.sub.6 haloalkyl, NH.sub.2, C.sub.1-C.sub.6 alkyl-NH.sub.2 (preferably C.sub.1-C.sub.3 alkyl-NH.sub.2), N(C.sub.1-C.sub.6 alkyl).sub.2 (preferably N(C.sub.1-C.sub.3 alkyl).sub.2), NH(C.sub.1-C.sub.6 alkyl) (preferably NH(C.sub.1-C.sub.3 alkyl)), N(C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6 alkylphenyl), NH(C.sub.1-C.sub.6 alkylphenyl), nitro, C(O)OH, C(O)OC.sub.1-C.sub.6 alkyl (preferably C(O)OC.sub.1-C.sub.3 alkyl), CONRiRii (wherein Ri and Rii are each independently H, D or C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.3 alkyl), NHC(O)(C.sub.1-C.sub.6 alkyl), NHC(O)(phenyl), N(C.sub.1-C.sub.6 alkyl)C(O)(C.sub.1-C.sub.6 alkyl), N(C.sub.1-C.sub.6 alkyl)C(O) (phenyl), C(O)C.sub.1-C.sub.6 alkyl, C(O)heteroaryl (preferably C(O)-5-7-numbered heteroaryl), C(O)C.sub.1-C.sub.6 alkylphenyl, C(O)C.sub.1-C.sub.6 haloalkyl, OC(O)C.sub.1-C.sub.6 alkyl (preferably OC(O)C.sub.1-C.sub.3 alkyl), S(O).sub.2C.sub.1-C.sub.6 alkyl, S(O)C.sub.1-C.sub.6 alkyl, S(O).sub.2-phenyl, S(O).sub.2C.sub.1-C.sub.6 haloalkyl, S(O).sub.2NH.sub.2, S(O).sub.2NH(C.sub.1-C.sub.6 alkyl), S(O).sub.2NH(phenyl), NHS(O).sub.2(C.sub.1-C.sub.6 alkyl), NHS(O).sub.2(phenyl) and NHS(O).sub.2(C.sub.1-C.sub.6 haloalkyl), wherein each of said alkyl, cycloalkyl, phenyl, aryl, heterocyclyl and heteroaryl is optionally further substituted by one or more substituents selected from the following substituents: halogen, OH, NH.sub.2, cycloalkyl, 3-8 membered heterocyclyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl-, OC.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyl-OH, C.sub.1-C.sub.4 alkyl-OC.sub.1-C.sub.4 alkyl, OC.sub.1-C.sub.4 haloalkyl, cyano, nitro, C(O)OH, C(O)OC.sub.1-C.sub.6 alkyl, CON(C.sub.1-C.sub.6 alkyl).sub.2, CONH(C.sub.1-C.sub.6 alkyl), CONH.sub.2, NHC(O)(C.sub.1-C.sub.6 alkyl), NH(C.sub.1-C.sub.6 alkyl)C(O)(C.sub.1-C.sub.6 alkyl), SO.sub.2(C.sub.1-C.sub.6 alkyl), SO.sub.2(phenyl), SO.sub.2(C.sub.1-C.sub.6 haloalkyl), SO.sub.2NH.sub.2, SO.sub.2NH(C.sub.1-C.sub.6 alkyl), SO.sub.2NH(phenyl), NHSO.sub.2(C.sub.1-C.sub.6 alkyl), NHSO.sub.2(phenyl) and NHSO.sub.2(C.sub.1-C.sub.6 haloalkyl). Herein when one atom or group is substituted with a plurality of substituents, the plurality of substituents may be identical or different.

[0073] As used herein, the term hydrocarbyl means the group remained after an aliphatic hydrocarbon loses one hydrogen atom, including straight-chain or branched-chain, saturated or unsaturated hydrocarbyl groups, including alkyl, alkenyl, and alkynyl.

[0074] As used herein, the term acyl refers to a hydrocarbyl-carbonyl group, preferably the acyl is C.sub.4-C.sub.24 acyl, C.sub.6-C.sub.18 acyl, C.sub.6-C.sub.12 acyl, C.sub.6-C.sub.10 acyl, C.sub.4-C.sub.6 acyl, C.sub.2-C.sub.12 acyl, or C.sub.2-C.sub.6 acyl.

[0075] As used herein, the term alkoxy refers to an alkyl-oxy group, preferably the alkoxy is C.sub.1-C.sub.10 alkoxy, more preferably, the alkoxy is C.sub.1-C.sub.6 alkoxy, most preferably, the alkoxy is C.sub.1-C.sub.3 alkoxy.

[0076] As used herein, the term heterocycle refers to a saturated or unsaturated cyclic group containing heteroatom(s) selected from N, O, S, and the like, and the heterocycle may be optionally substituted with one or more substituents.

[0077] In order to make the purposes, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention will be clearly and completely described below with reference to the accompanying drawings and specific examples. Obviously, the described examples are a part of rather than all of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Intermediate 1: 1-dodecyl-5-oxo-2-undecylpyrrolidine-3-carboxylic Acid

[0078] ##STR00042##

[0079] To a 250 mL reaction flask were successively added n-dodecyl amine (1.85 g, 10 mmol), n-dodecanal (1.84 g, 10 mmol), and absolute methanol (100 mL). The resulting mixture was stirred and reacted at room temperature for 12 hours. The solvent was removed by evaporation to dryness under reduced pressure. Then xylene (150 mL) and butanedioic anhydride (1.00 g, 10 mmol) were successively added. The resulting mixture was heated up to 140? C. and reacted for 10 hours. The solvent was removed by evaporation to dryness under reduced pressure, and then hexane (50 mL) was added. The resulting mixture was crystallized under stirring, filtered, washed with a small amount of hexane, and dried to produce 1-dodecyl-5-oxo-2-undecylpyrrolidine-3-carboxylic acid (3.75 g, 83%).

Intermediate 2: 1-hexadecyl-2-nonyl-6-oxopiperidine-3-carboxylic Acid

[0080] ##STR00043##

[0081] To a 250 mL reaction flask were successively added n-hexadecyl amine (2.42 g, 10 mmol), n-decyl aldehyde (1.56 g, 10 mmol), and absolute methanol (100 mL). The resulting mixture was stirred and reacted at room temperature for 12 hours. The solvent was removed by evaporation to dryness under reduced pressure. Then xylene (150 mL) and glutaric anhydride (1.14 g, 10 mmol) were successively added. The resulting mixture was heated up to 140? C. and reacted for 10 hours. The solvent was removed by evaporation to dryness under reduced pressure, and then hexane (50 mL) was added. The resulting mixture was crystallized under stirring, filtered, washed with a small amount of hexane, and dried to produce 1-hexadecyl-2-nonyl-6-oxopiperidine-3-carboxylic acid (3.51 g, 71%).

Intermediate 3: 1-((Z)-octadeca-9-en-1-yl)-5-oxo-2-nonylpyrrolidine-3-carboxylic Acid

[0082] ##STR00044##

[0083] To a 250 mL reaction flask were successively added oleylamine (2.68 g, 10 mmol), n-decyl aldehyde (1.56 g, 10 mmol), and absolute methanol (100 mL). The resulting mixture was stirred and reacted at room temperature for 12 hours. The solvent was removed by evaporation to dryness under reduced pressure. Then xylene (150 mL) and butanedioic anhydride (1.00 g, 10 mmol) were successively added. The resulting mixture was heated up to 140? C. and reacted for 10 hours. The solvent was removed by evaporation to dryness under reduced pressure, and then hexane (50 mL) was added. The resulting mixture was crystallized under stirring, filtered, washed with a small amount of hexane, and dried to produce 1-((Z)-octadeca-9-en-1-yl)-5-oxo-2-nonylpyrrolidine-3-carboxylic acid (4.54 g, 90%).

Intermediate 4: 1-dodecyl-2-((8Z,11Z)-heptadeca-8,11-dien-1-yl)-5-oxopyrrolidine-3-carboxylic Acid

[0084] ##STR00045##

[0085] To a 250 mL reaction flask were successively added n-dodecyl amine (1.85 g, 10 mmol), cis,cis-octadeca-9,12-dienal (2.64 g, 10 mmol), and absolute methanol (100 mL). The resulting mixture was stirred and reacted at room temperature for 12 hours. The solvent was removed by evaporation to dryness under reduced pressure. Then xylene (150 mL) and butanedioic anhydride (1.00 g, 10 mmol) were successively added. The resulting mixture was heated up to 140? C. and reacted for 10 hours. The solvent was removed by evaporation to dryness under reduced pressure, and then hexane (50 mL) was added. The resulting mixture was crystallized under stirring, filtered, washed with a small amount of hexane, and dried to produce 1-dodecyl-2-((8Z,11Z)-heptadeca-8,11-dien-1-yl)-5-oxopyrrolidine-3-carboxylic acid (4.31 g, 81%).

Intermediate 5: 1-((Z,Z)-octadeca-9,12-dien-1-yl)-5-oxo-2-undecylpyrrolidine-3-carboxylic Acid

[0086] ##STR00046##

[0087] To a 250 mL reaction flask were successively added linoleylamine (2.66 g, 10 mmol), n-dodecanal (1.84 g, 10 mmol), and absolute methanol (100 mL). The resulting mixture was stirred and reacted at room temperature for 12 hours. The solvent was removed by evaporation to dryness under reduced pressure. Then xylene (150 mL) and butanedioic anhydride (1.00 g, 10 mmol) were successively added. The resulting mixture was heated up to 140? C. and reacted for 10 hours. The solvent was removed by evaporation to dryness under reduced pressure, and then hexane (50 mL) was added. The resulting mixture was crystallized under stirring, filtered, washed with a small amount of hexane, and dried to produce 1-((Z,Z)-octadeca-9,12-dien-1-yl)-5-oxo-2-undecylpyrrolidine-3-carboxylic acid (4.31 g, 81%).

Intermediate 6: 1-dodecyl-2-((8Z)-heptadeca-8-en-1-yl)-5-oxopyrrolidine-3-carboxylic Acid

[0088] ##STR00047##

[0089] To a 250 mL reaction flask were successively added n-dodecyl amine (1.85 g, 10 mmol), cis-9-octadecenal (2.66 g, 10 mmol), and absolute methanol (100 mL). The resulting mixture was stirred and reacted at room temperature for 12 hours. The solvent was removed by evaporation to dryness under reduced pressure. Then xylene (150 mL), and butanedioic anhydride (1.00 g, 10 mmol) were successively added. The resulting mixture was heated up to 140? C. and reacted for 10 hours. The solvent was removed by evaporation to dryness under reduced pressure, and then hexane (50 mL) was added. The resulting mixture was crystallized under stirring, filtered, washed with a small amount of hexane, and dried to produce 1-dodecyl-2-((8Z)-heptadeca-8-en-1-yl)-5-oxopyrrolidine-3-carboxylic acid (4.17 g, 78%).

Intermediate 7: 1-hexadecyl-2-((8Z,11Z)-heptadeca-8,11-dien-1-yl)-5-oxopyrrolidine-3-carboxylic Acid

[0090] ##STR00048##

[0091] To a 250 mL reaction flask were successively added n-hexadecyl amine (2.42 g, 10 mmol), cis,cis-octadeca-9,12-dienal (2.64 g, 10 mmol), and absolute methanol (100 mL). The resulting mixture was stirred and reacted at room temperature for 12 hours. The solvent was removed by evaporation to dryness under reduced pressure. Then xylene (150 mL), and butanedioic anhydride (1.00 g, 10 mmol) were successively added. The resulting mixture was heated up to 140? C. and reacted for 10 hours. The solvent was removed by evaporation to dryness under reduced pressure, and then hexane (50 mL) was added. The resulting mixture was crystallized under stirring, filtered, washed with a small amount of hexane, and dried to produce 1-hexadecyl-2-((8Z,11Z)-heptadeca-8,11-dien-1-yl)-5-oxopyrrolidine-3-carboxylic acid (4.76 g, 81%).

EXAMPLE 1: SYNTHESIS OF COMPOUND N12A12C4O2

[0092] ##STR00049##

[0093] To a 250 mL reaction flask were successively added 1-dodecyl-5-oxo-2-undecylpyrrolidine-3-carboxylic acid (intermediate 1) (903 mg, 2 mmol), 3-dimethylamino-1-propanol (310 mg, 3 mmol), and dichloromethane (50 mL). The mixture was dissolved under stirring. Then dicyclohexylcarbodiimide (824 mg, 4 mmol) and 4-dimethylaminopyridine (5 mg, 0.04 mmol) were added. The resulting mixture was reacted at room temperature for 2 hours, washed with water three times, dried over anhydrous sodium sulfate, concentrated, and then purified with a flash column chromatography system (dichloromethane:methano=20:1 to 5:1) to obtain the compound N12A12C4O2 (1.03 g, 96%).

[0094] .sup.1H NMR (400 MHz, DMSO-d6): ?4.15 (m, 2H), 3.94 (m, 1H), 3.18 (m, 2H), 2.90 (m, 1H), 2.73 (m, 1H), 2.62 (m, 1H), 2.34 (t, 2H), 2.16 (s, 6H), 1.67 (m, 2H), 1.39-1.18 (m, 40H), 0.89 (m, 6H). ESI-MS calculated for C.sub.33H.sub.65N.sub.2O.sub.3.sup.+ [M+H].sup.+ 537.5, found 537.7

EXAMPLE 2: SYNTHESIS OF COMPOUND N16A10C5O10

[0095] ##STR00050##

[0096] To a 250 mL reaction flask were successively added 1-hexadecyl-2-nonyl-6-oxopiperidine-3-carboxylic acid (intermediate 2) (988 mg, 2 mmol), N-ethoxypiperidine (387 mg, 3 mmol), and dichloromethane (50 mL). The mixture was dissolved under stirring. Then dicyclohexylcarbodiimide (824 mg, 4 mmol) and 4-dimethylaminopyridine (5 mg, 0.04 mmol) were added. The resulting mixture was reacted at room temperature for 2 hours, washed with water three times, dried over anhydrous sodium sulfate, concentrated, and then purified with a flash column chromatography system (dichloromethane:methanol=20:1 to 5:1) to obtain the compound N16A10C5O10 (1.03 g, 85%).

[0097] .sup.1H NMR (400 MHz, DMSO-d6): ?4.15 (m, 2H), 3.94 (m, 1H), 3.18 (m, 2H), 2.90 (m, 1H), 2.73 (m, 1H), 2.62 (m, 1H), 2.34 (t, 2H), 2.16 (s, 6H), 1.67 (m, 2H), 1.39-1.18 (m, 40H), 0.89 (m, 6H). ESI-MS calculated for C.sub.33H.sub.65N.sub.2O.sub.3.sup.+ [M+H].sup.+ 606.0, found 606.3

EXAMPLE 3: SYNTHESIS OF COMPOUND N12A12C4D1

[0098] ##STR00051##

[0099] To a 250 mL reaction flask were successively added 1-dodecyl-5-oxo-2-undecylpyrrolidine-3-carboxylic acid (intermediate 1) (903 mg, 2 mmol), N,N-dimethylethylene diamine (353 mg, 4 mmol), and dichloromethane (50 mL). The mixture was dissolved under stirred. Then O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU, 1.14 g, 3 mmol) and N,N-diisopropylethylamine (516 mg, 4 mmol) were added. The resulting mixture was reacted at room temperature for 2 hours. After the completion of the reaction detected by TLC, dichloromethane (200 mL) was added. The resulting mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and then purified with a flash column chromatography system (dichloromethane:methanol=20:1 to 5:1) to obtain the compound N12A12C4D1 (982 mg, 94%).

[0100] .sup.1H NMR (400 MHz, DMSO-d6): ?4.17 (m, 2H), 3.95 (m, 1H), 3.19 (m, 2H), 2.91 (m, 1H), 2.72 (m, 1H), 2.63 (m, 1H), 2.34 (t, 2H), 2.16 (s, 6H), 1.67 (m, 2H), 1.39-1.18 (m, 38H), 0.89 (m, 6H). ESI-MS calculated for C.sub.32H.sub.64N.sub.3O.sub.2.sup.+ [M+H].sup.+ 522.5, found 522.9

EXAMPLE 4: SYNTHESIS OF COMPOUND N12A20C4O10

[0101] ##STR00052##

[0102] To a 250 mL reaction flask were successively added 1-dodecyl-2-((8Z,11Z)-heptadeca-8,11-dien-1-yl)-5-oxopyrrolidine-3-carboxylic acid (intermediate 4) (1064 mg, 2 mmol), N-ethoxypiperidine (387 mg, 3 mmol), and dichloromethane (50 mL). The mixture was dissolved under stirred. Then dicyclohexylcarbodiimide (824 mg, 4 mmol) and 4-dimethylaminopyridine (5 mg, 0.04 mmol) were added. The resulting mixture was reacted at room temperature for 2 hours, washed with water three times, dried over anhydrous sodium sulfate, concentrated, and then purified with a flash column chromatography system (dichloromethane:methanol=20:1 to 5:1) to obtain the compound N12A20C4O10 (1.03 g, 80%).

[0103] .sup.1H NMR (400 MHz, DMSO-d6): ?4.15 (m, 2H), 3.94 (m, 1H), 3.18 (m, 2H), 2.90 (m, 1H), 2.73 (m, 1H), 2.62 (m, 1H), 2.34 (t, 2H), 2.16 (s, 6H), 1.67 (m, 2H), 1.39-1.18 (m, 40H), 0.89 (m, 6H). ESI-MS calculated for C.sub.33H.sub.65N.sub.2O.sub.3.sup.+ [M+H].sup.+ 644.1, found 644.3.

EXAMPLE 5: SYNTHESIS OF LIPID 1

[0104] ##STR00053##

[0105] To a 250 mL reaction flask were successively added 1-((Z)-octadeca-9-en-1-yl)-5-oxo-2-nonylpyrrolidine-3-carboxylic acid (intermediate 3) (1.01 g, 2 mmol), N-ethoxypiperidine (387 mg, 3 mmol), and dichloromethane (50 mL). The mixture was dissolved under stirred. Then dicyclohexylcarbodiimide (824 mg, 4 mmol) and 4-dimethylaminopyridine (5 mg, 0.04 mmol) were added. The resulting mixture was reacted at room temperature for 2 hours, washed with water three times, dried over anhydrous sodium sulfate, concentrated, and then purified with a flash column chromatography system (dichloromethane:methanol=20:1 to 5:1) to obtain the LIPID 1 (1.16 g, 94%).

[0106] .sup.1H NMR (400 MHz, DMSO-d6): ?5.38 (m, 2H), 4.15 (m, 2H), 3.94 (m, 1H), 3.18 (m, 2H), 2.97 (m, 2H), 2.90 (m, 1H), 2.73 (m, 1H), 2.42 (m, 4H), 2.16 (m, 4H), 1.59-1.18 (m, 46H), 0.89 (m, 6H). ESI-MS calculated for C.sub.33H.sub.65N.sub.2O.sub.3.sup.+ [M+H].sup.+ 617.6, found 617.8.

[0107] Other amino lipid compounds were synthesized by using similar methods, and their structures were shown in Table 1. The compound named Dlin-MC3 in Table 1 was commercially available and is also known as MC3 herein.

Assay 1

[0108] Evaluation of Luciferase mRNA In Vivo Delivery Performance of Lipid Nanoparticles Prepared from Amino Lipid Compounds [0109] Preparation process: The delivery vector in the present invention, DSPC, cholesterol, and PEG2000-DMG were mixed and dissolved in absolute ethanol in a molar ratio of 50:10:38.5:1.5. Luciferase mRNA (Fluc mRNA) was dissolved in a sodium acetate solution (50 mM, pH=4.0). Two microinjection pumps were used, and the ratio of ethanol solution to sodium acetate solution (50 mM, pH=4.0) was controlled to be 1:3. A crude solution of lipid nanoparticles was prepared in a microflow channel chip, then dialyzed for 6 hours with 1?PBS (phosphate buffer solution) at a controlled temperature of 4? C. by using a dialysis cassette (Fisher, MWCO 20,000), and filtered with a 0.22 ?m microporous filter membrane before use. The mass ratio of the amino lipid compound to luciferase mRNA (Fluc mRNA) was about 10:1. [0110] Animal preparation: 6-week-old male BALB/c mice with body weights of about 20 g were selected, and fed in an SPF-grade feeding room. Animal experiments were strictly carried out according to the guidelines of the national health institution and the requirements of animal ethics. [0111] In vivo delivery: 3 mice were randomly selected per group and injected with lipid nanoparticles at a dose of 0.5 mg/kg by intramuscular injection. After 6 hours, 200 ?L of 10 mg/mL potassium D-fluorescein was injected into each mouse via the tail vein, and after 10 minutes, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed and recorded by photographing.

[0112] Table 1 provides the expression intensities of Fluc mRNA delivered by the intramuscular injection administration of the representative amino lipid compounds with DLin-MC3 as the control (two batches of tests in total). A plurality of the amino lipid compounds had similar expression intensities to DLin-MC3, and a plurality of the amino lipid compounds had significantly better expression intensities than the positive control.

TABLE-US-00002 Amino lipid Fluorescence compound Structure intensity batch N12A12C4O2 [00054] 2.8E+06 1 N16A10C5O10 [00055] 4.5E+06 1 3 [00056] 5.1E+06 1 4 [00057] 2.8E+06 1 6 [00058] 3.1E+05 1 7 [00059] 6.1E+05 1 N12A12C4D1 [00060] 2.9E+07 1 9 [00061] 6.1E+06 1 10 [00062] 8.1E+05 1 11 [00063] 2.1E+06 1 12 [00064] 2.1E+06 1 13 [00065] 2.1E+06 1 14 [00066] 4.1E+06 1 15 [00067] 2.8E+07 1 16 [00068] 1.4E+07 1 N12A20C4O10 [00069] 2.7E+06 1 19 [00070] 2.1E+06 1 20 [00071] 5.2E+06 1 Dlin-MC3 [00072] 7.7E+06 1 Lipid 1 [00073] 8.8E+06 2 Lipid 2 [00074] 4.9E+06 2 Lipid 3 [00075] 2.9E+07 2 Lipid 4 [00076] 1.6E+07 2 Lipid 5 [00077] 7.6E+06 2 Lipid 6 [00078] 3.7E+06 2 Lipid 7 [00079] 4.8E+07 2 Lipid 8 [00080] 2.3E+07 2 Lipid 9 [00081] 4.2E+06 2 Lipid 10 [00082] 3.6E+06 2 Dlin-MC3 [00083] 6.9E+06 2

Assay 2

[0113] Evaluation of Ovalbumin mRNA In Vivo Delivery and Immunization Performance of Lipid Nanoparticles (Delivery Vector) Prepared from Amino Lipid Compounds [0114] Preparation process: The amino lipid compound described in the present invention, DSPC, cholesterol, and PEG2000-DMG were mixed and dissolved in absolute ethanol in a molar ratio of 50:10:38.5:1.5. Ovalbumin mRNA (OVA mRNA) was dissolved in a sodium acetate solution (50 mM, pH=4.0). Two microinjection pumps were used, and the ratio of ethanol solution to sodium acetate solution (50 mM, pH=4.0) was controlled to be 1:3. A crude solution of lipid nanoparticles was prepared in a microflow channel chip, then dialyzed for 6 hours with 1?PBS at a controlled temperature of 4? C. by using a dialysis cassette (Fisher, MWCO 20,000), and filtered with a 0.22 ?m microporous filter membrane before use. The mass ratio of the amino lipid compound to ovalbumin mRNA (OVA mRNA) was about 10:1. [0115] Animal preparation: 6-week-old male BALB/c mice with body weights of about 20 g were selected, and fed in an SPF-grade feeding room. Animal experiments were strictly carried out according to the guidelines of the national health institution and the requirements of animal ethics. [0116] In vivo delivery: 3 mice were randomly selected per group and injected with lipid nanoparticles at a dose of 0.5 mg/kg by subcutaneous injection (Day 0). After 7 days, the same amount was used for another boost (Day 7). Tail vein blood was taken on the 21st day for the serological analysis. DLin-MC3 was used as the control. [0117] Enzyme-linked immunosorbent assay (ELISA): a flat-bottomed 96-well plate (Nunc) was pre-coated with a concentration of OVA protein of 0.5 ?g protein per well in 50 mM carbonate buffer (pH 9.6), and was allowed to stay overnight at 4? C., and then blocked with 5% glycine. Anti-serums, proteins obtained from immune animals, were diluted from 10.sup.2 to 10.sup.6 with PBS-0.05% Tween (PBS-T) at pH 7.4, added to the wells and incubated at room temperature, and placed at 37? C. for 1 hour. Horseradish peroxidase (HRP) coupled goat anti-mouse IgG was used for labeling in PBS-T-1% BSA at a dilution rate of 1:10,000. After the addition of the HRP substrate, the optical density was determined at a wavelength, and the absorbance at 450 nm was measured in an ELISA microplate reader (Bio-Rad).

[0118] IgG antibody titers produced by the delivery of OVA mRNA via the subcutaneous administration of LNPs prepared from representative compounds N12A12C4O2, N16A10C5O10, N12A12C4D1, and N12A20C4O10 were shown in FIG. 1, wherein IgG antibody titers derived from N12A12C4O2 and DLin-MC3 were comparable, while IgG antibody titers derived from N16A10C5O10, N12A12C4D1, and N12A20C4O10 were significantly superior to that of the DLin-MC3 control group.

[0119] As shown in FIG. 2, IgG antibody titers produced by the delivery of OVA mRNA via the subcutaneous administration of LNPs prepared with the representative compounds Lipid 1 to Lipid 10 were significantly superior to that of the DLin-MC3 control group. It will be appreciated that modifications and variations are possible to those skilled in the art in light of the above teachings, and it is intended that the present invention cover all such modifications and variations as fall within the scope of the appended claims.