Novel provitamin b5-based lipid, lipid nanoparticle comprising same, and uses thereof

Abstract

The present invention relates to a novel provitamin B5-based lipid derivative compound, a lipid nanoparticle composition comprising same, and the like. More specifically, the novel provitamin B5-based lipid compound functions as an ionizable lipid in the formation of lipid nanoparticles and forms stable lipid nanoparticles, which can be used for delivering pharmaceutically active ingredients such as mRNA into the body.

Description

Novel lipids based on provitamin B5, lipid nanoparticles containing the same, and uses thereof

[0001] The present invention relates to a novel lipid based on provitamin B5, lipid nanoparticles containing the same, and a vaccine or gene therapy composition further comprising a preventive and therapeutic agent such as a nucleic acid in the lipid nanoparticles.

[0002] Nucleic acid-based medicines, which began about 40 years ago by injecting plasmid DNA into the body to help produce deficient proteins, have since been reported to include various types, including antigene, decoy, antisense, siRNA, and miRNA, which inhibit gene transcription and translation. Nucleic acid-based medicines target DNA or RNA, rather than proteins, and have garnered attention as personalized treatments through complementary binding with specific DNA or RNA sequences. Nucleic acid-based medicines are utilized not only as therapeutic agents but also as preventive agents that protect against diseases by injecting genes that can express antigens for specific diseases. Gene-based vaccines are categorized as DNA vaccines, RNA vaccines, and viral vector vaccines. Among them, RNA vaccines inject mRNA encoding antigens into the body, causing the antigens to be expressed in the body and inducing antibody formation. RNA vaccines do not carry the potential risks of viral vector-based vaccines, such as infection, or the genetic mutations of DNA vaccines. They also offer the advantage of rapid development, garnering attention as an effective response to the COVID-19 outbreak in 2019.

[0003] Current lipid nanoparticles are typically composed of a mixture of four components: ionizable lipids, phospholipids (helper lipids), cholesterol (structural lipids), and PEGylated lipids, all in a specific ratio. Among these, ionizable lipids are known to possess a positive charge in low pH environments, allowing them to bind to negatively charged nucleic acids. At neutral pH, they lose their charge, maintaining the stability of LNPs and facilitating endosomal escape within cells, thereby enhancing delivery efficiency. While existing ionizable lipids exhibit high delivery efficiency and biocompatibility, improvements are still needed in biodegradability and mitigation of side effects. In particular, synthetic lipids have the potential to be metabolized and accumulated in the body, increasing the demand for ionizable lipids based on natural or biocompatible ingredients.

[0004] The technical problem to be achieved by the present invention is to provide a novel lipid derivative compound based on provitamin B5.

[0005] Another technical problem to be achieved by the present invention is to provide a lipid nanoparticle composition comprising the compound.

[0006] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0007] To solve the above problem, the present inventors provide a compound represented by the following chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof:

[0008]

[0009] In the above chemical formula,

[0010] X is any one of CH2, NH, O, and S,

[0011] n is an integer from 0 to 3,

[0012] R1 is a secondary amine, at least one of a secondary amine substituted with any one of methyl, ethyl, hydroxyethyl, and hydroxybutyl, a substituted or unsubstituted C3-C6 heterocycloalkyl, and a straight chain C1-C6 alkyl substituted or unsubstituted with a hydroxyl group or dimethylamine,

[0013] In the case where the above C3-C6 heterocycloalkyl is substituted, it is substituted with C1-C6 straight chain alkyl which is unsubstituted or substituted with hydroxy,

[0014] R x and R y Is and;

[0015] R2 and R3 are each independently C3-C 20 It is a saturated or unsaturated hydrocarbon.

[0016] According to one aspect, the above R2 and R3 can each independently be a C5-C6 saturated hydrocarbon.

[0017] According to one side, the above R1

[0018] and It may be at least one selected from the group consisting of .

[0019] According to one aspect, the compound may be at least one selected from the group consisting of:

[0020]

[0021]

[0022]

[0023] [Compound 20-4]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] [Compound 20-11]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] [Compound 22-1]

[0040]

[0041]

[0042]

[0043]

[0044] According to another embodiment of the present invention, a lipid nanoparticle composition comprising one or more of the compounds described above is provided.

[0045] According to one side, the compound may have a positive charge under slightly acidic conditions and bind to a nucleic acid having a negative charge.

[0046] According to one aspect, the composition may further comprise one or more selected from the group consisting of PEGylated lipids, helper lipids and structural lipids.

[0047] According to one side, the helper lipid is 6,6'-trehalose glycolipid, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Dieter PC), 1-Oleoyl-2-cholesterylhexyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Dilinoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), It may be at least one selected from the group consisting of dipalmitoyl-phosphatidyl-ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0048] According to one aspect, the structural lipid may be at least one selected from the group consisting of cholesterol, bile acid derivatives including alkyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene.

[0049] According to one aspect, the composition may comprise PEG lipids, helper lipids and structural lipids, and may comprise 30 to 50 mol% of the compound, 1 to 2 mol% of PEG lipids, 10 to 30 mol% of helper lipids and 38 to 39 mol% of structural lipids.

[0050] According to one aspect, the compound may be at least one selected from the group consisting of:

[0051]

[0052]

[0053]

[0054] [Compound 20-4]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] [Compound 20-11]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] [Compound 22-1]

[0071]

[0072]

[0073]

[0074]

[0075] According to one aspect, the lipid nanoparticle composition may contain a therapeutic or prophylactic agent therein.

[0076] According to one aspect, the therapeutic or prophylactic agent may be selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

[0077] According to one aspect, the therapeutic or preventive agent is messenger RNA, and the lipid nanoparticle may have a diameter of 70 to 200 nm and an internal zeta potential of -70 to -20 mV.

[0078] According to another embodiment of the present invention, a vaccine composition comprising the lipid nanoparticle composition is provided. Preferably, when used in a vaccine composition, the nucleic acid contained within the lipid nanoparticle may be a nucleic acid expressing an antigen protein, and most preferably, it may be OVA mRNA.

[0079] The present invention provides a novel provitamin B5-based lipid compound that functions as an ionizable lipid and can bind to nucleic acids and the like to form lipid nanoparticles. Lipid nanoparticles comprising the compound of the present invention exhibit excellent nucleic acid encapsulation performance, colloidal stability, and nucleic acid intracellular delivery efficiency.

[0080] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0081] Figure 1 shows the results of electrophoresis to measure the encapsulation efficiency of lipid nanoparticles.

[0082] Figure 2 shows the in vitro delivery efficiency of lipid nanoparticles C2001 to C2302 to the Huh7 cell line.

[0083] Figure 3 shows the in vitro delivery efficiency of lipid nanoparticles C2001 to C2302 to the HEK293 cell line.

[0084] Figure 4 shows the in vitro delivery efficiency of lipid nanoparticles 32001 to 32302 to the Huh7 cell line.

[0085] Figure 5 shows the in vitro delivery efficiency for lipid nanoparticles 32001 to 32302 to the HEK293 cell line.

[0086] Figure 6 shows the results of administering a tumor cell line to evaluate the vaccine applicability and confirming the tumor size compared to the control group.

[0087] Figure 7 shows [chemical formula 1] of the present invention.

[0088] The present inventors synthesized a novel lipid compound based on dexpanthenol, a precursor of vitamin B5 (pantothenic acid). This novel lipid compound exhibits cationic properties under mildly acidic conditions and is suitable for encapsulating negatively charged biomolecules, such as nucleic acids, into lipid nanoparticles (LNPs), thereby functioning as a so-called ionizable lipid. Accordingly, the present invention provides a novel provitamin B5-based lipid compound, a lipid nanoparticle composition comprising the same, a vaccine composition comprising the same, and a method for preparing the same.

[0089] To solve the above problem, the present inventors provide a compound represented by the following chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof:

[0090]

[0091] In the above chemical formula,

[0092] X is any one of CH2, NH, O, and S,

[0093] n is an integer from 0 to 3,

[0094] R1 is a secondary amine, at least one of a secondary amine substituted with any one of methyl, ethyl, hydroxyethyl, and hydroxybutyl, a substituted or unsubstituted C3-C6 heterocycloalkyl, and a straight chain C1-C6 alkyl substituted or unsubstituted with a hydroxyl group or dimethylamine,

[0095] In the case where the above C3-C6 heterocycloalkyl is substituted, it is substituted with C1-C6 straight chain alkyl which is unsubstituted or substituted with hydroxy,

[0096] R x and R y Is and;

[0097] R2 and R3 are each independently C3-C 20 It is a saturated or unsaturated hydrocarbon.

[0098] The term 'saturated hydrocarbon' used in the present invention means a hydrocarbon composed of single bonds, and 'unsaturated hydrocarbon' means a hydrocarbon having one or more double bonds or triple bonds.

[0099] In the present invention, the term “substitution” refers to a reaction in which an atom or atomic group included in a molecule of a compound is replaced with another atom or atomic group.

[0100] In the present invention, the term “chain-like” refers to a molecule having a chain-like structure, and the chain-like structure is a chemical structure in which carbon atoms are connected in a chain shape, and there are straight chain-like structures and branched structures.

[0101] The term “alkyl group” used in the present invention means an alkane with one hydrogen atom removed, preferably -(CH2) a-Can be expressed as CH3.

[0102] In the present invention, the term “cyclo or ring” refers to a structure in which both ends of the skeleton of an organic compound are connected to form a ring.

[0103] In the present invention, the term “chain or cyclic alkyl group” means a monovalent linear or branched or cyclic saturated hydrocarbon residue consisting solely of carbon and hydrogen atoms, having 1 to 20 carbon atoms. Examples of such alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 2-butyl, 3-butyl, pentyl, n-hexyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0104] In the present invention, the term "heterocycloalkyl group" typically refers to a saturated or unsaturated (but not aromatic) cyclohydrocarbon, which may be optionally unsubstituted, monosubstituted or polysubstituted, in the structure of which at least one is selected from a heteroatom of N, O or S.

[0105] According to one aspect, the above R4 and R5 can each independently be a C5-C6 saturated hydrocarbon.

[0106] According to one side, the above R2 and R3 are each independently and It may be at least one selected from the group consisting of .

[0107] According to one aspect, the compound may be at least one selected from the group consisting of:

[0108]

[0109]

[0110]

[0111] [Compound 20-4]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] [Compound 20-11]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] [Compound 22-1]

[0127]

[0128]

[0129]

[0130]

[0131] According to another embodiment of the present invention, a lipid nanoparticle composition comprising one or more of the compounds described above is provided. The lipid nanoparticle refers to a nano-sized spherical particle composed of lipid, and for the purposes of the present invention, it may preferably contain a nucleic acid therein.

[0132] According to one aspect, the compound has a positive charge under slightly acidic conditions and can bind to a nucleic acid having a negative charge to play the role of an ionizable lipid in the composition of lipid nanoparticles. The slightly acidic conditions may be that the pH of the entire solution containing the compound is 3 to 6.9, preferably 4.5 to 6.5. The ionizable lipid is an ionizable compound having properties similar to lipids and can play a role in highly efficiently encapsulating a drug (e.g., an anionic drug and / or nucleic acid) into lipid nanoparticles through electrostatic interaction with the drug. The ionizable lipid can be protonated (positively charged) at a pH lower than the pKa of the ionizable lipid and can be substantially neutral at a pH higher than the pKa.

[0133] According to one aspect, the composition may further comprise at least one selected from the group consisting of PEG lipids, helper lipids and structural lipids, and preferably may comprise all of PEG lipids, helper lipids and structural lipids.

[0134] According to one aspect, the helper lipid may promote the fusion of lipid nanoparticles, and may be 6,6'-trehalose glycolipid, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octa Decenyl-sn-glycero-3-phosphocholine (18:0 Dieter PC), 1-Oleoyl-2-cholesterylhexyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Dilinoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), It may be at least one selected from the group consisting of dipalmitoyl-phosphatidyl-ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE), and preferably any one of 6,6'-trehalose glycolipid or DSPC.

[0135] According to one aspect, the structural maintenance lipid refers to a lipid that provides morphological rigidity to lipid charging within a lipid nanoparticle and plays a role in improving the stability of the nanoparticle by being dispersed in the core and surface of the nanoparticle, and may be at least one selected from the group consisting of cholesterol, bile acid derivatives including alkyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene, and preferably may be any one of cholesterol and alkyl lithocholate.

[0136] According to one aspect, the composition may comprise PEG lipids, helper lipids and structural lipids, and may comprise 30 to 50 mol% of the compound, 1 to 2 mol% of PEG lipids, 10 to 30 mol% of helper lipids and 38 to 39 mol% of structural lipids.

[0137] Most preferably, the composition may comprise 50 mol% of the compound, 1.5 mol% of PEG lipid, 10 mol% of helper lipid and 38.5 mol% of structural lipid, in which case the helper lipid used may be DSPC and the structural lipid may be cholesterol.

[0138] Additionally, as another most preferred example, the compound may be comprised of 30 mol%, PEG lipid of 1.5 mol%, helper lipid of 30 mol%, and structural lipid of 38.5 mol%, in which case the helper lipid used may be 6,6'-trehalose dioleate, and the structural lipid may be alkyl lithocholate.

[0139] As shown in the examples below, the compositions described above showed optimal RNA loading efficiency and intracellular mRNA translation efficiency and stability.

[0140] According to one aspect, the compound may be at least one selected from the group consisting of:

[0141]

[0142]

[0143]

[0144] [Compound 20-4]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152] [Compound 20-11]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] [Compound 22-1]

[0161]

[0162]

[0163]

[0164]

[0165] According to one aspect, the lipid nanoparticle composition may contain a therapeutic or prophylactic agent therein.

[0166] According to one aspect, the therapeutic or prophylactic agent may be selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

[0167] According to one aspect, the therapeutic or preventive agent is messenger RNA, and the lipid nanoparticle may have a diameter of 70 to 200 nm and an internal zeta potential of -70 to -20 mV.

[0168] According to another embodiment of the present invention, a vaccine composition comprising the lipid nanoparticle composition is provided. The vaccine composition may preferably be an mRNA vaccine and may further comprise a pharmaceutically acceptable salt or adjuvant.

[0169] The vaccine composition of the present invention may be administered via, for example, the oral or parenteral route. Here, parenteral refers to a broad route of administration, and includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intranasal, sublingual, intrathecal, inhalation, ocular, rectal, vaginal, and intracerebroventricular administration.

[0170] When formulating the above composition, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0171] Solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc., and these solid preparations are prepared by mixing one or more compounds according to the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

[0172] Preparations for parenteral administration include sterile aqueous solutions, lyophilized preparations, suppositories, etc. The lyophilized preparation may be a solid state obtained by freezing a drug and then sublimating the moisture in a vacuum. Preferably, a disaccharide compound such as sucrose, maltose, or trehalose may be used alone or in combination of two or more. The base for the suppository may be witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, or gelatin.

[0173] The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The composition of the present invention 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 singly or in multiple doses. It is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0174] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0175] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0176] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0177]

[0178] Example 1. Synthesis of a novel lipid compound based on provitamin B5

[0179]

[0180] Compound 8 was synthesized as described above. Thereafter, a final provitamin B5-based novel lipid compound was synthesized through one of the following processes.

[0181] 1) Esterification of alcohol

[0182]

[0183] 2) Amine alkylation after esterification

[0184]

[0185] 3) Michael addition after alcoholysis of acyl halide

[0186]

[0187] 4) Synthetic route involving carbamates, carbonates, and thiocarbonates via carbonyldiimidazole

[0188]

[0189] A novel lipid compound based on provitamin B5 was synthesized as shown in the above reaction scheme. The specific synthetic method according to the above reaction scheme is described below.

[0190]

[0191] Example 1-1. Synthesis of compounds 2 to 8

[0192] 5-((2-Hexyldecyl)oxy)-5-oxopentanoic acid (compound 2)

[0193]

[0194] In a 100 mL single-necked flask, solid glutaric anhydride 1 (470 mg, 2 equiv) and DMAP (630 mg, 2.5 equiv) were added to a solution of 2-hexyl-1-decanol (500 mg, 1 equiv) in DCM (50 mL) at room temperature. The reaction mixture was stirred vigorously for approximately 13 h, and the reaction was monitored by TLC (SiO₂; hexane / ethyl acetate, 5:5; PMA stain). The reaction was quenched with 2 N HCl solution (10 mL) and extracted with DCM (3 x 25 mL). The combined organic layers were washed with 2 N HCl solution (2 x 20 mL), brine (1 x 30 mL), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the crude product was purified by flash column chromatography (SiO₂; hexane / ethyl acetate, 9:1 to 8:2, v / v) to give acid 2 (603 mg, 82%) as a clear liquid.

[0195] ¹H NMR (CDCl₃, 400 MHz): δ 0.80 (t,J= 5.1 Hz, 6H), 1.17-1.25 (m, 25H), 1.54 (bs, 1H), 1.87-1.92 (qnt,J= 5.2 Hz, 2H), 2.31-2.38 (m, 4H), 3.92 (d,J= 7.2 Hz, 2H);

[0196] ¹³C NMR (CDCl₃, 100 MHz): δ 14.07, 14.08, 19.86, 22.64, 22.67, 26.69, 29.30, 29.54, 29.60, 29.93, 31.25, 31.80, 31.89, 33.06, 33.25, 37.27, 67.39, 173.10, 179.25.

[0197]

[0198] (R)-N-(3-Hydroxypropyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (Compound 4)

[0199]

[0200] D-Panthenol 3 (4.0 g, 1 eq) was dissolved in anhydrous acetone (150 mL), and anhydrous sodium sulfate (20 g) was added. After stirring for about 10 minutes, p-toluenesulfonic acid monohydrate (0.3 g, 0.08 eq) was added, and the mixture was stirred vigorously for 48 hours at room temperature under an argon atmosphere. The disappearance of panthenol was monitored by TLC (SiO₂; ethyl acetate / methanol, 9:1; KMnO₄ stain) until the reaction was complete. After the reaction, acetone was removed under reduced pressure, and the residue was suspended in a saturated sodium bicarbonate solution (200 mL) and extracted with ethyl acetate (6 x 50 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 4:6 to 8:2, v / v) to obtain ketal 4 (4.20 g, 88%) as a white crystalline solid.

[0201] ¹H NMR (CDCl₃, 400 MHz): δ 0.94 (s, 3H), 0.98 (s, 3H), 1.36 (s, 3H), 1.39 (s, 3H), 1.60-1.67 (m, 2H), 3.22 (d,J= 11.7 Hz, 1H), 3.26-3.34 (m, 1H), 3.36-3.43 (m, 1H), 3.46 (t,J= 6.5 Hz, 1H), 3.55 (q,J= 5.6 Hz, 2H), 3.69 (d,J= 11.6, 1H), 6.77 (bs, 1H), 4.12 (s, 1H);

[0202] ¹³C NMR (CDCl₃, 100 MHz): δ 18.9, 19.1, 22.3, 29.6, 32.7, 33.2, 35.3, 59.2, 71.7, 77.4, 99.3, 171.3.

[0203]

[0204] (R)-(9H-Fluoren-9-yl)methyl (3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propyl) carbonate (Compound 5)

[0205]

[0206] Alcohol 4 (1.0 g, 1 eq) was dissolved in DCM (40 mL), pyridine (1.30 mL, 4 eq) was added, and the mixture was stirred for about 5 minutes. Then, fluorenylmethoxycarbonyl chloride (2.10 g, 2 eq) was slowly added using a dropping funnel at 5°C under an argon atmosphere over 30 minutes. The mixture was stirred for 8 hours, and the progress of the reaction was monitored by TLC (SiO₂; ethyl acetate / hexane, 3:7; PMA stain). After completion of the reaction, the reaction was quenched with water (10 mL) and concentrated. The pyridine residue was evaporated and removed with toluene (3 x 15 mL). The resulting black material was dissolved in DCM (50 mL), washed with saturated sodium bicarbonate solution (2 x 50 mL) and brine (1 x 20 mL), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 1:9 to 2:8, v / v) to obtain acetonide 5 (1.62 g, 73%) as a clear oil.

[0207] ¹H NMR (CDCl₃, 400 MHz): δ 0.99 (s, 3H), 1.03 (s, 3H), 1.35 (s, 3H), 1.43 (s, 3H), 1.83-1.86 (m, 2H), 3.21-3.28 (m, 2H), 3.34-3.40 (m, 1H), 3.64 (d,J= 11.7 Hz, 1H), 4.07 (s, 1H), 4.22 (t,J= 7.2 Hz, 3H), 4.36 (d,J= 7.2 Hz, 2H), 6.79 (t,J= 6.0 Hz, 1H), 7.25 (t,J= 6.9 Hz, 2H), 7.32 (t,J= 7.4 Hz, 2H), 7.56 (d,J= 7.4 Hz, 2H), 7.68 (d,J= 8.2 Hz, 2H);

[0208] ¹³C NMR (CDCl₃, 100 MHz): δ 18.65, 18.96, 22.12, 28.84, 29.49, 32.92, 35.40, 46.73, 65.75, 69.61, 71.35, 98.94, 120.04, 125.07, 127.12, 127.83, 141.22, 143.34, 155.14, 169.70.

[0209]

[0210] (R)-(9H-Fluoren-9-yl)methyl (3-(2,4-dihydroxy-3,3-dimethylbutanamido)propyl) carbonate (화합물6)

[0211]

[0212] Acetonide 5 (1.60 g, 1 eq) and DL-1,4-dithiothreitol (DTT; 1.15 g, 2 eq) were dissolved in DCM, followed by addition of p-toluenesulfonic acid monohydrate (427 mg, 0.6 eq) at room temperature. The reaction mixture was stirred for 1 h and the progress of the reaction was monitored by TLC (SiO₂; hexane / ethyl acetate, 1:1; UV activated). After completion of the reaction, saturated sodium bicarbonate solution (100 mL) was added and extracted with DCM (3 x 50 mL). The residue was washed with brine (1 x 50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 1:1 to 7:3, v / v) to give diol 6 (1.23 g, 77%) as a clear oil.

[0213] ¹H NMR (CDCl₃, 400 MHz): δ 0.90 (s, 3H), 0.99 (s, 3H), 1.25 (t,J= 6.4 Hz, 2H), 3.31-3.40 (m, 2H), 3.47-3.50 (m, 2H), 4.00 (s, 1H), 4.22 (t,J= 7.2 Hz, 3H), 4.43 (d,J= 7.2 Hz, 2H), 7.14 (t,J= 5.6 Hz, 1H), 7.31 (t,J= 7.4 Hz, 2H), 7.39 (t,J= 7.4 Hz, 2H), 7.61 (d,J= 7.4 Hz, 2H), 7.76 (d,J= 7.6 Hz, 2H);

[0214] ¹³C NMR (CDCl₃, 100 MHz): δ 18.75, 18.97, 22.13, 28.83, 32.91, 46.74, 65.78, 69.63, 71.37, 98.95, 120.06, 125.07, 127.24, 127.85, 141.23, 143.55, 155.15, 169.80.

[0215]

[0216] (((8R)-19-Hexyl-8-((5-((2-hexyldecyl)oxy)-5-oxopentanoyl)oxy)-9,9-dimethyl-7,12,16-trioxo-2,11,17-trioxa-6-azaheptacosanoyl)oxy)fermium (Compound 7)

[0217]

[0218] A single-necked flask (100 mL) was charged with diol 6 (1.20 g, 1 eq), EDCI·HCl (1.61 g, 3 eq), and 4-dimethylaminopyridine (DMAP; 100 mg, 0.3 eq) along with DCM (50 mL). The mixture was stirred vigorously at room temperature under argon for 10 min, and then acid 2 (2.50 g, 2.5 eq) dissolved in DCM (20 mL) was added at room temperature. The reaction mixture was stirred at room temperature for approximately 22 h, and the disappearance of the alcohol was monitored by TLC (SiO₂; hexane / ethyl acetate, 8:2; UV activated). After completion of the reaction, DCM (100 mL) was added, and the residue was washed with saturated sodium bicarbonate solution (3 x 60 mL) and brine (1 x 50 mL), and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure, and the residue was purified through flash column chromatography (SiO₂; ethyl acetate / hexane, 1:9, v / v) to obtain the target product 7 (2.21 g, 64%) as a clear liquid.

[0219] ¹H NMR (CDCl₃, 400 MHz): δ 0.87 (t,J= 6.5 Hz, 12H), 1.02 (s, 3H), 1.07 (s, 3H), 1.19–1.30 (m, 41H), 1.57 (m,82H), 1.57 (m.66). (m, 6H), 2.34-2.41 (m, 6H), 2.48 (t,J= 7.1 Hz, 2H), 3.34 (q,J= 7.6 Hz, 2H), 3.88 (d,J= 11.0 Hz, 1H), 3.97 (d,J= 3.3). 8.0 Hz, 1H), 4.20–4.27 (m, 3H), 4.44 (d,J= 7.2 Hz, 2H), 4.99 (s, 1H), 6.57 (t,J= 5.8 Hz, 1H), 7.32 (t,J7= 4. 1H), 7.4 Hz, 2H), 7.62 (d,J= 7.5 Hz, 2H), 7.78 (d,J= 7.5 Hz, 2H);

[0220] ¹³C NMR (CDCl₃, 100 MHz): δ 14.12, 14.13, 20.01, 20.04, 20.82, 21.44, 22.66, 22.68, 26.65, 29.68, 29.69, 29.62, 29.96, 31.18, 31.21, 32.95, 33.15, 33.23, 33.37, 25.74, 37.26, 37.46, 46.79, 69.54, 67.34, 69.81, 76.61, 120.10, 125.08, 127.18, 127.93, 141.32, 143.28, 155.48, 172.73.2,

[0221]

[0222] Bis(2-hexyldecyl) O,O'-((R)-4-((3-hydroxypropyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) diglutarate (화합물8)

[0223]

[0224] A 100 mL single-necked flask was prepared with a piperidine / DMF (20:80, v / v) solution and cooled to 0°C. 7 (2.20 g) and DMF (20 mL) were added to the same flask to form a solution, which was then cooled to 0°C. Then, 20% piperidine / DMF (50 mL) was slowly poured under an argon atmosphere. The mixture was stirred for approximately 5–10 minutes and the reaction was monitored by TLC (SiO₂; hexane / ethyl acetate, 7:3; PMA staining). After completion of the reaction, the mixture was acidified to pH 3 by adding 3 M HCl solution. The reaction mixture was diluted with DCM (200 mL) and washed with water (3 x 60 mL) and brine (1 x 50 mL). The solution was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The produced oil was purified through flash column chromatography (SiO₂; ethyl acetate / hexane, 1:9, v / v) to obtain alcohol 8 (1.29 g, 79%) in the form of a transparent liquid.

[0225] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.0 Hz, 12H), 1.03 (s, 3H), 1.09 (s, 3H), 1.21-1.31 (m, 51H), 1.58-1.65 (m, 2H), 1.66-1.72 (m, 2H), 1.92-2.00 (m, 4H), 2.35-2.41 (m, 6H), 2.47 (t,J= 7.0 Hz, 2H), 3.25 (t,J= 5.6 Hz, 1H), 3.42 (q,J= 5.7 Hz, 2H), 3.70 (q,J= 5.0 Hz, 2H), 3.86 (d,J= 11.0 Hz, 1H), 2.96-2.98 (m, 4H), 4.08 (d,J= 11.0 Hz, 1H), 5.00 (s, 1H), 6.97 (t,J= 5.4 Hz, 1H);

[0226] ¹³C NMR (CDCl₃, 100 MHz): δ 14.11, 14.12, 19.97, 20.01, 20.88, 21.50, 22.65, 22.68, 26.64, 26.69, 29.31, 29.57, 29.61, 29.95, 31.20, 31.42, 31.81, 31.90, 32.96, 33.05, 33.23, 33.34, 37.17, 37.25, 37.41, 60.54, 67.39, 67.61, 69.28, 168.66, 171.56, 172.68, 173.28, 173.68.

[0227]

[0228] Example 1-2. Synthesis through esterification of alcohol

[0229]

[0230]

[0231] O,O'-((R)-4-((3-((Dimethylglycyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl)bis(2-hexyldecyl) diglutarate (Compound 20-10)

[0232]

[0233] Dimethylglycine (30 mg, 1.3 equiv), EDCIㆍHCl (65 mg, 1.5 equiv), and 4-dimethylaminopyridine (DMAP; 6 mg, 0.2 equiv) were added to a 50 mL single-necked flask along with DCM (20 mL). The mixture was stirred vigorously for 5 min at room temperature under an argon atmosphere. Afterwards, alcohol 8 (200 mg, 1 equiv) was added and stirred at room temperature for 14 h. The disappearance of the alcohol was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After the reaction was completed, DCM (50 mL) was added and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 10:0 to 9:1, v / v) to obtain 20-10 (186 mg, 85%) as a clear oil.

[0234] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.0 Hz, 12H), 1.03 (s, 3H), 1.07 (s, 3H), 1.19-1.30 (m, 49H), 1.61 (bs, 2H), 1.82-1.87 (m, 2H), 1.91-2.01 (m, 4H), 2.35 (s, 6H), 2.37-2.41 (m, 6H), 2.49-2.53 (m, 2H), 3.18 (s, 2H), 3.23-3.57 (m, 2H), 3.88 (d,J= 11.0 Hz, 1H), 3.98 (d,J= 5.7 Hz, 4H), 4.07 (d,J= 11.0 Hz, 1H), 4.13-4.25 (m, 2H), 4.98 (s, 1H), 6.63 (t,J= 5.9 Hz, 1H).

[0235] ¹³C NMR (CDCl₃, 100 MHz): δ 14.09, 14.10, 20.01, 20.11, 20.80, 21.43, 22.63, 22.66, 26.63, 26.67, 28.69, 29.30, 29.55, 29.60, 29.94, 31.19, 31.80, 31.88, 32.96, 33.16, 33.21, 33.35, 35.64, 37.25, 37.41, 45.36, 60.43, 61.69, 67.31, 67.38, 69.23, 76.64, 168.00, 171.14, 171.75, 172.64, 173.13.

[0236]

[0237] O,O'-(2,2-Dimethyl-4-((3-(2-(4-methylmorpholin-2-yl)acetoxy)propyl)amino)-4-oxobutane-1,3-diyl)bis(2-hexyldecyl) diglutarate (Compound 20-11)

[0238]

[0239] A magnetic stirrer was placed in a dried 50 mL test tube, and alcohol 8 (200 mg, 1 eq), 4-methyl-2-morpholinacetic acid (72 mg, 2 eq), EDCIㆍHCl (109 mg, 2.5 eq), 4-dimethylaminopyridine (DMAP; 11 mg, 0.4 eq), and DCM (20 mL) were added. The vessel was purged with argon, sealed, and heated in a silicone oil bath at 60°C for 3 days. The disappearance of alcohol was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After the reaction was completed, the reaction solution was cooled to room temperature, DCM (50 mL) was added, and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 10:0 to 9:1, v / v) to obtain 20-11 (153 mg, 66%) as a clear oil.

[0240] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.6 Hz, 12H), 1.03 (s, 3H), 1.07 (s, 3H), 1.18-1.29 (m, 50H), 1.60 (bs, 2H), 1.84-1.84 (m, 2H), 1.90-2.02 (m, 4H), 2.36-2.46 (m, 6H), 2.51 (t,J= 7.2 Hz, 2H), 2.52-2.59 (m, 4H), 2.22 (s, 3H), 3.22 (s, 2H), 3.31 (q,J= 6.2 Hz, 2H), 3.76 (t,J= 4.3 Hz, 4H), 3.87 (d,J= 11.0 Hz, 1H), 3.96 (d,J= 5.5 Hz, 4H), 4.05 (d,J= 11.0 Hz, 1H), 4.16-4.21 (m, 2H), 4.97 (s, 1H), 6.55 (t,J= 5.8 Hz, 1H).

[0241] ¹³C NMR (CDCl₃, 100 MHz): δ 14.08, 14.11, 20.04, 20.12, 20.82, 21.42, 22.62, 22.67, 26.64, 26.69, 28.71, 29.28, 29.54, 29.58, 29.96, 31.18, 31.81, 31.87, 32.98, 33.52, 33.22, 33.36, 35.68, 37.24, 37.42, 53.31, 59.52, 61.73, 66.76, 67.33, 67.42, 69.21, 76.62, 168.02, 170.48, 171.71, 172.63, 173.13.

[0242]

[0243] O,O'-(2,2-Dimethyl-4-oxo-4-((3-(2-thiomorpholinoacetoxy)propyl)amino)butane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 20-12)

[0244]

[0245] 2-Thiomorpholinoacetic acid (43 mg, 1.3 equiv), EDCIㆍHCl (59 mg, 1.5 equiv), and 4-dimethylaminopyridine (DMAP; 5 mg, 0.2 equiv) were added to a 50 mL single-necked flask along with DCM (20 mL). The mixture was stirred vigorously for 5 min at room temperature under an argon atmosphere. Afterwards, alcohol 8 (180 mg, 1 equiv) was added and stirred at room temperature for 11 h. The disappearance of the alcohol was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After the reaction was completed, DCM (50 mL) was added and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 5:5 to 10:0, v / v) to give 20-12 (173 mg, 83%) as a pale yellow oil.

[0246] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 7.0 Hz, 12H), 1.03 (s, 3H), 1.07 (s, 3H), 1.19-1.31 (m, 51H), 1.19-1.31 (m, 51H), 1.2-H1 (bs. 1. 1.93–2.01 (m, 4H), 2.36–2.42 (s, 6H), 2.50 (t,J= 7.2 Hz, 2H), 2.70 (t,J= 4.6 Hz, 4H), 2.84 (t,J= 4.5 Hz, 3. 2H), (q,J= 6.5 Hz, 2H), 3.87 (d,J= 11.0 Hz, 1H), 3.97 (d,J= 5.8 Hz, 4H), 4.07 (d,J= 11.0 Hz, 1H), 4.14-4.19 (m. 3, 2H), 4.14-4.19 (m. 3, 2H). 1H).

[0247] ¹³C NMR (CDCl₃, 100 MHz): δ 14.12, 20.04, 20.14, 20.83, 21.45, 22.65, 22.67, 26.64, 26.69, 29.81, 29.86, 29.61, 29.95, 31.20, 31.81, 31.89, 32.98, 33.19, 33.22, 33.36, 35.68, 37.26, 37.44, 54.574, 61.08. 67.42, 69.22, 76.63, 168.04, 170.77, 171.72, 172.64, 173.51.

[0248]

[0249] O,O'-(2,2-Dimethyl-4-((3-(2-morpholinoacetoxy)propyl)amino)-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (화합물20-13)

[0250]

[0251] 2-Morpholinoacetic acid (41 mg, 1.3 equiv), EDCIㆍHCl (62 mg, 1.5 equiv), and 4-dimethylaminopyridine (DMAP; 5 mg, 0.2 equiv) were added to a 50 mL single-necked flask together with DCM (20 mL). The mixture was stirred vigorously for 5 min at room temperature under an argon atmosphere. Afterwards, alcohol 8 (190 mg, 1 equiv) was added and stirred at room temperature for 19 h. The reaction was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After the reaction was completed, DCM (50 mL) was added and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 5:5 to 10:0, v / v) to give 20-13 (176 mg, 81%) as a pale yellow oil.

[0252] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.8 Hz, 12H), 1.03 (s, 3H), 1.07 (s, 3H), 1.21-1.31 (m, 53H), 1.61 (bs, 2H), 1.84-1.85 (m, 2H), 1.91-2.01 (m, 4H), 2.36-2.41 (m, 6H), 2.50 (t,J= 7.2 Hz, 2H), 2.56-2.59 (m, 4H), 3.22 (s, 2H), 3.30 (q,J= 6.2 Hz, 2H), 3.75 (t,J= 4.2 Hz, 4H), 3.87 (d,J= 11.0 Hz, 1H), 3.97 (d,J= 5.6 Hz, 4H), 4.07 (d,J= 11.0 Hz, 1H), 4.15-4.20 (m, 2H), 4.98 (s, 1H), 6.56 (t,J= 5.8 Hz, 1H).

[0253] ¹³C NMR (CDCl₃, 100 MHz): δ 14.09, 14.10, 20.03, 20.13, 20.81, 21.43, 22.63, 22.66, 26.63, 26.68, 28.70, 29.29, 29.55, 29.59, 29.94, 31.19, 31.80, 31.88, 32.97, 33.51, 33.21, 33.35, 35.67, 37.25, 37.43, 53.30, 59.53, 61.74, 66.75, 67.32, 67.41, 69.20, 76.63, 168.01, 170.49, 171.70, 172.62, 173.12.

[0254]

[0255] O,O'-(4-((3-((3-(Dimethylamino)propanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 20-1)

[0256]

[0257] 3-(Dimethylamino)propionic acid (40 mg, 1.3 equiv), EDCI.HCl (33 mg, 1.5 equiv), and 4-dimethylaminopyridine (DMAP; 3 mg, 0.25 equiv) were added to a 50 mL single-necked flask together with DCM (20 mL). The mixture was stirred vigorously for 5 min at room temperature under an argon atmosphere. Afterwards, alcohol 8 (100 mg, 1 equiv) was added and stirred at room temperature for 3.4 h. The disappearance of the alcohol was monitored by TLC (SiO₂; ethyl acetate / methanol, 9.5:0.5; PMA stain). After the reaction was completed, DCM (50 mL) was added and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 10:0 to 9.5:0.5, v / v) to obtain 20-1 (79 mg, 71%) as a clear oil.

[0258] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.2 Hz, 12H), 1.03 (s, 3H), 1.07 (s, 3H), 1.19-1.31 (m, 50H), 1.61 (bs, 2H), 1.81-1.84 (m, 2H), 1.93-2.01 (m, 4H), 2.23 (s, 6H), 2.35-2.42 (m, 6H), 2.46-2.53 (m, 4H), 2.60 (t,J= 7.1 Hz, 2H), 3.31 (q,J= 6.1 Hz, 2H), 3.87 (d,J=11.0 Hz, 1H), 3.97 (d,J= 5.7 Hz, 4H), 4.06 (d,J= 11.0 Hz, 1H), 4.10-4.17 (m, 2H), 4.99 (s, 1H), 6.62 (bs, 1H).

[0259] ¹³C NMR (CDCl₃, 100 MHz): δ 14.11, 14.12, 20.02, 20.13, 20.83, 21.44, 22.65, 22.68, 26.64, 26.69, 28.65, 29.32, 29.57, 29.61, 29.96, 31.20, 31.82, 31.90, 32.98, 33.02, 33.19, 33.22, 33.37, 35.42, 37.26, 37.43, 45.26, 54.87, 61.30, 67.32, 67.39, 69.25, 76.62, 167.99, 171.74, 172.91, 173.14.

[0260]

[0261] O,O'-((R)-2,2-Dimethyl-4-((3-((3-morpholinopropanoyl)oxy)propyl)amino)-4-oxobutane-1,3-diyl)bis(2-hexyldecyl) diglutarate (Compound 20-3)

[0262]

[0263] 3-Morpholinopropionic acid (47 mg, 1.3 equiv), EDCIㆍHCl (65 mg, 1.5 equiv), and 4-dimethylaminopyridine (DMAP; 6 mg, 0.2 equiv) were added to a 50 mL single-necked flask together with DCM (20 mL). The mixture was stirred vigorously for 5 min at room temperature under an argon atmosphere. Afterwards, alcohol 8 (200 mg, 1 equiv) was added and stirred at room temperature for 9 h. The reaction was monitored for alcohol disappearance by TLC (SiO₂; hexane / ethyl acetate, 9:1; PMA stain). After completion of the reaction, DCM (50 mL) was added and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 10:0 to 9.5:0.5, v / v) to obtain 20-3 (206 mg, 89%) as a clear oil.

[0264] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.1 Hz, 12H), 1.03 (s, 3H), 1.08 (s, 3H), 1.18-1.29 (m, 49H), 1.61 (bs, 2H), 1.79-1.84 (m, 2H), 1.93-2.01 (m, 4H), 2.35-2.40 (m, 6H), 2.42-2.45 (m, 4H), 2.49-2.52 (m, 4H), 2.67 (t,J= 7.0 Hz, 2H), 3.33 (q,J= 6.3 Hz, 2H), 3.67 (t,J= 4.4 Hz, 4H), 3.88 (d,J= 11.0 Hz, 1H), 3.98 (d,J= 5.7 Hz, 4H), 4.08 (d,J= 11.0 Hz, 1H), 4.11-4.18 (m, 2H), 4.99 (s, 1H), 6.63 (t,J= 5.9 Hz, 1H).

[0265] ¹³C NMR (CDCl₃, 100 MHz): δ 14.09, 14.10, 20.00, 20.11, 20.83, 21.40, 22.63, 22.66, 26.62, 26.67, 28.78, 29.29, 29.55, 29.59, 29.94, 31.18, 31.80, 31.88, 32.18, 32.95, 33.17, 33.20, 33.34, 35.38, 37.24, 37.41, 53.38, 54.00, 61.29, 68.88, 67.30, 67.37, 69.23, 76.62, 167.97, 171.72, 172.63, 172.83, 173.11.

[0266]

[0267] O,O'-(2,2-Dimethyl-4-oxo-4-((3-((3-(pyrrolidin-1-yl)propanoyl)oxy)propyl)amino)butane-1,3-diyl)bis(2-hexyldecyl) diglutarate (화합물20-5)

[0268]

[0269] 3-(Pyrrolidin-1-yl)propionic acid (42 mg, 1.3 equiv), EDCIㆍHCl (65 mg, 1.5 equiv), and 4-dimethylaminopyridine (DMAP; 6 mg, 0.2 equiv) were added to a 50 mL single-necked flask together with DCM (20 mL). The mixture was stirred vigorously for 5 min at room temperature under an argon atmosphere. Afterwards, alcohol 8 (200 mg, 1 equiv) was added and stirred at room temperature for 9 h. The disappearance of the alcohol was monitored by TLC (SiO₂; DCM / methanol, 9:1; PMA stain). After completion of the reaction, DCM (50 mL) was added and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; DCM / methanol, 10:0 to 8:2, v / v) to obtain 20-5 (217 mg, 85%) as a clear oil.

[0270] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.0 Hz, 12H), 1.03 (s, 3H), 1.07 (s, 3H), 1.19-1.29 (m, 50H), 1.61 (bs, 2H), 1.76-1.85 (m, 6H), 1.93-2.01 (m, 4H), 2.35-2.42 (m, 6H), 2.51-2.55 (m, 8H), 2.77 (t,J= 7.3 Hz, 2H), 3.30 (q,J= 6.2 Hz, 2H), 3.88 (d,J= 11.0 Hz, 1H), 3.88 (d,J= 5.64 Hz, 4H), 4.06 (d,J= 11.0 Hz, 1H), 4.11-4.18 (m, 2H), 4.99 (s, 1H), 6.63 (bs, 1H).

[0271] ¹³C NMR (CDCl₃, 100 MHz): δ 14.11, 14.12, 20.00, 20.12, 20.83, 21.41, 22.65, 22.68, 23.48, 26.64, 26.68, 28.71, 29.31, 29.57, 29.61, 29.95, 31.19, 31.81, 31.89, 32.97, 33.19, 33.21, 33.36, 34.23, 37.25, 37.43, 51.44, 53.97, 61.24, 67.31, 67.38, 69.25, 76.60, 167.99, 171.74, 172.66, 172.98, 173.13.

[0272]

[0273] O,O'-(2,2-Dimethyl-4-oxo-4-((3-((3-(piperidin-1-yl)propanoyl)oxy)propyl)amino)butane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 20-6)

[0274]

[0275] 3-(Piperidin-1-yl)propionic acid (46 mg, 1.3 equiv), EDCIㆍHCl (65 mg, 1.5 equiv), and 4-dimethylaminopyridine (DMAP; 6 mg, 0.2 equiv) were added to a 50 mL single-necked flask together with DCM (20 mL). The mixture was stirred vigorously for 5 min at room temperature under an argon atmosphere. Afterwards, alcohol 8 (200 mg, 1 equiv) was added and stirred at room temperature for 6 h. The disappearance of the alcohol was monitored by TLC (SiO₂; DCM / methanol, 9:1; PMA stain). After completion of the reaction, DCM (50 mL) was added and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; DCM / methanol, 10:0 to 8:2, v / v) to obtain 20-6 (174 mg, 75%) as a clear oil.

[0276] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.9 Hz, 12H), 1.03 (s, 3H), 1.07 (s, 3H), 1.19-1.30 (m, 51H), 1.41 (m, 4H), 1.51-1.57 (m, 4H), 1.62 (bs, 2H), 1.72-1.86 (m, 2H), 1.91-2.04 (m, 4H), 2.35-2.42 (m, 10H), 2.48-2.54 (m, 4H), 2.63 (t,J= 7.1 Hz, 2H), 3.32 (q,J=6.3 Hz, 2H), 3.87 (d,J= 11.0 Hz, 1H), 3.97 (d,J= 2.4 Hz, 4H), 4.07 (d,J= 10.9 Hz, 1H), 4.10-1.17 (m, 2H), 5.00 (s, 1H), 6.64 (t,J= 6.0 Hz, 1H).

[0277] ¹³C NMR (CDCl₃, 100 MHz): δ 14.11, 14.12, 19.99, 20.12, 20.84, 21.40, 22.65, 22.67, 24.27, 25.93, 26.64, 26.68, 28.76, 29.31, 29.56, 29.62, 29.95, 31.20, 31.81, 31.89, 32.46, 32.97, 33.20, 33.21, 33.36, 35.28, 37.25, 37.43, 54.29, 54.36, 61.11, 67.31, 67.37, 69.26, 76.61, 167.98, 171.74, 172.65, 173.11, 173.13, 173.27.

[0278]

[0279] O,O'-(2,2-Dimethyl-4-((3-((3-(4-methylpiperazin-1-yl)propanoyl)oxy)propyl)amino)-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 20-7)

[0280]

[0281] 3-(4-methylpiperazin-1-yl)propionic acid (51 mg, 1.3 equiv), EDCIㆍHCl (65 mg, 1.5 equiv), and 4-dimethylaminopyridine (DMAP; 5.53 mg, 0.2 equiv) were added to a 50 mL single-necked flask along with DCM (20 mL). The mixture was stirred vigorously for 5 min at room temperature under an argon atmosphere. Afterwards, alcohol 8 (200 mg, 1 equiv) was added and stirred at room temperature for 12 h. The disappearance of the alcohol was monitored by TLC (SiO₂; ethyl acetate / methanol, 9:1; PMA stain). After completion of the reaction, DCM (50 mL) was added and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 9:1 to 8:2, v / v) to give 20-7 (168 mg, 71%) as a clear oil.

[0282] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.9 Hz, 12H), 1.03 (s, 3H), 1.08 (s, 3H), 1.19-1.29 (m, 51H), 1.61 (bs, 2H), 1.80-1.83 (m, 2H), 1.91-2.01 (m, 4H), 2.27 (s, 3H), 2.35-2.44 (m, 10H), 2.46-2.52 (m, 7H), 2.68 (t,J= 7.2 Hz, 2H), 3.36 (q,J= 6.2 Hz, 2H), 3.88 (d,J=10.9 Hz, 1H), 3.97 (d,J= 5.6 Hz, 4H), 4.07 (d,J= 11.0 Hz, 1H), 4.10-1.18 (m, 2H), 5.00 (s, 1H), 6.64 (bs, 1H).

[0283] ¹³C NMR (CDCl₃, 100 MHz): δ 14.11, 19.99, 20.11, 20.85, 21.41, 22.64, 22.67, 26.69, 26.67, 28.77, 29.30, 29.56, 29.60, 29.94, 31.19, 31.80, 31.88, 32.41, 32.96, 33.18, 33.21, 33.35, 33.32, 37.24, 37.42, 46.02, 52.89, 53.59, 55.05, 61.19, 67.30, 67.37, 69.25, 76.60, 167.98, 171.73, 172.64, 173.01, 173.10, 173.12.

[0284]

[0285] O,O'-(4-((3-((4-(Dimethylamino)butanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (compound 20-9)

[0286]

[0287] 4-(Dimethylamino)butyric acid (39 mg, 1.3 equiv), EDCI.HCl (65 mg, 1.5 equiv), and 4-dimethylaminopyridine (DMAP; 5.53 mg, 0.2 equiv) were added to a 50 mL single-necked flask along with DCM (20 mL). The mixture was stirred vigorously for 5 min at room temperature under an argon atmosphere. Afterwards, alcohol 8 (200 mg, 1 equiv) was added and stirred at room temperature for 8 h. The disappearance of the alcohol was monitored by TLC (SiO₂; ethyl acetate / methanol, 9:1; PMA stain). After completion of the reaction, DCM (50 mL) was added and washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 10:0 to 9:1, v / v) to obtain 20-9 (194 mg, 86%) as a clear oil.

[0288] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 6.8 Hz, 12H), 1.03 (s, 3H), 1.07 (s, 3H), 1.19-1.29 (m, 50H), 1.61 (bs, 2H), 1.76-1.84 (m, 4H), 1.93-2.01 (m, 4H), 2.21 (s, 6H), 2.28 (t,J= 7.2 Hz, 2H), 2.33-2.41 (m, 8H), 2.51 (t,J= 7.2 Hz, 2H), 3.29 (q,J= 6.2 Hz, 2H), 3.87 (d,J= 11.0 Hz, 1H), 3.96-3.983.97 (d,J= 5.6 Hz, 4H), 4.07 (d,J= 11.0 Hz, 1H), 4.11-4.12 (m, 2H), 4.99 (s, 1H), 6.61 (t, J = 5.76 Hz, 1H).

[0289] ¹³C NMR (CDCl₃, 100 MHz): δ 14.10, 20.01, 20.12, 20.82, 21.42, 22.63, 22.66, 22.92, 26.63, 26.67, 28.75, 29.30, 29.55, 29.60, 29.94, 31.19, 31.80, 31.88, 31.99, 32.97, 33.17, 33.21, 33.35, 35.59, 37.25, 37.44, 45.39, 58.80, 61.31, 67.31, 67.38, 69.23, 76.60, 167.96, 171.70, 172.63, 173.10, 174.00.

[0290]

[0291] Example 1-3. Synthesis via amine alkylation after esterification

[0292]

[0293]

[0294] O,O'-(4-((3-((3-Bromopropanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 9)

[0295]

[0296] A 50 mL single-necked flask was charged with alcohol 8 (300 mg, 1 eq), EDCIㆍHCl (196 mg, 3 eq), and 4-dimethylaminopyridine (DMAP; 8 mg, 0.2 eq) together with DCM (20 mL). The mixture was cooled to 0°C, and 4-(dimethylamino)butyric acid (39 mg, 4 eq) dissolved in DCM was slowly added to the flask over 20 min. The mixture was then stirred vigorously at 60°C for 19 h. The disappearance of alcohol was monitored by TLC (SiO₂; ethyl acetate / hexane, 3:7; PMA stain). After completion of the reaction, DCM (50 mL) was added, and the residue was washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 1:9 to 5:5, v / v) to obtain bromide 9 (262 mg, 86%) as a clear oil.

[0297] ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 7.0 Hz, 12H), 1.03 (s, 3H), 1.08 (s, 3H), 1.19-1.31 (m, 50H), 1.61 (bs, 2H), 1.83-1.88 (bs, 2H), 1.91-2.01 (m, 4H), 2.36-2.42 (s, 6H), 2.49-2.51 (m, 2H), 2.94 (t,J= 6.6 Hz, 2H), 3.29-3.34 (m, 2H), 3.59 (t,J= 6.6 Hz, 2H), 3.87 (d,J= 11.0 Hz, 1H), 3.97 (d,J= 5.8 Hz, 4H), 4.06 (d,J= 10.9 Hz, 1H), 4.17-4.21 (m, 2H), 4.99 (s, 1H), 6.60 (bs, 1H).

[0298] ¹³C NMR (CDCl₃, 100 MHz): δ 14.11, 14.12, 20.03, 20.14, 20.86, 21.44, 22.65, 22.68, 26.00, 26.65, 26.69, 28.59, 29.31, 29.57, 29.61, 29.96, 31.21, 31.82, 31.90, 32.99, 33.19, 33.23, 33.37, 35.53, 37.25, 37.45, 37.68, 62.02, 67.33, 67.41, 69.24, 76.64, 168.06, 170.91, 171.74, 172.65, 173.15.

[0299]

[0300] O,O'-(4-((3-((3-(Diethylamino)propanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 20-2)

[0301]

[0302] A solution of bromide 9 (200 mg, 1 eq) in DCM (10 mL) was slowly added to a solution of triethylamine (60 mg, 3 eq) and diethylamine (29 mg, 2 eq) in DCM (20 mL) over 20 minutes. The resulting solution was stirred vigorously at room temperature for 5 hours, and the disappearance of bromide 9 was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After completion of the reaction, DCM (50 mL) was added, and the mixture was washed with water (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, from 10:0 to 9:1, v / v) to give 20-2 (196 mg, 99%) as a clear oil.

[0303] 1H NMR (CDCl₃, 400 MHz): δ 0.88 (t,J= 7.0 Hz, 12H), 1.00 (t,J= 7.2 Hz, 6H), 1.03 (s, 3H), 1.07 (s, 3H, 1.6H), 1.6H (1.19), 2H), 1.84–1.76 (m, 2H), 1.91–2.01 (m, 4H), 2.36–2.42 (m, 6H), 2.44–2.25 (m, 8H), 2.77 (t,J= 7.2 Hz, 2H (1q).8 Hz),J. 3.88 (d,J= 11.0 Hz, 1H), 3.97 (d,J= 5.8 Hz, 4H), 4.06 (d,J= 11.0 Hz, 1H), 4.10–4.17 (m, 2H), 5.00 (s, 6 Hz (H. 0,8).

[0304] ¹³C NMR (CDCl₃, 100 MHz): δ 11.82, 14.11, 14.13, 20.00, 20.12, 20.84, 21.41, 22.66, 22.68, 26.63, 26.64, 29.57, 29.62, 29.96, 31.20, 31.82, 31.90, 32.60, 32.97, 33.20, 33.22, 33.37, 35.29, 37.27, 34.48, 61.16, 67.31, 67.37, 69.26, 76.61, 167.98, 171.74, 172.66, 173.12, 173.14, 173.43.

[0305]

[0306] O,O'-(4-((3-((3-(1,4-Oxazepan-4-yl)propanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (화합물20-4)

[0307]

[0308] A solution of bromide 9 (150 mg, 1 eq) in DCM (10 mL) was slowly added to a solution of triethylamine (45 mg, 3 eq) and 1,4-oxaziphane (30 mg, 2 eq) in DCM (20 mL) over 20 minutes. The resulting solution was stirred vigorously at room temperature for 9 hours, and the disappearance of bromide 9 was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After completion of the reaction, DCM (50 mL) was added, and the residue was washed with water (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 5:5 to 10:0, v / v) to obtain 20-4 (151 mg, 99%) as a clear oil.

[0309] ¹H NMR (CDCl₃, 400 MHz): δ 0.82 (t,J= 6.9 Hz, 12H), 0.96 (s, 3H), 1.00 (s, 3H), 1.14-1.27 (m, 50H), 1.54 (bs, 2H), 1.71-1.74 (m, 2H), 1.75-1.77 (m, 2H), 1.82-1.96 (m, 4H), 2.28-2.34 (m, 6H), 4.43-2.46 (m, 4H), 2.63-2.67 (m, 4H), 2.81 (t,J= 6.8 Hz, 2H), 3.25 (q,J= 6.3 Hz, 2H), 3.63-3.65 (m, 2H), 3.70 (t,J= 5.9 Hz, 2H), 3.81 (d,J= 11.0 Hz, 1H), 3.90 (d,J= 5.6 Hz, 4H), 3.99 (d,J= 11.0 Hz, 1H), 4.05-4.09 (m, 2H), 4.91 (s, 1H), 6.59 (bs, 1H).

[0310] ¹³C NMR (CDCl₃, 100 MHz): δ 14.08, 14.09, 20.01, 20.12, 20.82, 21.41, 22.62, 22.65, 26.63, 26.67, 28.78, 29.29, 29.54, 29.59, 29.93, 31.20, 31.79, 31.87, 32.97, 33.19, 33.21, 33.35, 35.44, 37.26, 37.42, 53.29, 53.63, 57.37, 61.34, 67.31, 67.38, 68.53, 68.86, 69.24, 76.65, 168.00, 171.72, 172.62, 172.89, 173.10.

[0311]

[0312] Example 1-4. Synthesis via Michael addition after alcoholysis of acyl halide

[0313]

[0314]

[0315] 1-(2-((Tert-butyldiphenylsilyl)oxy)ethyl)piperazine (Compound 10)

[0316]

[0317] A solution of tert-butylchlorodiphenylsilane (2.53 g, 1.2 equiv) in DCM (10 mL) was added dropwise at 0°C to a solution of 2-(piperazin-1-yl)ethan-1-ol 9 (1 g, 1 equiv), DMAP (9 mg, 0.1 equiv), and pyridine (1 g, 1.6 equiv) in DCM (40 mL). The mixture was warmed to room temperature and stirred for 13 h. The progress of the reaction was monitored by TLC (SiO₂; DCM / MeOH, 9:1; PMA stain). After completion of the reaction, the reaction mixture was diluted with DCM (50 mL) and washed with water (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained oil was purified through flash column chromatography (SiO₂; DCM / MeOH, 10:0 to 9:1, v / v) to obtain the target substance 10 (2 g, 71%) as a white solid.

[0318] ¹H NMR (CDCl₃, 400 MHz): δ 1.04 (s, 9H), 2.67 (s, 2H), 2.88 (s, 4H), 3.22 (s, 4H), 3.81 (s, 2H), 7.45-7.37 (m, 6H), 7.65 (dd,J= 7.8, 1.2 Hz, 4H), 9.61 (bs, 2H).

[0319] ¹³C NMR (CDCl₃, 100 MHz): δ 19.15, 26.81, 43.56, 50.26, 59.74, 62.11, 76.70, 127.73, 129.77, 133.48, 135.54.

[0320]

[0321] Bis(2-((tert-butyldimethylsilyl)oxy)ethyl)amine (compound 12)

[0322]

[0323] Diethanolamine 11 (1 g, 1 equiv) was dissolved in anhydrous DCM (40 mL), and imidazole (2 g, 3 equiv) was added while stirring at 0°C. Then, tert-butyldimethylsilyl chloride (3.6 g, 2.5 equiv) was dissolved in DCM (10 mL) and added dropwise. The mixture was allowed to warm to room temperature and stirred for 13 h while reacting. The progress of the reaction was monitored by TLC (SiO₂; EA / hexane, 5:5; PMA stain). After completion of the reaction, the reaction mixture was diluted with DCM (50 mL) and washed with ammonia water (2 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained crude product was purified through flash column chromatography (SiO₂; EA / hexane, 1:9 to 5:5, v / v) to obtain the target substance 12 (2.82 g, 89%) as a colorless viscous oil.

[0324] ¹H NMR (CDCl₃, 400 MHz): δ 0.00 (s, 12H), 0.85 (s, 18H), 1.85 (s, 1H), 2.66 (t,J= 5.5 Hz, 4H), 3.67 (t,J= 5.4 Hz, 4H).

[0325] ¹³C NMR (CDCl₃, 100 MHz): δ -5.34, 18.31, 25.95, 51.81, 65.61.

[0326]

[0327] 2-((Tert-butyldimethylsilyl)oxy)-N-methylethan-1-amine (Compound 14)

[0328]

[0329] 2-(Methylamino)ethan-1-ol 13 (1 g, 1 equiv) was dissolved in anhydrous DCM (40 mL), stirred at 0°C, and imidazole (2.71 g, 3 equiv) was added. Tert-butyldimethylsilyl chloride (4 g, 2 equiv) dissolved in DCM (10 mL) was then added dropwise. The mixture was allowed to warm to room temperature and stirred for 11 h. The reaction progress was monitored by TLC (SiO₂; DCM / MeOH, 9:1; PMA stain). After completion of the reaction, the reaction mixture was diluted with DCM (50 mL) and washed with saturated sodium bicarbonate solution (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained crude product was purified through flash column chromatography (SiO₂; DCM / MeOH, 10:0 to 9:1, v / v) to obtain the target substance 14 (1.91 g, 76%) as a clear oil.

[0330] ¹H NMR (CDCl₃, 400 MHz): δ 0.00 (s, 6H), 0.83-0.84 (m, 9H), 2.38 (s, 3H), 2.60 (t,J= 5.2 Hz, 2H), 3.65 (t,J= 5.3 Hz, 2H).

[0331] ¹³C NMR (CDCl₃, 100 MHz): δ -5.37, 18.30, 25.91, 36.21, 33.76, 62.08.

[0332]

[0333] O,O'-(4-((3-(Acryloyloxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 15)

[0334]

[0335] Alcohol 8 and triethylamine were added to anhydrous DCM (40 mL), and the mixture was cooled to 0°C in an ice-water bath. The heterogeneous mixture was stirred for 15 minutes, and then acryloyl chloride (23.41 mL, 0.288 mol) dissolved in DCM (10 mL) was added slowly over 30 minutes. The flask was removed from the ice-water bath, and the reaction was allowed to proceed at room temperature for 3 hours. The progress of the reaction was monitored by TLC (SiO₂; hexane / EA, 8:2; PMA staining). After complete consumption of alcohol 8, the reaction mixture was washed with water (2 x 50 mL) and saturated sodium bicarbonate solution (2 x 50 mL) to remove the formed triethylamine hydrochloride and unreacted acryloyl chloride. The organic layer was dried over anhydrous Na₂SO₄, and DCM was removed under reduced pressure to obtain the crude product. This preparation was purified by flash column chromatography (SiO₂; hexane / EA, from 8:2 to 6:4, v / v) to obtain the target substance 15 (1.91 g, 76%) as a clear oil.

[0336] ¹H NMR (CDCl₃, 400 MHz): δ 0.81 (t,J= 6.0 Hz, 12H), 0.97 (s, 3H), 1.01 (s, 3H), 1.15-1.29 (m, 50H), 1.54 (bs, 2H), 1.77-1.84 (m, 2H), 1.85-1.94 (m, 4H), 2.28-2.34 (m, 6H), 4.44 (t,J= 7.0 Hz, 2H), 3.21-3.26 (m, 2H), 3.80 (d,J= 12.0 Hz, 1H), 3.90 (d,J= 5.7 Hz, 4H), 4.00 (d,J= 11.0 Hz, 1H), 4.14 (t,J= 5.9 Hz, 2H), 4.93 (s, 1H), 5.78 (dd,J= 10.4, 1.1 Hz, 1H), 6.06 (dd,J= 17.3, 10.4 Hz, 1H), 6.35 (dd,J= 17.3, 1.2 Hz, 1H), 6.61 (t,J= 6.0 Hz, 1H).

[0337] ¹³C NMR (CDCl₃, 100 MHz): δ 14.07, 19.99, 20.11, 20.81, 21.42, 22.61, 22.64, 26.61, 26.65, 28.69, 29.27, 29.53, 29.57, 29.91, 31.18, 31.78, 31.86, 32.94, 33.15, 33.19, 33.33, 35.59, 37.24, 37.42, 31.57, 67.28, 67.35, 69.22, 76.59, 128.16, 131.14, 166.45, 168.00, 171.69, 172.60, 173.09.

[0338]

[0339] Bis(2-hexyldecyl) O,O'-(4-((3-((3-(4-(2-hydroxyethyl)piperazin-1-yl)propanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) diglutarate (화합물20-8)

[0340]

[0341] 1-(2-((Tert-butyldiphenylsilyl)oxy)ethyl)piperazine 10 (94 mg, 1.2 equiv, Michael donor) and methanol / DCM (7:3, 20 mL) were added to a single-necked flask prepared in an anhydrous state, and the heterogeneous mixture was stirred at room temperature for 10 min. Subsequently, alkene 15 (200 mg, 1 equiv, Michael acceptor) dissolved in DCM (10 mL) was slowly added over 10 min. The reaction mixture was heated to 35°C and stirred under a nitrogen atmosphere for 13 h. After completion of the reaction, the mixture was diluted with DCM (50 mL) and washed with saturated sodium bicarbonate solution (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue (Michael adduct) was dissolved in THF (20 mL) and stirred at 20°C for 5 min. 1 M nBu₄NF solution (1 mL, 1.5 equiv) dissolved in THF was slowly added at room temperature over 10 min, and the reaction mixture was stirred for 1 h. After completion of the reaction, the mixture was diluted with water (2 x 50 mL) and extracted with DCM (3 x 40 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the crude product. This crude product was purified by flash column chromatography (SiO₂; EA / MeOH, from 10:0 to 7:2, 1:19, v / v) to obtain the target substance 20-8 (139 mg, 61%, reaction yield in two steps) as a colorless oil.

[0342] ¹H NMR (CDCl₃, 400 MHz): δ 0.81 (t,J= 7.0 Hz, 12H), 0.97 (s, 3H), 1.01 (s, 3H), 1.14-1.29 (m, 50H), 1.55 (bs, 2H), 1.75-1.76 (m, 2H), 1.88-1.92 (m, 4H), 2.24-2.33 (m, 6H), 2.42-2.53 (m, 4H), 2.54-2.68 (m, 4H), 2.71 (m, 2H), 3.01 (bs, 1H), 3.21-3.25 (m, 2H), 3.55 (t,J= 5.2 Hz, 4H), 3.82 (d,J= 10.0 Hz, 1H), 3.75 (d,J= 5.8 Hz, 4H), 4.00 (d,J= 10.1 Hz, 1H), 4.01-4.02 (m, 2H), 4.86 (s, 1H), 6.77 (bs, 1H).

[0343] ¹³C NMR (CDCl₃, 100 MHz): δ 14.07, 19.97, 20.06, 20.77, 21.45, 22.64, 22.65, 26.64, 26.66, 28.44, 29.27, 29.55, 29.58, 29.91, 31.18, 31.75, 31.85, 32.85, 32.96, 33.16, 33.17, 33.38, 35.49, 37.20, 37.34, 41.63, 52.66, 58.37, 58.78, 61.43, 67.24, 67.35, 69.26, 76.67, 168.11, 171.71, 172.62, 172.72, 173.03, 173.13.

[0344]

[0345] O,O'-(4-((3-((3-(Bis(2-hydroxyethyl)amino)propanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (화합물20-14)

[0346]

[0347] Bis(2-((tert-butyldimethylsilyl)oxy)ethyl)amine 12 (85 mg, 1.2 equiv, Michael donor) and methanol / DCM (7:3, 20 mL) were added to a dried single-necked flask, and the heterogeneous mixture was stirred at room temperature for 10 min. Then, alkene 15 (200 mg, 1 equiv, Michael acceptor) dissolved in DCM (10 mL) was slowly added over 10 min. The reaction mixture was heated to 35°C and stirred under a nitrogen atmosphere for 8 h. After the reaction was completed, the mixture was diluted with DCM (50 mL) and washed with saturated sodium bicarbonate solution (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue (Michael adduct) was dissolved in THF (20 mL) and stirred at 20°C for 5 min. 1 M nBu₄NF solution (1 mL, 1.5 equiv) dissolved in THF was slowly added at room temperature over 10 min, and the reaction mixture was stirred for 1 h. After completion of the reaction, the mixture was diluted with water (2 x 50 mL) and extracted with DCM (3 x 40 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated to obtain the crude product. This crude product was purified by flash column chromatography (SiO₂; EA / MeOH, from 10:0 to 9:1, 1:19, v / v) to obtain the target compound 20-14 (158 mg, 71%, yield in 2 steps) as a colorless oil.

[0348] ¹H NMR (CDCl₃, 400 MHz): δ 0.81 (t,J= 6.1 Hz, 12H), 0.96 (s, 3H), 0.99 (s, 3H), 1.15-1.28 (m, 51H), 1.54 (bs, 2H), 1.77-1.78 (m, 2H), 1.83-1.93 (m, 4H), 2.28-3.34 (m, 6H), 2.40-2.45 (m, 2H), 2.49-2.52 (m, 2H), 2.65-2.66 (m, 4H), 2.85-2.88 (m, 2H), 3.32-3.28 (m, 2H), 3.44 (bs, 2H), 3.58 (t,J= 4.5 Hz, 4H), 3.80 (d,J= 11.0 Hz, 1H), 3.90 (d,J= 5.1 Hz, 4H), 3.97 (d,J= 10.0 Hz, 1H), 4.04-4.05 (m, 2H), 4.85 (s, 1H), 6.60 (bs, 1H).

[0349] ¹³C NMR (CDCl₃, 100 MHz): δ 14.03, 19.91, 20.02, 20.72, 21.42, 22.64, 22.66, 26.63, 26.67, 28.78, 29.30, 29.60, 29.94, 31.19, 31.80, 31.88, 32.19, 32.95, 33.17, 33.35, 35.38, 37.25, 37.42, 53.39, 54.00, 61.30, 66.89, 67.31, 67.38, 69.23, 76.62, 167.98, 171.73, 172.64, 172.84, 173.12.

[0350]

[0351] Bis(2-hexyldecyl)O,O'-(4-((3-((3-((2-hydroxyethyl)(methyl)amino)propanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) diglutarate (화합물20-15)

[0352]

[0353] 2-((Tert-butyldimethylsilyl)oxy)-N-methylethan-1-amine 14 (49 mg, 1.2 equiv, Michael donor) and methanol / DCM (7:3, 20 mL) were added to a dried single-necked flask, and the heterogeneous mixture was stirred at room temperature for 10 min. Then, alkene 15 (200 mg, 1 equiv, Michael acceptor) dissolved in DCM (10 mL) was slowly added over 10 min. The reaction mixture was heated to 35 °C and stirred under a nitrogen atmosphere for 10 h. After the reaction was completed, the mixture was diluted with DCM (50 mL) and washed with saturated sodium bicarbonate solution (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue (Michael adduct) was dissolved in THF (20 mL) and stirred at 20°C for 5 min. 1 M nBu₄NF solution (1 mL, 1.5 equiv) dissolved in THF was slowly added thereto at room temperature over 10 min, and the reaction mixture was stirred for 1 h. After completion of the reaction, the mixture was diluted with water (2 x 50 mL) and extracted with DCM (3 x 40 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated to obtain the crude product. This crude product was purified by flash column chromatography (SiO₂; EA / MeOH, from 10:0 to 9.75:0.25, v / v) to obtain the target compound 20-15 (162 mg, 75%, yield in two steps) as a colorless oil.

[0354] ¹H NMR (CDCl₃, 400 MHz): δ 0.80 (t,J= 7.0 Hz, 12H), 0.96 (s, 3H), 1.00 (s, 3H), 1.14-1.28 (m, 50H), 1.54 (bs, 2H), 1.74-1.77 (m, 2H), 1.85-1.91 (m, 4H), 2.23 (s, 3H), 2.23-2.34 (m, 6H), 2.41-2.50 (m, 4H), 2.51-2.69 (m, 2H), 2.70 (t,J= 6.9 Hz, 2H), 3.02 (bs, 1H), 3.24 (q,J= 6.2 Hz, 2H), 3.53 (t,J= 5.2 Hz, 2H), 3.80 (d,J= 11.0 Hz, 1H), 3.90 (d,J= 5.8 Hz, 4H), 3.99 (d,J= 11.0 Hz, 1H), 4.08-4.04 (m, 2H), 4.89 (s, 1H), 6.76 (bs, 1H).

[0355] ¹³C NMR (CDCl₃, 100 MHz): δ 14.06, 19.98, 20.09, 20.74, 21.41, 22.60,

[0356]

[0357] Example 1-5. Synthesis of carbamates, carbonates, and thiocarbonates using carbonyldiimidazole as a mediator.

[0358]

[0359]

[0360] 2-(Dimethylamino)ethane-1-thiol (compound 16)

[0361]

[0362] A 50 mL round-bottomed flask was dried overnight, degassed, filled with nitrogen, and placed in an ice-brine bath. Dimethylamine (5 mL, 2 M, THF solution, 1 equiv) was added to the flask, followed by dropwise addition of ethylene sulfide (0.80 g, 1.3 equiv) dissolved in 20 mL of THF. The reaction mixture was stirred for 2 h under nitrogen while slowly heating from 0°C to room temperature. The reaction progress was monitored by TLC (SiO2; DCM / hexane, 9:1; KMnO4 stain). After completion of the reaction, the slightly yellow solution was anaerobically passed through Celite to remove solid impurities formed during the reaction. The pale yellow filtrate was concentrated to a pale yellow oil, which was then purified by fractional distillation to give compound 16 (0.82 g, 78%). This compound was used immediately without further purification.

[0363] ¹H NMR (CDCl₃, 400 MHz): δ 2.25 (s, 6H), 2.48-2.50 (m, 2H), 2.60 (t,J= 6.9 Hz, 2H).

[0364] ¹³C NMR (CDCl₃, 100 MHz): δ 23.90, 46.21, 62.22.

[0365]

[0366] Bis(2-hexyldecyl)O,O'-(2,14,14-trimethyl-6,12-dioxo-7-oxa-2,5,11-triazapentadecane-13,15-diyl) diglutarate (Compound 21-1)

[0367]

[0368] A 50 mL single-necked flask was charged with 1,1'-carbonyldiimidazole (CDI; 55 mg, 1.5 equiv) and anhydrous DCM (10 mL) to form a white suspension, which was then cooled to 0 °C in an ice bath. A solution of alcohol 8 (200 mg, 1 equiv) in DCM (15 mL) was added using a dropping funnel over 30 min to form a homogeneous solution. Subsequently, dimethylethanolamine (DMEA; 61 mg, 3 equiv) and triethylamine (69 mg, 3 equiv) were added, and the mixture was stirred vigorously at room temperature for 18 h. The reaction mixture was diluted with DCM (50 mL), washed with water (3 × 30 mL), saturated brine (1 × 30 mL), dried over Na₂SO₄, filtered, and the solvent was evaporated in vacuo. The resulting oil was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, from 10:0 to 9:1) to obtain clear oil 21-1 (160 mg, 71%).

[0369] ¹H NMR (CDCl₃, 400 MHz): δ 0.83 (t,J= 6.2 Hz, 12H), 0.99 (s, 3H), 1.04 (s, 3H), 1.20-1.29 (m, 50H), 1.58 (bs, 2H), 1.74 (bs, 2H), 1.89-1.94 (m, 4H), 2.20 (s, 6H), 2.33-2.46 (m, 8H), 2.44-2.52 (m, 2H), 3.20-3.26 (m, 4H), 3.85 (d,J= 10.8 Hz, 1H), 3.93 (d,J= 4.5 Hz, 4H), 4.01-4.07 (m, 3H), 4.96 (s, 1H), 5.39 (bs, 1H).

[0370] ¹³C NMR (CDCl₃, 100 MHz): δ 14.04, 19.96, 20.09, 35.40, 156.96, 173.03.

[0371]

[0372] O,O'-(2,2-Dimethyl-4-((3-(((2-morpholinoethyl)carbamoyl)oxy)propyl)amino)-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 21-2)

[0373]

[0374] A 50 mL single-necked flask was equipped with a stirrer and a 25 mL equilibrium pressure separatory funnel, which was then dried over fire in an argon atmosphere. 1,1'-Carbonyldiimidazole (CDI, 55 mg, 1.5 equivalents) and anhydrous DCM (10 mL) were quickly added to the flask to form a white suspension, which was then cooled to 0°C in an ice bath. The flask was opened with a syringe, and alcohol 8 (200 mg, 1 equivalent) was dissolved in DCM (15 mL), charged into the separatory funnel, and stirred dropwise for 30 minutes to ensure that the mixture became a homogeneous solution. After rinsing the separatory funnel with additional DCM (5 mL) and stirring at room temperature for approximately 3-5 h, it was confirmed that alcohol 8 and CDI had reacted to form the alkoxycarbonylimidazole intermediate 17 (TLC, SiO₂; ethyl acetate / hexane, 7:3; PMA staining). 4-(2-aminoethyl)morpholine (89 mg, 3 eq) and triethylamine (92 mg, 4 eq) were added to intermediate 17 and stirred at room temperature for 13 h. The reaction progress was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After completion of the reaction, the mixture was diluted with DCM (50 mL) and washed with water (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, from 10:0 to 9:1, v / v) to obtain 21-2 (172 mg, 73%) as a clear oil.

[0375] <h2 style=";text-align:left;direction:ltr">¹H NMR (CDCl₃, 400 MHz): δ 0.81 (t,J= 6.0 Hz, 12H), 0.96 (s, 3H), 1.01 (s, 3H), 1.15-1.27 (m, 51H), 1.55 (bs, 2H), 1.71 (bs, 2H), 1.84-1.94 (m, 4H), 2.28-2.38 (m, 6H), 2.40-2.50 (m, 8H), 3.25 (m, 4H), 3.69 (bs, 4H), 3.82 (d,J= 11.0 Hz, 1H), 3.90 (d,J= 6.0 Hz, 4H), 3.99 (d,J= 11.0 Hz, 1H), 4.41-4.08 (m, 2H), 4.93 (s, 1H), 6.73 (bs, 1H), 7.20 (bs, 1H).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0376] <h2 style=";text-align:left;direction:ltr"> ¹³C NMR (CDCl₃, 100 MHz): δ 13.95, 19.89, 20.01, 20.72, 21.34, 22.48, 22.51, 26.50, 26.53, 29.14, 29.40, 29.44, 29.78, 31.09, 31.65, 31.74, 32.84, 33.04, 33.17, 35.28, 35.28, 37.07, 37.14, 37.25, 53.24, 57.47, 61.52, 66.47, 67.06, 67.10, 69.14, 76.62, 156.84, 167.80, 171.66, 172.37, 172.82.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0377] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0378] <h2 style=";text-align:left;direction:ltr"> Bis(2-hexyldecyl)O,O'-(2,14,14-trimethyl-6,12-dioxo-5,7-dioxa-2,11-diazapentadecane-13,15-diyl) diglutarate (화합물22-1)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0379] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0380] A 50 mL single-necked flask was equipped with a stirrer and a 25 mL equilibrium pressure dropping funnel, which was then dried over fire in an argon atmosphere. 1,1'-Carbonyldiimidazole (CDI, 55 mg, 1.5 equivalents) and anhydrous DCM (10 mL) were quickly added to the flask to form a white suspension, which was then cooled to 0°C in an ice bath. The flask was opened, and alcohol 8 (200 mg, 1 equivalent) was dissolved in DCM (15 mL), charged into the dropping funnel, and stirred dropwise for 30 minutes to ensure that the mixture became a homogeneous solution. After rinsing the dropping funnel with additional DCM (5 mL), it was removed and stirred at room temperature for approximately 3–5 h to confirm that alcohol 8 and CDI had reacted to form the alkoxycarbonylimidazole intermediate 17 (TLC, SiO₂; ethyl acetate / hexane, 7:3; PMA staining). Dimethylethanolamine (DMEA, 61 mg, 3 equiv), triethylamine (69 mg, 3 equiv), and DMAP (33 mg, 1.2 equiv) were added to the reaction mixture, and the mixture was stirred at room temperature for 18 h. After the reaction was completed, the mixture was diluted with DCM (50 mL) and washed with water (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, from 10:0 to 9:1, v / v) to obtain 22-1 (163 mg, 72%) as a clear oil.

[0381] 1H NMR (CDCl₃, 400 MHz): δ 0.83 (t,J= 6.1 Hz, 12H), 0.98 (s, 3H), 1.03 (s, 3H), 1.15–1.30 (m, 50H), 1.57 (b, 1. 2 1.86–1.96 (m, 4H), 2.24 (s, 6H), 2.31–2.37 (m, 6H), 2.46 (t,J= 7.3 Hz, 2H), 2.55 (t,J= 5.6 Hz, 2H), 3.21 (d,J=3,3.3. 11.0 Hz, 1H), 3.93 (d,J= 5.6 Hz, 4H), 4.03 (d,J= 11.0 Hz, 1H), 4.14 (t,J= 5.9 Hz, 2H), 4.94 (s, 1H), 6.6= 1.8 (J).

[0382] ¹³C NMR (CDCl₃, 100 MHz): δ 14.03, 19.97, 20.09, 20.73, 21.40, 22.57, 22.60, 26.58, 26.62, 4 29.53, 29.57, 29.88, 31.16, 31.74, 31.82, 32.90, 33.11, 33.15, 33.29, 35.60, 37.22, 37.38, 45.55, 57.61, 3.65.21. 67.30, 69.16, 76.56, 155.42, 167.97, 171.61, 172.52, 173.00.

[0383]

[0384] O,O'-(2,2-Dimethyl-4-((3-(((2-morpholinoethoxy)carbonyl)oxy)propyl)amino)-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (화합물22-2)

[0385]

[0386] A 50 mL single-necked flask was equipped with a stirrer and a 25 mL equilibrium pressure dropping funnel, which was then dried over fire in an argon atmosphere. 1,1'-Carbonyldiimidazole (CDI, 55 mg, 1.5 equivalents) and anhydrous DCM (10 mL) were quickly added to the flask to form a white suspension, which was then cooled to 0°C in an ice bath. The flask was opened, and alcohol 8 (200 mg, 1 equivalent) was dissolved in DCM (15 mL), filled into the dropping funnel, and stirred dropwise for 30 minutes to ensure that the mixture became a homogeneous solution. After rinsing the dropping funnel with additional DCM (5 mL), it was removed and stirred at room temperature for approximately 3–5 h to confirm that alcohol 8 and CDI had reacted to form the alkoxycarbonylimidazole intermediate 17 (TLC, SiO₂; ethyl acetate / hexane, 7:3; PMA staining). 2-Morpholinoethanol (89 mg, 3 equiv), triethylamine (69 mg, 3 equiv), and DMAP (83 mg, 3 equiv) were then added to intermediate 17 and stirred at 40°C for 18 h. After completion of the reaction, the mixture was diluted with DCM (50 mL) and washed with water (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 5:5 to 10:0, v / v) to obtain 22-2 (162 mg, 69%) as a clear oil.

[0387] <h2 style=";text-align:left;direction:ltr">¹H NMR (CDCl₃, 400 MHz): δ 0.81 (t,J= 6.9 Hz, 12H), 0.96 (s, 3H), 1.00 (s, 3H), 1.14-1.28 (m, 51H), 1.54 (bs, 2H), 1.79-1.84 (m, 2H), 1.85-1.94 (m, 4H), 2.28-2.34 (m, 6H), 2.41-2.45 (m, 6H), 2.59 (t,J= 5.5 Hz, 2H), 3.25-3.29 (m, 2H), 3.63 (t,J= 4.4 Hz, 4H), 3.80 (d,J= 11.0 Hz, 1H), 3.91 (d,J= 5.6 Hz, 4H), 3.99 (d,J= 11.0 Hz, 1H), 4.11 (t,J= 6.0 Hz, 2H), 4.19 (t,J= 5.6 Hz, 2H), 4.91 (s, 1H), 6.53 (bs, 1H).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0388] <h2 style=";text-align:left;direction:ltr"> ¹³C NMR (CDCl₃, 100 MHz): δ 14.04, 14.14, 20.00, 20.11, 20.77, 21.41, 22.58, 22.61, 26.60, 26.64, 28.65, 29.24, 29.50, 29.89, 31.19, 31.75, 31.83, 32.93, 33.18, 33.32, 35.67, 37.25, 37.42, 53.75, 57.00, 64.84, 65.33, 66.74, 67.28, 67.36, 69.17, 76.62, 155.35, 167.99, 171.62, 172.54, 173.03.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0389] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0390] <h2 style=";text-align:left;direction:ltr"> Bis(2-hexyldecyl)O,O'-(2,14,14-trimethyl-6,12-dioxo-7-oxa-5-thia-2,11-diazapentadecane-13,15-diyl) diglutarate (화합물23-1)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0391] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0392] A 50 mL single-necked flask was equipped with a stirrer and a 25 mL equilibrium pressure dropping funnel, which was then dried over fire in an argon atmosphere. 1,1'-Carbonyldiimidazole (CDI, 55 mg, 1.5 equivalents) and anhydrous DCM (10 mL) were added to the flask to form a white suspension, which was then cooled to 0°C in an ice bath. The flask was opened, and alcohol 8 (200 mg, 1 equivalent) was dissolved in DCM (15 mL), filled into the dropping funnel, and stirred dropwise for 30 minutes to ensure that the mixture became a homogeneous solution. After rinsing the dropping funnel with additional DCM (5 mL), it was removed and stirred at room temperature for approximately 3–5 h to confirm that alcohol 8 and CDI had reacted to form the alkoxycarbonylimidazole intermediate 17 (TLC, SiO₂; ethyl acetate / hexane, 7:3; PMA staining). 2-(Dimethylamino)ethane-1-thiol (71 mg, 3 eq), triethylamine (69 mg, 3 eq), and DMAP (83 mg, 3 eq) were added to the mixture, which was stirred at room temperature for 10 h. The reaction progress was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After completion of the reaction, the mixture was diluted with DCM (50 mL) and washed with water (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 10:0 to 9.5:0.5, v / v) to obtain 23-1 (172 mg, 75%) as a clear oil.

[0393] 1H NMR (CDCl₃, 400 MHz): δ 0.81 (t,J= 6.2 Hz, 12H), 0.96 (s, 3H), 1.00 (s, 3H), 1.12-1.29 (m, 49H), 1.81-H NMR (bs, 1. 1. 1.84–1.94 (m, 4H), 2.23 (s, 6H), 2.28–2.34 (m, 6H), 2.43 (t,J= 7.2 Hz, 2H), 2.51 (t,J= 7.2 Hz, 2H), 2.3, 2.6 (J, J (q,J= 6.3 Hz, 2H), 3.80 (d,J= 11.0 Hz, 1H), 3.91 (d,J= 5.6 Hz, 4H), 4.00 (d,J= 11.0 Hz, 1H), 4.19 (t,J= 4. 2H (H), 4.19. 6.49 (bs, 1H).

[0394] ¹³C NMR (CDCl₃, 100 MHz): δ 14.04, 14.05, 20.04, 20.15, 20.80, 21.44, 22.60, 22.63, 26.63, 28.66, 28.67, 29.52, 29.57, 29.92, 31.22, 31.77, 31.86, 33.16, 33.22, 33.36, 35.87, 37.87, 37.29, 37.47, 54.03, 67.33, 67.42, 69.21, 76.65, 168.02, 171.40, 171.63, 172.56, 173.04.

[0395]

[0396] O,O'-(2,2-Dimethyl-4-((3-(((((2-morpholinoethyl)thio)carbonyl)oxy)propyl)amino)-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (화합물23-2)

[0397]

[0398] A 50 mL single-necked flask was equipped with a stirrer and a 25 mL equilibrium pressure dropping funnel, which was then dried over fire in an argon atmosphere. 1,1'-Carbonyldiimidazole (CDI, 55 mg, 1.5 equivalents) and anhydrous DCM (10 mL) were added to the flask to form a white suspension, which was then cooled to 0°C in an ice bath. The flask was opened, and alcohol 8 (200 mg, 1 equivalent) was dissolved in DCM (15 mL), filled into the dropping funnel, and stirred dropwise for 30 minutes to ensure that the mixture became a homogeneous solution. After rinsing the dropping funnel with additional DCM (5 mL), it was removed and stirred at room temperature for approximately 3–5 h to confirm that alcohol 8 and CDI had reacted to form the alkoxycarbonylimidazole intermediate 17 (TLC, SiO₂; ethyl acetate / hexane, 7:3; PMA staining). 2-Morpholinoethane-1-thiol (100 mg, 3 eq), triethylamine (69 mg, 3 eq), and DMAP (83 mg, 3 eq) were added to the mixture, which was stirred at 40°C for 20 h. The progress of the reaction was monitored by TLC (SiO₂; ethyl acetate / hexane, 7:3; PMA staining). After completion of the reaction, the mixture was diluted with DCM (50 mL) and washed with water (3 x 30 mL) and brine (1 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 5:5 to 10:0, v / v) to obtain 23-2 (168 mg, 69%) as a clear oil.

[0399] <h2 style=";text-align:left;direction:ltr">¹H NMR (CDCl₃, 400 MHz): δ 0.82 (t,J= 6.9 Hz, 12H), 0.97 (s, 3H), 1.01 (s, 3H), 1.12-1.30 (m, 51H), 1.55 (bs, 2H), 1.78-1.83 (m, 2H), 1.85-1.93 (m, 4H), 2.28-2.35 (m, 6H), 2.42-2.47 (m, 6H), 2.60 (t,J= 5.5 Hz, 2H), 2.93 (t,J= 6.0 Hz, 2H), 3.26-3.27 (m, 2H), 3.62 (t,J= 4.5 Hz, 4H), 8.80 (d,J= 11.0 Hz, 1H), 3.91 (d,J= 5.6 Hz, 4H), 3.96 (d,J= 11.0 Hz, 1H), 4.20 (t,J= 5.6 Hz, 2H), 4.92 (s, 1H), 6.53 (bs, 1H).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0400] <h2 style=";text-align:left;direction:ltr"> ¹³C NMR (CDCl₃, 100 MHz): δ 14.05, 14.12, 20.22, 20.12, 20.78, 21.42, 22.59, 22.62, 26.61, 26.65, 28.66, 29.25, 29.51, 29.88, 31.18, 31.74, 31.84, 32.94, 33.17, 33.31, 35.66, 37.24, 37.43, 53.74, 57.01, 64.85, 65.32, 66.74, 67.29, 67.36, 69.16, 76.62, 155.36, 167.99, 171.61, 172.53, 173.01.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0401] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0402] <h2 style=";text-align:left;direction:ltr"> 실시예 2. 실험방법<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0403] Lipid nanoparticle formulation: All lipid compounds were dissolved in a 1:1 mixture of chloroform and methanol at a concentration of 10 μg / μL, and then mixed according to the lipid nanoparticle composition. The chloroform and methanol were removed by vacuum distillation and dissolved again in ethanol. mRNA was prepared by dissolving it in a pH 4.0 buffer solution (sodium citrate, 50 mM). Lipid nanoparticles were prepared in two types: LNP Cxxxx (50:10:38.5:1.5, ionizable lipids:DSPC:cholesterol:DMG-PEG2000) and LNP 3xxxx (30:30:38.5:10, ionizable lipids:6,6'-trehalose dioleate(TDO):methylpentyl lithocholate:DMG-PEG2000), and the N / P ratio was 6 and 3, respectively. Lipid nanoparticles were prepared using a vortex (<50 μg mRNA, mixing volume ratio 1:3 = lipid solution: mRNA solution) and NanoAssemblr® Ignite™ (≥50 μg mRNA, flow rate 10 mL / min, mixing volume ratio 1:3 = lipid solution: mRNA solution). The obtained lipid nanoparticles were washed twice with DPBS using an Amicon® Ultra-15 Centrifugal Filter tube and a refrigerated centrifuge, and then concentrated to a concentration of 0.25 μg / μL.

[0404] Measurement of Size, Polydispersity Index (PDI), and Zeta Potential: Lipid nanoparticles were diluted with DPBS to a concentration of 2.5 ng / μL, and the size and polydispersity index (PDI) of the nanoparticles were measured using a Zetasizer Ultra (Malvern) device. Zeta potential was measured after diluting the solution to the same concentration with distilled water.

[0405] Encapsulation efficiency: Encapsulation efficiency was determined by electrophoresis using a 1% agarose gel. After preparing a gel containing MOPS buffer, formaldehyde, RNA staining compound, and agarose, free mRNA and LNPs were electrophoresed (7 min, 100 V) to identify the free mRNA band observed without encapsulation.

[0406] Intracellular delivery efficiency: 8 × 10 HEK293 or Huh-7 cell lines were seeded in a 24-well plate. 4 Cells were seeded at a concentration of 10 cells / ml, treated with EGFP mRNA⊂LNP (0.5 μg RNA / well) the next day, and the culture medium was replaced after 6 hours. 24 hours after treatment, the fluorescence signal of intracellularly expressed GFP was measured using a flow cytometer. Delivery efficiency (%) was expressed as the percentage of the fluorescence signal of the novel lipid nanoparticles distributed within the gated area by gating on the area where the fluorescence signal of the control LNP appeared.

[0407] Evaluation of cancer prevention vaccine efficacy: Lipid nanoparticles loaded with OVA mRNA were administered subcutaneously twice (10 μg mRNA) at 4-day intervals to 6-8-week-old C57BL / 6 mice (n=6). Four days after the last administration, E.G7-OVA cancer cell line (1×10 6 cells) were administered subcutaneously, and the size of the growing cancer (mm) was monitored at 2-3 day intervals. 3 = 0.5 × length × width 2 ) is confirmed. The size of the cancer is 1,500 mm 3 If abnormalities or skin necrosis were observed, the mice were sacrificed and the experiment was terminated.

[0408]

[0409] The novel provitamin B5-based lipid prepared above was used to prepare lipid nanoparticles as follows. First, it was prepared as an ionizable lipid with DSPC, cholesterol, and PEG2000 at a ratio of 50:10:38.5:1.5. The specific composition, along with zeta potential and size, is shown in Table 1 below.

[0410] Lipid nanoparticle ionization lipid size (nm) PDI ζ potential ConSM-102 119.7±0.8 0.10 14.2±1.1 C2001 Compound 20-1 144.7±0.6 0.24 16.7±0.9 C2002 Compound 20-2 150.8±0.9 0.11 22.2±1.5 C2003 Compound 20-3 189.0±1.5 0.09-3.0±0.4 C2004 Compound 23-2 141.8±1.4 0.18 13.7±1.5 C2005 Compound 20-5 133.5±1.0 0.15 18.0±0.8 C2006 Compound 20-6 141.8±1.20.2019.0±2.0C2007 Compound 20-7 165.5±11.90.2518.0±0.9C2008 Compound 20-4 18.1±1.10.1913.7±0.7C2009 Compound 20-9 145.0±5.20.2218.7±1.0C2010 Compound 20-10 132.6±3.00.1713.8±1.1C2011 Compound 20-11 146.8±1.00.1917.2±3.0C2012 Compound 20-12 82.1±0.50.19-16.2±1.8C2013 Compound 20-13257.3±2.50.17-28.5±1.7C2014Compound 20-14145.5±1.00.165.9±3.1C2015Compound 20-15137.6±1.00.1218.8±1.9C2101Compound 21-1115.6±1.00.3018.4±3.0C2102Compound 21-2139.6±1.90.196.3±1.8C2201Compound 22-1114.0±0.90.1610.8±3.8C2202Compound 22-2161.4±2.10.12-16.8±0.5C2301Compound 23-1154.4±2.00.16-6.4±0.5C2302Compound 23-2154.8±1.30.14-19.1±2.3

[0411]

[0412] Second, ionized lipids, 6,6'-trehalose dioleate (TDO), methylpentyl lithocholate, and PEG2000 were prepared in a ratio of 30:30:38.5:1.5. The specific composition is shown in Table 2 below along with the zeta potential and size.

[0413]

[0414] Lipid nanoparticlesIonizationLipidSize (nm)PDIζ potential32001Compound 20-1104.0±2.00.1120.0±0.432002Compound 20-2109.7±2.00.1118.7±2.232003Compound 20-399.0±1.80.13-5.9±1.532004Compound 23-2109.2±0.60.0718.8±0.832005Compound 20-5107.1±2.00.1520.8±1.232006Compound 20-6110.9±2.10.1614.8±0.532007Compound 20-7109.3±1.60.1420.5±3.332008 Compound 20-493.5±2.40.1420.5±2.332009 Compound 20-9100.9±1.70.1217.4±2.632010 Compound 20-10110.3±0.50.137.3±0.732011 Compound 20-11101.4±1.50.1511.8±1.232012 Compound 20-12101.1±1.30.14-10.7±0.932013 Compound 20-13156.6±2.20.18-27.2±1.132014 Compound 20-1497.0±0.50.11-2.2±1.132015 Compound 20-15104.6±1.20.1014.9±1.732101 Compound 21-199.4±1.60.1118.5±2.432102 Compound 21-297.2±1.80.101.4±0.932201 Compound 22-191.6±0.70.1418.0±0.932202 Compound 22-293.1±0.20.10-10.0±0.332301 Compound 23-188.6±0.50.118.5±0.432302 Compound 23-294.9±1.00.11-13.8±0.8

[0415] Example 3. Measurement of encapsulation efficiency and transfer efficiency

[0416] Afterwards, the encapsulation efficiency was measured as described above, and the electrophoresis results are shown in Fig. 1. When the manufactured lipid nanoparticles were run on an agarose gel, if a band was observed in the free mRNA region, it was determined that unencapsulated mRNA was present. In the case of C2012 and C2013, almost no encapsulation occurred. In the case of 32012, 32013, 32102, 32202, and 32302, some encapsulation occurred, but free mRNA was weakly observed, so the encapsulation efficiency was relatively low. Among C2003, C2012, C2013, C2202, C2302, 32003, 32012, 32013, 32014, 32202, and 32302, which show negative zeta potentials, the rest, excluding C2003, 32003, and 32014, showed a tendency for mRNA encapsulation not to occur or for the encapsulation efficiency to be somewhat low.

[0417] Afterwards, the in vitro delivery efficiency of the LNPs shown in Table 1 was tested as described above, and the results for the Huh7 cell line are shown in Fig. 2. It was confirmed that C2012 and C2013, which were hardly encapsulated, were not delivered at all to the Huh7 cell line. C2003, C2202, and C2302 were well encapsulated, but were not delivered well to the Huh7 cell line when the zeta potential had a negative value. The lipid nanoparticles that had excellent delivery efficiency of more than 80% to the Huh7 cell line were C2002, C2004, C2005, C2006, C2011, C2201, and C2301, which were well encapsulated and had a positive zeta potential value.

[0418] Next, the in vitro delivery efficiency of the LNPs shown in Table 1 was tested as described above, and the results for the HEK293 cell line are shown in Fig. 3. C2012 and C2013, which were poorly encapsulated, did not deliver well. C2003, C2202, and C2302 were well encapsulated, but when the zeta potential had a negative value, delivery was almost non-existent. C2004, C2014, and C2102 were well encapsulated, but when the zeta potential had a positive value, C2004 showed excellent delivery efficiency in the Huh cell line, but almost no delivery efficiency in the HEK293 cell line. C2014 and C2102 showed delivery efficiencies of 68.7% and 39.4%, respectively, in the Huh7 cell line, but almost no delivery in the HEK293 cell line. Lipid nanoparticles showing excellent delivery efficiencies of over 80% were C2002, C2005, C2006, C2011, and C2201, which were well encapsulated and had positive zeta potentials. Lipid nanoparticles showing excellent delivery efficiencies in both Huh7 cell lines and HEK293 cell lines were C2002, C2005, C2011, and C2201.

[0419] Next, the in vitro delivery efficiency of the LNPs shown in Table 2 was tested as described above, and the results for the Huh7 cell line are shown in Fig. 4. 32012, 32013, 32102, 32202, and 32302, which had somewhat low encapsulation efficiencies, had negative zeta potentials and showed significantly reduced delivery efficiency for the Huh7 cell line. 32102, which had somewhat low encapsulation efficiency, had a positive zeta potential but a low value of 1.4±0.9, showing significantly reduced delivery efficiency in the Huh7 cell line. 32003 and 32014 had low encapsulation but negative zeta potentials and showed significantly reduced delivery efficiency for the Huh7 cell line. 32301 showed good encapsulation and positive zeta potential, but showed low delivery efficiency in the Huh7 cell line. When the encapsulation efficiency was somewhat low or the zeta potential had a negative value, the delivery ability to the Huh7 cell line tended to be significantly reduced. The lipid nanoparticles that showed excellent delivery efficiencies of over 80% were 32001, 32002, 32004, 32007, 32009, 32011, and 32015.

[0420] Finally, the in vitro delivery efficiency of the LNPs shown in Table 2 was tested as described above, and the results for the HEK293 cell line are shown in Fig. 5. Among the lipid nanoparticles with somewhat low encapsulation efficiency or negative zeta potential, 32003, 32012, 32013, 32014, and 32202 showed low delivery efficiency of less than 30%. 32015, which showed excellent delivery efficiency for the Huh7 cell line, showed a significant decrease in delivery efficiency in the HEK293 cell line, whereas 32006, 32020, 32102, and 32302, which showed low delivery efficiency in the Huh7 cell line, showed somewhat improved delivery efficiency in the HEK392 cell line. For 32001, 32002, 32004, 32007, 32009, and 32011, excellent delivery efficiencies of over 80% were observed in both Huh7 and HEK293 cell lines, and for 32015, although the delivery efficiency in the Huh7 cell line was excellent, the delivery efficiency in the HEK293 cell line was only 13.1%.

[0421]

[0422] Lipid nanoparticles that showed excellent transduction efficiency in Huh7 cells but significantly lower transduction efficiency in HEK293 cells were C2006 and 32015. Lipid nanoparticles that showed excellent transduction efficiency in Huh7 cells but slightly lower transduction efficiency in HEK293 cells were C2301. Lipid nanoparticles that showed excellent transduction efficiency in both Huh7 cells and HEK293 cells were as follows: 32001, 32002, 32004, 32007, 32009, and 32011.

[0423]

[0424] Example 4. Evaluation of vaccine applicability

[0425] As described above, the possibility of application as a vaccine composition was evaluated by encapsulating OVA mRNA into 32001 lipid nanoparticles. 32001 lipid nanoparticles encapsulating OVA mRNA, control lipid nanoparticles, PBS, etc. were administered twice at 4-day intervals (subcutaneous injection, sc), and then 4 days later, tumor cell lines were administered and the tumor size compared to the control group was checked. The results are shown in Fig. 6. In the group administered 32001 lipid nanoparticles encapsulating OVA mRNA, tumor growth was significantly suppressed, confirming that 32001 works well as a preventive vaccine.

[0426] OVA mRNA, well delivered by lipid nanoparticles, expresses OVA protein, and when administered to the E.G7-OVA cell line, a mouse cancer cell line that overexpresses OVA, antibodies against OVA are generated by the immune system, it was confirmed that the expression of cancer cells was inhibited.

[0427]

[0428] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0429] Therefore, other implementations, other manufacturing examples and equivalents to the patent claims also fall within the scope of the claims described below.

Claims

1. A compound represented by the following chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof: In the above chemical formula, X is any one of CH2, NH, O, and S, n is an integer from 0 to 3, R1 is a secondary amine, at least one of a secondary amine substituted with any one of methyl, ethyl, hydroxyethyl, and hydroxybutyl, a substituted or unsubstituted C3-C6 heterocycloalkyl, and a straight chain C1-C6 alkyl substituted or unsubstituted with a hydroxyl group or dimethylamine, In the case where the above C3-C6 heterocycloalkyl is substituted, it is substituted with C1-C6 straight chain alkyl which is unsubstituted or substituted with hydroxy, R x and R y Is and; R2 and R3 are each independently C3-C 20 It is a saturated or unsaturated hydrocarbon.

2. In paragraph 1, A compound represented by chemical formula 1, wherein R2 and R3 are each independently a C5-C6 saturated hydrocarbon, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.

3. In paragraph 1, The above R2 and R3 are each independently and A compound represented by chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is at least one selected from the group consisting of:

4. In paragraph 1, The compound is characterized by being at least one selected from the group consisting of the following compounds, a stereoisomer thereof, a racemate thereof or a pharmaceutically acceptable salt thereof: [Compound 20-4] [Compound 20-11] [Compound 22-1] 5. A lipid nanoparticle composition comprising the compound of claim 1.

6. A lipid nanoparticle composition according to claim 5, wherein the compound has a positive charge under weakly acidic conditions and binds to a nucleic acid having a negative charge.

7. A lipid nanoparticle composition according to claim 6, characterized in that the composition further comprises at least one selected from the group consisting of PEGylated lipids, helper lipids, and structural lipids.

8. In the 7th paragraph, the helper lipid is 6,6'-trehalose glycolipid, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octa Decenyl-sn-glycero-3-phosphocholine (18:0 Dieter PC), 1-Oleoyl-2-cholesterylhexyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Dilinoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), A lipid nanoparticle composition characterized by comprising at least one selected from the group consisting of dipalmitoyl-phosphatidyl-ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

9. A lipid nanoparticle composition according to claim 7, characterized in that the structural lipid is at least one selected from the group consisting of cholesterol, bile acid derivatives including alkyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene.

10. In paragraph 7, A lipid nanoparticle composition, characterized in that the composition comprises all of PEG lipid, helper lipid and structural maintenance lipid, and comprises 30 to 50 mol% of the compound, 1 to 2 mol% of PEG lipid, 10 to 30 mol% of helper lipid and 38 to 39 mol% of structural maintenance lipid.

11. A lipid nanoparticle composition according to claim 10, characterized in that the compound is at least one selected from the group consisting of: [Compound 20-4] [Compound 20-11] [Compound 22-1] 12. A lipid nanoparticle composition according to any one of claims 5 to 11, wherein the lipid nanoparticle composition comprises a therapeutic or preventive agent therein.

13. A lipid nanoparticle composition according to claim 12, wherein the therapeutic or preventive agent is selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

14. In paragraph 13, the therapeutic or preventive agent is messenger RNA, A lipid nanoparticle composition, characterized in that the lipid nanoparticle has a diameter of 90 to 150 nm and an internal zeta potential of 0 to 30 mV.

15. A vaccine composition comprising the lipid nanoparticle composition of Article 12.

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