Novel ionizable lipids and lipid nanoparticles comprising same

Abstract

The present invention relates to novel ionized lipids and lipid nanoparticles comprising the ionized lipids. A therapeutic agent using the lipid nanoparticles comprising the novel ionized lipids according to the present invention can minimizes in vivo side effects caused by components of the nanoparticles, effectively transfers the nanoparticles to target cells, and enables efficient transport of pharmacologically active substances such as nucleic acids into the cytoplasm by facilitating endosomal escape within the target cells.

Description

Novel ionized lipids and lipid nanoparticles containing the same

[0001] The present invention relates to novel ionized lipids and lipid nanoparticles comprising said ionized lipids, and more specifically, to a novel type of ionized lipid capable of minimizing in vivo side effects and effectively delivering pharmacologically active substances to target cells, and lipid nanoparticles comprising said lipid.

[0002]

[0003] Even for the same drug, efficacy varies widely depending on the delivery method. A drug delivery system (DDS) refers to the administration route and form of the drug that efficiently delivers the required amount of medication while minimizing side effects and maximizing efficacy. In the pharmaceutical industry, drug delivery systems can be considered a high-value-added core technology with a high probability of success, capable of generating economic benefits comparable to new drug development.

[0004] Recently, among various drug delivery systems, the development of gene therapies utilizing gene drug delivery systems is gradually expanding. To successfully and safely perform gene therapy, it is crucial to deliver genes or gene regulatory factors to the desired tissues. Representative examples of such genes or gene regulatory factors include mRNA, siRNA, and miRNA. Since nucleic acids such as mRNA perform the function of expressing specific proteins, they can play a role in supplementing proteins deficient due to genetic factors. Furthermore, using mRNA that expresses cancer markers or viral surface proteins enables the development of anticancer drugs and vaccines that activate the body's immune response. Additionally, nucleic acids such as siRNA and miRNA are substances capable of inhibiting the expression of specific proteins within the body, and are gaining attention as important tools for the treatment of cancer, genetic diseases, infectious diseases, and autoimmune diseases. However, since it is difficult to deliver these nucleic acids directly into cells and they are easily degraded by enzymes in the blood, extensive research is being conducted to overcome these limitations.

[0005] Non-viral gene carriers, such as cationic liposomes and polymers, are attracting attention for effectively delivering these substances, and the improved stability profile and ease of fabrication and manipulation of polymer carriers have accelerated research on the design and synthesis of non-toxic and biodegradable polymer carriers for effective and safe gene delivery. Poly(L-lysine), polyethyleneimine, starburst, polyamidoamine dendrimers, and cationic liposomes have been widely studied as non-viral gene carriers because they can self-assemble spontaneously and can be compressed into structures small enough to introduce plasmid DNA (pDNA) into cells via endocytosis.

[0006] In particular, research on lipid nanoparticles as non-viral gene delivery vehicles has recently been actively underway (Non-Patent Literature 1-9). Lipid nanoparticles (LNPs) are particulate drug delivery vehicles that have high bioavailability and affinity because they utilize substances present in the body, such as phospholipids and cholesterol, and allow for the release and control of drugs, while also possessing high stability against degradation by enzymes.

[0007]

[0008] Meanwhile, mRNA-based vaccines and therapeutics exert vaccine and therapeutic effects by administering mRNA encoding a specific antigen or a protein exhibiting a therapeutic effect into the body, thereby causing it to be expressed as that antigen or protein. Regarding mRNA vaccines, extensive research has been conducted on cancer vaccines using mRNA encoding cancer antigens, and they have garnered even more attention since the efficacy of COVID-19 vaccines was proven in 2020. Liposomes or LNP forms are generally used as carriers to deliver mRNA vaccines and therapeutics into the body (U.S. Patent Publication 2020-0405844 A).

[0009] The efficacy of lipid nanoparticles used for delivering oligos such as mRNA, siRNA, and miRNA is largely determined by the characteristics of ionized lipids, which are one of the components constituting the particles. In the early stages of lipid nanoparticle development, positively charged lipids were widely used rather than ionized lipids. Since positively charged lipids constantly carry a positive charge, they facilitate the binding of negatively charged oligos through electrostatic attraction, thereby forming nanoparticles. Furthermore, because the lipid membranes constituting cell membranes and endosomes carry a negative charge, it was known that this promotes the fusion of positively charged lipid nanoparticles with the cell membrane, resulting in high efficiency in delivering oligos into the cell (U.S. Patent Publication 2018-0221510 A, 2018-0369384 A). However, lipid nanoparticles manufactured using positively charged lipids also possess a positive charge, and thus a significant disadvantage has been highlighted regarding the occurrence of various side effects due to unnecessary interactions with unspecified cells while circulating through the bloodstream. Ionized lipids have been used to overcome these disadvantages of positively charged lipids.

[0010] Ionizable lipids are characterized by being non-charged at neutral pH but positively charged under acidic conditions. Since they maintain an electrically neutral state during circulation within the body, they can overcome the disadvantages of positively charged lipid nanoparticles. On the other hand, when lipid nanoparticles enter the cell's endosome, the ionizable lipids transition to a positively charged state due to the acidic pH environment inside the endosome. This enhances the interaction between the nanoparticles and the lipid membrane constituting the endosome, thereby promoting the fusion of the particles and the lipid membrane. To achieve this, it is desirable for ionizable lipids to be designed with a chemical structure having a pKa value that is non-charged at neutral pH but positively charged at acidic pH. Therefore, while a pKa range suitable for ionizable lipids is known to be between 5 and 7, this range is a necessary condition, not a sufficient one, for the efficient development of ionizable lipids. Ultimately, it is necessary to verify whether they demonstrate excellent efficiency in cell and animal experiments.

[0011] Accordingly, the inventors of the present invention have made diligent efforts to develop an optimal ionized lipid for manufacturing lipid nanoparticles that deliver pharmacologically active substances such as oligos, and have confirmed that lipid nanoparticles containing a novel ionized lipid exhibit excellent effects, thereby completing the present invention.

[0012]

[0013] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to those skilled in the art to which the present invention belongs.

[0014]

[0015] [Prior Art Literature]

[0016] [Patent Literature]

[0017] (Patent Document 1) U.S. Published Patent US 2020-0405844 A1

[0018] (Patent Document 2) U.S. Published Patent US 2018-0221510 A1

[0019] (Patent Document 3) U.S. Published Patent US 2018-0369384 A1

[0020]

[0021] [Non-patent literature]

[0022] (Non-patent Document 1) Moss, KH, Popova, P., Hadrup, SR, Astakhova, K. & Taskova, M. Lipid Nanoparticles for Delivery of Therapeutic RNA Oligonucleotides. Mol Pharm 16, 2265-2277 (2019)

[0023] (Non-patent Document 2) Kulkarni, JA, Witzigmann, D., Chen, S., Cullis, PR & van der Meel, R. Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics. ACC Chem Res 52, 2435-2444 (2019)

[0024] (Non-patent Document 3) Buck, J., Grossen, P., Cullis, PR, Huwyler, J. & Witzigmann, D. Lipid-Based DNA Therapeutics: Hallmarks of Non-Viral Gene Delivery. ACS Nano 13, 3754-3782 (2019)

[0025] (비특허문헌 4) Akinc, A. et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat Nanotechnol 14, 1084-1087 (2019)

[0026] (비특허문헌 5) Springer, A.D. & Dowdy, S.F. GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics. Nucleic Acid Ther 28, 109-118 (2018)

[0027] (비특허문헌 6) Kulkarni, J.A., Cullis, P.R. & van der Meel, R. Lipid Nanoparticles Enabling Gene Therapies: From Concepts to Clinical Utility. Nucleic Acid Ther 28, 146-157 (2018)

[0028] (비특허문헌 7) Rietwyk, S. & Peer, D. Next-Generation Lipids in RNA Interference Therapeutics. ACS Nano 11, 7572-7586 (2017)

[0029] (비특허문헌 8) Fang, Y. et al. Cleavable PEGylation: a strategy for overcoming the "PEG dilemma" in efficient drug delivery. Drug Deliv 24, 22-32 (2017)

[0030] (Non-patent Document 9) Cullis, PR & Hope, MJ Lipid Nanoparticle Systems for Enabling Gene Therapies. Mol Ther 25, 1467-1475 (2017)

[0031]

[0032] The objective of the present invention is to provide a new type of ionized lipid that delivers pharmacologically active substances, such as nucleic acids, into the cytoplasm to produce efficacy, and lipid nanoparticles containing the same.

[0033] Another objective of the present invention is to provide a drug delivery composition comprising the lipid nanoparticles.

[0034]

[0035] To achieve the above objective, the present invention provides an ionizable lipid compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0036] [Chemical Formula 1]

[0037]

[0038] In Chemical Formula 1,

[0039] R1 is selected from the following groups,

[0040]

[0041] R 2 and R 3 C independently 6-12 Alkyl, C 6-12 Alkenyl, C 6-12 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl,

[0042] L 1 and L 2is independently selected from a group consisting of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=S)O-, -OC(=S)-, -SC(=S)-, -C(=S)S-, -C(=O)NH-, -NHC(=O)-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -C(=O)NHNHC(=O)-, -C(=S)NH-, -NHC(=S)-, -NHC(=S)O-, -NHC(=S)S-, and -NHC(=S)S-.

[0043] R 4 is C 12-30 Alkyl, C 12-30 Alkenyl, C 12-30 Alkinyl, C 12-30 Cycloalkyl, C 12-30 Cycloalkenyl, C 12-30 It is a cycloalkynyl or sterol, and

[0044] Sterol is cholesterol or beta-sitosterol, and

[0045] R 5 is either without a bond or; C 2-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Cycloalkynyl; or -R 6 Selected from a group composed of C(=O)-,

[0046] R 6 is either without a bond or; C 2-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Selected from cycloalkynyl,

[0047] R 7 C 2-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C2-6 Cycloalkyl, C 2-6 Cycloalkenyl, C 2-6 Selected from cycloalkynyl,

[0048] m is an integer from 1 to 3, and n is an integer satisfying m + n = 3, and

[0049] R 8 and R 9 is independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl,

[0050] Here, the alkyl, alkenyl, or alkynyl is a straight chain or a branched chain.

[0051] The present invention also provides lipid nanoparticles comprising the ionized lipid compound, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0052] The present invention also provides a method for producing the lipid nanoparticles comprising the following steps:

[0053] (a) R 3 -L 2 -R 5 Halogenates containing -Sterol and R 1 -NH-R 2 -L 1 -R 4 Reacting amine compounds or R 2 -L 1 -R 4 Halogenates containing and R 1 -NH-R 3 -L 2 -R 5 - A step of reacting the amine compound of the sterol in a solvent; and

[0054] (b) Step of removing the above solvent.

[0055] Here, R 1 It is selected from the following military units,

[0056]

[0057] R 2 and R 3 C independently 6-12 Alkyl, C 6-12 Alkenyl, C 6-12 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl,

[0058] L 1 and L 2 is independently selected from a group consisting of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=S)O-, -OC(=S)-, -SC(=S)-, -C(=S)S-, -C(=O)NH-, -NHC(=O)-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -C(=O)NHNHC(=O)-, -C(=S)NH-, -NHC(=S)-, -NHC(=S)O-, -NHC(=S)S-, and -NHC(=S)S-.

[0059] R 4 is C 12-30 Alkyl, C 12-30 Alkenyl, C 12-30 Alkinyl, C 12-30 Cycloalkyl, C 12-30 Cycloalkenyl, C 12-30 It is a cycloalkynyl or sterol, and

[0060] Sterol is cholesterol or beta-sitosterol, and

[0061] R 5 is either without a bond or; C 2-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Cycloalkynyl; or -R 6 Selected from a group composed of C(=O)-,

[0062] R 6 is either without a bond or; C 2-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Selected from cycloalkynyl,

[0063] R 7 C 2-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 2-6 Cycloalkyl, C 2-6 Cycloalkenyl, C 2-6 Selected from cycloalkynyl,

[0064] m is an integer from 1 to 3, and n is an integer satisfying m + n = 3, and

[0065] R 8 and R 9 is independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl,

[0066] Here, the alkyl, alkenyl, or alkynyl is a straight chain or a branched chain.

[0067] The present invention also provides a drug delivery composition comprising the lipid nanoparticles; and an anionic drug, nucleic acid, or a combination thereof.

[0068]

[0069] The lipid nanoparticles according to the present invention contain a new type of ionized lipid and can minimize unnecessary interactions with surrounding components while circulating in the bloodstream within the body, and when they enter the endosome of a cell, they facilitate fusion with the endosome lipid membrane, thereby having the effect of safely delivering pharmacologically active substances into the cell.

[0070] The therapeutic agent using lipid nanoparticles according to the present invention minimizes in vivo side effects caused by the components of the nanoparticles, effectively delivers the nanoparticles to target cells, and can efficiently transport pharmacologically active substances such as nucleic acids into the cytoplasm by escaping from endosomes within the target cells.

[0071]

[0072] Figure 1 is data showing the activity of luciferase measured after preparing lipid nanoparticles containing ionized lipids according to the present invention and treating them with HepG2 and HEK293 cell lines, respectively.

[0073] Figure 2 is data showing the activity of luciferase measured after producing lipid nanoparticles that do not contain cholesterol when preparing lipid nanoparticles containing ionized lipids according to the present invention and treating them with a HepG2 cell line.

[0074] Figure 3 is data showing the activity of luciferase after preparing lipid nanoparticles containing ionized lipids according to the present invention and treating them with HepG2 and HEK293 cell lines, respectively.

[0075]

[0076] Specific details for implementing the invention

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0078]

[0079] It was confirmed that when lipid nanoparticles containing ionized lipids, which are novel compounds according to the present invention, are used as drug delivery vehicles, in vivo side effects caused by the nanoparticle components are minimized, the nanoparticles are effectively delivered to target cells, and pharmacologically active substances such as nucleic acids are suitable for efficiently escaping endosomes within target cells to transport them into the cytoplasm.

[0080]

[0081] Accordingly, in one aspect, the present invention relates to an ionized lipid compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0082] [Chemical Formula 1]

[0083]

[0084] In the above chemical formula 1,

[0085] The above R1 can be selected from a group composed of the following structures, and

[0086]

[0087] The above R 2 and R 3 C independently comprising a straight chain, branch, or loop 6-12 alkyl of, C 6-12 alkenyl or C 6-12 It can be selected from alkynes.

[0088] The above L 1 and L 2 are independently -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=S)O-, -OC(=S)-, -SC(=S)-, -C(=S)S-, -C(=O)NH-, -NHC(=O)-, -NHC(=O)O-,

[0089] It may be selected from the group consisting of -OC(=O)NH-, -NHC(=O)NH-, -C(=O)NHNHC(=O)-, -C(=S)NH-, -NHC(=S)-, -NHC(=S)O-, and -NHC(=S)S-.

[0090] The above R 4 is C 12-30 Alkyl, C 12-30 Alkenyl, C 12-30 Alkinyl, C 12-30 Cycloalkyl, C 12-30 Cycloalkenyl, C 12-30 It is a cycloalkynyl or sterol, and

[0091] Preferably C 15-28 Alkyl, C 15-28 Alkenyl, C 15-28 Alkinyl, C 15-28 Cycloalkyl, C 15-28 Cycloalkenyl, C 15-28 It is a cycloalkynyl or sterol, and

[0092] More preferably, C comprising a straight chain, branch, or loop 18-26 alkyl of, C 18-26 of alkenyl, C 18-26 It can be selected from alkynols; or sterols.

[0093] The above R 5 is either without a bond; or C including a straight chain, branch, or loop 2-12 alkyl of, C 2-12 of alkenyl, C 2-12 alkynyl of; or -R 6 It can be selected from a group composed of C(=O)-.

[0094] Sterol is cholesterol or beta-sitosterol, and R 4 If is not a sterol, the hydroxyl group (-OH) on the 3rd carbon of the above sterol and R 5 can be connected,

[0095] The above R6 is either without a bond; or C including a straight chain, branch, or loop 2-12 alkyl of, C 2-12 of alkenyl, C 2-12 It can be selected from alkynes.

[0096] The above R 7 C containing a straight chain, branch, or loop 2-6 alkyl of, C 2-6 of alkenyl, C 2-6 It can be selected from alkynes, and

[0097] m is an integer from 1 to 3, and n is an integer satisfying m + n = 3, and

[0098] R 8 and R 9 is independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is cycloalkinyl.

[0099]

[0100] R 2 and R 3 is independently a straight chain or branch C 6-12 alkyl of, C 6-12 alkenyl or C 6-12 It is preferable that it be an alkynyl, and more preferably a straight-chain C 6-12 It is an alkyl of

[0101] L 1 and L 2 It is preferable that it is independently -C(=O)-, -OC(=O)-, -C(=O)O-, or -OC(=O)O-, and more preferably -C(=O)- or -OC(=O)-.

[0102] R 4 is a straight chain or branch C 18-26 Alkyl, C 18-26 Alkenyl, C 18-26It is an alkynyl or sterol, and more preferably a branch C 18-26 It is an alkyl.

[0103] R 5 is without bonds or straight chain C 2-12 alkyl or -R 6 It is preferable that C(=O)-, where, the above R 6 is without bonds or straight chain C 2-12 It is preferable that it be an alkyl, and R 7 Silver is a straight chain or branch C 2-6 Alkyl, C 2-6 Alkenyl or C 2-6 It is preferable that it be alkynyl, and more preferably branch C 2-6 It is an alkyl, and m is an integer from 1 to 3.

[0104] In the present invention, the term "absent" means "single bond."

[0105] In the present invention, the ionized lipid represented by Formula 1 may be represented by any one of Formulas 2 to 13 below, but is not limited thereto.

[0106] [Chemical Formula 2] TRC-025

[0107]

[0108] [Chemical Formula 3] TRC-026

[0109]

[0110] [Chemical Formula 4] TRC-030

[0111]

[0112] [Chemical Formula 5] TRC-101

[0113]

[0114] [Chemical Formula 6] TRC-111

[0115]

[0116] [Chemical Formula 7] TRC-114

[0117]

[0118] [Chemical Formula 8] TRC-121

[0119]

[0120] [Chemical Formula 9] TRC-124

[0121]

[0122] [Chemical Formula 10] TRC-131

[0123]

[0124] [Chemical Formula 11] TRC-146

[0125]

[0126] [Chemical Formula 12] TRC-156

[0127]

[0128] [Chemical Formula 13] TRC-166

[0129]

[0130] [Chemical Formula 14] TRC-021

[0131]

[0132] [Chemical Formula 15] TRC-022

[0133]

[0134] [Chemical Formula 16] TRC-024

[0135]

[0136] [Chemical Formula 17] TRC-027

[0137]

[0138] [Chemical Formula 18] TRC-036

[0139]

[0140] [Chemical Formula 19] TRC-104

[0141]

[0142] [Chemical Formula 20] TRC-186

[0143]

[0144] [Chemical Formula 21] TRC-196

[0145]

[0146] [Chemical Formula 22] TRC-206

[0147]

[0148] [Chemical Formula 23] TRC-216

[0149]

[0150] [Chemical Formula 24] TRC-221

[0151]

[0152] [Chemical Formula 25] TRC-226

[0153]

[0154] [Chemical Formula 26] TRC-236

[0155]

[0156] [Chemical Formula 27] TRC-246

[0157]

[0158] [Chemical Formula 28] TRC-256

[0159]

[0160] [Chemical Formula 29] TRC-239

[0161]

[0162] [Chemical Formula 30] TRC-240

[0163]

[0164] In this specification, “ionizable lipid” means an amine-containing lipid that can be easily cationized, and may be, for example, a lipid whose charge state changes depending on the surrounding pH.

[0165] The above ionized lipid can be cationized at a pH below the pKa of the cationic lipid and can be substantially electrically neutral at a pH above the pKa.

[0166] In the present invention, the ionized lipid may be characterized as an ionizable compound having properties similar to lipids, and may play a role in encapsulating the drug (e.g., anionic drugs and / or nucleic acids) with high efficiency through electrostatic interaction with the drug.

[0167]

[0168] In another aspect, the present invention relates to lipid nanoparticles (LNPs) comprising the ionized lipid compound, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0169] In one embodiment of the present invention, the lipid nanoparticles may include cationized ionized lipids and / or neutral ionized lipids.

[0170] In the present invention, the size of the lipid nanoparticles may be characterized as being 20 to 200 nm, preferably 50 to 150 nm, more preferably 60 to 120 nm, but is not limited thereto.

[0171] In the present invention, the content of the ionized lipid in the lipid nanoparticles may be characterized as being 20 to 70 mol%, preferably 25 to 65 mol%, and more preferably 30 to 60 mol% based on the total lipid, but is not limited thereto.

[0172] If the content of the above ionized lipid is less than 20 mol%, there is a problem of reduced efficacy, and if it exceeds 70 mol%, there is a problem of reduced efficacy and difficulty in forming uniform particles.

[0173] In this specification, "mol% (mol%, mol percent)" indicates the relative molar ratio of a specific component.

[0174] In the present invention, the lipid nanoparticles may be characterized by further comprising phospholipids and / or cholesterol or PEG-bound lipids.

[0175] The content of phospholipid, cholesterol, or PEG-bound lipid in the above lipid nanoparticles may be characterized as 5-20 mol% phospholipid, 20-60 mol% cholesterol, and 0.5-20 mol% PEG-bound lipid, but is not limited thereto.

[0176] In the present invention, the “phospholipid” serves to surround and protect the core formed by the interaction of ionized lipids and a drug within lipid nanoparticles, and binds to the lipid bilayer of target cells to facilitate passage through the cell membrane and endosomal escape during intracellular delivery of the drug.

[0177] The above phospholipids may be used without limitation as long as they are phospholipids capable of promoting the fusion of lipid nanoparticles according to the present invention, for example, DOPE (dioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPC (distearoylphosphatidylcholine), POPC (palmitoyloleoylphosphatidylcholine; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), EPC (egg phosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), DSPE (distearoylphosphatidylethanolamine), PE (phosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), POPE It may be (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]), sphingomyelin, etc.The above phospholipid may preferably be characterized as being one or more selected from the group consisting of DSPC (distearoylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DSPE (distearoylphosphatidylethanolamine), DOPE (dioleoylphosphatidyethanolamine), egg SM (sphingomyelin), and brain SM (sphingomyelin), but is not limited thereto.

[0178] In the present invention, “cholesterol” provides structural robustness to the lipid filling within the lipid nanoparticles and plays a role in improving the stability of the nanoparticles by being dispersed in the core and surface of the nanoparticles.

[0179] In the present invention, lipid nanoparticles may or may not include cholesterol as one of the constituent components of the nanoparticles.

[0180] In this specification, “PEG-bound lipid” refers to a form in which PEG (polyethyleneglycol) and lipid are conjugated, and is used interchangeably with “lipid-PEG,” “PEG-lipid,” or “lipid-PEG,” and means a lipid in which polyethyleneglycol (PEG), a hydrophilic polymer, is attached to one end. The PEG-bound lipid contributes to the serum stability of the nanoparticles within the lipid nanoparticles and prevents aggregation between nanoparticles. Additionally, the PEG-bound lipid protects the nucleic acid from degrading enzymes during in vivo delivery, thereby enhancing the in vivo stability of the nucleic acid and can increase the half-life of the drug encapsulated within the nanoparticles.

[0181] In the above PEG-bound lipid, PEG can be directly conjugated to the lipid or linked to the lipid through a linker moiety. Any linker moiety suitable for binding PEG to the lipid may be used. The linker moiety includes, but is not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), and hydrazone (-C(O)NHN=R-, -C=NNH-), as well as combinations thereof (e.g., a linker containing both a carbamate linker moiety and an amido linker moiety). In addition, esters (-C(O)O-, -OC(O)-), carbonates (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters and combinations thereof are included, but not limited to.

[0182] In the present invention, the PEG may be a single-chain or multi-chain polymer derivative having polyethylene glycol as a basic backbone. One end of the PEG may include a ligand, or not include one, or include a mixture thereof.

[0183] In the present invention, the lipid within the PEG-bound lipid may be used without limitation as long as it is a lipid capable of binding to polyethylene glycol, and phospholipids and / or cholesterol, which are other components of the lipid nanoparticle, may also be used. Specifically, the lipid within the PEG-bound lipid may be dimyristoylglycerol (DMG), ceramide, succinoyl-diacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), or cholesterol, but is not limited thereto. In addition, the above lipid may be a lipid comprising a structure in which two types of lipids selected from C3-C30 single and branched chain forms of alkyl, alkenyl, and alkynyl hydrocarbons are connected to glycerol by a bond of ether (-O-) or ester (-C(O)O-, -OC(O)-).

[0184] In one embodiment of the present invention, the PEG-bound lipid may be characterized as PEG-DMG, but is not limited thereto.

[0185] The PEG in the above PEG-bound lipid is a hydrophilic polymer that has the ability to inhibit the adsorption of plasma proteins, thereby increasing the circulation time of lipid nanoparticles in the body and preventing aggregation between nanoparticles. In addition, the PEG-bound lipid exhibits a stealth function in vivo, which can prevent the degradation of nanoparticles.

[0186] The above PEG may include a functionalized PEG in which a functional group or ligand is attached to the side that is not bound to lipids. The functional groups or ligands available for use in this case are succinyl, carboxylic acid, maleimide, amine group, biotin, cyanur, N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-galactose, D-galactose, N-acetyl-D-glucosamine, N-acetyl-D-glucosamine, D-glucose, D-mannose, F-fucose, carbohydrate derivatives, folate, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CADY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single chain It may be any one or more selected from the group consisting of a single-chain variable fragment (scFv) and an aptamer.

[0187]

[0188] In another aspect, the present invention relates to a method for producing the lipid comprising the following steps:

[0189] (a) R 3 -L 2 -R 5 Halogenates containing -Sterol and R 1 -NH-R 2 -L 1 -R 4 Reacting amine compounds or R 2 -L 1 -R 4 Halogenates containing and R 1 -NH-R 3 -L 2 -R 5- A step of reacting the amine compound of the sterol in a solvent; and

[0190] (b) Step of removing the above solvent.

[0191] Here, R 1 It is selected from the following military units,

[0192]

[0193] R 2 and R 3 C independently 6-12 Alkyl, C 6-12 Alkenyl, C 6-12 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl,

[0194] L 1 and L 2 is independently selected from a group consisting of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=S)O-, -OC(=S)-, -SC(=S)-, -C(=S)S-, -C(=O)NH-, -NHC(=O)-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -C(=O)NHNHC(=O)-, -C(=S)NH-, -NHC(=S)-, -NHC(=S)O-, -NHC(=S)S-, and -NHC(=S)S-.

[0195] R 4 is C 12-30 Alkyl, C 12-30 Alkenyl, C 12-30 Alkinyl, C 12-30 Cycloalkyl, C 12-30 Cycloalkenyl, C 12-30 It is a cycloalkynyl or sterol, and

[0196] Sterol is cholesterol or beta-sitosterol, and

[0197] R 5 is either without a bond or; C 2-12 Alkyl, C2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Cycloalkynyl; or -R 6 Selected from a group composed of C(=O)-,

[0198] R 6 is either without a bond or; C 2-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Selected from cycloalkynyl,

[0199] R 7 C 2-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 2-6 Cycloalkyl, C 2-6 Cycloalkenyl, C 2-6 Selected from cycloalkynyl,

[0200] m is an integer from 1 to 3, and n is an integer satisfying m + n = 3, and

[0201] R 8 and R 9 is independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl,

[0202] Here, the alkyl, alkenyl, or alkynyl is a straight chain or a branched chain.

[0203] In the manufacturing method of the present invention, the R 3 -L 2 -R 5 Halogenates containing -Sterol and R 1 -NH-R 2 -L 1 -R 4The molar ratio of the amine compound may be 1:1.0 to 2.0, and the above R 2 -L 1 -R 4 Halogenates containing and R 1 -NH-R 3 -L 2 -R 5 The molar ratio of the amine compound of the sterol can be 1:0.5 to 1.0.

[0204] In the manufacturing method of the present invention, the reaction of step (a) can be carried out at a temperature of 60 to 90°C for 12 to 72 hours.

[0205] In the manufacturing method of the present invention, a washing or purification process may be additionally included after step (b).

[0206]

[0207] In another aspect, the present invention relates to a drug delivery composition comprising the lipid nanoparticles; and an anionic drug, nucleic acid, or a combination thereof.

[0208] In the present invention, the anionic drug, nucleic acid, or combination thereof may be characterized by being encapsulated inside the lipid nanoparticle.

[0209] In the present invention, the “drug delivery composition” or “drug delivery vehicle” may have a physiologically active substance, such as an anionic drug and / or nucleic acid, encapsulated inside the lipid nanoparticles, and the physiologically active substance, such as anionic drug and / or nucleic acid, is encapsulated stably and with high efficiency, thereby exhibiting an excellent therapeutic effect through the delivery composition. In addition, there is an advantage in that the type of drug encapsulated inside the lipid nanoparticles can be varied according to the therapeutic purpose.

[0210] In this specification, "encapsulation" means encapsulating a delivery substance to efficiently incorporate it into the body.

[0211] In the present invention, the anionic drug may be characterized as being one or more selected from the group consisting of peptides, protein drugs, protein-nucleic acid structures and anionic biopolymer-drug conjugates, but is not limited thereto.

[0212] In the present invention, the nucleic acid may be characterized as being one or more selected from the group consisting of single-stranded siRNA, double-stranded siRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, mRNA, tRNA, lncRNA, piRNA, circRNA, saRNA, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme, but is not limited thereto.

[0213] In one embodiment of the present invention, to confirm the efficacy of lipid nanoparticles according to the present invention as a drug delivery vehicle, the nanoparticles were prepared to include mRNA as a delivery target, and it was confirmed that the mRNA was effectively expressed. It is obvious to those skilled in the art that a drug delivery vehicle comprising various nucleic acids or anionic drugs and lipid nanoparticles can be prepared by the method described in Korean Patent Publication No. 10-2021-0135494 and Korean Patent Application No. 10-2025-0114871 filed by the applicant, and that nucleic acids or anionic drugs can be effectively delivered.

[0214]

[0215] In the present invention, the drug delivery composition can be used as a pharmaceutical composition for treating diseases.

[0216] The above pharmaceutical composition may be administered to mammals, including humans, by various routes including parenteral administration, which may be applied intravenously, subcutaneously, intraperitoneally, or topically, and the dosage may be appropriately selected by a person skilled in the art, although it depends on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time.

[0217] The above pharmaceutical composition is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The above pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above factors, and this can be easily determined by a person skilled in the art.

[0218] Specifically, the effective amount of the compound according to the present invention may vary depending on the patient's age, gender, and weight, and may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the amount may increase or decrease depending on the route of administration, the severity of obesity, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0219]

[0220] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0221]

[0222] Example 1: Synthesis of ionized lipids

[0223] Synthesis of TRC-025

[0224] <Chemical Equation 1>

[0225]

[0226] Bromine compound 2 containing cholesterol (120 mg, 0.16 mmole), K2CO3 (89 mg, 0.64 mmole), and KI (40 mg, 0.24 mmole) were added to a reaction vessel. A solution of amine compound 1 (92 mg, 0.21 mmole) dissolved in acetonitrile (2 mL) and isopropyl ether (2 mL) was added to the reaction vessel at room temperature. After stirring for 40 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Then, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-025 (lipid 1, 25 mg, 30%) in a clear oil state.

[0227] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.86 (m, 1H), 4.05 (t, 2H), 3.75-3.55 (bm, 2H), 3.44 (m, 2H), 3.13 (m, 1H), 2.79-2.53 (bm, 6H), 2.36 (m, 1H), 2.29 (m, 4H), 2.18 (m, 1H), 2.03-1.80 (m, 5H), 1.28-1.40 (m, 25H), 1.40-1.20 (m, 48H), 1.18-0.92 (m, 12H), 0.91-0.85 (m, 15H), 0.68 (s, 3H).

[0228]

[0229] Synthesis of TRC-026

[0230] <Chemical Equation 2>

[0231]

[0232] Bromine compound 3 containing cholesterol (120 mg, 0.16 mmole), K2CO3 (89 mg, 0.64 mmole), and KI (40 mg, 0.24 mmole) were added to a reaction vessel. A solution of amine compound 1 (92 mg, 0.21 mmole) dissolved in acetonitrile (2 mL) and isopropyl ether (2 mL) was added to the reaction vessel at room temperature. After stirring for 20 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Then, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-026 (lipid 2, 113 mg, 46%) in a clear oil state.

[0233] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.86 (m, 1H), 4.05(t, 2H), 3.75-3.55 (bm, 2H), 3.44 (m, 2H), 3.13 (m, 1H), 3.02-2.75 (bm, 6H), 2.34 (m, 1H), 2.28 (m, 4H), 2.18 (m, 1H), 2.03-1.80 (m, 5H), 1.65-1.40 (m, 25H), 1.40-1.20 (m, 52H), 1.18-0.92 (m, 12H), 0.91-0.85 (m, 15H), 0.68(s, 3H)

[0234]

[0235] Synthesis of TRC-030

[0236] <Chemical Equation 3>

[0237]

[0238] Bromine compound 4 containing cholesterol (70 mg, 0.09 mmole), K2CO3 (51 mg, 0.37 mmole), and KI (23 mg, 0.14 mmole) were added to a reaction vessel. A solution of amine compound 1 (53 mg, 0.12 mmole) dissolved in acetonitrile (1 mL) and isopropyl ether (1 mL) was added to the reaction vessel at room temperature. After stirring at 75 °C for 44 hours, the solvent was removed by vacuum distillation of the reaction mixture. Then, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-030 (lipid 3, 25 mg, 30%) in the form of a white powder.

[0239] 1H NMR (CDCl3, 500 MHz): δ5.37 (m, 1H), 4.86 (m, 1H), 4.61 (m, 1H), 4.04 (t, 2H), 3.60-3.52 (bm, 2H), 2.66-2.60 (bm, 2H), 2.55-2.47 (bm, 4H), 2.31-2.24 (m, 8H), 2.03-2.24 (m, 3H), 1.87-1.80 (m, 3H), 1.65-1.40 (m, 22H), 1.40-1.20 (m, 52H), 1.18-0.92 (m, 12H), 0.91-0.85 (m, 15H), 0.67 (s, 1H).

[0240]

[0241] Synthesis of TRC-010

[0242] <Chemical Equation 4>

[0243]

[0244] A cholesterol-containing amine compound 6 (57 mg, 0.09 mmole), K2CO3 (40 mg, 0.29 mmole), and KI (18 mg, 0.11 mmole) were added to a reaction vessel. A solution of bromine compound 5 (25 mg, 0.07 mmole) dissolved in acetonitrile (1 mL) and isopropyl ether (1 mL) was added to the reaction vessel at room temperature. After stirring for 46 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Then, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-010 (lipid 4, 12 mg, 15%) as a transparent oil.

[0245] 1H NMR (CDCl3, 500 MHz): δ5.37 (m, 1H), 4.61 (m, 1H), 4.04 (t, 2H), 2.72-2.64 (bm, 2H), 2.64-2.54 (bm, 4H), 2.50-2.42 (bm, 2H), 2.35-2.25 (m, 12H), 2.05-1.90 (m, 4H), 1.88-1.78 (m, 4H), 1.66-1.40 (m, 18H), 1.40-1.20 (m, 33H), 1.18-0.92 (m, 6H), 0.91-0.85 (12H), 0.67 (s, 3H)

[0246]

[0247] Synthesis of TRC-111

[0248] <Chemical Equation 5>

[0249]

[0250] A amine compound containing cholesterol 7 (77 mg, 0.12 mmole), K2CO3 (48 mg, 0.34 mmole), and KI (21 mg, 0.13 mmole) were added to a reaction vessel. A solution of bromine compound 5 (30 mg, 0.09 mmole) dissolved in acetonitrile (1 mL) and isopropyl ether (1 mL) was added to the reaction vessel at room temperature. After stirring at 75 °C for 45 h, the solvent was removed by vacuum distillation of the reaction mixture. Then, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-111 (lipid 5, 28 mg, 39%) in a clear oil state.

[0251] 1H NMR (CDCl3, 500 MHz): δ5.37 (m, 1H), 4.61 (m, 1H), 4.04 (t, 2H), 3.05-2.95 (bm, 4H), 2.92-2.84 (bm, 2H), 2.59 (t, 2H), 2.49 (m, 4H), 2.32-2.25 (m, 6H), 2.05-1.90 (m, 8H), 1.88-1.78 (m, 4H), 1.65-1.40 (m, 18H), 1.40-1.20 (m, 33H), 1.18-0.92 (m, 6H), 0.91-0.85 (m, 12H), 0.67 (s, 3H)

[0252]

[0253] Synthesis of TRC-114

[0254] Chemical Equation 6

[0255]

[0256] Amine compound 8 (43 mg, 0.11 mmole), K2CO3 (40 mg, 0.29 mmole), and KI (18 mg, 0.11 mmole) were added to a reaction vessel. A solution of cholesterol-containing bromine compound 9 (50 mg, 0.07 mmole) dissolved in acetonitrile (1 mL) and isopropyl ether (1 mL) was added to the reaction vessel at room temperature. After stirring at 75 °C for 44 hours, the solvent was removed by vacuum distillation of the reaction mixture. Then, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-114 (lipid 6, 28 mg, 39%) in a clear oil state.

[0257] 1H NMR (CDCl3, 500 MHz): δ5.33 (m, 1H), 4.04 (t, 4H), 3.44 (m, 2H), 3.12 (m, 1H), 3.05-2.95 (bm, 3H), 2.90-2.80 (bm, 2H), 2.57 (t, 2H), 2.47 (m, 4H), 2.35 (m, 1H), 2.29 (dd, 4H), 2.16 (m, 1H), 2.05-1.90 (m, 8H), 1.88-1.78 (m, 4H), 1.65-1.40 (m, 22H), 1.40-1.20 (m, 36H), 1.18-0.92 (m, 8H), 0.91-0.85 (m, 12H), 0.67 (s, 3H).

[0258]

[0259] Synthesis of TRC-121

[0260] Chemical Equation 7

[0261]

[0262] Amine compound 10 (63 mg, 0.18 mmole), K2CO3 (65 mg, 0.47 mmole), and KI (29 mg, 0.18 mmole) were added to a reaction vessel. A solution of cholesterol-containing bromine compound 11 (70 mg, 0.12 mmole) dissolved in acetonitrile (1.3 mL) and isopropyl ether (1.3 mL) was added to the reaction vessel at room temperature. After stirring for 44 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-121 (lipid 7, 51 mg, 50%) in a clear oil state.

[0263] 1H NMR (CDCl3, 500 MHz): δ5.37 (m, 1H), 4.61 (m, 1H), 4.05 (t, 2H), 3.57 (m, 2H), 2.62-2.24 (b, 6H), 2.32-2.25 (bm, 6H), 2.05-1.93 (m, 2H), 1.87-1.68 (m, 3H), 1.87-1.72 (m, 3H). 1.72-1.40 (m, 22H), 1.40-1.20 (m, 29H), 1.18-0.92 (m, 12H), 0.91-0.85 (m, 12H), 0.67 (s, 3H).

[0264]

[0265] Synthesis of TRC-124

[0266] Chemical Equation 8

[0267]

[0268] Amine compound 10 (60 mg, 0.15 mmole), K2CO3 (55 mg, 0.41 mmole), and KI (25 mg, 0.15 mmole) were added to a reaction vessel. A solution of cholesterol-containing bromine compound 9 (70 mg, 0.12 mmole) dissolved in acetonitrile (1.1 mL) and isopropyl ether (1.1 mL) was added to the reaction vessel at room temperature. After stirring at 75 °C for 44 hours, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-124 (lipid 8, 36 mg, 36%) in a clear oil state.

[0269] 1H NMR (CDCl3, 500 MHz): δ5.36 (m, 1H), 4.05 (t, 2H), 3.59-3.51 (m, 2H), 3.45 (m, 2H), 3.12 (m, 1H), 2.62-2.38 (m, 6H), 2.36 (m, 1H), 2.32-2.25 (m, 4H), 2.16 (m, 1H), 2.05-1.93 (m, 2H), 1.87-1.72 (m, 3H), 1.72-1.40 (m, 26H), 1.40-1.20 (m, 35H), 1.18-0.92 (m, 12H), 0.91-0.85 (m, 12H), 0.67 (s, 3H).

[0270]

[0271] Synthesis of TRC-131

[0272] <Chemical Equation 9>

[0273]

[0274] Bromine compound 11 containing cholesterol (121 mg, 0.205 mmole), K2CO3 (91 mg, 0.66 mmole), and KI (41 mg, 0.25 mmole) were added to a reaction vessel. A solution of ethanolamine (5.0 mg, 0.08 mmole) dissolved in acetonitrile (2.7 mL) and isopropyl ether (2.7 mL) was added to the reaction vessel at room temperature. After stirring for 46 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-131 (lipid 9, 36 mg, 36%) in a clear oil state.

[0275] 1H NMR (CDCl3, 500 MHz): δ5.36 (m, 2H), 4.61 (m, 2H), 3.63-3.56 (bm, 2H), 2.70-2.62 (bm, 2H), 2.58-2.49 (bm, 4H), 2.30 (m, 4H), 2.27 (t, 4H), 2.05-1.93 (m, 7H), 1.87-1.72 (m, 6H), 1.62-1.40 (m, 21H), 1.40-1.20 (m, 24H), 1.18-0.92 (m, 21H), 0.92 (s, 3H), 0.90 (s, 3H), 0.86 (m, 12H), 0.67 (s, 6H).

[0276]

[0277] Synthesis of TRC-146

[0278] <Chemical Equation 10>

[0279]

[0280] Bromine compound 3 containing cholesterol (50 mg, 0.07 mmole), K2CO3 (37 mg, 0.27 mmole), and KI (17 mg, 0.10 mmole) were added to a reaction vessel. A solution of amine compound 12 (41 mg, 0.10 mmole) dissolved in acetonitrile (1.0 mL) and isopropyl ether (1.0 mL) was added to the reaction vessel at room temperature. After stirring for 46 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-146 (lipid 10, 54 mg, 71%) in a clear oil state.

[0281] 1H NMR (CDCl3, 500 MHz): δ5.36 (m, 1H), 4.86 (m, 1H), 4.05 (t, 2H), 3.59-3.52 (bm, 2H), 3.45 (m, 2H), 3.12 (m, 1H), 2.67-2.38 (bm, 6H), 2.36 (m, 1H), 2.32-2.25 (m, 4H), 2.18 (m, 1H), 2.05-1.93 (m, 2H), 1.87-1.72 (m, 3H), 1.72-1.40 (m, 26H), 1.40-1.20 (m, 54H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.67 (s, 3H).

[0282]

[0283] Synthesis of TRC-156

[0284] Chemical Equation 11

[0285]

[0286] Bromine compound 3 containing cholesterol (50 mg, 0.07 mmole), K2CO3 (37 mg, 0.27 mmole), and KI (17 mg, 0.10 mmole) were added to a reaction vessel. A solution of amine compound 13 (51 mg, 0.10 mmole) dissolved in acetonitrile (1.0 mL) and isopropyl ether (1.0 mL) was added to the reaction vessel at room temperature. After stirring for 46 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-156 (lipid 11, 24 mg, 30%) in oil form.

[0287] 1H NMR (CDCl3, 500 MHz): δ5.36 (m, 1H), 4.86 (m, 1H), 4.05 (t, 2H), 3.45 (m, 2H), 3.12 (m, 1H), 3.02-2.92 (bm, 4H), 2.90-2.82 (bm, 2H), 2.64 (m, 2H), 2.53 (m, 4H), 2.36 (m, 1H), 2.32-2.25 (m, 4H), 2.18 (m, 1H), 2.05-1.93 (m, 10H), 1.91-1.72 (m, 3H), 1.65-1.40 (m, 22H), 1.40-1.20 (m, 56H), 1.18-0.92 (m, 8H), 0.91-0.85 (m, 15H), 0.67 (s, 3H)

[0288]

[0289] Synthesis of TRC-166

[0290] <Chemical Equation 12>

[0291]

[0292] Bromine compound 3 containing cholesterol (50 mg, 0.07 mmole), K2CO3 (37 mg, 0.27 mmole), and KI (17 mg, 0.10 mmole) were added to a reaction vessel. A solution of amine compound 14 (50 mg, 0.07 mmole) dissolved in acetonitrile (1.0 mL) and isopropyl ether (1.0 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-166 (lipid 12, 52 mg, 65%) in oil form.

[0293] 1 H NMR (CDCl3, 500 MHz): δ5.33 (m, 1H), 4.86 (m, 1H), 4.04 (t, 2H), 3.44 (m, 2H), 3.12 (m, 1H), 2.70-2.40 (bm, 12H), 2.36 (m, 1H), 2.32-2.25 (m, 4H), 2.18 (m, 1H), 2.05-1.93 (m, 3H), 1.91-1.75 (m, 5H), 1.75-1.65 (bm, 4H), 1.65-1.40 (m, 22H), 1.40-1.20 (m, 56H), 1.18-0.92 (m, 11H), 0.91-0.85 (15H), 0.67 (s, 3H).

[0294]

[0295] Synthesis of TRC-021

[0296] <Chemical Equation 13>

[0297]

[0298] Bromine compound 11 (130 mg, 0.22 mmole) containing cholesterol and DIPEA (34 mg, 0.27 mmole) were added to a reaction vessel. A solution of amine compound 1 (98 mg, 0.07 mmole) dissolved in acetonitrile (11.0 mL) was added to the reactor at room temperature. After stirring for 18 hours at 75 °C, the reaction mixture was vacuum distilled to remove the solvent. Subsequently, DCM (20 mL) was added, the mixture was transferred to a separatory funnel, and washed with water (20 mL) and a saturated aqueous solution of NaCl (20 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered filtrate was vacuum distilled to remove the solvent. The mixture was then purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-021 (59 mg, 28%) in oil form.

[0299] 1H NMR (CDCl3, 500 MHz): δ5.36 (m, 1H), 4.86 (m, 1H), 4.61 (m, 1H), 3.90-3.65 (bm, 2H), 2.95-2.55 (bm, 6H), 2.32-2.25 (m, 6H), 2.05-1.93 (m, 2H), 1.91-1.75 (m, 3H), 1.75-1.40 (m, 18H), 1.40-1.20 (m, 42H), 1.18-0.92 (m, 13H), 0.91-0.85 (15H), 0.67 (s, 3H).

[0300]

[0301] Synthesis of TRC-022

[0302] <Chemical Equation 14>

[0303]

[0304] Bromine compound 15 (155 mg, 0.244 mmole) containing cholesterol and DIPEA (51 mg, 0.29 mmole) were added to a reaction vessel. A solution of amine compound 1 (100 mg, 0.244 mmole) dissolved in acetonitrile (24.0 mL) was added to the reactor at room temperature. After stirring for 20 hours at 75 °C, the reaction mixture was vacuum distilled to remove the solvent. Subsequently, DCM (20 mL) was added, the mixture was transferred to a separatory funnel, and washed with water (20 mL) and a saturated aqueous solution of NaCl (20 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered filtrate was vacuum distilled to remove the solvent. The mixture was then purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-022 (96 mg, 40%) in oil form.

[0305] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.86 (m, 1H), 4.2 (t, 2H), 3.90-3.70 (bm, 2H), 3.67 (m, 2H), 3.18 (m, 1H), 3.05-2.55 (bm, 6H), 2.32-2.15 (m, 6H), 2.05-1.93 (m, 2H), 1.91-1.75 (m, 3H), 1.70-1.40 (m, 18H), 1.40-1.20 (m, 42H), 1.18-0.92 (m, 13H), 0.91-0.85 (15H), 0.67 (s, 3H).

[0306]

[0307]

[0308] Synthesis of TRC-024

[0309] <Chemical Equation 15>

[0310]

[0311] Bromine compound 9 containing cholesterol (200 mg, 0.29 mmole) and DIPEA (44 mg, 0.35 mmole) were added to a reaction vessel. A solution of amine compound 1 (150 mg, 0.35 mmole) dissolved in acetonitrile (30 mL) was added to the reactor at room temperature. After stirring at 75 °C for 20 h, the reaction mixture was vacuum distilled to remove the solvent. Subsequently, DCM (20 mL) was added, the mixture was transferred to a separatory funnel, and washed with water (20 mL) and a saturated aqueous solution of NaCl (20 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered filtrate was vacuum distilled to remove the solvent. The mixture was then purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-024 (170 mg, 56%) in oil form.

[0312] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.86 (m, 1H), 4.05 (t, 2H), 3.90-3.80 (bm, 2H), 3.46 (m, 2H), 3.13 (m, 1H), 3.08-2.80 (bm, 6H), 2.34 (m, 1H), 2.32-2.15 (m, 4H), 2.18 (m, 1H), 2.05-1.93 (m, 2H), 1.91-1.75 (m, 3H), 1.70-1.40 (m, 22H), 1.40-1.20 (m, 46H), 1.18-0.92 (m, 13H), 0.91-0.85 (15H), 0.67 (s, 3H).

[0313]

[0314] Synthesis of TRC-027

[0315] <Chemical Equation 16>

[0316]

[0317] Bromine compound 17 containing cholesterol (30 mg, 0.04 mmole), K2CO3 (21 mg, 0.16 mmole), and KI (10 mg, 0.106 mmole) were added to a reaction vessel. A solution of amine compound 1 (50 mg, 0.07 mmole) dissolved in acetonitrile (1.0 mL) and isopropyl ether (1.0 mL) was added to the reaction vessel at room temperature. After stirring for 45 h at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Then, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-166 (29 mg, 65%) in oil form.

[0318] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.86 (m, 1H), 4.05 (t, 2H), 3.75-3.55 (bm, 2H), 3.44 (m, 2H), 3.13 (m, 1H), 3.02-2.75 (bm, 6H), 2.34 (m, 1H), 2.28 (m, 4H), 2.18 (m, 1H), 2.03-1.80 (m, 7H), 1.65-1.40 (m, 25H), 1.40-1.20 (m, 54H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.67 (s, 3H).

[0319]

[0320] Synthesis of TRC-036

[0321] <Chemical Equation 17>

[0322]

[0323] Bromine compound 3 containing cholesterol (100 mg, 0.13 mmole), K2CO3 (74 mg, 0.54 mmole), and KI (33 mg, 0.20 mmole) were added to a reaction vessel. A solution of amine compound 18 (59 mg, 0.13 mmole) dissolved in acetonitrile (1.5 mL) and isopropyl ether (1.5 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-166 (29 mg, 65%) in oil form.

[0324] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.81 (m, 1H), 4.05 (t, 2H), 3.70-3.55 (bm, 2H), 3.44 (m, 2H), 3.12 (m, 1H), 2.64 (bm, 2H), 2.51 (bm, 4H), 2.34 (m, 1H), 2.28 (m, 4H), 2.18 (m, 1H), 2.03-1.92 (m, 2H), 1.90-1.78 (m, 3H), 1.65-1.40 (m, 25H), 1.40-1.20 (m, 40H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.68 (s, 3H).

[0325]

[0326] Synthesis of TRC-104

[0327] <Chemical Equation 18>

[0328]

[0329] Bromine compound 5 containing cholesterol (20 mg, 0.06 mmole), K2CO3 (32 mg, 0.23 mmole), and KI (14 mg, 0.09 mmole) were added to a reaction vessel. A solution of amine compound 19 (57 mg, 0.080 mmole) dissolved in acetonitrile (0.7 mL) and isopropyl ether (0.7 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-166 (5.0 mg, 9%) in oil form.

[0330] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.88 (bm, 2H), 3.44 (m, 2H), 3.12 (m, 1H), 2.90 (m, 2H), 2.78 (bm, 4H), 2.62 (bm, 2H), 2.40 (s, 6H), 2.38-2.05 (m, 12H), 2.05-1.90 (m, 8H), 1.90-1.78 (m, 4H), 1.70-1.40 (m, 20H), 1.40-1.20 (m, 30H), 1.18-0.92 (m, 5H), 0.91-0.85 (14H), 0.68 (s, 3H).

[0331]

[0332] Synthesis of TRC-186

[0333] <Chemical Equation 19>

[0334]

[0335] Bromine compound 3 containing cholesterol (53 mg, 0.070 mmole), K2CO3 (39 mg, 0.28 mmole), and KI (18 mg, 0.11 mmole) were added to a reaction vessel. A solution of amine compound 20 (57 mg, 0.080 mmole) dissolved in acetonitrile (0.7 mL) and isopropyl ether (0.7 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-186 (5.0 mg, 9%) in oil form.

[0336] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.86 (m, 1H), 4.05 (t, 2H), 3.61 (d, 4H), 3.44 (m, 2H), 3.12 (m, 1H), 2.99 (m, 1H), 2.59 (m, 4H), 2.36 (m, 1H), 2.28 (m, 4H), 2.18 (m, 1H), 2.03-1.92 (m, 2H), 1.90-1.78 (m, 3H), 1.65-1.40 (m, 28H), 1.40-1.20 (m, 52H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.68 (s, 3H).

[0337]

[0338] Synthesis of TRC-196

[0339] <Chemical Equation 20>

[0340]

[0341] Bromine compound 3 containing cholesterol (73 mg, 0.097 mmole), K2CO3 (54 mg, 0.39 mmole), and KI (24 mg, 0.15 mmole) were added to a reaction vessel. A solution of amine compound 21 (99 mg, 0.19 mmole) dissolved in acetonitrile (1.1 mL) and isopropyl ether (1.1 mL) was added to the reaction vessel at room temperature. After stirring for 45 h at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-196 (107 mg, 94%) in oil form.

[0342] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.86 (m, 1H), 4.05 (t, 2H), 3.70 (t, 4H), 3.44 (m, 2H), 3.12 (m, 1H), 2.70-2.32 (bm, 12H), 2.36 (m, 1H), 2.28 (m, 4H), 2.18 (m, 1H), 2.03-1.92 (m, 2H), 1.90-1.78 (m, 3H), 1.65-1.40 (m, 26H), 1.40-1.20 (m, 50H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.68 (s, 3H).

[0343]

[0344] Synthesis of TRC-206

[0345] <Chemical Equation 21>

[0346]

[0347] Bromine compound 3 containing cholesterol (50 mg, 0.067 mmole), K2CO3 (37 mg, 0.27 mmole), and KI (16 mg, 0.10 mmole) were added to a reaction vessel. A solution of amine compound 22 (46 mg, 0.087 mmole) dissolved in acetonitrile (1.0 mL) and isopropyl ether (1.0 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-206 (37 mg, 50%) in oil form.

[0348] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.86 (m, 1H), 4.05 (t, 2H), 3.80-3.50 (m, 12H), 3.44 (m, 2H), 3.12 (m, 1H), 2.70-2.32 (bm, 4H), 2.36 (m, 1H), 2.28 (m, 4H), 2.18 (m, 1H), 2.05-1.65 (m, 9H), 1.70-1.40 (m, 25H), 1.40-1.20 (m, 48H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.67 (s, 3H).

[0349]

[0350] Synthesis of TRC-216

[0351] <Chemical Equation 22>

[0352]

[0353] Bromine compound 23 containing beta-sitosterol (71 mg, 0.091 mmole), K2CO3 (50 mg, 0.37 mmole), and KI (23 mg, 0.14 mmole) were added to a reaction vessel. A solution of amine compound 1 (52 mg, 0.12 mmole) dissolved in acetonitrile (1.0 mL) and isopropyl ether (1.0 mL) was added to the reaction vessel at room temperature. After stirring for 45 h at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-206 (88 mg, 85%) in oil form.

[0354] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.86 (m, 1H), 4.05 (t, 2H), 3.75-3.55 (m, 2H), 3.44 (m, 2H), 3.12 (m, 1H), 2.85-2.35 (bm, 6H), 2.34 (m, 1H), 2.28 (m, 4H), 2.18 (m, 1H), 2.03-1.80 (m, 5H), 1.65-1.40 (m, 24H), 1.40-1.20 (m, 54H), 1.18-0.92 (m, 12H), 0.91-0.85 (18H), 0.68 (s, 3H).

[0355]

[0356] Synthesis of TRC-221

[0357] <Chemical Equation 23>

[0358]

[0359] Bromine compound 11 containing cholesterol (90 mg, 0.15 mmole), K2CO3 (37 mg, 0.27 mmole), and KI (16 mg, 0.10 mmole) were added to a reaction vessel. A solution of serinol (280 mg, 0.087 mmole) dissolved in acetonitrile (1.5 mL) and isopropyl ether (1.5 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-221 (24 mg, 25%) in oil form.

[0360] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 2H), 4.86 (m, 2H), 3.63 (d, 4H), 3.02 (m, 1H), 2.62 (m, 4H), 2.30 (m, 1H), 2.26 (m, 4H), 1.99 (m, 4H), 1.85 (m, 6H), 1.65-1.40 (m, 24H), 1.40-1.20 (m, 24H), 1.18-0.92 (m, 24H), 0.91-0.85 (18H), 0.68 (s, 6H).

[0361]

[0362] Synthesis of TRC-226

[0363] <Chemical Equation 24>

[0364]

[0365] Bromine compound 3 containing cholesterol (120 mg, 0.16 mmole), K2CO3 (89 mg, 0.64 mmole), and KI (40 mg, 0.24 mmole) were added to a reaction vessel. A solution of amine compound 24 (170 mg, 0.23 mmole) dissolved in acetonitrile (2 mL) and isopropyl ether (2 mL) was added to the reaction vessel at room temperature. After stirring at 75 °C for 45 h, the solvent was removed by vacuum distillation of the reaction mixture. Then, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-226 (24 mg, 25%) in oil form.

[0366] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 2H), 4.04 (t, 4H), 3.64 (d, 4H), 3.43 (m, 4H), 3.11 (m, 2H), 3.03 (m, 1H), 2.64 (m, 4H), 2.36 (m, 2H), 2.28 (t, 4H), 2.18 (m, 2H), 1.99 (m, 4H), 1.85 (m, 6H), 1.65-1.40 (m, 30H), 1.40-1.20 (m, 48H), 1.18-0.92 (m, 24H), 0.91-0.85 (18H), 0.68 (s, 6H).

[0367]

[0368] Synthesis of TRC-236

[0369] <Chemical Equation 25>

[0370]

[0371] Bromine compound 3 containing cholesterol (60 mg, 0.08 mmole), K2CO3 (44 mg, 0.32 mmole), and KI (20 mg, 0.12 mmole) were added to a reaction vessel. A solution of amine compound 16 (138 mg, 0.096 mmole) dissolved in acetonitrile (1.0 mL) and isopropyl ether (1.0 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-236 (63 mg, 73%) in oil form.

[0372] 1H NMR (CDCl3, 500 MHz): δ5.33 (m, 1H), 4.05 (m, 4H), 3.75-3.55 (bm, 2H), 3.44 (m, 2H), 3.12 (m, 1H), 2.80-2.35 (bm, 6H), 2.34 (m, 1H), 2.28 (m, 3H), 2.18 (m, 1H), 2.03-1.80 (m, 5H), 1.65-1.40 (m, 23H), 1.40-1.20 (m, 48H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.68 (s, 3H).

[0373]

[0374] Synthesis of TRC-246

[0375] <Chemical Equation 26>

[0376]

[0377] Bromine compound 3 containing cholesterol (60 mg, 0.08 mmole), K2CO3 (44 mg, 0.32 mmole), and KI (20 mg, 0.12 mmole) were added to a reaction vessel. A solution of amine compound 25 (40 mg, 0.096 mmole) dissolved in acetonitrile (1.0 mL) and isopropyl ether (1.0 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-246 (59 mg, 62%) in oil form.

[0378] 1H NMR (CDCl3, 500 MHz): δ5.33 (m, 1H), 4.05 (m, 4H), 3.80 (m, 2H), 3.44 (m, 2H), 3.12 (m, 1H), 2.80-2.35 (bm, 6H), 2.34 (m, 1H), 2.28 (m, 3H), 2.18 (m, 1H), 2.03-1.80 (m, 5H), 1.65-1.40 (m, 23H), 1.40-1.20 (m, 50H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.68 (s, 3H).

[0379]

[0380] Synthesis of TRC-256

[0381] <Chemical Equation 27>

[0382]

[0383] Bromine compound 3 containing cholesterol (60 mg, 0.08 mmole), K2CO3 (44 mg, 0.32 mmole), and KI (20 mg, 0.12 mmole) were added to a reaction vessel. A solution of amine compound 25 (40 mg, 0.096 mmole) dissolved in acetonitrile (1.0 mL) and isopropyl ether (1.0 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (10 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (10 mL) and a saturated aqueous solution of NaCl (10 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-256 (46 mg, 48%) in oil form.

[0384] 1H NMR (CDCl3, 500 MHz): δ5.33 (m, 1H), 4.05 (m, 4H), 3.59 (bm, 2H), 3.44 (m, 2H), 3.12 (m, 1H), 2.80-2.35 (bm, 6H), 2.34 (m, 1H), 2.28 (m, 3H), 2.18 (m, 1H), 2.03-1.80 (m, 5H), 1.65-1.40 (m, 23H), 1.40-1.20 (m, 52H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.68 (s, 3H).

[0385]

[0386] Synthesis of TRC-239

[0387] <Chemical Equation 28>

[0388]

[0389] Cholesterol-containing sulfonic acid compound 28 (21 mg, 0.026 mmole), K2CO3 (14 mg, 0.10 mmole), and KI (6.5 mg, 0.039 mmole) were added to a reaction vessel. A solution of amine compound 27 (27 mg, 0.052 mmole) dissolved in acetonitrile (0.3 mL) and isopropyl ether (0.3 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (5 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (5 mL) and a saturated aqueous solution of NaCl (5 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-239 (17 mg, 64%) in oil form.

[0390] 1H NMR (CDCl3, 500 MHz): δ5.34 (m, 1H), 4.15 (bm, 1H), 4.05 (m, 4H), 3.78 (bm, 1H), 3.44 (m, 2H), 3.12 (m, 1H), 3.05-2.38 (bm, 6H), 2.37-2.10 (m, 5H), 2.08-1.75 (m, 8H), 1.65-1.40 (m, 20H), 1.40-1.20 (m, 44H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.67 (s, 3H).

[0391]

[0392] Synthesis of TRC-240

[0393] <Chemical Equation 29>

[0394]

[0395] Cholesterol-containing sulfonic acid compound 29 (30 mg, 0.036 mmole), K2CO3 (20 mg, 0.14 mmole), and KI (8.9 mg, 0.054 mmole) were added to a reaction vessel. A solution of amine compound 27 (17 mg, 0.043 mmole) dissolved in acetonitrile (0.5 mL) and isopropyl ether (0.5 mL) was added to the reaction vessel at room temperature. After stirring for 45 hours at 75 °C, the solvent was removed by vacuum distillation of the reaction mixture. Subsequently, DCM (5 mL) was added, the mixture was transferred to a separatory funnel, and washed with an aqueous solution of Na2S2O3 (5 mL) and a saturated aqueous solution of NaCl (5 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, DCM / MeOH) to obtain TRC-240 (13 mg, 34%) in oil form.

[0396] 1H NMR (CDCl3, 500 MHz): δ5.33 (m, 1H), 4.04 (m, 4H), 3.63 (bm, 2H), 3.44 (m, 2H), 3.12 (m, 1H), 2.75-2.35 (bm, 6H), 2.35-2.05 (m, 5H), 2.03-1.75 (m, 8H), 1.65-1.40 (m, 22H), 1.40-1.20 (m, 46H), 1.18-0.92 (m, 12H), 0.91-0.85 (15H), 0.67 (s, 3H).

[0397]

[0398] Example 2: Preparation of lipid nanoparticles

[0399] Two solutions were prepared to prepare lipid nanoparticles. One was an organic solution containing lipid components, prepared by dissolving the lipid components constituting the lipid nanoparticles in ethanol. In preparing lipid nanoparticles containing ionized lipids synthesized in Example 1, an ethanol solution was prepared with a total concentration of 4.5 to 6.5 mg / mL while maintaining a molar ratio of 50:10:38.5:1 for ionized lipids, DSPC (distearoylphosphatidylcholine), cholesterol, and PEG-DMG (1,2-dimyristoyl-3-PEG-glycerol), respectively.

[0400] Meanwhile, mRNA was prepared at a concentration of 0.07 mg / mL in an acetic acid buffer solution at pH 5. Lipid nanoparticles were prepared by mixing an ethanol solution and an aqueous buffer solution in a volume ratio of 1:3 using a micromixer chip, with the flow rate of the mixed solution set to 4 mL / min. The mixture was collected and dialyzed for 24 hours in a pH 7.5 PBS buffer solution. The particle size and poly dispersity index (PDI) of the prepared lipid nanoparticles were measured using a Dynamic Light Scattering (DLS) device (Table 1). It was confirmed that the size of the prepared lipid nanoparticles ranged between 70 nm and 130 nm, depending on the type of ionized lipid used.

[0401]

[0402] Example 3: Confirmation of RNA Encapsulation Rate

[0403] To verify the RNA encapsulation rate of lipid nanoparticles prepared using ionized lipids according to the present invention, the Quant-it RiboGreen RNA assay kit (Thermo Fisher Scientific) was used. Standard solutions of various RNAs were prepared in 1X TE buffer solution to fall within the range of 0 to 0.1 μM. Two types of lipid nanoparticle samples were prepared by diluting the lipid nanoparticle solutions so that the concentration of RNA contained in the lipid nanoparticles fell within the range of the RNA standard solutions. One of the lipid nanoparticle samples was diluted in 1X TE buffer solution, and the other was diluted in 2% TritonX-100 and 1X TE buffer solution. After mixing the RiboGreen reagent with the RNA standard solutions and the two types of lipid nanoparticle samples according to the method described in the RiboGreen Assay kit manual, fluorescence was measured at 480 nm for excitation and 520 nm for emission. The respective concentrations can be determined by substituting the fluorescence values ​​measured from the lipid nanoparticle samples into the calibration curve of the fluorescence values ​​measured from the RNA standard solutions. The RNA concentration measured in lipid nanoparticle samples containing TritonX-100 is the total RNA concentration (C_tot), and the RNA concentration measured in samples not containing TritonX-100 is the RNA concentration not encapsulated in the lipid nanoparticles (C_out). Therefore, the proportion of RNA encapsulated in the nanoparticles can be calculated as follows.

[0404] [Mathematical Formula 1]

[0405] Encapsulation Rate (%) = { 1 - C_out / C_tot} x 100

[0406] In the above formula

[0407] C_tot: Total concentration of RNA

[0408] C_out: Concentration of RNA not encapsulated in lipid nanoparticles.

[0409]

[0410] The RNA encapsulation rate calculated according to Equation 1 is as shown in Table 2.

[0411]

[0412] Example 4: Measurement of in vitro efficacy of lipid nanoparticles

[0413] To measure the in vitro efficacy of lipid nanoparticles according to the present invention, lipid nanoparticles containing each ionized lipid were prepared by the method described in Example 2. Each lipid nanoparticle was prepared to contain the same amount of mRNA expressing luciferase. Luciferase activity was measured after treating HepG2 and HEK293 cell lines with lipid nanoparticles at mRNA concentrations of 6.25, 12.5, 25, and 50 ng for 24 hours (Fig. 1). Measurements at each concentration were repeated three times, and the average value was calculated and expressed as a relative value. As a result of the measurement, it was confirmed that luciferase activity was measured in proportion to the RNA concentration treated in the cells.

[0414]

[0415] Example 5: Preparation of lipid nanoparticles not containing cholesterol as a component

[0416] The lipid portion of the ionized lipid according to the present invention has a structure containing cholesterol rather than a conventional alkyl chain. Therefore, it is possible to manufacture lipid nanoparticles without including cholesterol as a constituent among the four common lipid components that constitute lipid nanoparticles: ionized lipid, cholesterol, phospholipid, and PEG-lipid. To verify this, lipid nanoparticles were manufactured by excluding only the cholesterol component, in the same manner as described in Example 2. In Example 2, ionized lipid, DSPC, cholesterol, and PEG-DMG were included in a molar ratio of 50:10:38.5:1, respectively; however, in this example, a lipid-ethanol solution was prepared with ionized lipid, DSPC, cholesterol, and PEG-DMG in a molar ratio of 50:10:0:1, respectively, and used for the manufacture of lipid nanoparticles. All other conditions were carried out in the same manner as in Example 2. The results of measuring the size and dispersion of the manufactured lipid nanoparticles are as follows (Table 3). Through this example, it was confirmed that lipid nanoparticles not containing cholesterol can be manufactured using the ionized lipid of the present invention.

[0417]

[0418] Example 6: Confirmation of RNA encapsulation rate of lipid nanoparticles not containing cholesterol as a component

[0419] To confirm the RNA encapsulation rate of the lipid nanoparticles prepared in Example 5, the experiment was conducted in the same manner as described in Example 3. The measured RNA encapsulation rates are as follows (Table 4).

[0420]

[0421] Example 7: Measurement of in vitro efficacy of cholesterol-free lipid nanoparticles

[0422] To evaluate the in vitro efficacy of the lipid nanoparticles prepared in Example 5, lipid nanoparticles containing each ionized lipid were measured using the method described in Example 4. Luciferase activity was measured after treating HepG2 cell lines with lipid nanoparticles at mRNA concentrations of 6.25, 12.5, 25, and 50 ng for 24 hours (Fig. 2). Measurements at each concentration were repeated three times, and the average value was calculated and expressed as a relative value. It was confirmed that luciferase activity was measured in proportion to RNA concentration.

[0423]

[0424] Example 8: Measurement of in vitro efficacy of lipid nanoparticles

[0425] To prepare lipid nanoparticles containing the ionized lipids synthesized in Example 1, lipid nanoparticles were prepared using the method described in Example 2. The basic physical properties of the prepared nanoparticles, namely particle size, PDI, and RNA encapsulation rate, were measured using the methods described in Examples 2 and 3. The ionized lipids used for nanoparticle preparation and the physical properties of the nanoparticles are shown in Table 5. An experiment to measure the in vitro efficacy of the lipid nanoparticles according to the present invention was conducted using the method described in Example 4. For this purpose, each lipid nanoparticle was prepared to contain the same amount of mRNA expressing luciferase. HepG2 and HEK293 cell lines were treated with lipid nanoparticles at mRNA concentrations of 6.25, 12.5, 25, and 50 ng for 24 hours, and then luciferase activity was measured. Measurements at each concentration were repeated three times, and the average value was calculated and expressed as a relative value (Fig. 3). As a result of the measurement, it was confirmed that luciferase activity was measured in proportion to the RNA concentration treated in the cells.

[0426]

[0427] Example 9: Measurement of in vivo efficacy of lipid nanoparticles

[0428] Nanoparticles were prepared to evaluate the efficacy of the ionized lipids synthesized in Example 1 in an animal model. Since the efficacy of lipid nanoparticles does not always match the results measured in an in vitro environment and the results measured in an in vivo environment, it is desirable to confirm the efficiency in vivo using an animal model.

[0429] Nanoparticles for measuring in vivo efficiency were prepared according to the method described in Korean Patent Application No. 10-2025-0114871 filed by the applicant. Two solutions were prepared in the same manner as conventional lipid nanoparticle preparation methods. One was an organic solution containing lipid components, prepared by dissolving the lipid components constituting the lipid nanoparticles in ethanol. In preparing lipid nanoparticles containing ionized lipids synthesized in Example 1, an ethanol solution was prepared to have a total concentration of 4.5 to 6.5 mg / mL while maintaining the molar ratios of the ionized lipid, DSPC, cholesterol, TRP-133, and TRE-027 at 50:10:38.5:9:0.5, respectively. TRP-133 is a PEG-lipid in which PEG with a molecular weight of 550 is linked to two strands of C18 hydrocarbon chains, and TRE-027 has a molecular structure in which a GalNAc ligand is additionally bound to the opposite end of the PEG in addition to a PEG-lipid in which PEG with a molecular weight of 2000 is linked to two strands of C18 hydrocarbon chains. The GalNAc ligand plays a role in delivering lipid nanoparticles to liver tissue.

[0430] Meanwhile, mRNA expressing hEPO was prepared at a concentration of 0.1 mg / mL in a pH 7 Hepes buffer solution. Lipid nanoparticles were prepared by mixing an ethanol solution and an aqueous buffer solution in a volume ratio of 1:3 using a micromixer chip, with the flow rate of the mixed solution set to 4 mL / min. The mixture was collected and dialyzed for 24 hours in a pH 7.5 PBS buffer solution.

[0431] The particle size, PDI, and RNA encapsulation rate of lipid nanoparticles prepared using each ionized lipid are as shown in Table 6.

[0432]

[0433] Each LNP was intravenously administered to mice (n = 3) at a dose of 0.5 mpk, and serum was collected after 6 hours. The hEPO content in the serum was analyzed using the ELISA method. The measurement results are listed in Table 7.

[0434]

[0435] Example 10: Measurement of in vivo efficacy of lipid nanoparticles

[0436] Lipid nanoparticles were prepared using the method described in Example 9, and their in vivo efficacy was evaluated. The lipid nanoparticles prepared in this example were prepared in the same manner, except that a TRP-133 content of 4.5% was used. The ionized lipids used for nanoparticle preparation, the physical properties of the prepared particles, and the concentration of hEPO measured in animal serum are listed in Table 8.

[0437]

[0438] Example 11: Measurement of in vivo efficacy of lipid nanoparticles

[0439] Lipid nanoparticles were prepared using the method described in Example 9, and their in vivo efficacy was evaluated. The procedure was identical to that of this example, except that the lipid nanoparticles prepared in this example were prepared in an acetic acid buffer solution at pH 5. The ionized lipids used for nanoparticle preparation, the physical properties of the prepared particles, and the concentration of hEPO measured in animal serum are listed in Table 9.

[0440]

[0441] Example 12: Measurement of in vivo efficacy of lipid nanoparticles

[0442] Lipid nanoparticles were prepared using the method described in Example 10, and their in vivo efficacy was evaluated. The ionized lipids used to prepare the nanoparticles, the physical properties of the prepared particles, and the concentration of hEPO measured in animal serum are listed in Table 10.

[0443]

[0444] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Ionized lipid compounds represented by Chemical Formula 1, pharmaceutically acceptable salts thereof, or stereoisomers thereof: [Chemical Formula 1] In Chemical Formula 1, R1 is selected from the following groups, R 2 and R 3 C independently 6-12 Alkyl, C 6-12 Alkenyl, C 6-12 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl, L 1 and L 2 is independently selected from a group consisting of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=S)O-, -OC(=S)-, -SC(=S)-, -C(=S)S-, -C(=O)NH-, -NHC(=O)-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -C(=O)NHNHC(=O)-, -C(=S)NH-, -NHC(=S)-, -NHC(=S)O-, -NHC(=S)S-, and -NHC(=S)S-. R 4 is C 12-30 Alkyl, C 12-30 Alkenyl, C 12-30 Alkinyl, C 12-30 Cycloalkyl, C 12-30 Cycloalkenyl, C 12-30 It is a cycloalkynyl or sterol, and Sterol is cholesterol or beta-sitosterol, and R 5 is either without a bond or; C 2-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Cycloalkynyl; or -R 6 Selected from a group composed of C(=O)-, R 6 is either without a bond or; C 2-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Selected from cycloalkynyl, R 7 C 2-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 2-6 Cycloalkyl, C 2-6 Cycloalkenyl, C 2-6 Selected from cycloalkynyl, m is an integer from 1 to 3, and n is an integer satisfying m + n = 3, and R 8 and R 9 is independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl, Here, the alkyl, alkenyl, or alkynyl is a straight chain or a branched chain.

2. The above R 2 and R 3 is independently a straight chain or branch C 6-12 alkyl of, C 6-12 alkenyl or C 6-12 It is an alkinyl of, The above L 1 and L 2 is independently -C(=O)-, -OC(=O)-, -C(=O)O- or -OC(=O)O-, and The above R 4 is a straight chain or branch C 18-26 Alkyl, C 18-26 Alkenyl, C 18-26 It is an alkynyl or sterol, and The above R 5 is without bonds or straight chain C 2-12 alkyl or -R 6 C(=O)-is, The above R 6 is without bonds or straight chain C 2-12 It is an alkyl, The above R 7 Silver is a straight chain or branch C 2-6 Alkyl, C 2-6 Alkenyl or C 2-6 An ionized lipid compound characterized by being alkynyl, where m is an integer from 1 to 3 and n is an integer satisfying m + n = 3, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

3. The ionized lipid compound according to claim 1, characterized by being selected from the group consisting of formulas 2 to 30, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] [Chemical Formula 15] [Chemical Formula 16] [Chemical Formula 17] [Chemical Formula 18] [Chemical Formula 19] [Chemical Formula 20] [Chemical Formula 21] [Chemical Formula 22] [Chemical Formula 23] [Chemical Formula 24] [Chemical Formula 25] [Chemical Formula 26] [Chemical Formula 27] [Chemical Formula 28] [Chemical Formula 29] [Chemical Formula 30] 4. A lipid nanoparticle (LNP) comprising the ionized lipid compound of claim 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

5. Lipid nanoparticles according to claim 4, characterized in that the content of the ionized lipid compound is 20 to 70 mol% based on the total lipid.

6. Lipid nanoparticles according to claim 4, further comprising one or more selected from the group consisting of phospholipids, cholesterol, and PEG-bound lipids.

7. Lipid nanoparticles according to claim 6, further comprising phospholipids and PEG-bound lipids, or further comprising phospholipids, cholesterol, and PEG-bound lipids.

8. The method of claim 6, wherein the phospholipid is DOPE (dioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPC (distearoylphosphatidylcholine), POPC (palmitoyloleoylphosphatidylcholine; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), EPC (egg phosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), DSPE (distearoylphosphatidylethanolamine), PE (phosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), POPE Lipid nanoparticles characterized by being one or more selected from the group consisting of (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]), and sphingomyelin.

9. In claim 6, the lipid nanoparticle is characterized in that the PEG-bound lipid is directly bonded to the PEG or connected to the lipid through a linker moiety.

10. A lipid nanoparticle according to claim 9, wherein the linker moiety is selected from the group consisting of amido(-C(O)NH-), amino(-NR-), carbonyl(-C(O)-), carbamate(-NHC(O)O-), urea(-NHC(O)NH-), disulfide(-SS-), ether(-O-), succinyl(-(O)CCH2CH2C(O)-), succinamidyl(-NHC(O)CH2CH2C(O)NH-), hydrazone(-C(O)NHN=R-, -C=NNH-), ester(-C(O)O-), carbonate(-OC(O)O-), succinoyl, phosphate ester(-O-(O)POH-O-), sulfonate ester, and combinations thereof.

11. Lipid nanoparticles according to claim 9, characterized in that the PEG is a single-chain or multi-chain polymer derivative having a basic backbone of polyethylene glycol or polyethylene glycol.

12. Lipid nanoparticles according to claim 9, characterized in that the lipid comprises a structure in which two types of lipids selected from hydrocarbons of C3-C30 single and branched chain forms of alkyl, alkenyl, alkynyl, and sterol are linked to glycerol by bonding of ether (-O-), ester (-C(O)O-, -OC(O)-), or amide (-NHC(O)-, -C(O)NH-).

13. A lipid nanoparticle according to claim 9, characterized in that one end of the PEG comprises a ligand, or does not comprise a ligand, or comprises a mixture thereof.

14. A lipid nanoparticle according to claim 13, wherein the ligand is selected from the group consisting of N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-galactose, D-galactose, N-acetyl-D-glucosamine, N-acetyl-D-glucosamine, D-glucose, D-mannose, F-fucose, carbohydrate derivatives, folate, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CADY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single-chain variable fragment (scFv), and aptamer.

15. A method for producing the lipid of claim 1 comprising the following steps: (a) R 3 -L 2 -R 5 Halogenates containing -Sterol and R 1 -NH-R 2 -L 1 -R 4 Reacting amine compounds or R 2 -L 1 -R 4 Halogenates containing and R 1 -NH-R 3 -L 2 -R 5 - A step of reacting the amine compound of the sterol in a solvent; and (b) Step of removing the above solvent. Here, R 1 It is selected from the following military units, R 2 and R 3 C independently 6-12 Alkyl, C 6-12 Alkenyl, C 6-12 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl, L 1 and L 2 is independently selected from a group consisting of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=S)O-, -OC(=S)-, -SC(=S)-, -C(=S)S-, -C(=O)NH-, -NHC(=O)-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -C(=O)NHNHC(=O)-, -C(=S)NH-, -NHC(=S)-, -NHC(=S)O-, -NHC(=S)S-, and -NHC(=S)S-. R 4 is C 12-30 Alkyl, C 12-30 Alkenyl, C 12-30 Alkinyl, C 12-30 Cycloalkyl, C 12-30 Cycloalkenyl, C 12-30 It is a cycloalkynyl or sterol, and Sterol is cholesterol or beta-sitosterol, and R 5 is either without a bond or; C 2-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Cycloalkynyl; or -R 6 Selected from a group composed of C(=O)-, R 6 is either without a bond or; C 2-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkinyl, C 2-12 Cycloalkyl, C 2-12 Cycloalkenyl, C 2-12 Selected from cycloalkynyl, R 7 C 2-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 2-6 Cycloalkyl, C 2-6 Cycloalkenyl, C 2-6 Selected from cycloalkynyl, m is an integer from 1 to 3, and n is an integer satisfying m + n = 3, and R 8 and R 9 is independently H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkinyl, C 6-12 Cycloalkyl, C 6-12 Cycloalkenyl or C 6-12 It is a cycloalkynyl, Here, the alkyl, alkenyl, or alkynyl is a straight chain or a branched chain.

16. In Paragraph 15, the above R 3 -L 2 -R 5 Halogenates containing -Sterol and R 1 -NH-R 2 -L 1 -R 4 The molar ratio of the amine compound is 1:1.0 to 2.0, and the above R 2 -L 1 -R 4 Halogenates containing and R 1 -NH-R 3 -L 2 -R 5 A method for preparing lipids characterized by the molar ratio of the amine compound of the sterol being 1:0.5 to 1.

0.

17. A method for producing lipids according to claim 15, characterized in that the reaction of step (a) above is carried out at a temperature of 60 to 90°C for 12 to 72 hours.

18. A method for producing lipids according to claim 15, further comprising a washing or purification process after step (b) above.

19. Lipid nanoparticles of any one of paragraphs 4 to 14; and A drug delivery composition comprising an anionic drug, a nucleic acid, or a combination thereof.

20. A drug delivery composition according to claim 19, characterized in that the anionic drug, nucleic acid, or combination thereof is encapsulated inside the lipid nanoparticles.

21. A drug delivery composition according to claim 19, characterized in that the anionic drug is one or more selected from the group consisting of peptides, protein drugs, protein-nucleic acid structures, and anionic biopolymer-drug conjugates.

22. A drug delivery composition according to claim 19, wherein the nucleic acid is one or more selected from the group consisting of single-stranded siRNA, double-stranded siRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, mRNA, tRNA, lncRNA, piRNA, circRNA, saRNA, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme.

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