Novel ionizable lipid and lipid nanoparticle composition using same

Abstract

The present invention relates to a novel ionizable lipid and a lipid nanoparticle composition using same. Lipid nanoparticles formed using the novel ionizable lipid exhibit an excellent gene encapsulation rate and intracellular / in vivo gene delivery rate, and exhibit excellent muscle delivery of genes through local injection, and thus can be effectively used as a composition for drug delivery.

Description

Novel ionized lipid and lipid nanoparticle composition using the same

[0001] The present invention relates to a novel ionized lipid and a lipid nanoparticle composition using the same.

[0002] Nucleic acid-based medicines are being used not only as therapeutic agents but also as preventative agents that protect against diseases by injecting genes that can express antigens for specific diseases. Among these, gene-based vaccines are categorized into DNA vaccines, RNA vaccines, and viral vector vaccines. Among them, RNA vaccines inject mRNA encoding an antigen into the body, causing the antigen to be expressed and antibodies to be formed. RNA vaccines do not have the potential risks of infection associated with viral vector-based vaccines or genetic mutation associated with DNA vaccines. Furthermore, they can be developed quickly, garnering attention as an effective response to the COVID-19 outbreak in 2019.

[0003] However, genes are easily broken down by nucleases in the human body and, as negatively charged macromolecules, are not easily delivered into cells. Therefore, a method to deliver them stably and efficiently to the desired location is needed. Delivery techniques based on various materials such as lipids, polymers, dendrimers, and inorganic metal materials have been reported as gene delivery systems. Not only patisiran, the first siRNA new drug to receive FDA approval in 2018, but also the mRNA vaccine for COVID-19, which received emergency use approval in 2020, used lipid nanoparticles. Current lipid nanoparticles are generally used in a form in which four components are mixed in a certain ratio: ionizable lipids, phospholipids (helper lipids), cholesterol (structural maintenance lipids), and PEG-lipids.

[0004] With the commercialization of siRNA drugs and mRNA vaccines, lipid nanoparticle-based delivery systems face a variety of challenges. Beyond the challenges of traditional nucleic acid drug delivery systems, such as increased stability and cell permeability, they also face challenges such as targeting, ease of storage and distribution, mitigation of side effects, cost reduction, and response to breakthrough infections. Consequently, the nucleic acid drug market continues to require the development of lipid nanoparticles that can address these new challenges.

[0005] Recently, various studies and extensive clinical trials on mRNA vaccines have established that the efficacy of lipid nanoparticle-based mRNA vaccines is achieved through a three-step mechanism of action through local reactions at the injection site. That is, after intramuscular injection of lipid nanoparticles containing antigen mRNA, the final immune system is completed through the following stages: 1) antigen expression in nearby muscle cells, 2) antigen expression in antigen-presenting cells (dendritic cells, macrophages, Langerhans cells) residing around muscle tissue, and 3) B-cell and T-cell activation in lymph nodes around muscle tissue.

[0006] Meanwhile, when developing mRNA vaccines based on lipid nanoparticles, it is very important to control the immune response (immunogenicity) as a therapeutic effect of the vaccine and the systemic inflammatory response (reactogenicity) as a secondary side effect. In particular, it has been reported that side effects such as excessive systemic inflammatory response occurring in mRNA vaccines are related to the systemic circulation of lipid nanoparticles.

[0007] In other words, lipid nanoparticle-based mRNA vaccines exert their immune effects through a localized mechanism, but unnecessary systemic exposure of locally administered lipid nanoparticles can cause various adverse effects. Considering this, muscle-delivered lipid nanoparticle technology is required for optimal mRNA vaccine development. However, to date, no lipid nanoparticle technology has been developed that minimizes systemic exposure and achieves localized, concentrated delivery. Therefore, this remains an unmet fundamental technology in the development of mRNA infectious disease and cancer vaccines.

[0008] An object of the present invention is to provide a compound selected from novel compounds, pharmaceutically acceptable salts, tautomers, prodrugs or stereoisomers thereof.

[0009] Another object of the present invention is to provide a lipid nanoparticle composition comprising the compound.

[0010] Another object of the present invention is to provide a drug delivery composition comprising the lipid nanoparticle composition and a preventive or therapeutic agent.

[0011] To achieve the above purpose, the present invention provides a compound selected from the compounds represented by the following chemical formula 1, pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0012] [Chemical Formula 1]

[0013]

[0014] In the above chemical formula 1,

[0015] n is one of 1 to 6,

[0016] m1 and m3 may be the same or different and are one of 0 to 6,

[0017] m2 is 0 or 1,

[0018] L 11 , L 12 and L 13may be the same or different, and is one of (C1-C10)alkylene, (C2-C10)alkenylene and (C2-C10)alkynylene, and in said alkylene, alkenylene and alkynylene, at least one -CH2- is substituted or unsubstituted with -O-, -S- or -SS-,

[0019] A1 to A4 may be the same or different, and -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OP(=O)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-,-C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -C(=O)NR 11 -, -NR 11 C(=O)-, -OC(=O)NH-, -NHC(=O)O-, -OC(=O)NR 11 - and -NR 11 One of C(=O)O-, and the above R 11 is one of (C1-C10)alkyl, (C2-C10)alkenyl and (C2-C10)alkynyl, and in said alkyl, alkenyl and alkynyl, at least one -CH2- is substituted or unsubstituted with -O-, -S- or -SS-,

[0020] R 1 , R 2 , R 3 and R 4are each the same or different, and are one of 4 to 20-membered heteroaryl containing at least one heteroatom selected from the group consisting of hydroxy, (C1-C20)alkyl, (C2-C20)alkenyl, (C2-C20)alkynyl, (C3-C20)cycloalkyl, (C3-C20)aryl and N, O and S, wherein at least one -CH2- in said alkylene, alkenylene and alkynylene is unsubstituted or substituted with -O-, -S- or -SS-, and said (C3-C20)cycloalkyl, (C3-C20)aryl and 4 to 20-membered heteroaryl are unsubstituted or substituted with at least one of hydroxy, amino, halogen and (C1-C5)alkyl.

[0021] In addition, the present invention provides a lipid nanoparticle composition comprising a compound selected from the compound represented by the above chemical formula 1, a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0022] In addition, the present invention provides a drug delivery composition comprising the lipid nanoparticle composition and a preventive or therapeutic agent.

[0023] According to the present invention, it has been confirmed that lipid nanoparticles using novel ionized lipids exhibit excellent gene encapsulation rates and cell / in vivo gene delivery rates, and on the other hand, exhibit excellent muscle delivery by local injection, and thus can be usefully utilized as a drug delivery composition.

[0024] Figure 1 shows the manufacturing process of compound 1 of the present invention (Example 1-1).

[0025] Figure 2 shows the manufacturing process of compound 2 of the present invention (Example 1-2).

[0026] Figure 3 shows the manufacturing process of compound 3 of the present invention (Example 1-3).

[0027] Figure 4 shows the manufacturing process of compound 4 of the present invention (Example 1-4).

[0028] Figure 5 shows the manufacturing process of compound 5 of the present invention (Example 1-5).

[0029] Figure 6 shows the manufacturing process of compound 6 of the present invention (Example 1-6).

[0030] Figure 7 shows the manufacturing process of compound 7 of the present invention (Example 1-7).

[0031] Figure 8 shows the manufacturing process of compound 8 of the present invention (Example 1-8).

[0032] Figure 9 shows the manufacturing process of compound 9 of the present invention (Example 1-9).

[0033] Figure 10 shows the manufacturing process of compound 10 of the present invention (Example 1-10).

[0034] Figure 11 shows the manufacturing process of compound 11 of the present invention (Example 1-11).

[0035] Figure 12 shows the manufacturing process of compound 12 of the present invention (Example 1-12).

[0036] Figure 13 shows the manufacturing process of compound 13 of the present invention (Example 1-13).

[0037] Hereinafter, the present invention will be described in more detail.

[0038]

[0039] The present invention provides a compound selected from the compounds represented by the following chemical formula 1, pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0040] [Chemical Formula 1]

[0041]

[0042] In the above chemical formula 1,

[0043] n is one of 1 to 6,

[0044] m1 and m3 may be the same or different and are one of 0 to 6,

[0045] m2 is 0 or 1,

[0046] L 11 , L 12 and L 13 may be the same or different, and is one of (C1-C10)alkylene, (C2-C10)alkenylene and (C2-C10)alkynylene, and in said alkylene, alkenylene and alkynylene, at least one -CH2- is substituted or unsubstituted with -O-, -S- or -SS-,

[0047] A1 to A4 may be the same or different, and -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OP(=O)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-,-C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -C(=O)NR 11 -, -NR 11 C(=O)-, -OC(=O)NH-, -NHC(=O)O-, -OC(=O)NR 11 - and -NR 11 One of C(=O)O-, and the above R 11 is one of (C1-C10)alkyl, (C2-C10)alkenyl and (C2-C10)alkynyl, and in said alkyl, alkenyl and alkynyl, at least one -CH2- is substituted or unsubstituted with -O-, -S- or -SS-,

[0048] R 1 , R 2 , R 3 and R 4are each the same or different, and are one of 4 to 20-membered heteroaryl containing at least one heteroatom selected from the group consisting of hydroxy, (C1-C20)alkyl, (C2-C20)alkenyl, (C2-C20)alkynyl, (C3-C20)cycloalkyl, (C3-C20)aryl and N, O and S, wherein at least one -CH2- in said alkylene, alkenylene and alkynylene is unsubstituted or substituted with -O-, -S- or -SS-, and said (C3-C20)cycloalkyl, (C3-C20)aryl and 4 to 20-membered heteroaryl are unsubstituted or substituted with at least one of hydroxy, amino, halogen and (C1-C5)alkyl.

[0049] In the above chemical formula 1, n is one of 2 to 4, m1 and m3 may be the same or different, and are one of 0 to 4, and L 11 , L 12 and L 13 are each the same or different, and are one of (C3-C6)alkylene, (C3-C6)alkenylene and (C3-C6)alkynylene, A1 to A4 are each the same or different, and are -OC(=O)- or -C(=O)O-, and R 1 , R 2 and R 3 may be the same or different, and is one of (C5-C20)alkyl, (C5-C20)alkenyl and (C5-C20)alkynyl, and in the alkylene, alkenylene and alkynylene, at least one -CH2- is substituted or unsubstituted with -O-, -S- or -SS-, and R 4 is a 4 to 10-membered heteroaryl group containing at least one hydroxyl, (C3-C10)cycloalkyl, (C3-C10)aryl and N, wherein the (C3-C10)cycloalkyl, (C3-C10)aryl and 4 to 10-membered heteroaryl may be substituted or unsubstituted with amino,

[0050] Preferably, in the above chemical formula 1, n is one of 3 to 4, and L 11 , L 12 and L 13 may be the same or different, and are (C3-C6)alkylene, and R 1 , R 2 and R 3 may be the same or different, and is one of (C5-C20)alkyl, (C5-C20)alkenyl and (C5-C20)alkynyl, and in the alkylene, alkenylene and alkynylene, at least one -CH2- is substituted or unsubstituted with -S- or -SS-, and R 4 is a 4 to 10-membered heteroaryl containing at least one hydroxy or N, wherein the 4 to 10-membered heteroaryl may be substituted or unsubstituted with amino,

[0051] More preferably, the compound may be selected from the following group of compounds.

[0052] (1) (9Z,9'Z)-((3-((2-hydroxyethyl)(3-((Z)-tetra-9-decenoyloxy)propyl)amino)propyl)azane-3,1-diyl)bis(tetra-9-decenoate)

[0053] (2) (9Z,9'Z)-((3-((3-((Z)-hexa-9-decenoyloxy)propyl)((3-((3-hydroxyethyl)amino)propyl)azanediyl)bis(propane-3,1-diyl)bis(hexa-9-decenoate)

[0054] (3) Di((Z)-decenyl)4,4'-((3-((4-(Z)-4-decenyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azane-4-yl)dibutanoate

[0055] (4) Didodecyl 4,4'-((3-((4-(dodecyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azane-4-yl)dibutanoate

[0056] (5) Di((Z)-dec-4-en-1-yl)4,4'-((3-((2-((3-(6-aminopyridin-3-yl)propanoyl)oxy)ethyl)(4-((Z)-deceth-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azane-4-yl)dibutanoate

[0057] (6) Di((Z)-dec-4-en-1-yl)4,4'-((4-((4-((Z)-dec-4-en-1yloxy)-4-oxobutyl)(2-hydroxyethyl)amino)butyl)azane-dyl)dibutanoate

[0058] (7) Di((Z)-dec-4-en-1-yl)5,5'-((3-((5-((Z)-dec-4-en-1yloxy)-5-oxopentyl)(2-hydroxyethyl)amino)propyl)azanediyl)dipentanoate

[0059] (8) Dinonyl 6,6'-((4-((2hydroxyethyl)(6-(nonyloxy)-6-oxohexyl)amino)butyl)azane-2-yl)dihexanoate

[0060] (9) Heptadeca-9-yl 6-((4-((2-hydroxyethyl)(6-(nonyloxy)-6-oxohexyl)amino)butyl)(6-(nonyloxy)-6-oxohexyl)amino)hexanoate

[0061] (10) Didecyl 6,6'-((2-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)azane-2-yl)dihexanoate

[0062] (11) Decyl 6-((2-((6-decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)(6((2-hexyldecyl)oxy)-6-oxohexyl)amino)hexanoate

[0063] (12) Bis(2-hexyldecyl)6,6'-((2-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)azanedyl)dihexanoate

[0064] (13) Di((Z)-dec-4-en-1-yl)4,4'-((3-((4-((Z)-dec-4-en-1yloxy)-4-oxobutyl)(pyridin-4-yl)amino)propyl)azane-4-yl)dibutanoate

[0065] (14) Di((Z)-tetradec-9-en-1-yl)4,4'-((3-((2-hydroxyethyl)(4-oxo-4-((Z)-tetradec-9-en-1-yloxy)butyl)amino)propylazane-dyl)dibutanoate

[0066] (15) Bis(6-butyldisulfanyl)hexyl)4,4'-((3-((4-((6-butyldisulfanyl)hexyl)oxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azanedyl)dibutanoate

[0067] (16) Di((Z)-dec-4-en-1-yl)4,4'-((3-((2-((3)-(4-aminophenyl)propanoyl)oxy)ethyl)(4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azane-dibutanoate

[0068] (17) Di((Z))-dec-4-en-1-yl)4,4'-((3-((4-aminophenethyl)(4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azanedyl)dibutanoate;

[0069] (18) Di((Z)-dec-4-en-1-yl)4,4'-((3-((4-((Z)-dec-4-en-1yloxy)-4-oxobutyl)(pyridin-4-yl)amino)propyl)azane-4-yl)dibutanoate

[0070] (19) (9Z,9'Z)-((3-((2-hydroxyethyl)(4-((Z)-tetradec-9-enoyloxy)butyl)amino)propyl)azanedyl)bis(butane-4,1-diyl) bis(tetradec-9-enoate)

[0071] The above (9Z,9'Z)-((3-((2-hydroxyethyl)(3-((Z)-tetra-9-decenoyloxy)propyl)amino)propyl)azanediyl)bis(propane-3,1-diyl)bis(tetra-9-decenoate) [(9Z,9'Z)-((3-((2-hydroxyethyl)(3-((Z)-tetradec-9-enoyloxy)propyl)amino) propyl)azanediyl)bis(propane-3,1-diyl) bis(tetradec-9-enoate); hereinafter referred to as compound 1] has a structure as shown in the following chemical formula 2, and the molecular formula is C 56 H 104 It is N2O7 and its molecular weight is 917.43.

[0072] [Chemical Formula 2]

[0073]

[0074] The above (9Z,9'Z)-((3-((3-((Z)-hexa-9-decenoyloxy)propyl)((3-((3-hydroxyethyl)amino)propyl)azanediyl)bis(propane-3,1-diyl)bis(hexadec-9-enoate) [(9Z,9'Z)-((3-((3-((Z)-hexadec-9-enoyloxy)propyl)(2-hydroxyethyl)amino)propyl)azanediyl)bis(propane-3,1-diyl) bis(hexadec-9-enoate); hereinafter referred to as compound 2] has a structure as shown in the following chemical formula 3, and the molecular formula is C 62 H 116 It is N2O7 and its molecular weight is 1001.59.

[0075] [Chemical Formula 3]

[0076]

[0077] The above di((Z)-decenyl)4,4'-((3-((4-(Z)-4-decenyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azanediyl)dibutanoate [di((Z)-dec-4-en-1-yl)4,4'-((3-((4- ((Z)-dec-4-en-1-yloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azanediyl)dibutanoate; hereinafter referred to as compound 3] has a structure as shown in the following chemical formula 4, and its molecular formula is C 47 H 86 It is N2O7 and its molecular weight is 791.19.

[0078] [Chemical Formula 4]

[0079]

[0080] The above didodecyl 4,4'-((3-((4-(dodecyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azanediyl)dibutanoate [didodecyl 4,4'-((3-((4-(dodecyloxy)-4-oxobutyl) (2-hydroxyethyl)amino)propyl)azanediyl)dibutanoate; hereinafter referred to as compound 4] has a structure as shown in the following chemical formula 5, and its molecular formula is C 53 H 104 It is N2O7 and its molecular weight is 881.40.

[0081] [Chemical Formula 5]

[0082]

[0083] The above di((Z)-dec-4-en-1-yl)4,4'-((3-((2-((3-(6-aminopyridin-3-yl)propanoyl)oxy)ethyl)(4-((Z)-deceth-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azanediyl)dibutanoate [di((Z)-dec-4-en-1-yl)4,4'-((3- ((2-((3-(6-aminopyridin-3-yl) propanoyl)oxy)ethyl)(4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azanediyl)dibutanoate; hereinafter referred to as compound 5] has a structure as shown in the following chemical formula 6, and its molecular formula is C 55 H 94 It is N4O8 and its molecular weight is 939.36.

[0084] [Chemical Formula 6]

[0085]

[0086] The above di((Z)-dec-4-en-1-yl)4,4'-((4-((4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)(2-hydroxyethyl)amino)butyl)azanediyl)dibutanoate [di((Z)-dec-4-en-1-yl)4,4'-((4-((4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl) (2-hydroxyethyl)amino)butyl)azanediyl)dibutanoate; hereinafter referred to as compound 6] has a structure as shown in the following chemical formula 7, and its molecular formula is C 48 H 88 It is N2O7 and its molecular weight is 805.22.

[0087] [Chemical Formula 7]

[0088]

[0089] The above di((Z)-dec-4-en-1-yl)5,5'-((3-((5-((Z)-dec-4-en-1-yloxy)-5-oxopentyl)(2-hydroxyethyl)amino)propyl)azanediyl)dipentanoate [di((Z)-dec-4-en-1-yl)5,5'-((3-((5-((Z)-dec-4-en-1-yloxy)-5-oxopentyl) (2-hydroxyethyl)amino)propyl)azanediyl)dipentanoate; hereinafter referred to as compound 7] has a structure as shown in the following chemical formula 8, and its molecular formula is C 50 H 92 It is N2O7 and its molecular weight is 833.27.

[0090] [Chemical Formula 8]

[0091]

[0092] The above-mentioned dinonyl 6,6'-((4-((2-hydroxyethyl)(6-(nonyloxy)-6-oxohexyl)amino)butyl)azanediyl)dihexanoate [dinonyl6,6'-((4-((2-hydroxyethyl)(6-(nonyloxy) -6-oxohexyl)amino)butyl)azanediyl)dihexanoate; hereinafter referred to as compound 8] has a structure as shown in the following chemical formula 9, and its molecular formula is C 51 H 100 It is N2O7 and its molecular weight is 853.35.

[0093] [Chemical Formula 9]

[0094]

[0095] The above heptadeca-9-yl 6-((4-((2-hydroxyethyl)(6-(nonyloxy)-6-oxohexyl)amino)butyl)(6-(nonyloxy)-6-oxohexyl)amino)hexanoate [heptadecan-9-yl 6-((4-((2-hydroxyethyl)(6-(nonyloxy)-6-oxohexyl)amino)butyl)(6-(nonyloxy)-6-oxohexyl)amino)hexanoate; hereinafter referred to as compound 9] has a structure as shown in the following chemical formula 10, and its molecular formula is C59 H 116 It is N2O7 and its molecular weight is 965.56.

[0096] [Chemical Formula 10]

[0097]

[0098] The above didecyl 6,6'-((2-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)azanediyl)dihexanoate [didecyl 6,6'-((2-((6-(decyloxy)-6-oxohexyl) (2-hydroxyethyl)amino)ethyl)azanediyl)dihexanoate; hereinafter referred to as compound 10] has a structure as shown in the following chemical formula 11, and its molecular formula is C 52 H 102 It is N2O7 and its molecular weight is 867.38.

[0099] [Chemical Formula 11]

[0100]

[0101] The above decyl 6-((2-((6-decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)(6((2-hexyldecyl)oxy)-6-oxohexyl)amino)hexanoate [decyl 6-((2-((6-(decyloxy) -6-oxohexyl)(2-hydroxyethyl)amino)ethyl)(6-((2-hexyldecyl)oxy)-6-oxohexyl)amino)hexanoate; hereinafter referred to as compound 11] has a structure as shown in the following chemical formula 12, and its molecular formula is C 58 H 114 It is N2O7 and its molecular weight is 951.53.

[0102] [Chemical Formula 12]

[0103]

[0104] The above bis(2-hexyldecyl)6,6'-((2-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)azanediyl)dihexanoate [bis(2-hexyldecyl) 6,6'-((2-((6-(decyloxy) -6-oxohexyl)(2-hydroxyethyl)amino)ethyl)azanediyl)dihexanoate; hereinafter referred to as compound 12] has a structure as shown in the following chemical formula 13, and its molecular formula is C 64 H 126 It is N2O7 and its molecular weight is 1035.69.

[0105] [Chemical Formula 13]

[0106]

[0107] The above di((Z)-dec-4-en-1-yl)4,4'-((3-((4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)(pyridin-4-yl)amino)propyl)azanediyl)dibutanoate [di((Z)-dec-4-en-1-yl) 4,4'-((3-((4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)(pyridin-4-yl)amino)propyl)azanediyl)dibutanoate; hereinafter referred to as compound 13] has a structure as shown in the following chemical formula 14, and its molecular formula is C 50 H 85 It is N3O6 and its molecular weight is 824.23.

[0108] [Chemical Formula 14]

[0109]

[0110] The above di((Z)-tetradec-9-en-1-yl)4,4'-((3-((2-hydroxyethyl)(4-oxo-4-((Z)-tetradec-9-en-1-yloxy)butyl)amino)propylazanediyl)dibutanoate [di((Z)-tetradec-9-en-1-yl) 4,4'-((3-((2-hydroxyethyl)(4-oxo-4-((Z)-tetradec-9-en-1-yloxy)butyl)amino)propyl)azanediyl)dibutanoate; hereinafter referred to as compound 14] has a structure as shown in the following chemical formula 15, and its molecular formula is C 59 H 110 It is N2O7 and its molecular weight is 959.51.

[0111] [Chemical Formula 15]

[0112]

[0113] The above bis(6-butyldisulfanyl)hexyl)4,4'-((3-((4-((6-butyldisulfanyl)hexyl)oxy)-4-oxobutyl)(3-hydroxypropyl)amino)propyl)azanediyl)dibutanoate [bis(6-(butyldisulfanyl)hexyl) 4,4'-((3-((4-((6-(butyldisulfanyl)hexyl)oxy)-4-oxobutyl)(3-hydroxypropyl)amino)propyl)azanediyl)dibutanoate; hereinafter referred to as compound 15] has a structure as shown in the following chemical formula 16, and its molecular formula is C 48 H 94 It is N2O7S6 and its molecular weight is 1003.66.

[0114] [Chemical Formula 16]

[0115]

[0116] The above di((Z)-dec-4-en-1-yl)4,4'-((3-((2-((3)-(4-aminophenyl)propanoyl)oxy)ethyl)(4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azanediyl)dibutanoate [di((Z)-dec-4-en-1-yl)4,4'-((3-((2-((3-(4-aminophenyl)propanoyl)oxy) ethyl)(4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azanediyl)dibutanoate; hereinafter referred to as compound 16] has a structure as shown in the following chemical formula 17, and its molecular formula is C 56 H 95 It is N3O8 and its molecular weight is 938.37.

[0117] [Chemical Formula 17]

[0118]

[0119] The above di((Z))-dec-4-en-1-yl)4,4'-((3-((4-aminophenethyl)(4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azanediyl)dibutanoate [di((Z)-dec-4-en-1-yl) 4,4'-((3-((4-aminophenethyl)(4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azanediyl)dibutanoate; hereinafter referred to as compound 17] has a structure as shown in the following chemical formula 18, and its molecular formula is C 53 H 91 It is N3O6 and its molecular weight is 866.31.

[0120] [Chemical Formula 18]

[0121]

[0122] The above di((Z)-dec-4-en-1-yl)4,4'-((3-((4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)(pyridin-4-yl)amino)propyl)azanediyl)dibutanoate [di((Z)-dec-4-en-1-yl) 4,4'-((3-((4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)(4-hydroxybutyl)amino)propyl)azanediyl)dibutanoate; hereinafter referred to as compound 18] has a structure as shown in the following chemical formula 19, and its molecular formula is C 49 H 90 It is N2O7 and its molecular weight is 819.25.

[0123] [Chemical Formula 19]

[0124]

[0125] The above (9Z,9'Z)-((3-((2-hydroxyethyl)(4-((Z)-tetradec-9-enoyloxy)butyl)amino)propyl)azanediyl)bis(butane-4,1-diyl) bis(tetradec-9-enoate) [(9Z,9'Z)-((3-((2-hydroxyethyl)(4-((Z)-tetradec-9-enoyloxy)butyl)amino)propyl)azanediyl)bis(butane-4,1-diyl) bis(tetradec-9-enoate); hereinafter referred to as compound 19] has a structure as shown in the following chemical formula 20, and its molecular formula is C 59 H 110 It is N2O7 and its molecular weight is 959.51.

[0126] [Chemical Formula 20]

[0127]

[0128]

[0129] In addition, the present invention provides a lipid nanoparticle composition comprising a compound selected from the compound represented by the above chemical formula 1, a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0130] The lipid nanoparticles described above possess excellent safety and stability, and can function as delivery vehicles to enable genes containing RNA, DNA, or a mixture thereof as active ingredients to effectively exert their effects within cells. Accordingly, the lipid nanoparticles comprising ionized lipids exhibit excellent gene encapsulation rates and cell / in vivo gene delivery rates, and on the other hand, exhibit excellent muscle delivery of genes through local injection, and thus can be usefully utilized as drug delivery compositions.

[0131] The compound may be an ionizable lipid, and the ionizable lipid may comprise a gene, a protein or a compound, preferably a gene or a protein, and more preferably a gene.

[0132] The composition may further comprise, but is not limited to, one or more selected from the group consisting of neutral lipids, steroids, and polymeric lipids.

[0133] The above neutral lipid may be a phospholipid or a glycolipid. Specifically, the phospholipids include dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), It may be at least one selected from the group consisting of phosphatidylethanolamine (PE) and dipalmitoylphosphatidylethanolamine, and the glycolipid may be at least one selected from the group consisting of glucosylceramide, galactosylceramide, glucosylsphingosine, galactosylsphingosine, and phosphoglycoceramide, but is not limited thereto.

[0134] The above steroid may be one or more selected from the group consisting of cholesterol, bile acid derivatives and cholic acid derivatives, but is not limited thereto.

[0135] The above polymer lipid may be a pegylated lipid having a structure in which a water-soluble polymer and a lipid are combined, and the pegylated lipid may be at least one selected from the group consisting of PEG bound to dialkyloxypropyl (PEG-DAA); PEG bound to diacylglycerol (PEG-DAG) such as PEG-c-DOMG and PEG-DMG; PEG bound to phospholipids such as phosphatidylethanolamine (PEG-DLPE, PEG-DMPE and PEG-DSPE); PEG bound to ceramide (PEG-CER); PEG bound to cholesterol or a derivative thereof; PEG-modified phosphatidic acid; PEG-modified dialkylamine; and PEG-modified dialkylglycerol, but is not limited thereto. In addition, the pegylated lipid may be a functionalized PEG in which a functional group is combined on a side that is not combined with lipid. The functional group that can be used at this time may be one or more selected from the group consisting of succinyl, carboxylic acid, maleimide, n-hydroxysuccinimide, amine, biotin, cyanuric, and folate, but is not limited thereto.

[0136] The above ionized lipid may be manufactured into lipid nanoparticles, including neutral lipids, steroids, polymerized lipids, etc. In order to have a composition suitable for manufacturing lipid nanoparticles, the lipid composition excluding the active ingredient may include 20 to 65 mol% of ionized lipids, 2.5 to 30 mol% of neutral lipids, 20 to 60 mol% of steroids, and 0.5 to 5 mol% of polymerized lipids. For example, ionized lipid: neutral lipid: steroid: polymer polymerized lipid is 60:5:33.5:1.5, 50:10:38.5:1.5, 40:15:43.5:1.5, 30:20:48.5:1.5, 25:25:48.5:1.5, 60:5:33:2, 50:10:38:2, 40:15:43:2, 30:20:48:2, 25:25:48:2, 60:5:32.5:2.5, 50:10:37.5:2.5, 40:15:42.5:2.5, 30:20:47.5:2.5, 25:25:47.5:2.5, It can be composed in one mol% ratio selected from the group consisting of 60:5:32:3, 50:10:37:3, 40:15:42:3, 30:20:47:3 and 25:25:47:3.

[0137] In addition, the composition may further include a preventive or therapeutic agent, and the agent may be a gene including RNA, DNA or a mixture thereof composed of a single strand or a double strand, and specifically, may be at least one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme) and guide ribonucleic acid for gene correction (sgRNA), etc., but It is not limited.

[0138] In addition, the composition may include, but is not limited to, various anionic peptides, protein drugs, protein-nucleic acid structures, or anionic biopolymer-drug conjugates such as hyaluronic acid-peptide conjugates, hyaluronic acid-protein conjugates, antibodies, etc. as active ingredients.

[0139] The lipid nanoparticle composition including ionized lipids, neutral lipids, steroids, polymerized lipids, etc. of the present invention can be prepared as a lipid solution by dissolving in a solvent miscible with ethanol or water. Separately, the active ingredient can be prepared as an active ingredient solution by dissolving in a citric acid or acetic acid buffer having a pH of 4.0±1.0. In addition, the lipid solution and the active ingredient solution can be mixed at a volume ratio of 1:3 at a flow rate of about 10 to 15 mL / min using a microfluidic mixing device (Benchtop Nanoassemblr, Precision Nanosystems) to prepare lipid nanoparticles.

[0140]

[0141] In addition, the present invention provides a lipid nanoparticle composition comprising a compound selected from the compound represented by the above chemical formula 1, a pharmaceutically acceptable salt, tautomer or stereoisomer thereof; and a drug delivery composition comprising a preventive or therapeutic agent.

[0142] The above preventive or therapeutic agent may be at least one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribozyme (DNAzyme), guide ribonucleic acid for gene correction (sgRNA), and mixtures thereof, but is not limited thereto.

[0143] The above drug delivery composition can be administered to mammals including humans by various routes, including parenteral administration, and parenteral administration can be applied intravenously, subcutaneously, intraperitoneally, or locally, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time, but can be appropriately selected by those skilled in the art.

[0144] When formulating the above drug delivery composition according to an example, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, lyophilizing agents, binders, wetting agents, disintegrating agents, and surfactants.

[0145] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0146] Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0147] The drug delivery composition of the present invention can be administered while containing a pharmaceutically effective amount of a preventive or therapeutic agent. The effective dosage level of the preventive or therapeutic agent can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route and excretion rate, the duration of treatment, factors including concomitant medications, and other factors well known in the medical field. In one embodiment, the composition can be administered as an individual therapeutic agent or in combination with another therapeutic agent, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art. For example, the composition can be administered at 0.01 to 100 mg / kg, 0.1 to 50 mg / kg, or 1 to 10 mg / kg.

[0148] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0149]

[0150] [Example 1] Preparation of compound

[0151] 1-1. Preparation of compound 1

[0152] As shown in Fig. 1, compound 1-a (3.00 g, 13.2 mmol), compound 1-b (2.76 g, 19.9 mmol), and dimethylaminopyridine (hereinafter referred to as DMAP) (810 mg, 6.63 mmol) were dissolved in dichloromethane (hereinafter referred to as DCM) (60 mL), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide; hereinafter referred to as EDCI) (5.59 g, 29.2 mmol) was added at 0°C, followed by stirring at 20°C for 15 hours. The mixture was diluted with purified water (100 mL), extracted with DCM (80 mL × 3 times), washed with saturated sodium chloride solution (hereinafter referred to as brine) (100 mL), dried with sodium sulfate (hereinafter referred to as Na2SO4), filtered, and the filtrate was concentrated under reduced pressure and purified on silica gel (petroleum ether (hereinafter referred to as PE) / ethyl acetate (hereinafter referred to as EtOAc) = 100 / 1 to 19 / 1) to obtain compound 1-c as a colorless oil.

[0153] Compound 1-c (2.00 g, 5.76 mmol), compound 1-d (206 mg, 1.74 mmol), sodium carbonate (Na2CO3) (555 mg, 5.23 mmol), and sodium iodide (NaI) (262 mg, 1.74 mmol) were added to acetonitrile (MeCN; hereinafter referred to as MeCN) (25 mL) and stirred at 80°C for 15 hours. The mixture was concentrated under reduced pressure, diluted with purified water (40 mL), extracted with DCM (40 mL × 3 times), and washed with brine (60 mL). After drying over Na2SO4 and filtering, the solution was concentrated under reduced pressure, purified twice on silica gel (PE / EtOAc = 100 / 1 to 24 / 1), and lyophilized to obtain compound 1 as a colorless oil. 1 The results of H NMR analysis were as follows.

[0154] 1 H NMR (400 MHz, CDCl3) δ 5.31-5.39 (m, 6H), 4.08-4.13 (m, 6H), 3.57-3.58 (m, 2H), 2.42-2.59 (m, 12H), 2.29 (t,J= 7.6 Hz, 6H), 2.00-2.03 (m, 12H), 1.73-1.81 (m, 8H), 1.60-1.64 (m, 6H), 1.31-1.35 (m, 36H), 0.89-0.92 (m, 9H)

[0155]

[0156] 1-2. Preparation of Compound 2

[0157] As shown in Fig. 2, compound 2-a (3.00 g, 11.8 mmol) was dissolved in DCM (30 mL), dimethyl formamide (hereinafter referred to as DMF) (86.2 mg, 1.18 mmol) and oxalyl chloride ((COCl)2) (3.34 g, 26.3 mmol) were added at 0°C, and the mixture was stirred at 20°C for 1 hour. The mixture was concentrated under reduced pressure, diluted with DCM (20 mL), and then a mixture of compound 2-b (2.13 g, 15.3 mmol, 1.39 mL), DMAP (144 mg, 1.18 mmol), and triethylamine (TEA) (5.97 g, 59.0 mmol, 8.21 mL) dissolved in DCM (20 mL) at 0 °C was added and stirred at 20 °C for 15 hours. The mixture was concentrated under reduced pressure, diluted with purified water (50 mL), extracted with EtOAc (50 mL × 2), and washed with brine (60 mL). After drying over Na2SO4 and filtering, the solution was concentrated under reduced pressure, and purified on silica gel (PE / EtOAc = 100 / 1 to 20 / 1) to obtain compound 2-c as a colorless oil.

[0158] Compound 2-c (2.00 g, 5.33 mmol), compound 2-d (197 mg, 1.66 mmol), Na2CO3 (529 mg, 4.99 mmol), and NaI (249 mg, 1.66 mmol) were added to MeCN (25 mL) and stirred at 80°C for 15 hours. The mixture was diluted with purified water (30 mL), extracted with EtOAc (30 mL × 3 times), and washed with brine (40 mL). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (DCM / methanol (Methaol; hereinafter referred to as MeOH) = 100 / 1 to 24 / 1) to obtain compound 2 as a colorless oil. 1 The results of H NMR analysis were as follows.

[0159] 1 H NMR (500 MHz, CD3OD)δ 5.32-5.38 (m, 6H), 4.11-4.15 (m, 6H), 3.62 (t,J= 6.0 Hz, 2H), 2.51-2.64 (m, 12H), 2.32 (t,J= 7.0 Hz, 6H), 2.00-2.05 (m, 12H), 1.76-1.85 (m, 6H), 1.60-1.66 (m, 8H), 1.30-1.33 (m, 48H), 0.89-0.92 (m, 9H).

[0160]

[0161] 1-3. Preparation of compound 3

[0162] As shown in Fig. 3, compound 3-a (5.00 g, 32.0 mmol), DMAP (301 mg, 2.46 mmol), and diisopropylethylamine (hereinafter referred to as DIEA) (9.54 g, 73.8 mmol) were dissolved in DCM (50 mL), and compound 3-b (4.56 g, 24.6 mmol, 2.85 mL) dissolved in DCM (50 mL) was added at 0°C, followed by stirring at 20°C for 16 hours. The mixture was diluted with purified water (100 mL), adjusted to pH 3 using hydrochloric acid solution (2 M), extracted with DCM (100 mL × 2), washed with saturated sodium bicarbonate solution (hereinafter referred to as sat.aq.NaHCO3, 200 mL × 2), dried over Na2SO4, and filtered. After concentrating the filtrate under reduced pressure, the residue was purified on silica gel (PE / EtOAc = 100 / 1 to 20 / 1) to obtain compound 3-c as a colorless oil.

[0163] Compound 3-c (2.00 g, 6.55 mmol), compound 3-d (235 mg, 1.99 mmol), Na2CO3 (631 mg, 5.96 mmol), and NaI (297.6 mg, 1.99 mmol) were added to MeCN (25 mL) and stirred at 80°C for 15 hours. The mixture was diluted with purified water (30 mL), extracted with EtOAc (30 mL × 2), and washed with brine (40 mL). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (DCM / MeOH = 100 / 1 to 24 / 1) to obtain compound 3 as a colorless oil. 1 The results of H NMR analysis were as follows.

[0164] 1H NMR (400 MHz, CD3OD)δ 5.34-5.44 (m, 6H), 4.07 (t,J= 6.4 Hz, 6H), 3.63 (t,J= 6.0 Hz, 2H), 2.66 (t,J= 6.0 Hz, 2H), 2.50-2.63 (m, 10H), 2.37-2.39 (m, 6H), 2.10-2.14 (m, 6H), 2.04-2.06 (m, 6H), 1.76-1.83 (m, 6H), 1.64-1.73 (m, 8H), 1.31-1.35 (m, 18H), 0.89-0.93 (m, 9H).

[0165]

[0166] 1-4. Preparation of compound 4

[0167] As shown in Fig. 4, compound 4-a (200 mg, 1.0 mmol), DMAP (65 mg, 0.53 mmol), and N,N'-dicyclohexylcarbodiimide (hereinafter referred to as DCC) (65 mg, 0.53 mmol) were dissolved in DCM (20 mL), compound 4-b (270 mg, 1.6 mmol) was added at 0°C, and the mixture was stirred at 25°C (RT) for 15 hours. The mixture was diluted with purified water (20 mL), extracted with DCM (20 mL x 2), washed with brine (40 mL), dried with Na2SO4, and filtered. The resulting solution was concentrated under reduced pressure and purified on silica gel (n-hexane / EtOAc = 100 / 1 to 24 / 1) to obtain compound 4-c as a colorless oil.

[0168] Compound 4-c (121 mg, 0.363 mmol), compound 4-d (13 mg, 0.11 mmol), Na2CO3 (35 mg, 0.33 mmol), and NaI (16.2 mg, 0.11 mmol) were added to MeCN (2.5 mL) and stirred at 80°C for 15 hours. The mixture was diluted with purified water (10 mL), extracted with EtOAc (30 mL × 3 times), and washed with brine (50 mL). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (DCM / MeOH = 100 / 1 to 19 / 1) to obtain compound 4 as a colorless oil. 1 The results of H NMR analysis were as follows.

[0169] 1 H NMR (400 MHz, Methanol-d4)δ4.05(t,J= 6.6 Hz, 6H), 3.59 (t,J= 6.2 Hz, 2H), 2.60 (t,J= 6.2 Hz, 2H), 2.52 (dd,J= 8.4, 6.2 Hz, 4H), 2.49 - 2.42 (m, 6H), 2.33 (td,J= 7.1, 3.9 Hz, 6H), 1.75 (h,J= 7.5 Hz, 6H), 1.67 - 1.54 (m, 8H), 1.41 - 1.19 (m, 56H), 0.92 - 0.83 (m, 9H).

[0170]

[0171] 1-5. Preparation of compound 5

[0172] As shown in Fig. 5, compound 5-a (315 mg, 1.90 mmol), compound 3 (1.00 g, 1.26 mmol), and DMAP (386 mg, 3.16 mmol) were dissolved in DMF (10 mL), EDCI (363 mg, 1.90 mmol) was added, and the mixture was stirred at 25°C for 12 hours. DMAP (386 mg, 3.16 mmol), EDCI (363 mg, 1.90 mmol), and DMF (10 mL) were added to compound 5-a (315 mg, 1.90 mmol), and the mixture was stirred at 25°C for 24 hours. Saturated sodium carbonate solution (hereinafter referred to as sat. Na2CO3, 30 mL) was added to the reaction mixture, and the solution was extracted with DCM (50 mL × 2 times), and washed with brine (50 mL × 2). After drying with Na2SO4, the filtered solution was concentrated under reduced pressure and purified on silica gel (DCM / MeOH = 100 / 1 to 100 / 3) to obtain compound 5 as a yellow oil. 1 The results of H NMR analysis were as follows.

[0173] 1 H NMR (400 MHz, CDCl3)δ7.92(d,J= 2.0 Hz, 1H), 7.30 (dd,J= 8.4, 2.4 Hz, 1H), 6.46 (d,J= 8.4 Hz, 1H), 5.27 - 5.48 (m, 6H), 4.34 (s, 2H), 4.02-4.14 (m, 8H), 2.77-2.85 (m, 2H), 2.65 (t,J= 6.4 Hz, 2H), 2.52-2.60 (m, 2H), 2.36-2.50 (m, 10H), 2.28-2.36 (m, 6H), 2.04-2.12 (m, 6H), 1.95-2.02 (m, 6H), 1.65-1.79 (m, 12H), 1.48-1.57 (m, 2H), 1.26-1.39 (m, 18H), 0.89 (t,J= 6.8 Hz, 9H).

[0174]

[0175] 1-6. Preparation of compound 6

[0176] As shown in Fig. 6, compound 6-a (8.55 g, 46.1 mmol) dissolved in DCM (80 mL) was added to a solution of compound 6-b (5.54 g, 35.5 mmol) and TEA (10.8 g, 106 mmol) dissolved in DCM (60 mL) at 0°C, and the mixture was stirred at 30°C for 2 hours. The mixture was diluted with purified water (100 mL), extracted with DCM (100 mL × 3 times), washed with brine (200 mL), dried and filtered over Na2SO4, and concentrated under reduced pressure. The resulting solution was purified on silica gel (PE / EtOAc = 100 / 1 to 100 / 3) to obtain compound 6-c as a colorless oil. Compound 6-c (3.70 g, 12.1 mmol), compound 6-d (734 mg, 5.51 mmol), Na2CO3 (1.75 mg, 16.5 mmol), and NaI (826 mg, 5.51 mmol) were added to MeCN (50 mL) and stirred at 80°C for 18 hours. The mixture was diluted with purified water (100 mL), extracted with EtOAc (100 mL × 3 times), and the solution was filtered and washed with aq. Na2CO3 (100 mL). And washed with brine (100 mL). After drying with Na2SO4, the filtered solution was concentrated under reduced pressure and purified on silica gel (DCM / MeOH = 100 / 1 to 25 / 1) to obtain compound 6-e as a pale yellow oil.

[0177] Compound 6-e (1.30 g, 2.23 mmol) was dissolved in DCM (15 mL), trifluoroacetic acid (TFA) (1.5 mL) was added, and the mixture was stirred at 20°C for 16 hours. The mixture was concentrated under reduced pressure, diluted with DCM (40 mL), washed with sat.aq. NaHCO3 (40 mL × 3 times), dried over Na2SO4, and filtered. The solution was concentrated under reduced pressure to obtain compound 6-f as a yellow oil.

[0178] Compound 6-c (1.44 g, 4.72 mmol) and compound 6-h (2.88 g, 47.2 mmol) were added to MeCN (20 mL) and stirred at 20°C for 1 hour. The mixture was diluted with purified water (50 mL), extracted with DCM (40 mL × 3 times), washed with aq. Na2CO3 (50 mL), dried over Na2SO4, and the filtrate was concentrated under reduced pressure to obtain compound 6-g.

[0179] Compound 6-f (1.15 g, 2.15 mmol), compound 6-g (1.32 g, 3.24 mmol), and acetic acid (AcOH, 64.5 mg, 1.07 mmol) were added to dichloroethane (DCE) (20 mL) and stirred at 20°C for 0.5 h. At 0°C, sodium cyanoborohydride (NaBH3CN, 270 mg, 4.29 mmol) was added and stirred at 20°C for 2 h. The mixture was diluted with purified water (40 mL) and extracted with DCM (30 mL × 3 times) and the solution was washed with aq. Na2CO3 (40 mL). And washed with brine (40 mL). After drying with Na2SO4, the filtered solution was concentrated under reduced pressure, purified twice on silica gel (DCM / MeOH = 100 / 1 to 100 / 7), purified by prep-HPLC (C4 150 mm*19 mm; purified water: ACN = 100 / 10 to 100 / 80), and lyophilized. The dried product was diluted with DCM (30 mL) and washed with aq.Na2CO3 (30 mL). After washing with brine (30 mL), drying with Na2SO4, and filtering, the solution was concentrated under reduced pressure and purified on silica gel (DCM / MeOH = 100 / 1 to 100 / 6) to obtain compound 6 as a colorless oil, and compound 6 1 The results of H NMR analysis were as follows.

[0180] 1H NMR (400 MHz, CDCl3)δ5.26-5.50(m,6H),4.00-4.15(m,6H),3.48-3.67 (m,2H),2.40-2.71(m,12H),2.34(t,J= 7.2 Hz, 6H), 2.09-2.13 (m, 6H), 2.00-2.04 (m, 6H), 1.74-1.89 (m, 6H), 1.65-1.73 (m, 6H), 1.40-1.58 (m, 4H), 1.25-1.39 (m, 18H), 0.80-0.96 (m, 9H).

[0181]

[0182] 1-7. Preparation of compound 7

[0183] As shown in Fig. 7, compound 7-b (5.00 g, 32.0 mmol) and TEA (9.71 g, 96.0 mmol) were dissolved in DCM (50 mL), and then compound 7-a (7.02 g, 35.2 mmol) dissolved in DCM (70 mL) was added at 0°C and stirred at 20°C for 16 hours. The mixture was diluted with purified water (30 mL), extracted with EtOAc (30 mL × 2), washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting solution was purified on silica gel (PE / EtOAc = 100 / 1 to 100 / 6) to obtain compound 7-c as a colorless oil.

[0184] Compound 7-c (3.00 g, 9.40 mmol), compound 7-d (336 mg, 2.85 mmol), K2CO3 (1.06 mg, 9.97 mmol), and NaI (427 mg, 2.85 mmol) were added to MeCN (30 mL), filled with nitrogen gas, and stirred at 80°C for 16 hours. The mixture was diluted with purified water (50 mL), extracted with EtOAc (50 mL × 2), and the solution was washed with aq. Na2CO3 (30 mL). And washed with brine (30 mL). After drying with Na2SO4, the filtered solution was concentrated under reduced pressure, purified twice on silica gel (DCM / MeOH = 100 / 1 to 100 / 4), purified by prep-HPLC (C4 150 mm*19 mm; purified water:MeCN = 100 / 10 to 100 / 80), and lyophilized. The dried product was diluted with DCM (30 mL), washed with aq.Na2CO3 (30 mL), dried with Na2SO4, and the filtered solution was concentrated under reduced pressure to obtain compound 7 as a yellow oil. 1 The results of H NMR analysis were as follows.

[0185] 1 H NMR (400 MHz, CDCl3)δ5.29-5.46(m,6H),4.07(t,J= 6.8 Hz, 6H), 3.47 - 3.63 (m, 2H), 2.37-2.60 (m, 12H), 2.33 (t,J= 7.6 Hz, 6H), 2.08-2.14 (m, 6H), 1.99-2.04 (m, 6H), 1.58-1.73 (m, 14H), 1.45-1.53 ​​(m, 6H), 1.26-1.37 (m, 18H), 0.90 (t,J= 7.2 Hz, 9H).

[0186]

[0187] 1-8. Preparation of compound 8

[0188] As shown in Fig. 8, compound 8-a (25.0 g, 108 mmol) and compound 8-b (17.2 g, 119 mmol) were dissolved in DCM (300 mL), and a mixture of DMAP (13.5 g, 110 mmol) and EDCI (27.1 g, 142 mmol) dissolved in DCM (300 mL) was added, followed by stirring at 20°C for 14 hours. The mixture was diluted with purified water (300 mL), extracted with DCM (400 mL × 2), washed with brine (300 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure and purified on silica gel (PE / EtOAc = 100 / 1 to 12 / 1) to obtain compound 8-c as a colorless oil.

[0189] Compound 8-c (34.7 g, 97.1 mmol) was dissolved in DCM (175 mL), TFA (175 mL) was added, and the mixture was stirred at 20°C for 2 hours. The mixture was concentrated under reduced pressure, diluted with DCM (200 mL) and purified water (200 mL), adjusted to pH 10 with 1 M NaOH aqueous solution, and extracted with DCM (200 mL × 3 times). The solution was washed with 0.1 M NaOH aqueous solution (250 mL × 2 times) and brine (300 mL), dried over Na2SO4, and filtered. The solution was concentrated under reduced pressure to obtain compound 8-d as a colorless oil.

[0190] Compound 8-k (17.6 g, 90.1 mmol) was dissolved in DCM (200 mL), and DMF (507 mg, 6.93 mmol) and oxalyl chloride ((COCl)2, 26.4 g, 208 mmol) were added at 0 °C. The mixture was stirred at 20 °C for 1 hour, concentrated under reduced pressure, and diluted with DCM (100 mL). A mixture of compound 8-b (10.0 g, 69.3 mmol), DMAP (847 mg, 6.93 mmol), and TEA (35.1 g, 347 mmol) dissolved in DCM (150 mL) was added at 0 °C, and stirred at 20 °C for 14 hours. After that, diluted with purified water (200 mL), adjusted to pH 3 using 2 M hydrochloric acid solution, extracted with DCM (200 mL x 2), and washed with sat.NaHCO3 (300 mL x 2) and brine (300 mL). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (PE / EtOAc = 100 / 1 to 16 / 1) to obtain compound 8-e as a colorless oil.

[0191] Compound 8-d (9.59 g, 37.3 mmol), compound 8-e (3.99 g, 12.4 mmol), potassium carbonate (K2CO3, 5.15 g, 37.3 mmol), cesium carbonate (Cs2CO3, 1.21 g, 3.73 mmol), and NaI (558 mg, 3.73 mmol) were added to MeCN (200 mL) and stirred at 20°C for 24 hours. The mixture was filtered, the filter residue was washed with DCM (200 mL), and the filtrate was filtered under reduced pressure and purified on silica gel (DCM / MeOH = 100 / 1 to 32 / 1) to obtain compound 8-f as a pale yellow solid.

[0192] Compound 8-f (2.34 g, 4.70 mmol) and compound 8-g (2.15 g, 14.1 mmol) were dissolved in MeCN (50 mL), and potassium carbonate (K2CO3, 1.30 g, 9.40 mmol), cesium carbonate (Cs2CO3, 1.53 g, 4.70 mmol), and NaI (705 mg, 4.70 mmol) were added, and the mixture was stirred at 80°C for 24 hours. The mixture was concentrated to remove MeCN, diluted with DCM (50 mL) and purified water (50 mL), extracted with DCM (50 mL × 3), washed with brine (80 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure and purified on silica gel (DCM / MeOH = 100 / 1 to 32 / 1) to obtain compound 8-h as a pale yellow oil.

[0193] Compound 8-h was dissolved in DCM (18 mL), TFA (1.80 mL) was added, and the mixture was stirred at 20°C for 14 hours, 30°C for 2 hours, and then concentrated under reduced pressure. The mixture was diluted with DCM (50 mL), washed with aq. Na2CO3 (50 mL × 3 times), dried over Na2SO4, and filtered. The resulting solution was concentrated under reduced pressure to obtain compound 8-i as a yellow oil.

[0194] Compound 8-e (3.00 g, 9.34 mmol) was dissolved in ethanol (EtOH, 80 mL), compound 8-l (8.56 g, 14.0 mmol) was added at 20°C, and the mixture was stirred at 80°C for 14 hours. The mixture was concentrated, diluted with purified water (200 mL), extracted with EtOAc (30 mL), washed with sat.NaHCO3 (30 mL) and brine (40 mL), dried over Na2SO4, and filtered. The resulting solution was concentrated under reduced pressure to obtain compound 8-j as a white solid.

[0195] Compound 8-i (800 mg, 1.41 mmol), compound 8-j (425 mg, 1.41 mmol), and acetic acid (AcOH, 42.3 mg, 0.704 mmol) were added to DCE (10 mL) and stirred at 20°C for 1 hour. Sodium triacetosyborohydride (NaBH(OAc)3) (597 mg, 2.82 mmol) was added and stirred at 20°C for 15 hours. The mixture was diluted with purified water (30 mL), extracted with DCM (30 mL × 3), and washed with brine (50 mL). The solution, dried and filtered with Na2SO4, was concentrated under reduced pressure and purified on silica gel (DCM / MeOH = 100 / 1 to 10 / 1) to obtain compound 8 as a colorless oil, and compound 8 1 The results of H NMR analysis were as follows.

[0196] 1 H NMR (400 MHz, CD3OD) δ 4.07 (t, J= 6.8 Hz, 6H), 3.77-3.80 (m, 2H), 2.99-3.03 (m, 12H), 2.34-2.39 (m, 6H), 1.59-1.73 (m, 22H), 1.31-1.45 (m, 42H), 0.89-0.92 (m, 9H).

[0197]

[0198] 1-9. Preparation of compound 9

[0199] As shown in Fig. 9, compound 9-h (7.42 g, 38.0 mmol) was dissolved in DCM (200 mL), and DMF (214 mg, 2.92 mmol) and oxalyl chloride ((COCl)2, 11.1 g, 87.7 mmol) were added at 0°C, followed by stirring at 20°C for 1 hour. The mixture was concentrated under reduced pressure, diluted with DCM (100 mL), and then a mixture of compound 9-i (7.50 g, 29.2 mmol, 1.39 mL), DMAP (357 mg, 2.92 mmol), and TEA (14.8 g, 146 mmol) dissolved in DCM (120 mL) was added at 0°C, followed by stirring at 20°C for 14 hours. After that, the mixture was diluted with purified water (200 mL), adjusted to pH 3 using 2 M hydrochloric acid solution, extracted with DCM (200 mL x 2), and washed with sat.NaHCO3 (300 mL x 2). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (PE / EtOAc = 100 / 1 to 16 / 1) to obtain compound 9-b as a colorless oil.

[0200] Compound 9-a (9.59 g, 37.3 mmol) was added to MeCN (200 mL), and compound 9-b (5.38 g, 12.4 mmol), potassium carbonate (K2CO3, 5.15 g, 37.3 mmol), cesium carbonate (Cs2CO3, 1.21 g, 3.73 mmol), and NaI (558 mg, 3.73 mmol) were added, and the mixture was stirred at 20°C for 16 hours. The mixture was filtered, and the filtrate was filtered under reduced pressure and purified twice on silica gel (DCM / MeOH = 100 / 1 to 32 / 1) to obtain compound 9-c as a colorless oil.

[0201] Compound 9-c (600 mg, 984 μmol) and compound 9-d (450 mg, 2.95 mmol) were added to MeCN (10 mL), potassium carbonate (K2CO3, 408 mg, 2.95 mmol), cesium carbonate (Cs2CO3, 96.1 mg, 0.295 mmol), and NaI (147 mg, 0.984 mmol) were added, and the mixture was stirred at 80°C for 8 hours. The mixture was concentrated, diluted with purified water (40 mL), extracted with EtOAc (40 mL), washed with brine, dried over Na2SO4, and filtered. The solution was concentrated under reduced pressure and purified on silica gel (DCM / MeOH = 100 / 1 to 32 / 1) to obtain compound 9-e as a colorless oil.

[0202] Compound 9-e (200 mg, 0.275 mol) was dissolved in DCM (2 mL), TFA (307 mg, 2.69 mmol) was added at 20°C, and the mixture was stirred at 20°C for 28 hours. The mixture was concentrated under reduced pressure, washed with sat.aq.NaHCO3 (20 mL), extracted with EtOAc (20 mL), washed with brine, dried over Na2SO4, and filtered. The resulting solution was concentrated under reduced pressure to obtain compound 9-f as a colorless oil.

[0203] Compound 9-f (150 mg, 0.221 mmol) and compound 9-g (79.8 mg, 0.265 mmol) were dissolved in DCE (3 mL), and acetic acid (42.3 mg, 0.704 mmol) was added dropwise at 20°C, and stirred at 20°C for 1 hour. NaBH(OAc)3 (93.5 mg, 0.441 mmol) was added, and stirred at 20°C for 15 hours. The mixture was diluted with purified water (20 mL), extracted with DCM (20 mL × 3 times), and washed with brine (30 mL). The solution, dried and filtered over Na2SO4, was concentrated under reduced pressure, and purified on silica gel (DCM / MeOH = 100 / 1 to 19 / 1) to obtain compound 9 as a pale yellow oil. 1 The results of H NMR analysis were as follows.

[0204] 1 H NMR (400 MHz, CDCl3)δ4.83-4.88(m,1H),4.06(t,J=6.8 Hz, 4H), 3.70-3.71 (m, 2H), 2.69-2.79 (m, 12H), 2.30-2.32 (m, 6H), 1.60-1.71 (m, 20H), 1.50-1.52 (m, 4H), 1.26-1.39 (m, 54H), 0.87-0.90 (m, 12H).

[0205]

[0206] 1-10. Preparation of compound 10

[0207] As shown in Fig. 10, compound 10-a (2.99 g, 8.91 mmol), compound 10-b (290 mg, 2.78 mmol), Na2CO3 (885 mg, 8.35 mmol), and NaI (417 mg, 2.78 mmol) were added to MeCN (30 mL) and stirred at 80°C for 14 hours. The mixture was diluted with purified water (30 mL), extracted with EtOAc (30 mL × 3 times), and washed with brine (50 mL). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified twice on silica gel (DCM / MeOH = 100 / 1 to 20 / 1) to obtain compound 10 as a pale yellow oil. 1 The results of H NMR analysis were as follows.

[0208] 1 H NMR (500 MHz, CD3OD)δ 4.05-4.08 (m, 6H), 3.61 (t,J= 5.5 Hz, 2H), 2.83-2.86 (m, 2H), 2.69-2.75 (m, 4H), 2.67 (t,J= 5.5 Hz, 2H), 2.57-2.62 (m, 2H), 2.32-2.36 (m, 6H), 1.49-1.66 (m, 18H), 1.30-1.37 (m, 50H), 0.89-0.92 (m, 9H).

[0209]

[0210] 1-11. Preparation of compound 11

[0211] As shown in Fig. 11, compound 11-f (10.5 g, 53.6 mmol) was dissolved in DCM (100 mL), and DMF (301 mg, 4.12 mmol) and oxalyl chloride (15.7 g, 124 mmol) were added at 0°C, followed by stirring at 20°C for 1 hour. The mixture was concentrated under reduced pressure, diluted with DCM (80 mL), and then a mixture of compound 11-g (10.0 g, 41.2 mmol), DMAP (504 mg, 4.12 mmol), and TEA (20.9 g, 206 mmol) dissolved in DCM (120 mL) was added at 0°C, followed by stirring at 20°C for 14 hours. After that, the mixture was diluted with purified water (100 mL), adjusted to pH 3 using 2 M hydrochloric acid solution, and extracted with DCM (150 mL x 2), and washed with sat.NaHCO3 (200 mL) and brine (150 mL). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (PE / EtOAc = 100 / 1 to 16 / 1) to obtain compound 11-b as a colorless oil.

[0212] Compound 11-a (14.0 g, 29.4 mmol), compound 11-b (4.11 g, 9.79 mmol), Na2CO3 (2.08 g, 19.6 mmol), and NaI (1.47 g, 9.79 mmol) were added to MeCN (210 mL) and stirred at 50°C for 6 hours (2 batches). The 2-batch preparation was filtered and concentrated under reduced pressure, and then purified on silica gel (DCM / MeOH = 100 / 1 to 24 / 1) to obtain compound 11-c as a yellow oil.

[0213] Compound 11-c (790 mg, 1.37 mmol) was added to MeOH (10 mL), and a palladium / carbon catalyst (Pd / C, 80 mg, purity 10%) was added under nitrogen gas. The mixture was replaced with hydrogen (H2) and stirred at 25°C for 4 hours under hydrogen gas (15 Psi), and then stirred at 35°C for 14 hours. The reaction mixture was filtered, and the residue was washed with MeOH (20 mL), concentrated under reduced pressure, and purified on silica gel (0.2% NH3.MeOH in DCM / MeOH = 100 / 1 to 3 / 1) to obtain compound 11-d as a yellow oil.

[0214] Compound 11-d (300 mg, 0.678 mmol), compound 11-e (682 mg, 2.03 mmol), K2CO3 (281 mg, 2.03 mmol), and NaI (50.8 mg, 0.339 mmol) were added to MeCN (13 mL) and stirred at 80°C for 6 hours. The mixture was diluted with purified water (50 mL), extracted with DCM (50 mL × 2), and washed with brine (50 mL). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure, purified on silica gel (DCM / MeOH = 100 / 1 to 19 / 1), and lyophilized to obtain compound 11 as a colorless oil. 1 The results of H NMR analysis were as follows.

[0215] 1 H NMR (400 MHz, CDCl3) δ 4.06 (t,J= 6.8 Hz, 4H), 3.97 (d,J= 6.0 Hz, 2H), 3.60-3.61 (m, 2H), 2.47-2.68 (m, 12H), 2.31 (t,J= 7.2 Hz, 6H), 1.59-1.69 (m, 13H), 1.51-1.52 (m, 4H), 1.27-1.31 (m, 58H), 0.87-0.90 (m, 12H).

[0216]

[0217] 1-12. Preparation of compound 12

[0218] As shown in Fig. 12, compound 12-b (23.9 g, 96 mmol) was dissolved in DCM (800 mL), and compound 12-a (10.0 g, 96.0 mmol) dissolved in DCM (800 mL) was added at -40°C and under nitrogen gas, stirred for 3 hours, and then concentrated under reduced pressure to obtain compound 12-c as a white solid.

[0219] Compound 12-c (20.0 g, 42.0 mmol), compound 12-d (4.69 g, 14.0 mmol), Na2CO3 (2.97 g, 28.0 mmol), and NaI (2.10 g, 14.0 mmol) were added to MeCN (200 mL) and stirred at 50°C for 8 hours. After filtration and concentration under reduced pressure, the mixture was purified on silica gel (0.2% NH3.MeOH in DCM / MeOH = 100 / 1 to 24 / 1), diluted with DCM (20 mL), and washed with sat.aq.NaHCO3 (30 mL × 2). The dried and filtered solution was concentrated under reduced pressure to obtain compound 12-e.

[0220] Compound 12-e (1.50 g, 3.04 mmol) was added to isopropyl alcohol (I-PrOH) (15 mL), and a palladium / carbon catalyst (Pd / C, 150 mg, purity 10%) was added under nitrogen gas, followed by replacement with hydrogen, and stirred at 25°C for 4 hours under hydrogen gas (15 Psi), and then at 35°C for 14 hours. The reaction mixture was filtered, and the residue was washed with isopropyl alcohol (20 mL), concentrated under reduced pressure, and purified on silica gel (0.2% NH3.MeOH in DCM / MeOH = 100 / 1 to 1 / 100) to obtain compound 12-f as a colorless oil.

[0221] Compound 12-h (16.0 g, 82.1 mmol) was dissolved in DCM (200 mL), and DMF (462 mg, 6.32 mmol) and oxalyl chloride ((COCl)2, 24.1 g, 190 mmol) were added at 0°C, and the mixture was stirred at 20°C for 1 hour. The mixture was concentrated under reduced pressure, diluted with DCM (150 mL), and then a mixture of compound 12-i (10.0 g, 63.2 mmol), DMAP (772 mg, 6.32 mmol), and TEA (32.0 g, 316 mmol) dissolved in DCM (100 mL) was added at 0°C, and the mixture was stirred at 20°C for 14 hours. After the reaction was completed, the mixture was diluted with purified water (200 mL), adjusted to pH 3 using 2 M hydrochloric acid solution, extracted with DCM (200 mL x 2), and washed with sat.NaHCO3 (300 mL x 2). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (PE / EtOAc = 100 / 1 to 24 / 1) to obtain compound 12-d as a colorless oil.

[0222] Compound 12-j (10.5 g, 53.6 mmol) was dissolved in DCM (200 mL), and DMF (301 mg, 4.12 mmol) and oxalyl chloride ((COCl)2, 15.7 g, 124 mmol) were added at 0°C, and the mixture was stirred at 20°C for 1 hour. The mixture was concentrated under reduced pressure, diluted with DCM (100 mL), and then a mixture of compound 12-k (10.0 g, 41.3 mmol), DMAP (504 mg, 4.12 mmol), and TEA (20.9 g, 206 mmol) dissolved in DCM (100 mL) was added at 0°C, and the mixture was stirred at 20°C for 14 hours. After the reaction was completed, the mixture was diluted with purified water (200 mL), adjusted to pH 3 using 2 M hydrochloric acid solution, extracted with DCM (200 mL x 2), and washed with sat.NaHCO3 (300 mL x 2). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (PE / EtOAc = 100 / 1 to 19 / 1) to obtain compound 12-g as a colorless oil.

[0223] Compound 12-f (250 g, 0.697 mmol), compound 12-g (877 mg, 2.09 mmol), K2CO3 (289 mg, 2.09 mmol), and NaI (52.3 mg, 0.349 mmol) were added to MeCN (10 mL) and stirred at 80°C for 4 hours. The reaction mixture was concentrated under reduced pressure, diluted with purified water (30 mL), extracted with EtOAc (30 mL × 2 times), and washed with brine (30 mL). The solution, dried and filtered over Na2SO4, was concentrated under reduced pressure, and purified on silica gel (DCM / MeOH = 100 / 1 to 19 / 1) to obtain compound 12 as a colorless oil. 1 The results of H NMR analysis were as follows.

[0224] 1 H NMR (400 MHz, CD3OD) δ 4.07 (t,J= 6.8 Hz, 2H), 4.00 (d,J= 5.6 Hz, 4H), 3.47-3.72 (m, 2H), 2.41-3.20 (m, 12H), 2.27-2.37 (m, 6H), 1.63-1.68 (m, 10H), 1.48-1.55 (m, 6H), 1.28-1.37 (m, 68H), 0.89-0.92 (m, 15H).

[0225]

[0226] 1-13. Preparation of compound 13

[0227] As shown in Fig. 13, compound 13-a (15.6 g, 69.8 mmol) was dissolved in DCM (100 mL), and DMF (392 mg, 5.37 mmol) and oxalyl chloride ((COCl)2, 20.4 g, 161 mmol) were added at 0°C, followed by stirring at 20°C for 1 hour. The mixture was concentrated under reduced pressure, diluted with DCM (80 mL), and then a mixture of compound 13-b (10.0 g, 54 mmol), DMAP (656 mg, 5.37 mmol), and TEA (27.2 g, 268 mmol) dissolved in DCM (100 mL) was added at 0°C, followed by stirring at 20°C for 14 hours. After completion of the reaction, the mixture was diluted with purified water (100 mL), adjusted to pH 3 using 2 M hydrochloric acid solution, and extracted with DCM (150 mL x 2), washed with sat.NaHCO3 (150 mL x 2) and brine (150 mL). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (PE / EtOAc = 100 / 1 to 50 / 1) to obtain compound 13-c as a colorless oil.

[0228] Compound 13-c (6.01 g, 15.4 mmol), compound 13-d (500 mg, 4.80 mmol), Na2CO3 (1.53 g, 14.4 mmol), and NaI (719 g, 4.80 mmol) were added to MeCN (50 mL) and stirred at 80°C for 14 hours. The mixture was diluted with purified water (30 mL) at room temperature and extracted with EtOAc (30 mL × 3 times). The solution was washed with brine (50 mL). The solution was dried over Na2SO4 and filtered, and then concentrated under reduced pressure. The resulting solution was purified on silica gel (DCM / MeOH = 100 / 1 to 20 / 1) to obtain compound 13 as a yellow oil. 1 The results of H NMR analysis were as follows.

[0229] 1H NMR (400 MHz, CD3OD)δ 4.02 (d,J=5.6 Hz, 6H), 3.65 (t,J=5.2 Hz, 2H), 2.79-3.14 (m, 8H), 2.63-2.76 (m, 4H), 2.30-2.39 (m, 6H), 1.62-1.70 (m, 12H), 1.50-1.60 (m, 3H), 1.30-1.43 (m, 66H), 0.88-1.00 (m, 18H).

[0230]

[0231] [Example 2] Manufacturing of lipid nanoparticles

[0232] Lipid nanoparticles encapsulating Firefly Luciferase mRNA (RNA Gene, Korea) were prepared using the compound (ionized lipid) prepared in Example 1, dioleoylphosphatidylethanolamine (hereinafter referred to as DOPE) or distearoylphosphatidylcholine (hereinafter referred to as DSPC) (neutral lipid), cholesterol (steroid), and PEG 2000-DMG (polymer lipid). However, in the case of compound 7, lipid nanoparticles encapsulating Firefly Luciferase saRNA (Microuni, Korea) were prepared instead of Firefly Luciferase mRNA. Ionized lipids, DOPE / DSPC, cholesterol, and PEG 2000-DMG were mixed in a molar ratio of 40:15:43.5:1.5 and dissolved in ethanol at a concentration of 4.8 mg / mL to prepare a lipid solution. Separately, Firefly Luciferase mRNA was dissolved in a citric acid buffer solution (pH 4.0±1.0) or an acetic acid buffer solution (pH 5.0±1.0) at a weight ratio of 1:10 for mRNA:ionized lipid to prepare an active ingredient solution. Then, lipid nanoparticles were prepared by passing the lipid solution:active ingredient solution through a microfluidic mixing device (Benchtop Nanoassemblr, Precision Nanosystems) at a flow rate of approximately 15 mL / min at a volume ratio of 1:3. The manufactured lipid nanoparticles were diluted with Tris buffer containing 8.7% sucrose and dialyzed using a dialysis centrifuge tube to ensure that the ethanol content was less than 1%, and the final concentration was prepared to be 0.2 mg / mL based on mRNA.

[0233] For comparison, lipid nanoparticles encapsulating Firefly Luciferase mRNA (RNA Gene, Korea) were prepared using ALC-0315 ionized lipid, DSPC (neutral lipid), cholesterol (steroid), and ALC-0159 (polymeric lipid). ALC-0315, DSPC, cholesterol, and ALC-0159 were prepared in the same molar % ratio of 46.3:9.4:42.7:1.6 and used as ALC-0315 lipid nanoparticles for comparison.

[0234]

[0235] [Example 3] Confirmation of lipid nanoparticle size and polydispersity

[0236] The size and surface charge of lipid nanoparticles prepared according to the ionized lipid in Example 1 were measured. To measure the size of lipid nanoparticles, each lipid nanoparticle was diluted with PBS to a concentration of 1 μg / mL of mRNA, and the diameter and polydispersity index (PDI) of the lipid nanoparticles were measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nano (Malvern Instruments, UK).

[0237] As a result, as shown in Table 1, all lipid nanoparticles exhibited a size of approximately 120 nm or less and a good polydispersity of less than 0.2.

[0238] Compound NumberParticle Size (nm)PDI11080.0721130.073990.0541020.1351220.0361040.067890.068980.1191180.1101060.1111980.05121040.1

[0239]

[0240] [Example 4] Confirmation of gene inclusion rate

[0241] To confirm the gene encapsulation rate of lipid nanoparticles, Quant-iTTM RibogreenTM RNA Reagent and Kit was used. 5 μL of ribosomal RNA standard (100 μg / mL in TE buffer) was diluted with 245 μL TE (Tris-EDTA) buffer or 0.4% Triton-TE buffer to prepare a stock solution for a calibration curve. The stock solutions (2, 5, 10, 25, and 50 μL) were diluted with TE buffer or 0.4% Triton-TE buffer to make a total volume of 100 μL, and 100 μL of Quant-iTTM RibogreenTM RNA Reagent was added, mixed, and the fluorescence intensity (excitation 475 nm and emission 500–550 nm) was measured to prepare a calibration curve. 5 μL of the lipid nanoparticle solution prepared in Example 2 was diluted with 245 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5 in DEPC-treated water). 50 μL of the diluted lipid nanoparticle solution was added with 50 μL of TE buffer or 2% Triton-TE buffer, respectively, and incubated at 37°C for approximately 10 minutes. Then, 40 μL of each culture medium was added to a microplate, and 60 μL of TE buffer and 100 μL of Quant-iT™ Ribogreen™ RNA Reagent were sequentially added, mixed, and the fluorescence intensity was measured. The measured fluorescence intensity was substituted into the calibration curve to calculate the gene amount (TO) of the TE buffer and the gene amount (T2) of the 2% Triton-TE buffer, and then the gene inclusion rate was calculated using the following mathematical equation 1.

[0242] [Mathematical Formula 1]

[0243]

[0244] As a result, as shown in Table 2, it was confirmed that all compounds showed a good gene encapsulation rate of 85% or more.

[0245] Compound number Gene inclusion rate (%) 188.7289.2386.1495.4571.2684.4793.0895.9995.51091.61190.31292.1

[0246]

[0247] [Example 5] Confirmation of lipid nanoparticle gene transfer efficiency

[0248] 5-1. Confirmation of intracellular gene transfer efficiency

[0249] To confirm the intracellular gene delivery efficiency of lipid nanoparticles, lipid nanoparticles (FLuc mRNA-LNP) were used. To evaluate the gene delivery efficiency, HeLa cells (Korea Cell Line Bank, Seoul), a human cervical cancer cell line expressing the low-density lipoprotein receptor (LDL receptor) on the cell surface, were used. They were cultured in MEM medium supplemented with 10% fetal bovine serum and penicillin / streptomycin at 37°C and 5% CO2. HeLa cells were seeded at 2 × 10 per well in a 96-well plate. 4 The cells were divided into cell numbers and cultured, and after 16 to 24 hours, the lipid nanoparticles were diluted in MEM medium containing 10% FBS at a concentration of 0.25 μg / ml based on the encapsulated mRNA, and 100 μl was added per well to treat the cells.

[0250] To confirm the expression of intracellular luciferase delivered via lipid nanoparticles, the culture medium in the wells was removed after 6 hours, washed with PBS, and 100 μl of Glo-Lysis Buffer (Promega, WI, USA) was added to lyse the cells at room temperature for 5 minutes. 100 μl of Steady-Glo™ Luciferase Assay solution (Promega) was added to the lysed cells at a 1:1 ratio, incubated for 5 minutes at room temperature, and then transferred to a 96-well white plate. The luminescence value was detected using a GloMax Discover microplate reader (Promega). A calibration curve was generated using recombinant luciferase (Promega) as a standard, and the luciferase concentration (pg / ml) of the sample was derived from this.

[0251] As a result, as shown in Table 3, it was confirmed that the lipid nanoparticles using the ionized lipid of the present invention exhibited a high gene delivery effect in vitro. From the above results, it was confirmed that the lipid nanoparticles can be utilized as gene delivery vehicles.

[0252] Compound numberLuciferase expression level(Mean±SD, pg / ml)3228,260 ± 18,4727338,847 ± 4,760915,811 ± 3,398103,545 ± 2171267,540 ± 1,204

[0253] 5-2. Confirmation of in vivo gene transfer efficiency

[0254] To confirm the in vivo gene delivery efficiency and distribution of lipid nanoparticles, 7-week-old C57BL / 6 mice (Daehan Biolink) were injected intravenously at a single dose of 1 mg / kg based on the encapsulated gene of lipid nanoparticles or intramuscularly at 0.5 mg / kg each in both thighs. However, in the case of the lipid nanoparticle of compound 7 (containing saRNA), 0.05 mg / kg each was injected intramuscularly in both thighs. In the case of intravenous injection, the mice were sacrificed 4 hours after administration, and the livers were removed, and in the case of intramuscular injection, the organs including the thigh muscles were removed 4 or 6 hours after administration.

[0255] To confirm the level of luciferase expression in tissue samples, the weight of the tissue sections was measured. For liver tissue, it was transferred to a tube containing 3 mm metal beads, and 500 μl of Glo-Lysis Buffer (Promega) was added per 50 mg, and the tissue was homogenized using a bead homogenizer. For thigh muscle, 500 μl of Glo-Lysis Buffer (Promega) was added per 100 mg, and the tissue was homogenized using a rotor / stator homogenizer. The supernatant of the centrifuged homogenate was taken and diluted with Glo-Lysis Buffer at an appropriate ratio, and 50 μl each of the diluted tissue homogenate and Steady-Glo Luciferase Assay solution were reacted at a 1:1 ratio. After leaving for 5 minutes, the luminescence value of luciferase was measured in the same manner as in Example 3-1, and the expression amount of luciferase (ng / g tissue) was calculated.

[0256] As a result, as shown in Table 4, it was confirmed that the lipid nanoparticles using the ionized lipid of the present invention exhibited a good protein expression rate in liver tissue when injected intravenously, thereby exhibiting a good gene transfer effect when administered systemically.

[0257] Compound number: Luciferase expression in the liver after intravenous injection (Mean ± SD, ng / g liver) 312,066 ± 3,744 92,658 ± 881 101,311 ± 424

[0258] In addition, as shown in Table 5, it was confirmed that the lipid nanoparticles using the ionized lipid of the present invention have the characteristics of showing direct gene delivery ability in muscle tissue when injected intramuscularly and minimizing diffusion to liver tissue. Compared to the problem that existing ionized lipids have a problem of inducing high gene expression, especially in the liver, by leaking from the injection site even when injected intramuscularly and circulating a large amount of the drug systemically, it was confirmed that the lipid nanoparticles of the present invention show excellent gene delivery ability in muscle tissue, which is the injection site, when injected intramuscularly.

[0259] Compound number, time after administration, luciferase expression level in muscle and liver after intramuscular injection (Mean ± SD, ng / g tissue) Muscle (sum of both thighs) Liver 4 a) 4437 ± 1360.3 ± 0.085 a) 42,078 ± 31831 ± 156 a) 62,321 ± 677384 ± 3467 b) 6397 ± 2401.8 ± 1.0

[0260] a) 0.5 mg / kg administration, b) 0.05 mg / kg administration

[0261] In addition, as shown in Table 6, the lipid nanoparticles of the compound 3 of the present invention exhibited high muscle transmission upon intramuscular injection, and the ratio of the expression amount in the liver divided by the expression amount in the muscle was less than 5%, confirming that the systemic exposure was very low. On the other hand, the lipid nanoparticles of the ionizable lipid ALC-0315 used as a comparison leaked a large amount from the injection site, confirming that the ratio of the expression amount in the liver divided by the expression amount in the muscle exceeded 100%, confirming that the systemic exposure was very high. Considering that most of the lipid nanoparticles are delivered to the liver upon systemic exposure due to the nature of the lipid nanoparticles, it was confirmed that the ionizable lipid of the present invention has the characteristic of mainly expressing genes in muscle tissue upon intramuscular injection.

[0262] Compound number Time after administration Luciferase expression level in muscle and liver after local administration (Mean ± SD, ng / g tissue) Muscle (sum of both thighs) Liver 365,764 ± 1,989 118 ± 87 ALC-0315 62,264 ± 1,092 2,748 ± 1,979

[0263] For reference, mRNA vaccines using lipid nanoparticle technology are known to 1) primarily deliver genes to muscle cells after intramuscular injection, 2) secondarily deliver genes to dendritic cells and macrophages within muscle tissue, and 3) finally complete the immune system of B cells and T cells within the lymph nodes of muscle tissue. Therefore, since all immune responses of mRNA vaccines are completed in local muscle tissue, it is important to minimize unnecessary systemic exposure of lipid nanoparticles, which can cause excessive systemic immune responses (reactogenicity), in order to reduce systemic side effects of the vaccine and achieve optimal efficacy. In this regard, the ionized lipid of the present invention not only effectively delivers mRNA by encapsulating it in lipid nanoparticles, but also minimizes systemic exposure through muscle-selective delivery, thereby providing useful clinical benefits in the development of mRNA infectious disease vaccines and cancer vaccines, and it has been confirmed that it can also be utilized in the treatment of genetic muscle diseases.

[0264]

[0265] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A compound represented by the following chemical formula 1, a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, or a compound selected from the group consisting of: [Chemical Formula 1] In the above chemical formula 1, n is one of 1 to 6, m1 and m3 may be the same or different and are one of 0 to 6, m2 is 0 or 1, L 11 , L 12 and L 13 may be the same or different, and is one of (C1-C10)alkylene, (C2-C10)alkenylene and (C2-C10)alkynylene, and in said alkylene, alkenylene and alkynylene, at least one -CH2- is substituted or unsubstituted with -O-, -S- or -SS-, A1 to A4 may be the same or different, and -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OP(=O)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-,-C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -C(=O)NR 11 -, -NR 11 C(=O)-, -OC(=O)NH-, -NHC(=O)O-, -OC(=O)NR 11 - and -NR 11 One of C(=O)O-, and the above R 11 is one of (C1-C10)alkyl, (C2-C10)alkenyl and (C2-C10)alkynyl, and in said alkyl, alkenyl and alkynyl, at least one -CH2- is substituted or unsubstituted with -O-, -S- or -SS-, R 1 , R 2 , R 3 and R 4 are each the same or different, and are one of 4 to 20-membered heteroaryl containing at least one heteroatom selected from the group consisting of hydroxy, (C1-C20)alkyl, (C2-C20)alkenyl, (C2-C20)alkynyl, (C3-C20)cycloalkyl, (C3-C20)aryl and N, O and S, wherein at least one -CH2- in said alkylene, alkenylene and alkynylene is unsubstituted or substituted with -O-, -S- or -SS-, and said (C3-C20)cycloalkyl, (C3-C20)aryl and 4 to 20-membered heteroaryl are unsubstituted or substituted with at least one of hydroxy, amino, halogen and (C1-C5)alkyl.

2. In claim 1, in the chemical formula 1, n is one of 2 to 4, m1 and m3 may be the same or different and are one of 0 to 4, L 11 , L 12 and L 13 may be the same or different, and is one of (C3-C6)alkylene, (C3-C6)alkenylene and (C3-C6)alkynylene, A1 to A4 may be the same or different, and are -OC(=O)- or -C(=O)O-, R 1 , R 2 and R 3 may be the same or different, and is one of (C5-C20)alkyl, (C5-C20)alkenyl and (C5-C20)alkynyl, and in the alkylene, alkenylene and alkynylene, at least one -CH2- is substituted or unsubstituted with -O-, -S- or -SS-, R 4 A compound selected from a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that the compound is a 4 to 10-membered heteroaryl group containing at least one of hydroxy, (C3-C10)cycloalkyl, (C3-C10)aryl and N, wherein the (C3-C10)cycloalkyl, (C3-C10)aryl and the 4 to 10-membered heteroaryl are substituted or unsubstituted with amino.

3. In claim 1, in the chemical formula 1, n is one of 3 or 4, L 11 , L 12 and L 13 may be the same or different, and are (C3-C6)alkylene, R 1 , R 2 and R 3 may be the same or different, and is one of (C5-C20)alkyl, (C5-C20)alkenyl and (C5-C20)alkynyl, and in the alkylene, alkenylene and alkynylene, at least one -CH2- is substituted or unsubstituted with -S- or -SS-, R 4 A compound selected from a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that it is a 4 to 10-membered heteroaryl group containing at least one hydroxy or N, wherein said 4 to 10-membered heteroaryl group is substituted or unsubstituted with amino.

4. In claim 1, the compound is a compound selected from the group of compounds below, a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: (1) (9Z,9'Z)-((3-((2-hydroxyethyl)(3-((Z)-tetra-9-decenoyloxy)propyl)amino)propyl)azane-3,1-diyl)bis(tetra-9-decenoate); (2) (9Z,9'Z)-((3-((3-((Z)-hexa-9-decenoyloxy)propyl)((3-((3-hydroxyethyl)amino)propyl)azane-3,1-diyl)bis(hexa-9-decenoate); (3) Di((Z)-decenyl)4,4'-((3-((4-(Z)-4-decenyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azane-4-yl)dibutanoate; (4) Didodecyl 4,4'-((3-((4-(dodecyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azane-4-yl)dibutanoate; (5) Di((Z)-dec-4-en-1-yl)4,4'-((3-((2-((3-(6-aminopyridin-3-yl)propanoyl)oxy)ethyl)(4-((Z)-deceth-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azanedyl)dibutanoate; (6) Di((Z)-dec-4-en-1-yl)4,4'-((4-((4-((Z)-dec-4-en-1yloxy)-4-oxobutyl)(2-hydroxyethyl)amino)butyl)azanedyl)dibutanoate; (7) Di((Z)-dec-4-en-1-yl)5,5'-((3-((5-((Z)-dec-4-en-1yloxy)-5-oxopentyl)(2-hydroxyethyl)amino)propyl)azanedyl)dipentanoate; (8) Dinonyl 6,6'-((4-((2hydroxyethyl)(6-(nonyloxy)-6-oxohexyl)amino)butyl)azane-2-yl)dihexanoate; (9) Heptadeca-9-yl 6-((4-((2-hydroxyethyl)(6-(nonyloxy)-6-oxohexyl)amino)butyl)(6-(nonyloxy)-6-oxohexyl)amino)hexanoate; (10) Didecyl 6,6'-((2-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)azane-2-yl)dihexanoate; (11) Decyl 6-((2-((6-decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)(6((2-hexyldecyl)oxy)-6-oxohexyl)amino)hexanoate; (12) Bis(2-hexyldecyl)6,6'-((2-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)azanedyl)dihexanoate; (13) Di((Z)-dec-4-en-1-yl)4,4'-((3-((4-((Z)-dec-4-en-1yloxy)-4-oxobutyl)(pyridin-4-yl)amino)propyl)azanedyl)dibutanoate; (14) Di((Z)-tetradec-9-en-1-yl)4,4'-((3-((2-hydroxyethyl)(4-oxo-4-((Z)-tetradec-9-en-1-yloxy)butyl)amino)propylazanedyl)dibutanoate; (15) Bis(6-butyldisulfanyl)hexyl)4,4'-((3-((4-((6-butyldisulfanyl)hexyl)oxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azanedyl)dibutanoate; (16) Di((Z)-dec-4-en-1-yl)4,4'-((3-((2-((3)-(4-aminophenyl)propanoyl)oxy)ethyl)(4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azane-4-enyl)dibutanoate; (17) Di((Z))-dec-4-en-1-yl)4,4'-((3-((4-aminophenethyl)(4-((Z)-dec-4-en-1-yloxy)-4-oxobutyl)amino)propyl)azanedyl)dibutanoate; (18) di((Z)-dec-4-en-1-yl)4,4'-((3-((4-((Z)-dec-4-en-1yloxy)-4-oxobutyl)(pyridin-4-yl)amino)propyl)azane-1-yl)dibutanoate; and (19) (9Z,9'Z)-((3-((2-hydroxyethyl)(4-((Z)-tetradec-9-enoyloxy)butyl)amino)propyl)azanedyl)bis(butane-4,1-diyl) bis(tetradec-9-enoate).

5. A lipid nanoparticle composition comprising the compound of claims 1 to 4.

6. A lipid nanoparticle composition according to claim 5, characterized in that the compound is an ionized lipid.

7. A lipid nanoparticle composition according to claim 5, characterized in that the composition further comprises at least one selected from the group consisting of neutral lipids, steroids, and polymerized lipids.

8. A lipid nanoparticle composition according to claim 7, characterized in that the neutral lipid is a phospholipid or a glycolipid.

9. A lipid nanoparticle composition according to claim 7, characterized in that the steroid is at least one selected from the group consisting of cholesterol, bile acid derivatives, and cholic acid derivatives.

10. A lipid nanoparticle composition according to claim 7, wherein the polymer lipid is a PEGylated lipid, and is characterized in that at least one selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

11. A lipid nanoparticle composition according to claim 7, characterized in that the composition comprises 20 to 65 mol% of ionized lipid, 2.5 to 30 mol% of neutral lipid, 25 to 60 mol% of steroid, and 0.5 to 5 mol% of polymerized lipid.

12. A lipid nanoparticle composition according to claim 5, characterized in that the composition further comprises a preventive or therapeutic agent.

13. A lipid nanoparticle composition according to claim 12, wherein the preventive or therapeutic agent is at least one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribozyme (DNAzyme), guide ribonucleic acid for gene correction (sgRNA), and mixtures thereof.

14. A lipid nanoparticle composition according to claim 5; and a drug delivery composition comprising a preventive or therapeutic agent.

15. A drug delivery composition according to claim 14, wherein the preventive or therapeutic agent is at least one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribozyme (DNAzyme), guide ribonucleic acid for gene correction (sgRNA), and mixtures thereof.

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