Novel lipid nanoparticle composition for genetic manipulation in immune cells

Abstract

The present invention relates to: a lipid nanoparticle (LNP) composition for producing NK cells or T cells transformed with CAR, wherein the LNP composition for transforming natural killer (NK) cells or T cells is characterized by comprising a nucleic acid and a novel ionizable lipid; CAR-NK cells and CAR-T cells transformed thereby; and uses thereof. According to the present invention, mRNA can be stably delivered into immune cells through the LNP composition, and the transformed NK cells and T cells exhibit high viability and can highly express the target protein CAR.

Description

Novel lipid nanoparticle composition for genetic manipulation within immune cells

[0001] The present invention relates to a lipid nanoparticle (LNP) composition for the production of immune cells transformed by CAR, characterized by comprising a lipid or cholesterol substitute based on an oligo-gamma-glutamic acid derivative that replaces CAR-expressed nucleic acid and PEGylated lipids, a helper lipid based on vitamin C, and an ionized lipid based on pantothenic acid and panthenol.

[0002] Over the decades, methods for treating cancer have steadily changed and evolved. From the 1800s to the 1900s, surgical procedures, chemotherapy, and radiation therapy were primarily used; however, as their limitations began to emerge, cell therapy methods involving the extraction of immune cells from the body, their enhancement or genetic modification, and their subsequent reintroduction are being developed. Representative examples include Tumor Infiltrating Lymphocytes (TILs), Chimeric Antigen Receptors (CARs), and T-Cell Receptors (TCRs). In particular, research and clinical trials utilizing CARs, which are artificial receptors created through genetic recombination, are currently underway. However, cell therapy methods or gene therapies employing the aforementioned genetic modifications face numerous challenges, including undesirable immune responses and safety issues, due to the incorporation of genes at random locations within the genome. In this regard, DNA-transformed immune cells (CAR-NK cells (Chimeric Antigen Receptor Natural Killer cells) or CAR-T cells (Chimeric Antigen Receptor T cells)) have a fundamental problem in that safety is not guaranteed because the CAR gene is integrated into the genome or mutations occur, and NK cells, in particular, are known to be difficult to transfect. Since receptor expression is low in NK cells, the transfection efficiency within NK cells of lenti or retroviruses pseudotyped with VSV-G glycoprotein, which are mainly used in existing CAR-T therapy clinical trials or pharmaceuticals, is extremely low. Instead, RD114 or BaEV pseudotyped lenti / retroviruses can be used for the transfection of NK cells, but they have the disadvantage of being difficult to produce GMP-level viral titers.

[0003] To address this, the design of transgenic immune cells mediated by non-viral delivery methods using mRNA is required. Delivery methods for mRNA in non-viral gene manipulation include direct injection, electroporation, physical methods using gene guns, and various chemical delivery methods based on lipid complexes, peptides, polymers, inorganic materials, and metals. Among these, electroporation is widely used for the non-viral transfection of immune cells, including NK cells. However, it has disadvantages such as limited gene delivery capacity for mass production, low cell viability, and batch-specific variability due to electroporation. Therefore, establishing an efficient non-viral delivery system is essential for large-scale immune cell engineering.

[0004] The technical problem that the present invention aims to solve is to provide an LNP composition for immune cell transformation comprising nucleic acid, resveratrol as a lipid or cholesterol substitute based on oligo-gamma-glutamic acid, vitamin C-based helper lipid, and a novel ionized lipid.

[0005] Another technical problem that the present invention aims to solve is to provide CAR-expressing immune cells transformed with the above composition.

[0006] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below.

[0007] In order to solve the above problem,

[0008] The inventors provide an LNP composition for immune cell transformation characterized by comprising: a nucleic acid; a lipid based on an oligo-gamma-glutamic acid derivative; a helper lipid based on vitamin C or gallic acid; and an ionized lipid represented by the following chemical formula 3:

[0009]

[0010] In the above chemical formula 3,

[0011] R 1 to R 4 They may be the same or different from each other, and each C1 to C 12 It is an alkyl of, and

[0012] R 5 and R 6 They may be the same or different from each other, and each is a C1 to C6 alkyl or combines with each other to form a C3 to C6 cycloalkyl or heterocycloalkyl, and

[0013] A is any one selected from the group consisting of -C(=O)-NH-, -OC(=O)-, -C(=O)-O- and -OC(=O)-O-, and

[0014] n and m are integers from 1 to 6, respectively.

[0015] According to one side, in the above chemical formula 3,

[0016] R 1 to R 4 They may be the same or different from each other, and each C6 to C 12 It is a straight-chain alkyl, and

[0017] R 5 and R 6 Each is methyl or ethyl, or combines with each other to form morpholine or oxazepane, and

[0018] n and m can each be integers from 1 to 3.

[0019] According to one side, the ionized lipid represented by the above [Chemical Formula 3] may be any one selected from the group consisting of the following:

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] According to one side, the nucleic acid may be mRNA expressing CAR (Chimeric antigen receptors).

[0036] According to one side, if the helper lipid is vitamin C-based, it may be any one selected from the group consisting of the following:

[0037] and

[0038]

[0039] In addition, if the above helper lipid is gallic acid-based, it may be any one selected from the group consisting of the following:

[0040]

[0041]

[0042]

[0043]

[0044] According to one aspect, the lipid based on the oligo-gamma-glutamic acid derivative may be any one selected from the group consisting of the following:

[0045]

[0046] and

[0047] .

[0048] According to one aspect, the LNP composition further comprises a structure-maintaining lipid, and the molar ratios of the lipid based on an oligo-gamma-glutamic acid derivative excluding nucleic acid, the helper lipid based on vitamin C or gallic acid, the ionized lipid, and the structure-maintaining lipid may be 1.5 to 10%, 10 to 15%, 44 to 50%, and 30 to 38.5%, respectively. The structure-maintaining lipid may be selected from cholesterol or resveratrol, but is not limited thereto.

[0049] According to another embodiment of the present invention, a method for transforming immune cells is provided, comprising the step of treating an immune cell with any one of the above compositions.

[0050] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of cancer is provided, comprising as an active ingredient an immune cell that expresses CAR on its surface, as an immune cell produced by the above method.

[0051] According to one aspect, the immune cell may preferably be an NK cell or a T cell.

[0052] According to one aspect, the cancer may be selected from the group consisting of pancreatic cancer, breast cancer, ovarian cancer, glioma, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, liver cancer, lung cancer, colorectal cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, blood cancer, and melanoma.

[0053] According to one side, the pharmaceutical composition may additionally include other anticancer agents.

[0054] According to one aspect, the pharmaceutical composition can inhibit the proliferation, survival, metastasis, recurrence, or anticancer drug resistance of cancer.

[0055] According to another embodiment of the present invention, a method for preventing or treating cancer is provided, comprising the step of administering to a subject a composition containing, as an active ingredient, an immune cell having an immune cell expressing a CAR on its surface, as an immune cell produced by the above method.

[0056] According to another embodiment of the present invention, an immune cell produced by the above method, which expresses CAR on its surface, is provided for use in cancer prevention or treatment.

[0057] According to another embodiment of the present invention, an immune cell produced by the above method is provided for use in manufacturing a drug for cancer prevention or treatment, wherein the immune cell expresses CAR on its surface.

[0058] The present invention provides a novel ionized lipid compound, and through an LNP composition containing the same, mRNA can be stably delivered into immune cells, and the transformed immune cells can exhibit a high survival rate and express a target protein.

[0059] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by a person skilled in the art from the description below.

[0060] Figure 1 shows the results of measuring the GFP mRNA delivery and translation efficiency and cell viability in NK cells using SM-102 or lipid nanoparticles based on the chemical formula 3-1 of pantothenic acid as ionized lipids using a flow cytometer.

[0061] Figure 2 shows the results of measuring the GFP mRNA delivery and translation efficiency and cell viability in NK cells using lipid nanoparticles with chemical formula 1-1, an oligo-gamma-glutamic acid derivative, as a substitute for PEGylated lipids, using a flow cytometer.

[0062] Figure 3 shows the results of measuring the GFP mRNA delivery and translation efficiency and cell viability in NK cells of lipid nanoparticles using SM-102 ionized lipids and vitamin C-based lipids as substitutes for helper lipids using a flow cytometer.

[0063] Figure 4 shows the results of measuring the GFP mRNA delivery and translation efficiency and cell viability in NK cells of lipid nanoparticles using pantothenic acid-based ionized lipid of formula 3-1 and vitamin C-based lipid as substitutes for helper lipids using a flow cytometer.

[0064] Figure 5 shows the results of measuring the NK cell viability of lipid nanoparticles containing pantothenic acid-based ionized lipids of formulas 3-2 to 3-3, using vitamin C-based lipids as substitutes for helper lipids, or using oligo-gamma-glutamic acid derivative formula 1-1 as a substitute for PEGylated lipids using a flow cytometer.

[0065] Figure 6 shows the results of measuring the GFP mRNA delivery and translation efficiency within NK cells in the same experiment as Figure 5.

[0066] Figure 7 shows the results of the correlation between the GFP mRNA delivery and translation efficiency and cell viability in NK cells by lipid nanoparticles GFP021 to GFP034.

[0067] Figure 8 shows the results of measuring the GFP mRNA delivery and translation efficiency and cell viability in NK cells of lipid nanoparticles GFP035 to GFP040, which contain selected pantothenic acid and panthenol-based ionized lipids and use vitamin C or gallic acid-based lipids as helper lipids or resveratrol or cholesterol as structure-maintaining lipids, using a flow cytometer.

[0068] Figure 9 shows the results of measuring the CAR mRNA delivery and translation efficiency and cell viability within NK cells and summarizing the correlations using a flow cytometer with lipid nanoparticles containing selected pantothenic acid-based ionized lipids, using vitamin C-based lipids as substitutes for helper lipids, or using the oligo-gamma-glutamic acid derivative Formula 1-1 as a PEGylated lipid substitute.

[0069] Figure 10 shows the results of measuring the CAR mRNA delivery and translation efficiency and cell viability in NK cells of lipid nanoparticles containing pantothenic acid and panthenol-based ionized lipids, using vitamin C-based lipids as substitutes for helper lipids, and using oligo-gamma-glutamic acid derivatives such as formulas 1-1 to 1-2 as PEGylated lipid substitutes, using a flow cytometer.

[0070] Figure 11 shows the results of measuring the CAR mRNA delivery and translation efficiency and cell viability within NK cells and summarizing the correlations using a flow cytometer with lipid nanoparticles containing pantothenic acid and panthenol-based ionized lipids, using vitamin C-based lipids as substitutes for helper lipids, or using the oligo-gamma-glutamic acid derivative Formula 1-2 as a PEGylated lipid substitute.

[0071] Figure 12 shows the cell viability and CAR mRNA delivery and translation efficiency after treatment for the transformation of NK cells with lipid nanoparticles CAR017 and CAR029 using a flow cytometer.

[0072] Figure 13 shows the results of measuring the CAR mRNA delivery and translation efficiency of lipid nanoparticles CAR027 and CAR029 into T cells within PBMCs using a flow cytometer.

[0073] Figure 14 shows the results of measuring the CAR mRNA delivery and translation efficiency and cell viability in NK cells of lipid nanoparticles CAR030 to CAR040, which contain pantothenic acid and panthenol-based ionized lipids, use vitamin C-based lipids as substitutes for helper lipids, use resveratrol as a cholesterol substitute, or use oligo-gamma-glutamic acid derivatives such as formulas 1-1 to 1-3 as PEGylated lipid substitutes, using a flow cytometer.

[0074] The inventors intend to provide this invention by confirming that a lipid nanoparticle (LNP) composition containing novel ionized lipids has characteristics suitable for transfecting NK cells or T cells and expressing CAR in the production of CAR-NK or CAR-T by introducing CAR-expressing mRNA into NK cells or T cells.

[0075] To solve the above problem, the inventors provide an LNP composition for immune cell transformation characterized by comprising: a nucleic acid; a lipid based on an oligo-gamma-glutamic acid derivative; a helper lipid based on vitamin C or gallic acid; and an ionized lipid represented by the following chemical formula 3:

[0076]

[0077] In the above chemical formula 3,

[0078] R 1 to R 4 They may be the same or different from each other, and each C1 to C 12 It is an alkyl of, and

[0079] R 5 and R 6 They may be the same or different from each other, and each is a C1 to C6 alkyl or combines with each other to form a C3 to C6 cycloalkyl or heterocycloalkyl, and

[0080] A is any one selected from the group consisting of -C(=O)-NH-, -OC(=O)-, -C(=O)-O- and -OC(=O)-O-, and

[0081] n and m are integers from 1 to 6, respectively.

[0082] According to one side, in the above chemical formula 3,

[0083] R 1 to R 4 They may be the same or different from each other, and each C6 to C 12 It is a straight-chain alkyl, and

[0084] R 5 and R 6 Each is methyl or ethyl, or combines with each other to form morpholine or oxazepane, and

[0085] n and m can each be integers from 1 to 3.

[0086] According to one side, the ionized lipid represented by the above [Chemical Formula 3] may be any one selected from the group consisting of the following:

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] According to one aspect, the immune cell may preferably be an NK cell or a T cell.

[0104] The term “ionized lipid” as used in the present invention refers to an ionizable compound having properties similar to lipids, which can perform the role of enabling a drug (e.g., anionic drugs and / or nucleic acids) to be encapsulated within lipid nanoparticles with high efficiency through electrostatic interaction with the drug. The ionizable lipid is the pK of the ionized lipid a It can be protonated (positively charged) at pH below, and pK a At pH values ​​above this level, it may be substantially neutral. In one example, the lipid nanoparticles may comprise protonated ionizable lipids and / or ionizable lipids exhibiting neutrality. The ionizable lipids may be cationic lipids.

[0105] In the present invention, “helper lipid” may be one that promotes the fusion of lipid nanoparticles, preferably may be a vitamin C or gallic acid-based helper lipid, and in the case of a vitamin-based helper lipid, may be any one selected from the group consisting of the following:

[0106] and

[0107]

[0108] In addition, in the case of a gallic acid-based helper geology, it may be any one selected from the group consisting of the following:

[0109] and

[0110] .

[0111]

[0112] The above “LNP” or “lipid nanoparticle” refers to nano-sized spherical particles composed of lipids.

[0113] According to one side, the composition may further include a structural maintenance material.

[0114] The above-mentioned structure-maintaining lipid refers to a lipid that provides structural rigidity to the lipid packing within the lipid nanoparticles and plays a role in improving the stability of the nanoparticles by being dispersed in the core and surface of the nanoparticles. It may be one or more selected from the group consisting of cholesterol, resveratrol, bile acid derivatives including butyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene, but preferably it may be either cholesterol or resveratrol. Most preferably, when the above-mentioned gallic acid-based lipid is used as a helper lipid, resveratrol may be used as the structure-maintaining lipid, but is not limited thereto.

[0115] According to one aspect, the LNP composition further comprises a structure-maintaining lipid, and the molar ratios of the lipid based on an oligo-gamma-glutamic acid derivative excluding nucleic acid, the vitamin C-based helper lipid, the ionized lipid, and the structure-maintaining lipid may be 1.5 to 10%, 10 to 15%, 44 to 50%, and 30 to 38.5%, respectively.

[0116] The term “transformation” as used in the present invention refers to a process in which a nucleic acid containing a type of gene different from that of the original cell is introduced between cells and combined with DNA, etc., of the original cell, or transcribed and translated using the cell’s protein expression system. In the present invention, transformation may preferably involve introducing mRNA encoded to express CAR into a cell so that CAR is expressed on the surface of the cell.

[0117] According to one aspect, the nucleic acid may be mRNA expressing a CAR (Chimeric antigen receptor). The term "Chimeric antigen receptor" or "CAR" is defined as a cell surface receptor comprising an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain. The chimeric antigen receptor of the present invention is intended for use primarily with lymphocytes, such as T cells and natural killer (NK) cells, and is exemplified for use most preferably with NK cells.

[0118] According to one aspect, the lipid based on the oligo-gamma-glutamic acid derivative can improve the in vivo stability of lipid nanoparticles, can be used as a substitute for PEG lipids, and preferably can be any one selected from the group consisting of the following:

[0119]

[0120] and

[0121] .

[0122] In a preferred embodiment of the present invention, the composition of the LNP may consist of an oligo-gamma-glutamic acid-based lipid as a substitute for PEG lipids, cholesterol as a structural maintenance lipid, a vitamin C or gallic acid-based lipid as a helper lipid, and a pantothenic acid or panthenol-based lipid as an ionized lipid.

[0123] In addition, in another preferred embodiment of the present invention, the composition of the LNP may consist of a PEG lipid, resveratrol as a structural maintenance lipid, a vitamin C or gallic acid-based lipid as a helper lipid, and a pantothenic acid or panthenol-based lipid as an ionized lipid.

[0124] According to another embodiment of the present invention, a method for transforming NK cells or T cells is provided, comprising the step of treating any one of the above compositions to NK cells or T cells.

[0125] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of cancer is provided, comprising as an active ingredient NK cells or T cells that express CAR on their surface, as NK cells or T cells prepared by the above method.

[0126] According to one aspect, the cancer may be selected from the group consisting of pancreatic cancer, breast cancer, ovarian cancer, glioma, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, liver cancer, lung cancer, colorectal cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, blood cancer, and melanoma.

[0127] According to one side, the pharmaceutical composition may additionally include other anticancer agents.

[0128] According to one aspect, the pharmaceutical composition can inhibit the proliferation, survival, metastasis, recurrence, or anticancer drug resistance of cancer.

[0129] According to another embodiment of the present invention, a method for preventing or treating cancer is provided, comprising the step of administering to a subject a composition containing, as an active ingredient, NK cells or T cells expressing a CAR on their surface, as NK cells or T cells produced by the above method.

[0130] The term "anticancer agent" is a collective term for known drugs used in existing cancer treatments that act on various metabolic pathways of cells to exhibit cytotoxic or cytostatic effects on cancer cells, and includes chemotherapy agents, targeted anticancer agents, and immunotherapy agents.

[0131] The term "immunotherapy" refers to a drug that kills cancer cells by activating immune cells. In this specification, "immunotherapy" refers to a method of treating cancer that helps the immune system eliminate cancer. Immunotherapy is classified into active immunotherapy and passive immunotherapy. Active immunotherapy includes i) cancer vaccine therapy, which activates the immune system by injecting cancer cells or substances produced by cancer cells into the human body, and ii) immunomodulating therapy, which activates specific white blood cells by administering immune-modulating agents such as cytokines (interferon, interleukin, etc.) and growth factors. Passive immunotherapy includes therapeutic antibodies that bind to specific cancer cells and immune cell therapy. Immunotherapy specifically includes, but is not limited to, dendritic cell vaccine therapy, CAR-T (chimeric antigen receptor T cell) therapy, NK cell therapy (natural killer cell therapy), CTL therapy (cytotoxic T lymphocyte therapy), and adoptive cell transfer. In the present invention, immunotherapy may mainly refer to the aforementioned immunotherapy.

[0132] The term "subject" is used interchangeably with "patient" and may be a mammal requiring prevention or treatment of cancer, e.g., primates (e.g., humans), companion animals (e.g., dogs, cats, etc.), livestock animals (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). In one embodiment of the present invention, the subject is a human.

[0133] The term "treatment" generally means achieving desired pharmacological and / or physiological effects. These effects are therapeutic in that they partially or completely cure the disease and / or harmful effects resulting from such disease. Desirable therapeutic effects include, but are not limited to, prevention of the onset or recurrence of the disease, improvement of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or alleviation of the disease state, and remission or improved prognosis. Preferably, "treatment" may refer to medical intervention for an already manifested disease or disorder.

[0134] The term "prevention" refers to preventive treatment, that is, measures or procedures aimed at preventing rather than treating a disease. "Prevention" means obtaining the intended preventive pharmacological and / or physiological effects in the sense of partially or completely preventing a disease or its symptoms.

[0135] The term "administration" means providing a substance (e.g., CAR-expressing NK cells or T cells) to a subject for a preventive or therapeutic purpose (e.g., prevention or treatment of cancer).

[0136] The term “biological sample” encompasses various types of samples obtained from a subject and may be used in diagnostic or monitoring analysis methods. Biological samples include, but are not limited to, liquid samples of biological origin such as blood and other biological samples, biopsy samples, tissue cultures, or solid tissue samples such as cells and their offspring derived therefrom. Accordingly, biological samples include clinical samples, cells in cultures, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples, particularly tumor samples.

[0137] According to another embodiment of the present invention, an NK cell or T cell produced by the above method is provided for use in cancer prevention or treatment, wherein the NK cell or T cell expresses CAR on its surface.

[0138] According to another embodiment of the present invention, an NK cell or T cell produced by the above method is provided for use in manufacturing a drug for cancer prevention or treatment, wherein the NK cell or T cell expresses CAR on its surface.

[0139]

[0140] The terms used in the embodiments are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0141] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0142] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0143]

[0144] Example 1. Synthesis of lipids based on a novel oligo-gamma-glutamic acid derivative

[0145] Oligo-gamma-glutamic acid derivatives (the following 12 types) were designed as follows. After first designing the gamma-glutamic acid monomer A and its derivative monomers B and F, F, which is not commercially available, was synthesized as follows. A was purchased and used as Fmoc-Glu-OtBu (Combi-Block, cat# QA-0753, CAS# 84793-07-7), and B was purchased and used as Fmoc-Glu-OMe (Combi-Block, cat# QN-5744, CAS# 145038-49-9).

[0146] Example 1-1: Synthesis of Formula F

[0147] Synthesis of 5-(tert-butyl) 1-(2-(2-methoxyethoxy)ethyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate

[0148]

[0149] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (500 mg), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide Hydrochloride (465.0 mg), and 4-Dimethylamino pyridine (31.5 mg) were dissolved in 85 mL of dichloromethane and stirred in an ice bath under argon gas for 10 minutes. Diethyleneglycol monomethyl ether (280 μL) was mixed with 35 mL of dichloromethane and slowly added dropwise to the reaction mixture over 30 minutes in an ice bath under argon gas. The reaction mixture was heated to room temperature and stirred for 17 hours. After washing the organic layer with a saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium chloride, the remaining water was removed with sodium sulfate. The reaction mixture was purified by column chromatography (SiO2, dichloromethane → dichloromethane:methanol 93:7) to obtain the compound 5-(tert-butyl) 1-(2-(2-methoxyethoxy)ethyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (621 mg, quantitative yield).

[0150]

[0151] Synthesis of (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(2-(2-methoxyethoxy)ethoxy)-5-oxopentanoic acid (F)

[0152]

[0153] 5-(tert-butyl) 1-(2-(2-methoxyethoxy)ethyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (824 mg) was dissolved in 7.5 mL of dichloromethane, and then 7.5 mL of trifluoroacetic acid was added. The mixture was stirred in an ice bath for 2 hours, and then 15 mL of a saturated aqueous solution of sodium bicarbonate was added and stirred for an additional 10 minutes. The organic layer was washed with distilled water and a saturated aqueous solution of sodium chloride, and the remaining water was removed with sodium sulfate. The reaction mixture was purified by column chromatography (SiO2, dichloromethane → dichloromethane:methanol 94:6) to obtain a compound of formula F (714 mg, 97.0%).

[0154] The oligo-gamma-glutamic acid derivative was composed of lipids consisting of alkyl chains and 15 gamma-glutamic acid monomers A, B, and F having a repeating pattern. All monomers, including the synthesized monomers, were provided to Dandicure Co., Ltd. to synthesize the designed oligo-gamma-glutamic acid derivative in compliance with general solid-phase peptide synthesis methods, and Chemical Formula 1-1, which repeats as AAB, and Chemical Formula 1-3, which repeats as AAF, were derived as follows.

[0155]

[0156]

[0157]

[0158] Example 2. Synthesis of novel pantothenic acid and panthenol-based ionized lipids

[0159] A novel ionized lipid optimized for delivering mRNA into NK cells was synthesized through the following synthesis method.

[0160] Example 2-1. Synthesis of Formulas 3-1 to 3-8

[0161]

[0162] Synthesis of 5-((2-Hexyldecyl)oxy)-5-oxopentanoic acid (5)

[0163] 2-hexyl-1-decanoic acid (500 mg, 1 equivalent) was placed in a reaction vessel and dissolved in DCM (50 ml). Then, glutaric anhydride (component 4), 470 mg, 2 equivalents) and DMAP (630 mg, 2.5 equivalents) were added, and the mixture was vigorously stirred at room temperature for 13 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc / Hexane 1:1, PMA stain), 2N HCl aqueous solution (10 mL) was added to the reaction mixture, followed by extraction with DCM (3 x 25 mL). The organic layer was collected and washed with 2N HCl aqueous solution (2 x 20 mL) to remove residual base, and then washed with a saturated NaCl aqueous solution (30 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered filtrate was subjected to vacuum distillation to remove the solvent. Then, the mixture was purified by flash column chromatography (SiO2, EtOAc / hexane 1:9 -> 2:8) to obtain Compound 5 (603 mg, 82%) in a clear liquid state.

[0164] 1H NMR (CDCl3, 400 MHz): δ 0.79-0.82 (t,J= 5.1 Hz, 6H), 1.19 (m, 25H), 1.54 (br. s, 1H), 1.87-1.92 (qui,J= 5.2 Hz, 2H), 2.31-2.38 (m, 4H), 3.92 (d,J= 7.2 Hz, 2H); 13 C 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.

[0165] Synthesis of 3-((2-Hexyldecyl)oxy)-3-oxopropanoic acid (7)

[0166] Equal amounts of Meldrum's acid 6 (500 mg, 1 equivalent) and 2-hexyl-1-decanol (841 mg, 1 equivalent) are added to a reaction vessel, dissolved in toluene (50 mL), and heated under reflux for 5 hours. After confirming that the reaction has finished using TLC (SiO2, EtOAc / hexane 5:5), the reaction mixture is cooled to room temperature and then cooled again to 4 ℃. A saturated aqueous solution of NaHCO3 is added to the reaction mixture, and a 3M aqueous solution of HCl is added to neutralize it. Subsequently, the reaction mixture is transferred to a separatory funnel and extracted with EtOAc (2 x 100 mL), and the organic layer is collected and washed with a saturated aqueous solution of NaCl (1 x 100 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered liquid was subjected to vacuum distillation to remove the solvent. Then, the remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 5:5, with 1% AcOH added) to obtain Compound 7 (1103 mg, 97%) in a clear oil state.

[0167] 1H NMR (CDCl3, 400 MHz): δ 0.87-0.92 (m, 6H), 1.30 (m, 26H), 1.68 (s, 1H), 3.44-3.47 (m, 2H), 4.08-4.13 (m, 2H), 10.68 (br. s, 1H); 13 C NMR (CDCl3, 100 MHz): δ 14.09, 22.66, 22.69, 26.61, 26.66, 29.32, 29.56, 29.93, 31.10, 31.10, 31.89, 31.91, 37.22, 40.80, 68.79, 167.04, 171.69.

[0168] Synthesis of (R)-3-(2,2,5,5-Tetramethyl-1,3-dioxane-4-carboxamido)propanoic acid (9)

[0169] p-toluenesulfonic acid (PTSA, 3.97 g, 1.1 equivalents) was added all at once while stirring D-pantothenic acid hemicalcium salt 8 (5 g, 1 equivalent) and 2,2-dimethoxypropane (DMP, 60 mL) in a reaction vessel. The reaction mixture was stirred at room temperature for 20 hours, after which the resulting white solid was washed with acetone and filtered. The yellow solid obtained by vacuum distillation of the filtrate was washed with warm n-hexane to obtain compound 9 (5.45 g, 100%) as a white solid.

[0170] 1 H NMR (CDCl3, 400 MHz): δ 0.98 (s, 3H), 1.04 (s, 3H), 1.43 (s, 3H), 1.46 (s, 3H), 2.62 (dt,J= 6.0, 2.0, 2H), 3.29 (d,J= 1.5, 1H), 3.45-3.53 (m, 1H), 3.56-3.64 (m, 1H), 3.70 (d,J= 11.5, 1H), 4.11 (s, 1H), 7.05 (app bs, 1H); 13C NMR (CDCl3, 100 MHz): δ 18.7, 18.8, 22.0, 29.4, 30.9, 33.0, 33.9, 34.1, 71.4, 77.1, 99.1, 170.2.

[0171]

[0172] Synthesis of (9H-Fluoren-9-yl)methyl(R)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propanoate (10)

[0173] Acetal-protected pantothenic acid compound 9 (476 mg, 1.2 equivalents) was placed in a reaction vessel and dissolved in DCM (30 mL); then, EDCI·HCl (438 mg, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP) (38 mg, 0.2 equivalents) were added, and 5 under argon gas o Stir vigorously at C for 10 minutes. Then, add a solution of 9-fluorenemethanol (300 mg, 1 equivalent) dissolved in DCM (10 mL) to the reaction mixture 5 o After adding dropwise at C, the mixture was stirred at room temperature for 1 hour. After confirming that the reaction was complete using TLC (SiO2, EtOAc / hexane 1:9, PMA stain), additional DCM (50 mL) was added, the mixture was transferred to a separatory funnel, and washed sequentially with saturated aqueous NaHCO3 solution (2 x 50 mL) and saturated aqueous NaCl solution (20 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtrate was filtered. The solvent was removed by vacuum distillation, and the resulting mixture was purified by flash column chromatography (SiO2, EtOAc / hexane 1:9) to obtain Compound 10 (541 mg, 81%) as a clear oil.

[0174] 1H NMR (CDCl3, 400 MHz): δ 0.86 (s, 3H), 0.94 (s, 3H), 1.27 (s, 3H), 1.31 (s, 3H), 2.54-2.57 (t,J= 8.2 Hz, 2H), 3.14-3.17 (d,J= 5.1 Hz, 1H), 3.38-3.52 (m, 2H), 3.54-3.57 (d,J= 5.5 Hz, 1H), 3.98 (s, 1H), 4.07-4.10 (t,J= 8.3 Hz, 1H), 4.27-4.29 (d,J= 7.1 Hz, 2H), 6.87-6.90 (s, 1H), 7.16-7.18 (t,J= 5.1 Hz, 2H), 7.20-7.27 (t,J= 5.5 Hz, 2H), 7.29-7.46 (d,J= 5.0 Hz, 2H), 7.47-7.66 (d,J= 4.8 Hz, 2H); 13 C NMR (CDCl3, 100 MHz): δ 14.23, 18.67, 18.89, 21.03, 22.14, 29.45, 32.79, 32.98, 34.21, 34.25, 46.72, 46.92, 60.36, 66.66, 71.45, 76.87, 77.19, 77.50, 99.02, 119.78, 120.09, 120.35, 124.69, 125.01, 127.16, 127.87, 141.29, 143.64, 169.82, 171.06.

[0175]

[0176] (9H-Fluoren-9-yl)methyl(R)-3-(2,4-dihydroxy-3,3-dimethylbutanamido)propanoate (11)의 합성

[0177] Compound 10 (500 mg, 1 equivalent) and DL-1,4-dithiothreitol (DTT, 352 mg, 2 equivalents) were dissolved in DCM, and p-toluenesulfonic acid (98 mg, 0.5 equivalents) was added at room temperature and stirred for 15 minutes. When the reaction was complete, the reaction mixture was extracted with EtOAc (30 mL x 2), and all organic layers were combined and washed with a saturated aqueous solution of NaCl. The separated organic layers were dried with anhydrous Na2SO4, and the filtrate was filtered. The solvent was removed by vacuum distillation, and the resulting mixture was purified by flash column chromatography (SiO2, EtOAc / hexane 1:1 -> 7:3) to obtain Compound 11 (277 mg, 61%) in a clear oil state.

[0178] 1 H NMR (CDCl3, 400 MHz): δ 0.90 (s, 3H), 0.97 (s, 3H), 2.61-2.64 (t,J= 8.1 Hz, 2H), 3.47-3.58 (m, 4H), 4.00 (s, 1H), 4.18-4.21 (t,J= 5.1 Hz, 1H), 4.40-4.42 (d, 2H), 7.27-7.30 (m, 3H), 7.32-7.34 (t,J= 5.5 Hz, 2H), 7.39-7.42 (d,J= 5.0 Hz, 2H), 7.56-7.77 (d,J= 4.8 Hz, 2H); 13 C NMR (CDCl3, 100 MHz): δ 14.19, 20.34, 21.05, 21.31, 34.06, 34.63, 39.26, 46.71, 53.47, 60.47, 66.62, 71.15, 76.82, 77.14, 77.45, 77.48, 120.09, 124.95, 127.18, 127.89, 141.29, 143.59, 171.33, 172.17, 173.54.

[0179]

[0180] Synthesis of 4-((3-((9H-Fluoren-9-yl)methoxy)-3-oxopropyl)amino)-3-hydroxy-2,2-dimethyl-4-oxobutyl (2-hexyldecyl) malonate (12)

[0181] Add Compound 7 (181 mg, 1.1 equiv) to a reaction vessel and dissolve it in DCM (20 mL); then add EDCI·HCl (154 mg, 1.6 equivalents) and 4-dimethylaminopyridine (DMAP) (13 mg, 0.2 equivalents) and stir for 20 minutes at room temperature under an argon gas atmosphere. Add Compound 11 (200 mg, 1 equivalent) to another reaction vessel and dissolve it in DCM (10 mL); then 5 o After cooling to C, the mixture of Compound 7 under argon gas 5 o Dropwise at C for 30 minutes, then stirred at room temperature for 2 hours. After confirming that the reaction was complete by TLC (SiO2, EtOAc / hexane 2:8, PMA stain), the solvent was removed by vacuum distillation, the solution was dissolved again in DCM (50 mL), transferred to a separatory funnel, and washed sequentially with a saturated aqueous solution of NaHCO3 (2 x 100 mL) and a saturated aqueous solution of NaCl (100 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtrate was filtered. The solvent was removed by vacuum distillation of the filtered solution, and the resulting mixture was purified by flash column chromatography (SiO2, DCM / hexane 1:9 -> 1:1) to obtain Compound 12 (256 mg, 72%) in a clear oil state.

[0182] 1H NMR (CDCl3, 400 MHz): δ 0.78-0.81 (t,J= 8.5 Hz, 6H), 0.95-0.95 (d,J= 8.9 Hz, 6H), 1.15-1.28 (m, 41H), 1.54 (s, 1H), 2.52-2.53 (t,J= 5.7 Hz, 2H), 3.32 (s, 2H), 3.35-3.51 (m, 2H), 3.83-3.99 (m, 4H), 4.12-4.26 (t,J= 5.0 Hz, 1H), 4.22-4.34 (m, 2H), 4.81 (s, 1H); 13 C NMR (CDCl3, 100 MHz): δ 14.10, 20.03, 20.94, 21.32, 22.63, 22.65, 26.61, 26.63, 26.68, 29.32, 29.56, 29.60, 29.61, 29.93, 29.96, 31.08, 31.21, 31.82, 31.89, 33.01, 33.18, 33.66, 34.75, 37.20, 37.26, 37.32, 41.37, 46.70, 66.70, 67.39, 68.61, 70.34, 76.79, 77.11, 77.31, 77.42, 120.05, 125.01, 125.03, 127.15, 129.84, 141.28, 143.62, 143.72, 166.13, 166.95, 167.84, 171.73.

[0183]

[0184] Synthesis of 1-(9H-Fluoren-9-yl)-17-hexyl-9,9-dimethyl-3,7,12,14-tetraoxo-2,11,15-trioxa-6-azapentacosan-8-yl(2-hexyldecyl)glutarate (13)

[0185] Add Compound 5 (164 mg, 1.3 equivalents) to a reaction vessel and dissolve in DCM (30 mL); then add EDCI·HCl (101 mg, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP) (7 mg, 0.2 equivalents) and 5 under argon gas.o Stir vigorously at C. Then, add a solution of Compound 12 (250 mg, 1 equivalent) dissolved in DCM (10 mL) to the reaction mixture 5 o Dropwise at C and stirred at room temperature for 4 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc / hexane 1:9, PMA stain), DCM (100 mL) was added, transferred to a separatory funnel, and washed sequentially with saturated aqueous NaHCO3 solution (2 x 200 mL) and saturated aqueous NaCl solution (200 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtrate was filtered. The solvent was removed by vacuum distillation, and the resulting mixture was purified by flash column chromatography (SiO2, EtOAc / hexane 1:9) to obtain Compound 13 (300 mg, 81%) as a clear oil.

[0186] 1 H NMR (CDCl3, 400 MHz): δ 0.78-0.81 (t,J= 8.5 Hz, 12H), 0.95-0.97 (d,J= 8.9 Hz, 6H), 1.15-1.27 (m, 53H), 1.53 (s, 2H), (qui,J= 5.1 Hz, 2H), 2.26-2.30 (t,J= 5.5 Hz, 2H), 2.37-2.40 (t,J= 5.1 Hz, 2H), 2.53-2.56 (t,J= 5.7 Hz, 2H), 3.32 (s, 2H), 3.35-3.52 (m, 2H), 3.83-3.99 (m, 6H), 4.10-4.26 (t,J= 5.0 Hz, 1H), 4.29-4.34 (m, 2H), 4.82 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.09, 14.11, 20.01, 20.92, 21.30, 22.65, 22.67, 26.60, 26.64, 26.69, 29.31, 29.56, 29.59, 29.61, 29.93, 29.95, 31.07, 31.21, 31.81, 31.89, 33.02, 33.18, 33.67, 34.75, 37.20, 37.28, 37.32, 41.36, 46.70, 66.70, 67.35, 68.61, 70.34, 76.79, 77.11, 77.31, 77.42, 120.05, 125.01, 125.03, 127.15, 129.84, 141.28, 143.62, 143.70, 166.19, 166.98, 167.85, 171.72, 172.30, 173.01.

[0187]

[0188] Synthesis of 3-(4-((3-((2-Hexyldecyl)oxy)-3-oxopropanoyl)oxy)-2-((5-((2-hexyldecyl)oxy)-5-oxopentanoyl)oxy)-3,3-dimethylbutanamido)propanoic acid (14) as a precursor of chemical formulas 3-1 to 3-4

[0189] Mix piperidine and DMF in a reaction vessel at a volume ratio of 2:8, and 0 o Prepare a 20% piperidine solution by cooling to C. Place Compound 13 (300 mg) in another reaction vessel and dissolve it in DMF (20 ml) 0 o Stir at C for 5 minutes. Then, add the prepared 20% piperidine solution (40 ml) under argon gas, and 0 oThe mixture was stirred at C for 5 minutes. After the reaction was complete, 3M aqueous HCl solution was added to adjust the pH to 7, diluted with EtOAc (200 mL), transferred to a separatory funnel, and washed sequentially with distilled water (2 x 200 mL) and saturated aqueous NaCl solution (2 x 200 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtrate was filtered. The solvent was removed by vacuum distillation, and the resulting mixture was purified by flash column chromatography (SiO2, EtOAc / hexane 0:1 -> 4:6, with 1% AcOH added) to obtain Compound 14 (226 mg, 91%) as a clear oil.

[0190] 1 H NMR (CDCl3, 400 MHz): δ 0.82 (t,J= 8.1 Hz, 12H), 0.97-1.00 (d,J= 8.3 Hz, 6H), 1.15-1.29 (m, 50H), 1.56 (s, 2H), 1.85-1.93 (m, 2H), 2.30-2.55 (m, 6H), 3.28-3.38 (m, 3H), 3.55-3.63 (m, 1H), 3.85-4.01 (m, 6H), 4.87 (s, 1H), 6.76 (t,J= 5.7 Hz, 1H); 13 C NMR (CDCl3, 100 MHz): δ 14.08, 14.09, 19.86, 20.99, 21.24, 22.64, 22.66, 26.60, 26.64, 26.68, 29.30, 29.35, 29.55, 29.59, 29.94, 31.07, 31.20, 31.80, 31.89, 32.87, 33.18, 33.39, 34.49, 37.21, 37.22, 37.25, 41.32, 67.79, 68.67, 70.34, 77.23, 166.30, 167.09, 167.97, 171.70, 173.96, 175.31.

[0191]

[0192] Synthesis of 20-Hexyl-2,12,12-trimethyl-6,10,15,17-tetraoxo-14,18-dioxa-2,5,9-triazaoctacosan-11-yl (2-hexyldecyl) glutarate (Chemical Formula 3-1)

[0193] Add Compound 14 (100 mg, 1 equivalent) to a reaction vessel and dissolve in DCM (50 mL), then add HATU (66 mg, 1.5 equivalents), H Nig's base (22.32 mg, 1.5 equivalents) and Compound 15 (12 mg, 1.1 equivalents) were added and vigorously stirred at room temperature for 11 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc / MeOH 9:1, PMA stain), DCM (100 mL) was added, transferred to a separatory funnel, and washed sequentially with saturated aqueous NaHCO3 solution (2 x 200 mL) and saturated aqueous NaCl solution (200 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtrate was filtered. The solvent was removed by vacuum distillation, and the resulting mixture was purified by flash column chromatography (SiO2, EtOAc / MeOH 10:0 -> 7:3) to obtain the compound of Formula 3-1 (89 mg, 82%) in a clear oil state.

[0194] 1 H NMR (CDCl3, 400 MHz): δ 0.81 (t,J= 7.1 Hz, 12H), 0.97-0.99 (d,J= 8.1 Hz, 6H), 1.14-1.30 (m, 50H), 1.55 (s, 2H), 1.87-1.93 (qui,J= 8.2 Hz, 2H), 2.14 (s, 6H), 2.17-2.45 (m, 8H), 3.15-3.51 (m, 6H), 3.87-4.02 (m, 6H), 4.78 (s, 1H), 6.24 (m, 1H), 7.16-7.22 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.07, 19.99, 21.03, 21.06, 22.62, 22.64, 26.58, 26.62, 26.67, 29.58, 26.62, 26.67, 29.28, 29.53, 29.57, 29.58, 29.91, 31.05, 31.20, 31.78, 31.86, 33.06, 33.21, 35.26, 35.56, 26.67, 37.19, 37.27, 41.37, 45.08, 57.67, 67.34, 68.50, 70.28, 166.27, 166.81, 167.80, 171.54, 171.99, 173.00.

[0195]

[0196]

[0197] Synthesis of 18-Hexyl-10,10-dimethyl-1-morpholino-4,8,13,15-tetraoxo-12,16-dioxa-3,7-diazahexacosan-9-yl (2-hexyldecyl) glutarate (Chemical Formula 3-2)

[0198] Add Compound 14 (100 mg, 1 equivalent) to a reaction vessel and dissolve in DCM (50 mL), then add HATU (66 mg, 1.5 equivalents), H Nig's base (22.32 mg, 1.5 equivalents) and Compound 16 (17 mg, 1.1 equivalents) were added and vigorously stirred at room temperature for 9 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc / MeOH 9:1, PMA stain), DCM (100 mL) was added, the mixture was transferred to a separatory funnel, and washed with saturated aqueous NaHCO3 solution (2 x 200 mL) and saturated aqueous NaCl solution (200 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtrate was filtered. The solvent was removed by vacuum distillation, and the resulting mixture was purified by flash column chromatography (SiO2, EtOAc / MeOH 10:0 -> 9:1) to obtain the compound of Formula 3-2 (99 mg, 88%) in a clear oil state.

[0199] 1 H NMR (CDCl3, 400 MHz): δ 0.81 (t,J= 7.9 Hz, 12H), 0.97-0.99 (d,J= 8.2 Hz, 6H), 1.15-1.31 (m, 51H), 1.55 (s, 2H), 1.85-1.93 (qui,J= 8.1 Hz, 2H), 2.29-2.44 (m, 10H), 3.22-3.30 (m, 2H), 3.32 (s, 2H), 3.35-3.51 (m, 2H), 3.63-3.65 (t,J= 4.1 Hz, 4H), 3.87-4.01 (m, 6H), 6.15 (s, 1H), 7.02-7.05 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.08, 19.99, 20.99, 21.20, 22.63, 22.65, 26.58, 26.62, 26.67, 29.29, 29.54, 29.57, 29.59, 29.92, 29.93, 31.05, 31.19, 31.79, 31.79, 31.87, 33.05, 33.21, 35.17, 35.42, 35.51, 37.18, 37.23, 37.26, 41.36, 53.32, 57.02, 66.78, 67.35, 66.53, 70.28, 76.95, 166.26, 166.87, 167.86, 171.32, 171.87, 172.99.

[0200]

[0201] Synthesis of 20-Hexyl-2,12,12-trimethyl-6,10,15,17-tetraoxo-5,14,18-trioxa-2,9-diazaoctacosan-11-yl (2-hexyldecyl) glutarate (Chemical Formula 3-3)

[0202] Add Compound 14 (100 mg, 1 equivalent) to a reaction vessel and dissolve in DCM (30 mL); then add EDCI·HCl (33 mg, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP) (3 mg, 0.2 equivalents) and 5 under argon gas. o Stir vigorously at C for 10 minutes. Then, add a solution of Compound 17 (11 mg, 1.1 equivalents) dissolved in DCM (10 mL) to the reaction mixture 5 oAfter adding dropwise at C, the mixture was stirred at room temperature for 4 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc 100%, PMA stain), DCM (100 mL) was added, the mixture was transferred to a separatory funnel, and washed sequentially with saturated aqueous NaHCO3 solution (2 x 200 mL) and saturated aqueous NaCl solution (200 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtered filtrate was subjected to vacuum distillation to remove the solvent. The resulting mixture was purified by flash column chromatography (SiO2, EtOAc / MeOH 20:0 -> 19:1) to obtain the compound of Formula 3-3 (97 mg, 90%) in a clear oil state.

[0203] 1 H NMR (CDCl3, 400 MHz): δ 0.81 (t,J= 7.5 Hz, 12H), 0.97-0.99 (d,J= 8.2 Hz, 6H), 1.13-1.31 (m, 50H), 1.56 (s, 2H), 1.88-1.93 (qui,J= 8.2 Hz, 2H), 2.22 (s, 6H), 2.31 (t,J= 7.6 Hz, 2H), 2.41(t,J= 7.1 Hz, 2H), 2.47-2.52 (m, 4H), 3.34 (s, 2H), 3.37-3.52 (m, 2H), 3.86-4.14 (m, 8H), 4.83 (s, 1H), 6.83 (t,J= 7.8 Hz, 1H); 13 C NMR (CDCl3, 100 MHz): δ 14.07, 14.08, 20.01, 20.98, 21.17, 22.63, 22.65, 26.59, 26.64, 26.68, 29.29, 29.55, 29.58, 29.60, 29.93, 29.94, 31.07, 31.21, 31.79, 31.88, 33.03, 33.20, 33.76, 34.75, 37.20, 37.29, 41.37, 45.58, 57.68, 62.14,67.37, 68.58, 70.32, 166.21, 166.91, 167.72, 171.72, 172.36, 173.02.

[0204]

[0205]

[0206] Synthesis of 18-Hexyl-10,10-dimethyl-1-morpholino-4,8,13,15-tetraoxo-3,12,16-trioxa-7-azahexacosan-9-yl (2-hexyldecyl) glutarate (Chemical Formula 3-4)

[0207] Add Compound 14 (100 mg, 1.3 equivalents) to a reaction vessel and dissolve in DCM (30 mL); then add EDCI·HCl (33 mg, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP) (3 mg, 0.2 equivalents) and 5 under argon gas. o Stir vigorously at C for 10 minutes. Then, add a solution of Compound 18 (17 mg, 1.1 equivalents) dissolved in DCM (10 mL) to the reaction mixture 5 o Add dropwise from C and stir at room temperature for 6 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc 100%, PMA stain), DCM (100 mL) was added, transferred to a separatory funnel, and washed with saturated aqueous NaHCO3 solution (2 x 200 mL) and saturated aqueous NaCl solution (200 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtered filtrate was subjected to vacuum distillation to remove the solvent. The resulting mixture was purified by flash column chromatography (SiO2, EtOAc / hexane 8:3 -> 10:0) to obtain the compound of Formula 3-4 (89 mg, 79%) in a clear oil state.

[0208] 1H NMR (CDCl3, 400 MHz): δ 0.82 (t,J= 7.1 Hz, 12H), 0.97–0.99 (d,J= 8.9 Hz, 6H), 1.13–1.31 (m, 51H), 1.51–1.1 (s 1. 88), Hz, 2H), 2.23-2.33 (t,J= 7.5 Hz, 2H), 2.39-2.49 (m, 8H), 2.53-2.56 (t,J= 7.0 Hz, 2H), 3.34 (s, 2H), 3-37.6.6, 3.4. (t,J= 3.9 Hz, 4H), 3.86–4.00 (m, 6H), 4.11–4.17 (m, 2H), 4.83 (s, 1H), 6.75 (t,J= 7.9 Hz, 1H); 13 C NMR (CDCl3, 100 MHz): δ 14.06, 19.99, 20.89, 21.28, 22.61, 22.63, 26.57, 29.55, 29.57, 29.90, 29.92, 31.05, 31.19, 31.77, 31.86, 33.00, 33.17, 33.65, 34.73, 37.18, 37.34, 43.28. 56.98, 61.99, 66.80, 67.69, 66.80, 67.34, 68.58, 70.30, 166.16, 166.94, 167.75, 171.68, 172.29.

[0209]

[0210]

[0211] O,O'-(4-((3-(((9H-yl)methoxy)-3-oxopropyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl)bis(2-hexyldecyl) diglutarate (19)의 합성

[0212] Add Compound 11 (164 mg, 1.3 equivalents) to a reaction vessel and dissolve in DCM (30 mL); then add EDCI·HCl (101 mg, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP) (7 mg, 0.2 equivalents) and 5 under argon gas. o Stir vigorously at C for 10 minutes. Then, add a solution of Compound 7 (250 mg, 1 equivalent) dissolved in DCM (10 mL) to the reaction mixture 5 o Dropwise at C and stirred at room temperature for 4 hours. After confirming that the reaction was complete by TLC (SiO2, EtOAc / hexane 1:9, PMA stain), additional DCM (100 mL) was added, and the mixture was washed sequentially with a saturated aqueous solution of NaHCO3 (2 x 200 mL) and a saturated aqueous solution of NaCl (200 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtrate was filtered to remove the solvent by vacuum distillation. The resulting mixture was purified by flash column chromatography (SiO2, EtOAc / hexane 1:9) to obtain Compound 19 (300 mg, 81%) in a clear oil state.

[0213] 1H NMR (CDCl3, 400 MHz): δ 0.80 (t,J= 8.0 Hz, 12H), 0.92–0.97 (d,J= 9.2 Hz, 6H), 1.11–1.28 (m, 50H), 1.52 (s,J=2H3H), 5.5 Hz, 4H), 2.26–2.33 (m, 6H), 2.38 (t,J= 5.7 Hz, 2H), 2.53–2.56 (t,J= 5.9 Hz, 2H), 3.38–3.51 (m, 2H, 3.76), 3.9H 4.12–4.31 (t,J= 5.0 Hz, 1H), 4.33 (d,J= 9.1 Hz, 2H), 4.88 (s, 1H), 6.61 (m, 1H), 7.24 (t,J= 7.1 Hz, 29 (2H), ,J=6. (d,J= 8.3 Hz, 2H), 7.68 (d,J= 8.2 Hz, 2H); 13 C NMR (CDCl3, 100 MHz): δ 14.08, 14.10, 20.00, 20.13, 20.90, 21.32, 22.64, 22.67, 26.65, 26.69, 29.36, 31.21, 31.24, 31.81, 31.89, 33.05, 33.17, 33.24, 33.37, 33.66, 34.65, 37.29, 37.38, 46.74, 66.71, 69.23, 120.08, 124.98, 125.00, 127.17, 127.88, 141.30, 143.57, 143.65, 167.95, 171.62, 171.17.236. 173.09.

[0214]

[0215] 화학식 3-5내지 3-8의 합성 전구체로서 3-(2,4-bis((5-((2-Hexyldecyl)oxy)-5-oxopentanoyl)oxy)-3,3,3-acpronaicpro (dimethylpa0) 합성

[0216] In a reaction vessel, mix piperidine and DMF in a volume ratio of 2:8 and stir, then 0 o Prepare a 20% piperidine solution by cooling to C. In another reaction vessel, prepare Compound 19 (300 mg) dissolved in DMF (20 ml), stir for 5 minutes, and then 0 o Cool to C. Add the previously prepared 20% piperidine solution (40 mL) under argon gas, and 0 o The mixture was stirred at C for 5 minutes. Once the reaction was complete, 3M aqueous HCl solution was added to the reaction mixture to adjust the pH to 7. After adding EtOAc (200 mL), the mixture was transferred to a separatory funnel and washed sequentially with distilled water (2 x 200 mL) and saturated aqueous NaCl solution (2 x 200 mL). The separated organic layer was dried with anhydrous Na2SO4, and the filtered filtrate was subjected to vacuum distillation to remove the solvent. The resulting mixture was purified by flash column chromatography (SiO2, EtOAc / hexane 0:1 -> 4:6, with the addition of 1% AcOH) to obtain Compound 20 (226 mg, 91%) in a clear oil state.

[0217] 1 H NMR (CDCl3, 400 MHz): δ 0.81 (t,J= 8.1 Hz, 12H), 0.91-0.92 (d,J= 9.0 Hz, 6H), 1.12-1.21 (m, 51H), 1.52 (s, 2H), 1.62 (s, 1H), (qui,J= 5.5 Hz, 4H), 2.27-2.33 (m, 6H), 2.39 (t,J= 5.8 Hz, 2H), 2.53-2.55 (t,J= 5.3 Hz, 2H), 3.38-3.52 (m, 2H), 3.77-3.97 (m, 6H), 4.89 (s, 1H), 6.62 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.09, 14.11, 20.02, 20.14, 20.91, 21.31, 22.66, 22.68, 26.61, 26.65, 29.31, 29.56, 29.61, 29.95, 31.22, 31.24, 31.81, 31.89, 33.05, 33.17, 33.24, 33.37, 33.66, 34.65, 37.29, 37.38, 46.70, 66.71, 67.35, 67.41, 69.23, 167.95, 171.62, 172.48, 172.62, 173.01, 173.09.

[0218]

[0219] Synthesis of O,O'-(4-((3-((2-(Dimethylamino)ethyl)amino)-3-oxopropyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl)bis(2-hexyldecyl) diglutarate (Formula 3-5)

[0220] Add Compound 20 (100 mg, 1 equivalent) to a reaction vessel and dissolve in DCM (50 mL), then add HATU (66 mg, 1.5 equivalents), H Nig's base (22.32 mg, 1.5 equivalents) and Compound 15 (12 mg, 1.1 equivalents) were added and vigorously stirred at room temperature for 11 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc / MeOH 9:1, PMA stain), DCM (100 mL) was added, and the mixture was washed sequentially with a saturated aqueous solution of NaHCO3 (2 x 200 mL) and a saturated aqueous solution of NaCl (200 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtered filtrate was subjected to vacuum distillation. The resulting mixture was purified by flash column chromatography (SiO2, EtOAc / MeOH 10:0 -> 7:3) to obtain the compound of Formula 3-5 (89 mg, 82%) in a clear liquid state.

[0221] 1 H NMR (CDCl3, 400 MHz): δ 0.81 (t,J= 7.1 Hz, 12H), 0.97-0.99 (d,J= 8.1 Hz, 6H), 1.14-1.30 (m, 50H), 1.55 (s, 2H), 1.87-1.93 (qui,J= 8.2 Hz, 4H), 2.14 (s, 6H), 2.17-2.45 (m, 8H), 3.38-3.51 (m, 2H), 3.37 (d,J= 9.6 Hz, 2H), 3.91 (d,J= 9.7 Hz, 4H), 3.96 (d,J= 9.9 Hz, 2H), 4.10-4.14 (m, 2H), 4.87 (s, 1H), 6.24 (m, 1H), 7.16-7.22 (m, 1H); 13 C NMR (CDCl3, 100 MHz): δ 14.07, 19.99, 21.03, 21.06, 22.62, 22.64, 26.58, 26.62, 26.67, 29.58, 26.62, 26.67, 29.28, 29.53, 29.57, 29.58, 29.91, 31.05, 31.20, 31.78, 31.86, 33.06, 33.21, 35.26, 35.56, 26.67, 37.19, 37.27, 41.37, 45.08, 57.67, 67.34, 68.50, 70.28, 166.27, 166.81, 167.80, 171.54, 171.99, 173.00.

[0222]

[0223]

[0224] Synthesis of O,O'-(2,2-Dimethyl-4-((3-((2-morpholinoethyl)amino)-3-oxopropyl)amino)-4-oxobutane-1,3-diyl)bis(2-hexyldecyl) diglutarate (Formula 3-6)

[0225] Add Compound 20 (100 mg, 1 equivalent) to a reaction vessel and dissolve in DCM (50 mL), then add HATU (66 mg, 1.5 equivalents), H Nig's base (22.32 mg, 1.5 equivalents) and Compound 16 (17 mg, 1.1 equivalents) were added and vigorously stirred at room temperature for 9 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc / MeOH 9:1, PMA stain), DCM (100 mL) was added, followed by washing with a saturated aqueous solution of NaHCO3 (2 x 200 mL) and a saturated aqueous solution of NaCl (200 mL). The separated organic layer was dried with MgSO4, and the filtered solution was subjected to vacuum distillation. The resulting mixture was then purified by flash column chromatography (SiO2, EtOAc / MeOH 10:0 -> 9:1) to obtain the compound of Formula 3-6 (99 mg, 88%) in a clear oil state.

[0226] 1 H NMR (CDCl3, 400 MHz): δ 0.81-0.82 (t,J= 7.0 Hz, 12H), 0.95-0.98 (d,J= 8.5 Hz, 6H), 1.11-1.35 (m, 50H), 1.54 (s, 2H), 1.83-1.92 (m, 4H), 2.28-2.47 (m, 16H), 3.21-2.32 (m, 4H), 3.40 (t,J= 3.2 Hz, 4H), 3.78-3.81 (d,J= 8.2 Hz, 1H), 3.89-3.91 (d,J= 8.0 Hz, 4H), 3.94-3.97 (d,J= 9.2 Hz, 1H), 4.84 (s, 1H), 6.18 (m, 1H), 7.00 (t,J= 6.9 Hz, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.04, 14.05, 19.97, 20.09, 20.90, 21.30, 22.59, 22.62, 26.61, 26.65, 29.25, 29.51, 29.56, 29.90, 31.19, 31.31.76, 31.84, 33.05, 33.18, 33.32, 35.07, 35.37, 35.54, 37.25, 53.33, 57.06, 66.76, 67.28, 67.34, 69.21, 76.77, 76.91, 77.09, 77.41, 167.99, 171.45, 191.77, 172.57, 172.95, 173.02.

[0227]

[0228]

[0229] Synthesis of O,O'-(4-((3-((2-(Dimethylamino)ethyl)amino)-3-oxopropyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl)bis(2-hexyldecyl) diglutarate (Formula 3-7)

[0230] Add Compound 20 (100 mg, 1 equivalent) to a reaction vessel and dissolve in DCM (30 mL); then add EDCI·HCl (33 mg, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP) (3 mg, 0.2 equivalents), and 5 under argon gas o Stir vigorously at C for 10 minutes. Then, add a solution of Compound 17 (11 mg, 1.1 equivalents) dissolved in DCM (10 mL) to the reaction mixture 5 oAfter adding dropwise at C, the mixture was stirred at room temperature for 4 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc 100%, PMA stain), DCM (100 mL) was added, and the mixture was washed sequentially with a saturated aqueous solution of NaHCO3 (2 x 200 mL) and a saturated aqueous solution of NaCl (200 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtered filtrate was subjected to vacuum distillation. The resulting solution was then purified by column chromatography (SiO2, EtOAc / MeOH 20:0 -> 19:1) to obtain the compound of Formula 3-7 (97 mg, 90%) in a clear oil state.

[0231] 1 H NMR (CDCl3, 400 MHz): δ 0.81-0.82 (t,J= 7.1 Hz, 12H), 0.95-0.98 (d,J= 8.1 Hz, 6H), 1.11-1.32 (m, 50H), 1.54 (s, 2H), 1.85-1.91 (qui,J= 8.0 Hz, 4H), 2.22 (s, 6H), 2.30-2.32 (t,J= 7.0 Hz, 6H), 2.39 (t,J= 7.9 Hz, 2H), 2.43-2.51 (m, 4H), 3.37 (d,J= 9.6 Hz, 2H), 3.38-3.51 (m, 2H), 3.91 (d,J= 9.7 Hz, 4H), 3.96 (d,J= 9.9 Hz, 2H), 4.10-4.14 (m, 2H), 4.87 (s, 1H), 6.77 (t,J= 6.9 Hz, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.04, 19.99, 20.09, 20.79, 21.32, 22.60.22.62, 26.61, 26.66, 29.26, 29.52, 29.56, 29.91, 31.20, 31.77, 31.85, 33.02, 33.16, 33.19, 33.33, 33.75, 34.66, 37.26, 37.30, 45.57, 57.68, 62.16, 67.28, 67.34, 69.21, 167.80, 171.59, 172.44, 192.55, 172.95, 173.01.

[0232]

[0233]

[0234] Synthesis of O,O'-(2,2-Dimethyl-4-((3-((2-morpholinoethyl)amino)-3-oxopropyl)amino)-4-oxobutane-1,3-diyl)bis(2-hexyldecyl) diglutarate (Formula 3-8)

[0235] Add Compound 20 (100 mg, 1.3 equivalents) to a reaction vessel and dissolve in DCM (30 mL); then add EDCI·HCl (33 mg, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP) (3 mg, 0.2 equivalents) and 5 under argon gas. o Stir vigorously at C for 10 minutes. Then, add a solution of Compound 18 (17 mg, 1.1 equivalents) dissolved in DCM (10 mL) to the reaction mixture 5 oAfter adding dropwise at C, the mixture was stirred at room temperature for 6 hours. After confirming that the reaction was complete using TLC (SiO2, EtOAc 100%, PMA stain), DCM (100 mL) was added, followed by washing with a saturated aqueous solution of NaHCO3 (2 x 200 mL) and a saturated aqueous solution of NaCl (1 x 200 mL). The separated organic layer was dried with anhydrous MgSO4, and the filtrate was filtered. The mixture obtained by vacuum distillation was purified by flash column chromatography (SiO2, EtOAc / hexane 8:3 -> 10:0) to obtain the compound of Formula 3-8 (89 mg, 79%) in a clear oil state.

[0236] 1 H NMR (CDCl3, 400 MHz): δ 0.81 (t,J= 7.6 Hz, 12H), 0.94-0.98 (d,J= 8.7 Hz, 6H), 1.10-1.31 (m, 47H), 1.54 (s, 2H), 1.85-1.93 (m, 4H), 2.28-2.57 (m, 16H), 2.33-2.50 (m, 2H), 3.65 (m, 4H), 3.76-3.79 (d, 1H), 3.89-3.98 (m, 5H), 4.13-1.19 (m,2H), 4.88 (s,1H); 13 C NMR (CDCl3, 100 MHz): δ 14.06, 20.01, 20.12, 20.89, 21.31, 22.62, 22.64, 26.64, 26.68, 29.28, 29.54, 29.58, 29.93, 31.22, 31.79, 31.87, 33.06, 33.18, 33.23, 33.35, 33.66, 34.68, 37.29, 37.35, 53.76, 56.97, 61.62, 66.72, 67.34, 37.41, 69.21, 167.88, 171.62, 172.38, 172.59, 173.00, 173.05.

[0237]

[0238] Example 2-2. Synthesis of Formula 3-9 and Formula 3-10

[0239]

[0240]

[0241] Synthesis of (R)-N-(3-Hydroxypropyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (22)

[0242] Anhydrous sodium sulfate (20 g) was added to D-panthenol 21 (4.0 g, 1 equivalent) while stirring in anhydrous acetone (150 mL) solution. After stirring for about 10 minutes, p-toluenesulfonic acid monohydrate (0.3 g, 0.08 equivalents) was added, and the reaction mixture was vigorously stirred at room temperature under an argon atmosphere for 48 hours. The reaction was monitored for the loss of D-panthenol using TLC (SiO2; ethyl acetate / methanol, 9:1; KMnO4 staining). After the reaction was complete, the acetone was removed by vacuum distillation, the residue was suspended in a saturated sodium bicarbonate solution (200 mL), and extracted six times with ethyl acetate (6 x 50 mL). The organic layer was dried with anhydrous magnesium sulfate and filtered, and the solvent was removed by vacuum distillation. The oily product of the preparation was purified by flash column chromatography (SiO2; ethyl acetate / hexane, 4:6 to 8:2, v / v) to obtain ketal-22 (4.20 g, 88%), a white crystalline solid.

[0243] 1H NMR (CDCl3, 400 MHz): δ 1.01 (s, 3H), 1.06 (s, 3H), 1.43 (s, 3H), 1.47 (s, 3H), 1.71 (dqd,J= 11.0, 5.5, 2.6 Hz, 2H), 3.22 (t,J= 6.5 Hz, 1H), 3.29 (d,J= 11.7 Hz, 1H), 3.43-3.33 (m, 1H), 3.48 (dtd,J= 13.9, 6.9, 5.5 Hz, 1H), 3.66-3.59 (m, 2H), 3.69 (dd,J= 11.6, 0.9 Hz, 1H), 6.77 (br s, 1H), 4.12 (s, 1H); 13 C 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.

[0244] (R)-(9H-Fluoren-9-yl)methyl(3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propyl) carbonate (23)의 합성

[0245] Pyridine (1.30 mL, 4 equivalents) was added to a 40 mL DCM solution of alcohol 22 (1.0 g, 1 equivalent). After stirring for about 5 minutes, fluorenylmethoxycarbonyl chloride (2.10 g, 2 equivalents) was slowly added over 30 minutes using a dropper funnel under an argon atmosphere at 5°C. The reaction mixture was stirred for 8 hours and monitored by TLC (SiO2; ethyl acetate / hexane, 3:7; PMA staining). Once the reaction was complete, H2O (10 mL) was added to the reaction mixture to terminate the reaction and concentrate the solution. The residue of pyridine was removed by co-evaporation with toluene (3 x 15 mL). The resulting gum was dissolved in 50 mL of DCM, washed with saturated sodium bicarbonate solution (2 x 50 mL) and saturated saline (1 x 20 mL), and dried with sodium sulfate. After concentration under reduced pressure, the resulting oil was purified by flash column chromatography (SiO2; ethyl acetate / hexane, 1:9 to 2:8, v / v) to obtain acetonide 23 (1.62 g, 73%) in a clear oil state.

[0246] 1 H 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 (sxt,J= 6.52 Hz, 1H), 3.64 (d,J= 11.68 Hz, 1H), 4.07 (s, 1H), 4.22 (t,J= 7.24 Hz, 3H), 4.36 (d,J= 7.24 Hz, 2H), 6.79 (t,J= 6.00 Hz, 1H), 7.25 (t,J= 6.92 Hz, 2H), 7.32 (t,J= 7.36 Hz, 2H), 7.56 (d,J= 7.41 Hz, 2H), 7.68 (d,J= 8.21 Hz, 2H); 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.

[0247] Synthesis of (R)-(9H-Fluoren-9-yl)methyl(3-(2,4-dihydroxy-3,3-dimethylbutanamido)propyl) carbonate (24)

[0248] p-toluenesulfonic acid monohydrate (427 mg, 0.6 equivalents) was added at room temperature to a DCM solution of acetonide 23 (1.60 g, 1 equivalent) and DL-1,4-dithiothreol (DTT; 1.15 g, 2 equivalents) while stirring. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, it was monitored by TLC (SiO2; hexane / ethyl acetate, 1:1; UV-active). After adding a saturated sodium bicarbonate solution (100 mL), the reaction mixture was extracted with DCM (3 x 50 mL), washed with saturated saline (1 x 50 mL), dried with anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The residue was purified by flash column chromatography (SiO2; ethyl acetate / hexane, 1:1 to 7:3, v / v) to obtain a transparent oil-state diol 24 (1.23 g, 77%).

[0249] 1H NMR (CDCl3, 400 MHz): δ 0.90 (s, 3H), 0.99 (s, 3H), 1.25 (t,J= 6.44 Hz, 2H), 3.31-3.40 (m, 2H), 3.47 (m, 2H), 4.00 (s, 1H), 4.22 (t,J= 7.24 Hz, 3H), 4.43 (d,J= 7.16 Hz, 2H), 7.14 (t,J= 5.64 Hz, 1H), 7.31 (t,J= 7.44 Hz, 2H), 7.39 (t,J= 7.44 Hz, 2H), 7.61 (d,J= 7.44 Hz, 2H), 7.76 (d,J= 7.60 Hz, 2H); 13 C 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.

[0250] (((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 (25)의 합성

[0251] A magnetic stirring device was fitted to a 100-mL single-neck round-bottom flask, and Diol 24 (1.20 g, 1 equivalent), EDCI·HCl (1.61 g, 3 equivalents), and 4-dimethylaminopyridine (DMAP; 100 mg, 0.3 equivalents) were added to 50 mL of DCM. The mixture was vigorously stirred at room temperature for 10 minutes under an argon atmosphere. Then, Acid 5 (2.50 g, 2.5 equivalents) dissolved in 20 mL of DCM at room temperature was slowly poured in, and the reaction mixture was stirred at room temperature until the reaction was complete (approximately 22 hours). The loss of alcohol was monitored by TLC (SiO2; hexane / ethyl acetate, 8:2; UV-active). After the reaction was complete, 100 mL of DCM was added, and the mixture was washed with saturated sodium bicarbonate solution (3 x 60 mL) and saturated saline solution (1 x 50 mL). The organic layer was dried with anhydrous magnesium sulfate and filtered, after which the solvent was removed by vacuum distillation. The preparation was purified by flash column chromatography (SiO2; ethyl acetate / hexane, 1:9, v / v) to obtain a clear liquid of 25 (2.21 g, 64%).

[0252] 1H NMR (CDCl3, 400 MHz): δ 0.87 (t,J= 6.48 Hz, 12H), 1.02 (s, 3H), 1.07 (s, 3H), 1.25 (m, 49H), 1.59 (m, 2H, 1.87), 1.6H-7 2.34-2.41 (m, 6H), 2.46-2.49 (t,J= 7.08 Hz, 2H), 3.34 (q,J= 7.60 Hz, 2H), 3.88 (d,J= 10.96 Hz, 1H), 3.93 (d,J=10-3). 8.00 Hz, 1H), 4.20-4.27 (m, 3H), 4.44 (d,J= 7.20 Hz, 2H), 4.99 (s, 1H), 6.57 (t,J= 5.84 Hz, 1H), 7.22,4.4 (t,J= Hz), 7.48 Hz, 2H), 7.62 (d,J= 7.48 Hz, 2H), 7.78 (d,J= 7.52 Hz, 2H); 13 C NMR (CDCl3, 100 MHz): δ 14.12, 14.13, 20.01, 20.04, 20.82, 21.44, 22.66, 22.68, 26.65, 26.69, 29.68, 29.68, 29.96, 31.18, 31.21, 32.95, 33.15, 33.23, 33.37, 25.74, 37.26, 37.46, 46.79, 65.56, 67.384, 76.61, 120.10, 125.08, 127.18, 127.93, 141.32, 143.28, 155.48, 168.06,

[0253] 화학식 3-9~10의 합성 전구체로서Bis(2-hexyldecyl)O,O'-((R)-4-((3-hydroxypropyl)amino)-2,2-dimethyl-4)dixotaradiyl (2,6-oxobutadiyl-1 합성

[0254] A piperidine / DMF (20:80, v / v) solution was prepared in a 100 mL single-neck round-bottom flask. The prepared solution was cooled to 0°C. 25 (2.20 g) and DMF (20 mL) were added to another flask of the same specifications. The mixture was stirred for 10 minutes to prepare the solution, after which it was cooled to 0°C. Subsequently, DMF (50 mL) containing 20% ​​piperidine was slowly poured in under an argon atmosphere. The solution was stirred under an argon atmosphere for only 5–10 minutes. TLC (SiO2; hexane / ethyl acetate, 7:3; PMA staining) was used to monitor the reaction. After the reaction was complete, 3 M HCl was added to the reaction mixture to acidify it to pH 3. The reaction mixture was diluted with DCM (200 mL) and washed with water (3 x 60 mL) and saturated saline (1 x 50 mL). The solution was dried with sodium sulfate, filtered, and then evaporated under reduced pressure. The resulting oil was purified by flash column chromatography (SiO2; ethyl acetate / hexane, 1:9, v / v) to obtain a clear liquid alcohol26 (1.29 g, 79%).

[0255] 1 H NMR (CDCl3, 400 MHz): δ 0.88 (t,J=6.04 Hz, 12H), 1.03 (s, 3H), 1.09 (s, 3H), 1.26 (m, 51H), 1.61 (m, 2H), 1.66-1.72 (m, 2H), 1.92-2.00 (m, 4H), 2.35-2.41 (m, 6H), 2.47 (t,J= 6.96 Hz, 2H), 3.25 (t,J= 5.60 Hz, 1H), 3.42 (q,J= 5.68 Hz, 2H), 3.70 (q,J= 5.04 Hz, 2H), 3.86 (d,J= 10.96 Hz, 1H), 2.96-2.98 (m, 4H), 4.08 (d,J= 11.00 Hz, 1H), 5.00 (s, 1H), 6.97 (t,J= 5.44 Hz, 1H); 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.

[0256]

[0257] Synthesis of O,O'-((R)-2,2-Dimethyl-4-((3-((3-morpholinopropanoyl)oxy)propyl)amino)-4-oxobutane-1,3-diyl)bis(2-hexyldecyl)diglutarate (화학식 3-9)

[0258] A magnetic stirring device was fitted to a 50-mL single-neck round-bottom flask, and 3-morpholinopropionic acid 27 (47 mg, 1.3 equivalents), EDCl·HCl (65 mg, 1.5 equivalents), and 4-dimethylaminopyridine (DMAP) (6 mg, 0.2 equivalents) were added to 20 mL of DCM. The mixture was vigorously stirred at room temperature for 5 minutes under an argon atmosphere. Then, alcohol 26 (200 mg, 1 equivalent) was added, and the reaction mixture was stirred at room temperature for 9 hours. The reaction was monitored for the loss of alcohol using TLC (SiO2; hexane / ethyl acetate, 9:1; PMA staining). After the reaction was complete, 50 mL of DCM was added, and the mixture was washed with saturated sodium bicarbonate solution (3 x 40 mL) and saturated saline (1 x 30 mL). The organic layer was dried with anhydrous magnesium sulfate and filtered, after which the solvent was removed by vacuum distillation. The preparation was purified by flash column chromatography (SiO2; ethyl acetate / methanol, 10:0 to 9.5:0.5, v / v) to obtain a clear liquid compound of Formula 3-9 (206 mg, 89%).

[0259] 1 H NMR (CDCl3, 400 MHz): δ 0.88 (t,J=6.08 Hz, 12H), 1.03 (s, 3H), 1.08 (s, 3H), 1.26 (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= 7.04 Hz, 2H), 3.33 (q,J= 6.32 Hz, 2H), 3.67 (t,J= 4.40 Hz, 4H), 3.88 (d,J= 10.96 Hz, 1H), 3.98 (d,J= 5.68 Hz, 4H), 4.08 (d,J= 10.96 Hz, 1H), 4.11-4.18 (m, 2H), 4.99 (m, 1H), 6.63 (t,J= 5.92 Hz, 1H);13 C 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. HRMS (ESI): [M+H] + m / zcalculated for C 58 H 107 N2O 12 + : 1023.7819; found: 1023.7832.

[0260]

[0261] Synthesis of O,O'-((R)-4-((3-((Dimethylglycyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl)bis(2-hexyldecyl) diglutarate (Formula 3-10)

[0262] A magnetic stirring device was fitted to a 50-mL single-neck round-bottom flask, and dimethylglycine 28 (30 mg, 1.3 equivalents), EDCl·HCl (65 mg, 1.5 equivalents), and 4-dimethylaminopyridine (DMAP) (6 mg, 0.2 equivalents) were added to 20 mL of DCM. The mixture was vigorously stirred at room temperature for 5 minutes under an argon atmosphere. Then, alcohol 26 (200 mg, 1 equivalent) was added, and the reaction mixture was stirred at room temperature for 14 hours. The reaction was monitored for the loss of alcohol using TLC (SiO2; ethyl acetate; PMA staining). After the reaction was complete, 50 mL of DCM was added, and the mixture was washed with saturated sodium bicarbonate solution (3 x 40 mL) and saturated saline (1 x 30 mL). The organic layer was dried with anhydrous magnesium sulfate and filtered, after which the solvent was removed by vacuum distillation. The preparation was purified by flash column chromatography (SiO2; ethyl acetate / methanol, 10:0 to 9:1, v / v) to obtain a clear liquid compound of formula 3-10 (186 mg, 85%).

[0263] 1 H NMR (CDCl3, 400 MHz): δ 0.88 (t,J=6.00 Hz, 12H), 1.03 (s, 3H), 1.07 (s, 3H), 1.26 (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= 11.00 Hz, 1H), 3.98 (d,J= 5.68 Hz, 4H), 4.07 (d,J= 10.96 Hz, 1H), 4.13-4.25 (m, 2H), 4.98 (m, 1H), 6.63 (t,J= 5.88 Hz, 1H); 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. HRMS (ESI): [M+H] + m / zcalculated for C 55 H 103 N2O 11 + : 967.7556; found: 967.7574.

[0264]

[0265]

[0266] Synthesis of O,O'-(4-((3-((4-(Dimethylamino)butanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 3-11)

[0267] 4-(dimethylamino)butyric acid 29 (39 mg, 1.3 equivalents), EDCl26 (65 mg, 1.5 equivalents), and 4-dimethylaminopyridine (DMAP; 5.53 mg, 0.2 equivalents) were added to a 50 mL single-neck flask along with DCM (20 mL). The mixture was vigorously stirred for 5 minutes under an argon atmosphere at room temperature. Subsequently, alcohol 26 (200 mg, 1 equivalent) was added, and the mixture was stirred for 8 hours at room temperature. Alcohol loss was monitored by TLC (SiO₂; ethyl acetate / methanol, 9:1; PMA staining). After the reaction was complete, DCM (50 mL) was added, and the mixture was washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried with anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The concentrated mixture was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 10:0 to 9:1, v / v) to obtain the compound of formula 3-11 (194 mg, 86%) in the form of a clear oil.

[0268] 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).

[0269] 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.

[0270]

[0271]

[0272] O,O'-(4-((3-((3-Bromopropanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Compound 31)

[0273] Alcohol 26 (300 mg, 1 equivalent), EDCI·HCl (196 mg, 3 equivalents), and 4-dimethylaminopyridine (DMAP; 8 mg, 0.2 equivalents) were added to a 50 mL single-neck flask along with DCM (20 mL). After cooling the mixture to 0°C, 4-(dimethylamino)butyric acid 30 (39 mg, 4 equivalents) was dissolved in DCM and slowly added to the flask over 20 minutes. Subsequently, the mixture was vigorously stirred at 60°C for 19 hours. Alcohol loss was monitored by TLC (SiO₂; ethyl acetate / hexane, 3:7; PMA staining). After the reaction was complete, DCM (50 mL) was added, and the mixture was washed with saturated sodium bicarbonate solution (3 x 40 mL) and brine (1 x 30 mL). The organic layer was dried with anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The concentrated mixture was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 1:9 to 5:5, v / v) to obtain bromide 31 (262 mg, 86%) in the form of a clear oil.

[0274] 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).

[0275] 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.

[0276]

[0277] Synthesis of O,O'-(4-((3-((3-(Diethylamino)propanoyl)oxy)propyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl) bis(2-hexyldecyl) diglutarate (Formula 3-12)

[0278]

[0279]

[0280] A solution of bromide 31 (200 mg, 1 equivalent) dissolved in DCM (10 mL) was slowly added over 20 minutes to a solution of triethylamine (60 mg, 3 equivalents) and diethylamine 32 (29 mg, 2 equivalents) dissolved in DCM (20 mL). The resulting solution was vigorously stirred at room temperature for 5 hours, and the loss of bromide 31 was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After the reaction was complete, 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 with anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The concentrated mixture was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 10:0 to 9:1, v / v) to obtain the compound of formula 3-12 (196 mg, 99%) in the form of a clear oil.

[0281] 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).

[0282] 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.

[0283]

[0284]

[0285] 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 (화학싕읩 읱 3-13

[0286] A solution of bromide 31 (150 mg, 1 equivalent) dissolved in DCM (10 mL) was slowly added over 20 minutes to a solution of triethylamine (45 mg, 3 equivalents) and 1,4-oxazipan 33 (30 mg, 2 equivalents) dissolved in DCM (20 mL). The resulting solution was vigorously stirred at room temperature for 9 hours, and the loss of bromide 31 was monitored by TLC (SiO₂; ethyl acetate; PMA staining). After the reaction was complete, 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 with anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The concentrated mixture was purified by flash column chromatography (SiO₂; ethyl acetate / hexane, 5:5 to 10:0, v / v) to obtain the compound of formula 3-13 (151 mg, 99%) in the form of a clear oil.

[0287] 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).

[0288] 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.

[0289]

[0290] Synthesis of bis(2-hexyldecyl)O,O'-(2,14,14-trimethyl-6,12-dioxo-5,7-dioxa-2,11-diazapentadecane-13,15-diyl)diglutarate (화학식 3-14)

[0291]

[0292] A stirrer was placed in a 50 mL wooden flask, fitted with a 25 mL equilibrium pressure dropping funnel, and then dried with fire under an argon atmosphere. 1,1'-carbonyldiimidazole (CDI, 55 mg, 1.5 equivalents) and anhydrous DCM (10 mL) were rapidly added to the flask to form a white suspension, and the flask was cooled to 0°C. The flask was opened, alcohol 26 (200 mg, 1 equivalent) was dissolved in DCM (15 mL), filled into the dropping funnel, and the mixture was dropped for 30 minutes to allow it to become a homogeneous solution. Subsequently, the dropping funnel was rinsed with an additional 5 mL of DCM and removed. The mixture was stirred at room temperature for approximately 3–5 hours to confirm that alcohol 26 and CDI had reacted to form the alkoxycarbonylimidazole intermediate 34 (TLC, SiO₂; ethyl acetate / hexane, 7:3; PMA staining). Dimethylethanolamine 17 (DMEA, 61 mg, 3 equivalents), triethylamine (69 mg, 3 equivalents), and DMAP (33 mg, 1.2 equivalents) were added to the reaction mixture and stirred at room temperature for 18 hours. After the reaction was complete, 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 with anhydrous sodium sulfate, filtered, and then vacuum distilled to remove the solvent. The concentrated mixture was purified by flash column chromatography (SiO₂; ethyl acetate / methanol, 10:0 to 9:1, v / v) to obtain the compound of formula 3-14 (163 mg, 72%) as a clear oil.

[0293] 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 (bs, 2H), 1.82 (m, 2H), 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), 3.27-3.31 (m, 2H), 3.82 (d,J= 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), 6.61 (t,J= 5.8 Hz, 1H).

[0294] ¹³C NMR (CDCl₃, 100 MHz): δ 14.03, 19.97, 20.09, 20.73, 21.40, 22.57, 22.60, 26.58, 26.62, 28.57, 29.23, 29.49, 29.54, 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, 65.21, 65.46, 67.23, 67.30, 69.16, 76.56, 155.42, 167.97, 171.61, 172.52, 173.00.

[0295]

[0296] Example 3. Synthesis of a novel gallic acid-based helper lipid

[0297] A novel helper lipid optimized for delivering GFP mRNA into NK cells was synthesized using the following synthesis method.

[0298] Example 3-1. Synthesis of Formulas 2-3 to 2-4

[0299]

[0300] Methyl 3,4,5-tris(methoxymethoxy)benzoate (36)

[0301] Methyl 3,4,5-trihydroxybenzoate 35 (1 g, 1 equivalent) was added to a 100 mL three-necked flask connected to a Teflon-coated elliptical magnetic stirrer, a 100 mL pressure-balancing dropping funnel fitted with a rubber stopper, a CaCl₂ drying tube, and a rubber stopper. Dichloromethane anhydrous (DCM, 20 mL) was injected into the reaction flask using a syringe, and stirring was started. The suspension was cooled to 0°C. Subsequently, H nig base (DMAP; 4 g, 6 equivalents) was added via a syringe over a period of 1 minute. Subsequently, methoxymethyl chloride (MOM-Cl; 1.75 g, 4 equivalents) and anhydrous DCM (30 mL) were placed in a dropping funnel and manually stirred. This solution was slowly dropwise added to the clear reaction mixture over 30 minutes, and after the dropping was complete, the reaction mixture was slowly raised to room temperature (25°C) while stirring was continued for 18 hours. The progress of the reaction was monitored by TLC (SiO₂; hexane / ethyl acetate 6:4, PMA staining). After the reaction was complete, the reaction mixture was poured with a saturated aqueous NH₄Cl solution (40 mL) and DCM (10 mL), and the layers were separated. The aqueous layer was further extracted with DCM (30 mL twice). The organic layer was bonded, washed with brine (30 mL), and dried with MgSO₄; the solvent was removed by vacuum distillation after filtration. The resulting residue was purified by flash column chromatography (SiO₂; hexane / ethyl acetate 8:2 to 5:5, v / v), and methyl 3,4,5-tris(methoxymethoxy)benzoate 36 (1.52 g, 89%) was obtained in the form of a white solid.

[0302] 1 H NMR (CDCl3, 400 MHz): δ 3.44 (s, 6H), 3.53 (s, 3H), 3.81 (s, 3H), 5.14 (s, 2H), 5.17 (s, 4H), 7.46 (s, 2H);13 C NMR (CDCl3, 100 MHz): δ 52.21, 56.39, 57.21, 95.27, 98.49, 111.61, 125.91, 140.70, 150.69, 166.36.

[0303]

[0304] 3,4,5-tris(methoxymethoxy)benzoic acid (37)

[0305] Methyl 3,4,5-tris(methoxymethoxy)benzoate 36 (1.50 g, 1 equivalent), potassium carbonate (K₂CO₃, 1.31 g, 2 equivalents), and methanol / water (1:1, 50 mL) were added to a 100 mL round-bottom flask equipped with a Teflon-coated magnetic stirrer and a reflux condenser. The mixture was heated at 50°C for 5 hours. The progress of the reaction was monitored using TLC (SiO₂; hexane / ethyl acetate / formic acid = 6:4:0.1; UV active). After the reaction was complete, the reaction mixture was vacuum distilled to evaporate the methanol, poured into ice water, and carefully acidified to pH 3 using 1 M hydrochloric acid (HCl). The resulting mixture was transferred to a separatory funnel and extracted with ethyl acetate (30 mL each, a total of 4 times). After bonding the organic layer, it was dried with magnesium sulfate (MgSO₄), filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (SiO₂; chloroform / acetone / toluene / formic acid = 85:5:10:1, v / v / v / v) and subsequently recrystallized using chloroform and hexane to obtain 3,4,5-tris(methoxymethoxy)benzoic acid 37 (1.16 g, 81%) as a white solid.

[0306] 1 H NMR (CDCl3, 400 MHz): δ 3.44 (s, 6H), 3.60 (s, 3H), 5.14 (s, 2H), 5.17 (s, 4H), 7.48 (s, 2H); 13C NMR (CDCl3, 100 MHz): δ 166.64, 150.39, 139.96, 126.23, 110.81, 97.80, 94.82, 56.56, 55.88.

[0307] 3-Bromopropane-1,2-diyl distearate (39)

[0308]

[0309] 3-bromopropane-1,2-diol 38 (1 g, 1 equivalent), EDCI·HCl (3.71 g, 3 equivalents), 4-dimethylaminopyridine (DMAP; 236 mg, 0.3 equivalents), and dichloromethane (DCM, 40 mL) were added to a 100 mL one-neck round-bottom flask equipped with a magnetic stirrer. This mixture was vigorously stirred at room temperature for 10 minutes under an argon atmosphere. Subsequently, a solution of stearic acid (4.59 g, 2.5 equivalents) dissolved in 10 mL of DCM was poured in, and the reaction mixture was stirred at room temperature. The progress of the reaction was confirmed by TLC (SiO₂; hexane / ethyl acetate = 1:29, PMA staining) to check for the loss of alcohol 38. The reaction was completed after approximately 9 hours, and after adding 30 mL of DCM, the reaction mixture was washed with saturated sodium bicarbonate solution (50 mL three times) and brine (50 mL once). The organic layer was dried with anhydrous magnesium sulfate, filtered, and the solvent was removed by vacuum distillation. The concentrated mixture was purified by flash column chromatography (SiO₂; ethyl acetate / hexane = 1:29, v / v) to obtain 3-bromopropane-1,2-diyl distearate39 (3.68 g, 83%) in the form of a clear oil.

[0310] 1H NMR (CDCl3, 400 MHz): δ 0.88 (t,J= 6.6 Hz, 6H), 1.25 (m, 56H), 1.61 (t,J= 6.9 Hz, 2H), 1.63 (t,J= 7.1 Hz, 2H), 2.34 (t,J= 8.1 Hz, 4H), 3.47 (dd,J= 10.9, 5.5 Hz, 1H), 3.53 (dd,J= 10.8, 5.6 Hz, 1H), 4.23 (dd,J= 11.9, 5.7 Hz, 1H), 4.34 (dd,J= 11.8, 4.2 Hz, 1H), 5.18-5.23 (m, 1H); 13 C NMR (CDCl3, 100 MHz): δ 14.14, 22.71, 24.92, 29.09, 29.29, 29.39, 29.50, 29.72, 29.91, 31.95, 34.08, 34.21, 63.00, 69.90, 172.80, 173.19.

[0311]

[0312]

[0313] 3-((3,4,5-tris(methoxymethoxy)benzoyl)oxy)propane-1,2-diyl distearate (40)

[0314] A Teflon-coated rod-magnetic stirrer was fitted into a dried 100 mL one-neck round-bottom flask (RBF), and 3-bromopropane-1,2-diyl distearate 39 (1 g, 1 equivalent), 3,4,5-tris(methoxymethoxy)benzoic acid 37 (527 mg, 1.2 equivalents), potassium carbonate (K₂CO₃, 402 mg, 2 equivalents), and DMF (40 mL) were added in sequence. The flask was fitted with a reflux condenser and heated to 60°C. The reaction mixture was stirred at 500 rpm for 13 hours at this temperature, and the progress of the reaction was monitored every hour using TLC (SiO₂; hexane / ethyl acetate = 8:2, PMA staining). After the reaction was complete, the reaction mixture was concentrated under reduced pressure. The concentrated mixture was washed sequentially with water (50 mL twice), saturated sodium bicarbonate solution (50 mL three times), and brine (50 mL once). The organic layer was bound and dried with anhydrous magnesium sulfate, filtered, and the solvent removed by vacuum distillation. The concentrated mixture was purified by flash column chromatography (SiO₂; hexane / ethyl acetate = 1:9 to 6:4, v / v) to obtain 3-((3,4,5-tris(methoxymethoxy)benzoyl)oxy)propane-1,2-diyl distearate 40 (1 g, 80%) in the form of a white solid wax.

[0315] 1 H NMR (CDCl3, 400 MHz): δ 0.86 (t,J= 6.8 Hz, 6H), 1.24 (m, 56H), 1.55 (m, 4H), 2.23-2.27 (m, 4H), 4.13 (dd,J= 11.9, 5.8 Hz, 1H), 4.27-4.31 (m, 2H), 4.41 (dd,J= 11.8, 4.3 Hz, 1H), 5.33-5.37 (m, 1H), 7.43 (s, 2H); 13C NMR (CDCl3, 100 MHz): δ 14.10, 22.68, 24.88, 29.12, 29.27, 29.35, 29.47, 29.65, 29.69, 31.92, 34.16, 34.21, 62.12, 62.96, 68.85, 111.85, 125.19, 141.09, 150.72, 165.28, 172.88, 173.25.

[0316]

[0317]

[0318] Synthesis of 3-((3,4,5-trihydroxybenzoyl)oxy)propane-1,2-diyl distearate (Chemical Formula 2-3)

[0319] In a 50 mL one-neck round-bottom dry flask equipped with a Teflon-coated rod-shaped magnetic stirrer, a solution of 3-((3,4,5-tris(methoxymethoxy)benzoyl)oxy)propane-1,2-diyl distearate 40 (500 mg, 1 equivalent) dissolved in isopropanol (i-PrOH, 35 mL) at room temperature was stirred, and a catalytic amount of concentrated sulfuric acid (HCl; 4.73 μL, 0.1 equivalent) was added. The reaction mixture was stirred at 500 rpm for 1 hour, and since the desired product decomposes if the reaction time is prolonged, the progress of the reaction was monitored every 20 minutes using TLC (SiO₂; hexane / ethyl acetate = 7:3, PMA staining). When it was confirmed by TLC that all starting materials had been consumed, the reaction mixture was poured into a saturated sodium bicarbonate aqueous solution (50 mL), and the aqueous layer was then extracted with dichloromethane (DCM, 40 mL three times) and subsequently washed with brine (30 mL once). The organic layer was dried with MgSO₄, filtered, and then the solvent was removed by vacuum distillation. The concentrated mixture was purified by flash column chromatography (SiO₂; hexane / ethyl acetate / acetic acid = 60:40:1 from 90:10:1, v / v / v) to obtain 3-((3,4,5-trihydroxybenzoyl)oxy)propane-1,2-diyl distearate (compound of Formula 2-3) in the form of a white powder (320 mg, yield 75%).

[0320] 1 H NMR (CDCl3, 400 MHz): δ 0.82 (t,J= 6.7 Hz, 6H), 1.18 (m, 62H), 1.60 (s, 6H), 2.31 (td,J= 7.5, 3.1 Hz, 4H), 3.50 (s, 6H), 3.60 (s, 3H), 4.19 (dd,J= 11.9, 5.8 Hz, 1H), 4.33-4.38 (m, 2H), 4.48 (dd,J= 11.8, 4.3 Hz, 1H), 5.20 (s, 2H), 5.22 (s, 4H), 5.37-5.43 (m, 1H), 7.50 (s, 2H); 13C NMR (CDCl3, 100 MHz): δ 14.11, 22.69, 24.89, 29.13, 29.28, 29.37, 29.49, 29.67, 29.71, 31.93, 34.07, 34.23, 56.39, 57.22, 62.13, 62.98, 68.87, 95.3, 98.50, 111.87, 125.20, 141.11, 150.74, 165.30, 172.89, 173.26.

[0321]

[0322] 2-hexyldecyl 2-bromoacetate (43)

[0323] 2-hexyldecane-1-ol 42 (200 mg, 1 equivalent), EDCI·HCl (237 mg, 1.5 equivalents), 4-dimethylaminopyridine (DMAP; 20 mg, 0.2 equivalents), and dichloromethane (DCM, 20 mL) were added to a 50 mL one-neck round-bottom flask equipped with a magnetic stirrer. The mixture was vigorously stirred at room temperature for 10 minutes under an argon atmosphere. Subsequently, 2-bromoacetic acid 41 (149 mg, 1.3 equivalents) was dissolved in 5 mL of DCM and added, and the reaction mixture was stirred at room temperature. The progress of the reaction was confirmed by TLC (SiO₂; hexane / ethyl acetate = 1:49, PMA staining) to check for the disappearance of alcohol 42. The reaction was completed after approximately 3 hours, and after adding 25 mL of DCM, the mixture was washed with saturated sodium bicarbonate solution (30 mL three times) and brine (30 mL once). The organic layer was dried with anhydrous magnesium sulfate, filtered, and the solvent removed by vacuum distillation. The concentrated mixture was purified by flash column chromatography (SiO₂; ethyl acetate / hexane = 1:39 from 1:49, v / v), yielding 2-hexyldecyl 2-bromoacetate 43 (261 mg, yield 87%) in the form of a clear oil.

[0324] 1H NMR (CDCl3, 400 MHz): δ 0.88 (s, 3H), 0.90 (s, 3H), 1.29 (m, 25H), 1.67 (br s, 2H), 3.83-4.10 (m, 4H); 13 C NMR (CDCl3, 100 MHz): δ 14.08, 22.66, 25.90, 26.63, 29.28, 29.55, 29.89, 31.09, 31.77, 31.88, 37.25, 40.93, 69.04, 167.47.

[0325]

[0326] 2-((2-hexyldecyl)oxy)-2-oxoethyl 3,4,5-tris(methoxymethoxy)benzoate (44)

[0327] In an oven-dried 50 mL one-neck round-bottom flask (RBF) equipped with a Teflon-coated rod-magnetic stirrer, 2-hexyldecyl 2-bromoacetate 43 (400 mg, 1 equivalent), 3,4,5-tris(methoxymethoxy)benzoic acid 37 (400 mg, 1.2 equivalents), potassium carbonate (K₂CO₃, 304 mg, 2 equivalents), and DMF (30 mL) were added in sequence. The flask was fitted with a reflux condenser and heated to 60°C, and the reaction mixture was stirred for 13 hours. The progress of the reaction was monitored every hour using TLC (SiO₂; hexane / ethyl acetate = 9:1, PMA staining).

[0328] The reaction mixture was concentrated under reduced pressure, and the concentrated mixture was washed sequentially with water (30 mL twice), saturated sodium bicarbonate solution (40 mL twice), and brine (40 mL once). The organic layer was bound, dried with anhydrous magnesium sulfate, filtered, and the solvent removed by vacuum distillation. The concentrated mixture was purified by flash column chromatography (SiO₂; hexane / ethyl acetate = 1:9 to 2:8, v / v) to obtain 2-((2-hexyldecyl)oxy)-2-oxoethyl 3,4,5-tris(methoxymethoxy)benzoate 44 (521 mg, yield 81%) in the form of a colorless oil.

[0329] 1 H NMR (CDCl3, 400 MHz): δ 0.81 (t,J= 5.8 Hz, 6H), 1.18 (m, 29H), 1.54 (br s, 1H), 3.43 (s, 6H), 3.54 (s, 3H), 4.02 (d,J= 5.7 Hz, 2H), 4.74 (s, 2H), 5.15 (s, 2H), 5.17 (s, 4H), 7.51 (s, 2H); 13 C NMR (CDCl3, 100 MHz): δ 14.08, 14.10, 22.63, 22.67, 26.61, 26.66, 29.29, 29.56, 29.90, 31.11, 31.78, 31.88, 37.25, 56.40, 57.23, 61.26, 68.15, 95.30, 98.52, 111.94, 124.91, 141.14, 150.76, 165.25, 167.96.

[0330]

[0331]

[0332] Synthesis of 2-((2-hexyldecyl)oxy)-2-oxoethyl 3,4,5-trihydroxybenzoate (GA13-1, chemical formula 2-4)

[0333] In a 50 mL oven-dried one-neck round-bottom flask equipped with a Teflon-coated rod-shaped magnetic stirrer, a solution of 2-((2-hexyldecyl)oxy)-2-oxoethyl 3,4,5-tris(methoxymethoxy)benzoate 44 (500 mg, 1 equivalent) dissolved in isopropanol (i-PrOH, 30 mL) was stirred, and a catalytic amount of concentrated hydrochloric acid (HCl, 7.33 μL, 0.1 equivalent) was added. The reaction mixture was stirred at 500 rpm for 2 hours at room temperature, and since the desired product decomposes if the reaction time is prolonged, the progress of the reaction was monitored every 20 minutes using TLC (SiO₂; hexane / ethyl acetate = 7:3, PMA staining).

[0334] Once it was confirmed via TLC analysis that the starting material was completely consumed, the reaction mixture was poured into a saturated sodium bicarbonate aqueous solution (50 mL), followed by extraction of the aqueous layer with dichloromethane (DCM, 40 mL three times) and washing with brine (30 mL once). The organic layer was dried with magnesium sulfate (MgSO₄), filtered, and the solvent removed by vacuum distillation. The concentrated mixture was purified by flash column chromatography (SiO₂; hexane / ethyl acetate / acetic acid = 70:30:1 from 90:10:1, v / v / v) to obtain 2-((2-hexyldecyl)oxy)-2-oxoethyl 3,4,5-trihydroxybenzoate (GA13-1, compound of chemical formula 2-4) (301 mg, yield 78%) as an off-white solid.

[0335] 1 H NMR (CDCl3, 400 MHz): δ 0.89 (td,J= 1.8, 7.1 Hz, 6H), 1.26 (m, 25H), 1.67 (br s, 1H), 4.12-4.14 (d,J= 5.7, 2H), 4.84 (s, 2H), 6.33 (br s, 3H), 7.19 (s, 2H); 13C NMR (CDCl3, 100 MHz): δ 14.06, 14.09, 22.64, 22.67, 26.61, 26.66, 29.31, 29.54, 29.57, 29.92, 31.05, 31.78, 31.89, 37.23, 61.11, 68.99, 110.31, 120.00, 137.31, 143.66, 166.30, 169.57.

[0336]

[0337] Example 4. Preparation of lipid nanoparticles containing novel lipids

[0338] Lipid nanoparticle carriers were prepared with the compositions shown in Tables 1 and 2 below using two novel oligo-gamma-glutamic acid derivatives represented by Chemical Formula 1-1 or Chemical Formula 1-2, two vitamin C-based helper lipids, and ten pantothenic acid and panthenol-based ionized lipids.

[0339] Lipid nanoparticles were prepared by rapidly mixing an RNA solution (50 mM sodium citrate buffer, 110 mM sodium chloride, pH=4.0) and a lipid mixture (ethanol, dimethyl sulfoxide) using a laboratory mixer and emulsifier (NanoAssemblr Spark, Precision Nanosystems, Inc.), and then the solvent was converted to physiological saline or phosphate buffered saline using a centrifugation filter tube (UFC5010, Amicon). Tables 1 and 2 show lipid nanoparticles prepared using mRNA expressing green fluorescence protein (GFP) and mRNA expressing chimeric antigen receptor (CAR), respectively.

[0340]

[0341] LNPNo.RNA capturingHelperMemb. stabilizingStabilizingCharacterizationGFP001SM-102 / Dioleyl ThrehaloseDOPElitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 251038.51.5125.1 ± 1.30.0617.1 ± 0.5GFP002SM-102DSPCCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)501038.51.5104.6 ± 0.30.1413.5 ± 1.2GFP003화학식3-1 / DioleylThrehaloseDOPEL-BuDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 251038.51.582.0 ± 0.90.1719.4 ± 1.2GFP004화학식3-1DSPCCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)501038.51.5109.7 ± 0.30.1019.9 ± 1.1GFP005화학식3-1DSPCCholesterol화학식 1-1Size (nm)PDIζ-potential (mV)501030.010.0161.1 ± 0.090.09-29.5 ± 1.0GFP008SM-102 / Dioleyl Threhalose6-palmtoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 251038.51.587.1 ± 0.10.1-12.7 ± 0.0GFP009SM-102 / Dioleyl Threhalose6-palmtoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 251533.51.589.0 ± 0.50.2-15.7 ± 1.8GFP010SM-102 / Dioleyl Threhalose6-palmtoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 201538.51.5105.9 ± 0.40.3-13.4 ± 0.7GFP011SM-102 / Dioleyl Threhalose2,6-dipalmitoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 251038.51.596.3 ± 0.30.13.2 ± 0.5GFP012SM-102 / Dioleyl Threhalose2,6-dipalmitoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 251533.51.593.8 ± 0.40.1-13.6 ± 1.4GFP013SM-102 / Dioleyl Threhalose2,6-dipalmitoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 201538.51.5118.4 ± 0.10.7-16.6 ± 0.6GFP014화학식3-16-palmtoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)303038.51.5135.2± 4.70.23-22.3± 0.2GFP015화학식3-1 / Dioleyl Threhalose2,6-dipalmitoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 251038.51.5129.7± 1.10.193.0± 0.7GFP016Formula 3-1 / Dioleyl Threhalose2,6-dipalmitoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 201538.51.5156.2± 1.80.16-14.0± 1.6GFP017Formula 3-1 / Dioleyl Threhalose6-palmtoyl ascorbic acidlitcholate butyl esterDMG-PEG2000Size (nm)PDIζ-potential (mV)25 / 152038.51.5136± 2.00.22-21.1± 0.8GFP018Formula 3-12,6-dipalmitoyl ascorbic acidcholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)50 10 38.5 1.5 11 2.1± 0.1 0.2 12 2± 1.1 GFP019 Formula 3-12,6-dipalmitoyl ascorbic acid cholesterol DMG-PEG2000 Size (nm) PDIζ-potential (mV)45 15 38.5 1.5 11 9.8± 13.8 0.2 32 0.4± 1.0 GFP020 Formula 3-12,6-dipalmitoyl ascorbic acid cholesterol DMG-PEG2000 Size (nm) PDIζ-potential (mV)40 20 38.5 1.5 13 9.4± 1.5 0.1 61 4.6± 0.5 GFP021 Formula 3-2 DSPC cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV)50 10 30.0 10.0 23 3.4 ± 11.1 0.11 -3 3.9 ± 2.1 GFP022 Formula 3-3 DSPC Cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV)50 10 30.0 10.0 26 6.4 ± 10.5 0.30 -3 8.2 ± 2.9 GFP023 Formula 3-4 DSPC Cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV)50 10 30.0 10.0 20 8.6 ± 8.4 0.0 5 -2 2.2 ± 0.1GFP024 Formula 3-5DSPC Cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV) 50 10 30.0 10.0 29 0.7 ± 7.1 0.25 -4 2.2 ± 1.3 GFP025 Formula 3-6DSPC Cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV) 50 10 30.0 10.0 20 7.8 ± 6.3 0.1 2 -27.0 ± 5.3 GFP026 Formula 3-7DSPC Cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV) 50 10 30.0 10.0 25 3 ± 5.3 0.2 4 -35.4 ± 1.1 GFP027 Formula 3-8DSPC Cholesterol Formula 1-1 Size (nm)PDIζ-potential (mV)501030.010.0217 ± 1.20.10-24.7 ± 0.5GFP028Chemical formula3-22,6-dipalmitoyl ascorbic acidCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.51.5108.1 ± 0.80.11-3.8 ± 0.6GFP029Formula 3-32,6-dipalmitoyl ascorbic acidCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.51.5107 ± 2.40.1628.7 ± 2.2GFP030Formula 3-42,6-dipalmitoyl ascorbic acidCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.51.5121.2 ± 1.90.12-27.5 ± 3.9GFP031Formula3-52,6-dipalmitoyl ascorbic acidCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.51.5104.7 ± 0.60.1519.8 ± 2.2GFP032Formula 3-62,6-dipalmitoyl ascorbic acidCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.51.5108.4 ± 1.40.14-3.6 ± 0.5GFP033Formula 3-72,6-dipalmitoyl ascorbic acidCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.51.5116.6 ± 1.50.1012.2 ± 3.9GFP034Formula3-82,6-dipalmitoyl ascorbic acidCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.51.5131.8 ± 1.40.09-26.6 ± 1.6GFP035Formula3-82,6-dipalmitoyl ascorbic acidCholesterolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.53118.5±4.30.24-23.4±1.6GFP036Chemical formula3-92,6-dipalmitoyl ascorbic acidResveratrolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.5390.7±2.40.28-10.1±0.7GFP037Formula3-102,6-dipalmitoyl ascorbic acidResveratrolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.5379.8±3.20.16-24.0±1.3GFP038 Formula 3-9 Formula 2-3ResveratrolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.53104.6±3.10.18-22.0±1.4GFP039 Formula 3-10 Formula 2-4ResveratrolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.5374.8±1.20.202.2±2.8GFP040화학식3-112,6-dipalmitoyl ascorbic acidResveratrolDMG-PEG2000Size (nm)PDIζ-potential (mV)451538.53115.7±4.00.12-13.8±5.4.

[0342]

[0343] LNPNo.RNA capturingHelperMemb. Stabilizing Characterization CAR-ConSM-102 DSPC Cholesterol DMG-PEG 2000 Size (nm) PDIζ-potential (mV) 50 10 38.5 1.5 10 4.6 ± 0.3 0.1 41 3.5 ± 1.2 CAR001 Formula 3-1 DSPC Cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV) 50 10 30.0 10.0 15 0.2 ± 1.1 0.0 6 -29.6 ± 0.7 CAR002 Formula 3-3 DSPC Cholesterol Formula 1-1 Size (nm) PDIζ(mV) 50 10 30.0 10.0 18 0.7 ± 2.1 0.1 1 -34.2 ± 0.4 CAR003 Formula 3-5 DSPC Cholesterol Formula 1-1 Size (nm)PDIζ(mV)50 10 30.0 10.0 19 1.6 ± 0.7 0.0 5 -3 3.5 ± 0.5 CAR004 Chemical Formula 3-12,6-dipalmitoyl ascorbic acid Cholesterol DMG-PEG2000 Size (nm)PDIζ(mV)45 15 38.5 1.5 9 2.2 ± 0.9 0.1 22 0.1 ± 0.5 CAR005 Chemical Formula 3-42,6-dipalmitoyl ascorbic acid Cholesterol DMG-PEG2000 Size (nm)PDIζ(mV)45 15 38.5 1.5 14 0.5 ± 1.6 0.0 9 -4 1.9 ± 1.1 CAR006 Chemical Formula 3-52,6-dipalmitoyl ascorbic acid Cholesterol DMG-PEG2000 Size (nm)PDIζ(mV)451538.51.5104.4 ± 2.60.1012.3 ± 0.4CAR007 Chemical Formula 3-82,6-dipalmitoyl ascorbic acid Cholesterol DMG-PEG2000 Size (nm)PDIζ(mV)451538.51.5138.2 ± 3.70.05-39.0 ± 0.6CAR008 Chemical Formula 3-12,6-dipalmitoyl ascorbic acid Cholesterol Chemical Formula 1-1 Size (nm) PDIζ-potential (mV) 50 10 30.0 10.0 2 5.6 ± 5.8 0.0 2 -40.2 ± 0.6 CAR009 Chemical Formula 3-32,6-dipalmitoyl ascorbic acid Cholesterol Chemical Formula 1-1 Size (nm) PDIζ-potential (mV) 50 10 30.0 10.0 2 0 4.1 ± 7.1 0.0 3 -40.2 ± 1.9 CAR010 Chemical Formula 3-42,6-dipalmitoyl ascorbic acid Cholesterol Chemical Formula 1-1 Size (nm) PDIζ-potential (mV) 50 10 30.0 10.0 1 33.8 ± 2.7 0.09 -45.5 ± 7.6 CAR011 Formula 3-5 2,6-dipalmitoyl ascorbic acid Cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV) 50 10 30.0 10.0 22 3.7 ± 1.3 0.09 -47.3 ± 2.2 CAR012 Formula 3-8 2,6-dipalmitoyl ascorbic acid Cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV) 50 10 30.0 10.0 112 ± 2.0 0.0 7 -39.7 ± 0.3 CAR013 Formula 3-9 2,6-dipalmitoyl ascorbic acid Cholesterol Formula 1-1 Size (nm) PDIζ-potential (mV)50 10 30.0 10.0 13 1.1 ± 0.8 0.0 9 -4 2.5 ± 2.1 CAR014 Chemical Formula 3-10 2,6-dipalmitoyl ascorbic acid Cholesterol Chemical Formula 1-1 Size (nm) PDIζ-potential (mV)50 10 30.0 10.0 16 5.7 ±0.2 0.0 4 -4 0.2 ±1.0 CAR015 Chemical Formula 3-8 2,6-dipalmitoyl ascorbic acid Cholesterol Chemical Formula 1-2 Size (nm) PDIζ-potential (mV)45 15 38.5 1.5 11 6.5 ±1.9 0.0 5 -4 1.2 ±1.1CAR016 Chemical Formula 3-9 2,6-dipalmitoyl ascorbic acid Cholesterol Chemical Formula 1-2 Size (nm) PDIζ-potential (mV) 45 15 38.5 1.5 119.4 ±2.2 0.12 -36.2 ±0.9 CAR017 Chemical Formula 3-10 2,6-dipalmitoyl ascorbic acid Cholesterol Chemical Formula 1-2 Size (nm) PDIζ-potential (mV) 45 15 38.5 1.5 123.9 ±0.3 0.02 -41.4 ±1.2 CAR018 Chemical Formula 3-8 DSPC Cholesterol DMG-PEG2000 Size (nm) PDIζ-potential (mV)44.3 14.8 37.9 3.0 95.7±1.2 0.13 -10.4±2.0 CAR019 Formula 3-9 DSPC Cholesterol DMG-PEG2000 Size (nm) PDIζ-potential (mV)44.3 14.8 37.9 3.0 95.1±2.4 0.12 -12.0±0.3 CAR020 Formula 3-10 DSPC Cholesterol DMG-PEG2000 Size (nm) PDIζ-potential (mV)44.3 14.8 37.9 3.0 81.5±0.8 0.10 -4.2±1.1 CAR021 Formula 3-8 2,6-dipalmitoyl ascorbic acid Cholesterol DMG-PEG2000 Size (nm) PDIζ-potential (mV)44.3 14.8 37.9 3.0 99.7±1.8 0.20 -24.5±3.5 CAR022 Formula 3-9 2,6-dipalmitoyl ascorbic acid Cholesterol DMG-PEG2000 Size (nm) PDIζ-potential (mV)44.3 14.8 37.9 3.0 93.4±0.9 0.0 9 -32.8±1.2 CAR023 Formula 3-10 2,6-dipalmitoyl ascorbic acid Cholesterol DMG-PEG2000 Size (nm) PDIζ-potential (mV)44.3 14.8 37.9 3.0 68.0±1.1 0.23 -12.0±5.1 CAR024 Formula 3-8 DSPC Cholesterol Formula 1-2 Size (nm) PDIζ-potential (mV)44.314.837.93.0112.8±1.40.02-27.4±0.7CAR025 Chemical Formula 3-9DSPC Cholesterol Chemical Formula 1-2 Size (nm)PDIζ-potential (mV)44.314.837.93.0105.3±0.40.10-29.7±0.4CAR026 Chemical Formula 3-10DSPC Cholesterol Chemical Formula 1-2 Size (nm)PDIζ-potential (mV)44.314.837.93.080.2±0.50.07-29.3±6.1CAR027 Chemical Formula 3-82,6-dipalmitoyl ascorbic acid Cholesterol Chemical Formula 1-2 Size (nm)PDIζ-potential (mV)44.3 14.8 37.9 3.0 97.6±0.2 0.0 3 -39.7±3.6 CAR028 Formula 3-9 2,6-dipalmitoyl ascorbic acid Cholesterol Formula 1-2 Size (nm) PDIζ-potential (mV)44.3 14.8 37.9 3.0 82.8±0.4 0.0 8 -27.2±4.6 CAR029 Formula 3-10 2,6-dipalmitoyl ascorbic acid Cholesterol Formula 1-2 Size (nm) PDIζ-potential (mV)44.3 14.8 37.9 3.0 106.8±1.0 0.0 6 -22.1±1.0 CAR030 Formula 3-8 2,6-dipalmitoyl ascorbic acidResveratrolDMG-PEG2000Size (nm)PDIζ-potential (mV)44.314.837.93.074.7±2.80.20-3.7±1.1CAR031Chemical formula3-102,6-dipalmitoyl ascorbic acidResveratrolDMG-PEG2000Size (nm)PDIζ-potential (mV)44.314.837.93.071.1±2.30.024.7±0.8CAR032Chemical formula3-122,6-dipalmitoyl ascorbic acidResveratrolDMG-PEG2000Size (nm)PDIζ-potential (mV)44.314.837.93.079.1±1.50.1921.1±1.9CAR033 Chemical Formula 3-132,6-dipalmitoyl ascorbic acid Resveratrol DMG-PEG2000 Size (nm) PDIζ-potential (mV) 44.3 14.8 37.9 3.0 88.6±0.7 0.18 3.2±0.2 CAR034 Chemical Formula 3-142,6-dipalmitoyl ascorbic acid Resveratrol DMG-PEG2000 Size (nm) PDIζ-potential (mV) 44.3 14.8 37.9 3.0 77.8±1.7 0.18 24.1±0.9 CAR035 Chemical Formula 3-102,6-dipalmitoyl ascorbic acid Cholesterol Chemical Formula 1-3 Size (nm) PDIζ-potential (mV)50 10 30 10 149.4±2.8 0.04 -41.1±2.7 CAR036 Formula 3-10 2,6-dipalmitoyl ascorbic acid Cholesterol Formula 1-2 Size (nm) PDIζ-potential (mV)50 10 30 10 189.4±3.0 0.03 -37.6±1.2 CAR037 Formula 3-13 2,6-dipalmitoyl ascorbic acid Cholesterol Formula 1-3 Size (nm) PDIζ-potential (mV)50 10 30 10 149.9±3.0 0.04 -41.6±0.7 CAR038 Formula 3-12 2,6-dipalmitoyl ascorbic acid Cholesterol Formula 1-2 Size (nm) PDIζ-potential (mV)50103010325.3±6.90.25-26.4±1.2CAR039Chemical Formula 3-132,6-dipalmitoyl ascorbic acidCholesterolChemical Formula 1-2Size (nm)PDIζ-potential (mV)50103010177.5±3.10.02-28.7±1.7CAR040Chemical Formula 3-142,6-dipalmitoyl ascorbic acidCholesterolChemical Formula 1-2Size (nm)PDIζ-potential (mV)50103010183.6±6.00.06-38.4±2.1.

[0344]

[0345]

[0346] Example 5. Primary screening of novel lipid nanoparticles via GFP expression

[0347] Lipid nanoparticles GFP001 with a composition developed by the inventor and GFP002 with a conventionally used composition were prepared using the commercially available ionized lipid SM-102. Additionally, lipid nanoparticles GFP003 and GFP004 were prepared by replacing SM-102 in lipid nanoparticles GFP001 and GFP002 with a novel pantothenic acid-based ionized lipid [Formula 3-1]. The composition and structural physicobiochemical characteristics of the lipid nanoparticles (GFP 001 to 004) encapsulating GFP mRNA are shown in Table 1 above. Specifically, the composition values ​​in the table represent mol% and include the diameter, polydispersity index (PDI), and zeta potential of the prepared particles measured using dynamic light scattering.

[0348] Using the prepared particles, experiments were conducted as follows to confirm the viability and intracellular GFP expression rate of peripheral blood natural killer (PBNK) cells, and the results are shown in Figure 1. First, primary PBNK cells (1 M cell / 500 μL) dispersed in MACS medium were inoculated into a 24-well cell culture plate. Then, lipid nanoparticles encapsulated with CAR mRNA were added to each well at a concentration of 3 μg / 50 μL based on RNA and mixed thoroughly. After culturing for 3 hours, serum equivalent to 5% of the medium volume was added, and the cells were cultured for another 3 hours. After washing the cells with phosphate-buffered saline, they were stained with Live / Dead dye. Subsequently, the green fluorescence of intracellularly expressed GFP and the far-infrared fluorescence intensity of the dead cells were measured using a flow cytometer. Lipid nanoparticles containing SM-102 (GFP 001, 002) showed cell viability ranging from 31.2% to 54.4%, but the GFP expression rate was significantly low at less than 6%. On the other hand, lipid nanoparticles containing novel ionized lipids (Chemical Formula 3-1) (GFP 003, 004) showed very low cell viability ranging from 5.98% to 2.41%, but the GFP expression rate increased significantly to 43.9–46.0% solely through the replacement of ionized lipids. Consequently, it was confirmed that SM-102 reduces the efficiency of mRNA delivery to primary NK cells, and that cell viability decreases when GFP mRNA is successfully delivered into cells by introducing novel ionized lipids.

[0349] The inventors intended to prepare lipid nanoparticles using an oligo-gamma-glutamic acid derivative to increase the activity of NK cells and introduce mRNA into the cells.

[0350] Lipid nanoparticles (GFP005) were prepared using the oligo-gamma-glutamic acid derivative of Formula 1-1 and a novel pantothenic acid-based ionized lipid (Formula 3-1), and their composition and structural physicobiochemical properties are shown in Table 1 above. Specifically, the composition values ​​in the table represent mol% and include the diameter, PDI, and zeta potential of the prepared particles measured using dynamic light scattering.

[0351] Experiments were conducted as described above using the prepared particles, and the viability of PBNK cells and intracellular GFP expression rates were confirmed through flow cytometry; the results are shown in Figure 2. Lipid nanoparticle GFP005, containing 10 mol% of the oligo-gammaglutamic acid derivative of Formula 1-1 by replacing all of DMG-PEG2000 and part of cholesterol in lipid nanoparticle GFP004, significantly improved cell viability to 76.0% and increased GFP expression efficiency to 70.1%. Consequently, the oligo-gammaglutamic acid derivative of Formula 1-1 significantly improved cell viability and GFP expression efficiency, while using ionized lipids of Formula 3-1 demonstrated high delivery efficiency.

[0352] A deficiency in vitamin C (ascorbic acid) can cause damage to NK cell function, and the presence of ascorbic acid is known to be beneficial for the proliferation of NK cells. Accordingly, the inventors intended to prepare lipid nanoparticles using vitamin C-based lipids to introduce mRNA into NK cells.

[0353] Experiments were conducted as described above using lipid nanoparticle GFP001, the control group in Table 1, and lipid nanoparticles GFP008 to GFP020 containing vitamin C-based lipids (6-palmitoyl ascorbic acid and 2,6-dipalmitoyl ascorbic acid). The viability of PBNK cells and the intracellular GFP expression rate were confirmed through flow cytometry, and the results are shown in Figures 3 and 4, respectively.

[0354] The results for lipid nanoparticles GFP008 to GFP013 containing the ionized lipid SM-102 are shown in Figure 3. Lipid nanoparticle GFP008, in which DOPE, the helper lipid of GFP001, was replaced with 6-palmitoyl ascorbic acid, did not significantly affect cell viability. Lipid nanoparticles GFP009 and GFP010, in which 6-palmitoyl ascorbic acid was increased from 10 mol% to 15 mol% and n-butyl lithocholate and 6,6'-trehalose dioleate were lowered by 5 mol%, respectively, showed a slight increase in cell viability to 32.8% and 32.2%. Lipid nanoparticle GFP011, in which DOPE, the helper lipid of GFP001, was replaced with 2,6-dipalmitoyl ascorbic acid, showed a significant increase in cell viability to 51.5%. Lipid nanoparticles GFP012 and GFP013, in which 2,6-dipalmitoyl ascorbic acid was increased from 10 mol% to 15 mol% in GFP011 and n-butyl lithocholate and 6,6'-trehalose dioleate were lowered by 5 mol%, respectively, showed cell viability that was slightly decreased or increased to 39.5% and 53.0%, respectively. Consequently, lipid nanoparticles containing 15 mol% or more of vitamin C-based lipid 6-palmitoyl ascorbic acid or containing 10 mol% to 15 mol% of 2,6-dipalmitoyl ascorbic acid can increase cell viability.

[0355] Based on the results of Figure 3, cell viability and GFP expression rates were confirmed upon the introduction of lipid nanoparticles GFP014 to GFP020 containing pantothenic acid ionized lipid formula 3-1 into cells, and the results are shown in Figure 4.

[0356] Lipid nanoparticle GFP014, in which the ionized lipid SM-102 and 6,6'-Dioleyl trehalose of lipid nanoparticle GFP010 were replaced with 30 mol% of the ionized lipid pantothenic acid formula 3-1 and 2,6-dipalmitoyl ascorbic acid was replaced with 30 mol% of 6-palmitoyl ascorbic acid, showed a slight increase in cell viability to 46.2%, but exhibited a low GFP expression rate of 27.1%. Lipid nanoparticles GFP015 and GFP016, in which the ionized lipid SM-102 of lipid nanoparticles GFP011 and GFP013 were replaced with the ionized lipid pantothenic acid formula 3-1, did not significantly affect cell viability to 54.5%–55.6%, but exhibited a low GFP expression rate of 9.26%–3.95%. Lipid nanoparticle GFP017, in which the ionized lipid SM-102 of lipid nanoparticle GFP010 was replaced with the ionized lipid pantothenic acid formula 3-1, 6,6'-Dioleyl trehalose was reduced by 5 mol%, and 6-palmitoyl ascorbic acid was increased by 5 mol%, showed a slightly increased cell viability of 50.4%, but a low GFP expression rate of 28.9%. Lipid nanoparticle GFP018, in which the ionized lipid SM-102 of lipid nanoparticle GFP002 was replaced with the ionized lipid pantothenic acid formula 3-1, showed a somewhat low cell viability of 30.0% but a high GFP expression rate of 86.8%. Lipid nanoparticle GFP019, in which the ratio of pantothenic acid ionized lipid formula 3-1 was lowered by 5 mol% and 2,6-dipalmitoyl ascorbic acid was increased by 5 mol% in lipid nanoparticle GFP018, showed a slightly increased survival rate of 42.4%, while the GFP expression rate was maintained at 88.2%. Lipid nanoparticle GFP020, in which the ratio of pantothenic acid ionized lipid formula 3-1 was lowered by 10 mol% and 2,6-dipalmitoyl ascorbic acid was increased by 10 mol% in lipid nanoparticle GFP018, showed a significantly increased survival rate of 70.4%, while the GFP expression rate decreased to 52.2%.As a result, it was confirmed that the lipid composition ratio of lipid nanoparticle GFP019, which improves GFP expression efficiency to nearly 90% while slightly improving cell viability, was optimized for lipid nanoparticles containing 15 mol% of 2,6-dipalmitoyl ascorbic acid, a vitamin C lipid.

[0357] Lipid nanoparticles (GFP021 to GFP035) were prepared using novel pantothenic acid-based ionized lipids of Chemical Formulas 3-2 to 3-8 to efficiently deliver mRNA into NK cells, and their composition and structural physicobiochemical characteristics are shown in Table 1 above. Specifically, the composition values ​​in the table represent mol% and include the diameter, PDI, and zeta potential of the prepared particles measured using dynamic light scattering.

[0358] Cell viability and GFP expression rates were confirmed upon intracellular introduction of lipid nanoparticles GFP021 to GFP035 containing pantothenic acid ionized lipid formulas 3-2 to 3-8, and the results are shown in Figures 5 to 7. Lipid nanoparticles GFP021 to GFP027, in which the pantothenic acid-based ionized lipid formula 3-1 of lipid nanoparticle GFP005 containing the oligo-gamma-glutamic acid derivative of formula 1-1 was replaced with pantothenic acid ionized lipid formulas 3-2 to 3-8, showed high cell viability of over 80%, except for GFP022, while GFP022 showed a relatively low cell viability of 52.5%. Lipid nanoparticles GFP021, 022, 024, and 025, in which the pantothenic acid-based ionized lipid formula 3-1 of lipid nanoparticle GFP005 was replaced with pantothenic acid-based ionized lipid formulas 3-2, 3-3, 3-5, and 3-6, showed higher GFP expression rates. Lipid nanoparticles GFP028 to GFP034, in which the pantothenic acid-based ionized lipid formula 3-1 of lipid nanoparticle GFP019 containing the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid was replaced with pantothenic acid ionized lipid formulas 3-2 to 3-8, showed increased cell viability of 60% to 80%, except for GFP029, which was significantly decreased. Lipid nanoparticles GFP030, 031, and 034, in which pantothenic acid-based ionized lipid formula 3-1 of lipid nanoparticle GFP019 was replaced with pantothenic acid-based ionized lipid formulas 3-4, 3-5, and 3-8, showed higher GFP expression rates.

[0359] As can be seen in Figure 7, there are a total of 5 types of lipid nanoparticles with a cell viability of 50% or more and a GFP expression rate of 70% or more, including lipid nanoparticles GFP022 and 024 which contain an oligo-gamma-glutamic acid derivative of formula 1-1 and pantothenic acid-based ionized lipids of formulas 3-3 and 3-5, and lipid nanoparticles GFP030, 031, and 034 which contain 2,6-dipalmitoyl ascorbic acid, a vitamin C-based lipid, and pantothenic acid-based ionized lipids of formulas 3-4, 3-5, and 3-8.

[0360] As a result, it was confirmed that lipid nanoparticles containing novel pantothenic acid-based ionized lipids with formulas 3-3, 3-4, 3-5, and 3-8, an oligo-gamma-glutamic acid derivative with formula 1-1, and a vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid showed high delivery and translation efficiency of GFP mRNA within NK cells.

[0361]

[0362] The inventors intended to prepare lipid nanoparticles containing vitamin or gallic acid-based lipids as helper lipids and resveratrol as a cholesterol substitute to increase the activity of NK cells and introduce mRNA into the cells.

[0363] First, lipid nanoparticle GFP035 was prepared by increasing the amount of DMG-PEG2000 to 3 mol% to reduce particle size and facilitate entry into NK cells. It was confirmed that the particle size was reduced by approximately 20 nm compared to lipid nanoparticle GFP034, which had 1.5 mol% of the same DMG-PEG2000, and that it was prepared uniformly. Accordingly, lipid nanoparticles GFP036 to GFP040 were further prepared, containing pantothenic acid ionized lipids with chemical formulas 3-8 to 3-11, and containing vitamin C-based lipids or gallic acid-based lipids of chemical formulas 2-3 and 2-4 as helper lipids, and containing cholesterol and resveratrol as substitutes. Cell viability and GFP expression rates were confirmed upon introduction into cells, and the results are shown in Figure 8.

[0364] Looking at Figure 8, it was confirmed that lipid nanoparticles 036, 037, and 040, which used chemical formulas 3-9 to 3-11 as ionized lipids, vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid as a helper lipid, and resveratrol as a cholesterol substitute, maintained a cell viability of over 60% and a GFP expression rate of over 60%. In particular, for lipid nanoparticle 040, which used chemical formula 3-11 as an ionized lipid, the intracellular GFP expression rate increased significantly to about 80% compared to lipid nanoparticles 036 and 037, which used chemical formulas 3-9 and 3-10 as ionized lipids, but the cell viability decreased by about 10%. In the case of lipid nanoparticles 038 and 039, which used chemical formulas 3-9 and 3-10 as ionized lipids and included resveratrol as a cholesterol substitute, and used gallic acid-based lipids chemical formulas 2-3 and 2-4 as helper lipids, the expression efficiency increased by about 10% while maintaining cell viability in the 70% range compared to lipid nanoparticles 036 and 037, which used vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid as a helper lipid.

[0365]

[0366] Example 6. Selection of Optimal Novel Lipid Nanoparticles via CAR-mRNA Expression

[0367] To select lipid nanoparticles capable of effectively expressing CAR mRNA in NK cells, lipid nanoparticles were prepared containing one or more or all of the pantothenic acid-based ionized lipids (Chemical Formulas 3-1, 3-3, 3-4, 3-5, and 3-8) selected in the first screening, the oligo-gamma-glutamic acid derivative (Chemical Formula 1-1), and the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid. The composition and structural physicobiochemical characteristics of the lipid nanoparticles (CAR 001 to 029) encapsulating CAR mRNA are shown in Table 2 above. Specifically, the composition values ​​in the table represent mol% and include the diameter, PDI, and zeta potential of the prepared particles measured using dynamic light scattering.

[0368] NK cell viability and CAR protein expression rates were verified through the following experiments. First, primary peripheral blood natural killer (PBNK) cells (1 M cell / 500 μL) dispersed in MACS medium were inoculated into a 24-well cell culture plate. Then, lipid nanoparticles encapsulated with CAR mRNA were added to each well at a concentration of 3 μg / 50 μL (based on RNA) and thoroughly mixed. The sequence of the CAR mRNA was denoted as SEQ ID NO. 1. After culturing for 3 hours, serum equivalent to 5% of the medium volume was added, and the cells were cultured for another 3 hours. After washing the cells with phosphate-buffered physiological saline, they were stained primarily by treating them with an antigen-biotin protein recognized by CAR and a Live / Dead dye for staining dead cells. Subsequently, streptavidin-APC was applied to the cells to finally stain the CAR expressed on the surface of the PBNK cells. Through flow cytometry, the red fluorescence labeling CAR expressed on the cell surface and the far-infrared fluorescence intensity stained on dead cells were measured.

[0369] For pantothenic acid-based ionized lipids with formulas 3-1, 3-3, 3-4, 3-5, and 3-8, lipid nanoparticles CAR001 to CAR012 containing either or both the oligo-gamma-glutamic acid derivative formula 1-1 and the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid were prepared, and after transfection into NK cells, cell viability and CAR expression rates were confirmed, and the results are shown in Fig. 9. The composition and structural physicobiochemical characteristics of the lipid nanoparticles (CAR 001 to 012) are shown in Table 2 above.

[0370] In the case of lipid nanoparticles CAR001 and CAR003 containing pantothenic acid-based ionized lipids with formulas 3-1 and 3-5 and formula 1-1, cell viability was excellent, but CAR expression was almost non-existent. Additionally, in the case of lipid nanoparticle CAR006 containing formula 3-5 and 2,6-dipalmitoyl ascorbic acid, CAR expression rate was excellent, but a very low cell viability of less than 20% was observed. On the other hand, lipid nanoparticles CAR010 and CAR012 containing pantothenic acid-based ionized lipids with formulas 3-4 and 3-8, formula 1-1, and 2,6-dipalmitoyl ascorbic acid showed high cell viability of over 80% and exhibited high CAR expression efficiencies of 68.7% and 72.9%, respectively. Therefore, as can be confirmed in the diagram showing the correlation between cell viability and CAR expression efficiency for each lipid nanoparticle, the leading lipid nanoparticles suitable for CAR-NK cell production were identified as CAR010 and CAR012.

[0371] To further optimize lipid nanoparticles for CAR-NK cell production, novel panthenol-based ionized lipids, formulas 3-9 and 3-10, were introduced along with a novel pantothenic acid-based ionized lipid, formula 3-8, and lipid nanoparticles were prepared containing both 2,6-dipalmitoyl ascorbic acid and the oligo-gamma-glutamic acid derivative formulas 1-1 or 1-2. The composition and structural physicobiochemical characteristics of the lipid nanoparticles (CAR013 to 017) encapsulating CAR mRNA are shown in Table 2 above. Specifically, the composition values ​​in the table represent mol% and include the diameter, PDI, and zeta potential of the prepared particles measured using dynamic light scattering.

[0372] Lipid nanoparticles CAR013 and CAR014 containing novel panthenol-based ionized lipids with formulas 3-9 and 3-10, 2,6-dipalmitoyl ascorbic acid, and an oligo-gamma-glutamic acid derivative with formula 1-1, and lipid nanoparticles CAR015 to CAR017 containing pantothenic acid and panthenol-based ionized lipids with formulas 3-8, 3-9, and 3-10, 2,6-dipalmitoyl ascorbic acid, and an oligo-gamma-glutamic acid derivative with formula 1-2 were prepared, and cell viability and CAR expression rates were confirmed after transfection of NK cells, and the results are shown in Fig. 10. Lipid nanoparticle CAR013, containing an oligo-gamma-glutamic acid derivative of chemical formula 1-1 and a panthenol-based ionized lipid of chemical formula 3-9, showed a very high cell viability of 91.1%, but the CAR expression efficiency dropped significantly to 3.41. Lipid nanoparticle CAR017, containing a panthenol-based ionized lipid of chemical formula 3-10, showed a slightly lower cell viability of 87.4%, but the CAR expression efficiency increased significantly compared to the lead lipid nanoparticle CAR012. CAR015 to CAR017, which contain novel ionized lipids 3-8, 3-9, and 3-10 based on pantothenic acid and panthenol and contain both 2,6-dipalmitoyl ascorbic acid and the oligo-gamma-glutamic acid derivative formula 1-2, showed improved CAR expression efficiency compared to CAR012 to CAR014 containing formula 1-1, while cell viability remained largely unaffected at over 80%.

[0373] As a result, by optimizing the structure of panthenol-based ionized lipids and oligo-gamma-glutamic acid derivatives, CAR015 and CAR017, which exhibit a high cell viability of over 80% and a CAR expression efficiency of over 50%, were additionally selected as lipid nanoparticles suitable for CAR-NK cell production.

[0374] To determine whether lipid nanoparticles must contain both the oligo-gammaglutamic acid derivative of Formula 1-2 and the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid to be suitable for CAR-NK cell production, lipid nanoparticles containing pantothenic acid and panthenol-based ionized lipids Formulas 3-8, 3-9, and 3-10, and containing either the oligo-gammaglutamic acid derivative of Formula 1-2 or the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid, either individually or together, were prepared at an optimized component ratio. In addition, CAR-Con lipid nanoparticles with a conventionally used composition were prepared using SM-102, a commercially available ionized lipid. The composition and structural physicobiochemical characteristics of the lipid nanoparticles (CAR 018 to 029) encapsulating CAR mRNA are shown in Table 2 above. Specifically, the composition values ​​in the table represent mole% and include the diameter, PDI, and zeta potential of the manufactured particles measured using dynamic light scattering.

[0375] Lipid nanoparticles CAR018 to CAR020 comprising pantothenic acid and panthenol-based ionized lipids Formulas 3-8, 3-9, and 3-10, excluding the oligo-gamma-glutamic acid derivative of Formula 1-2 and the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid; lipid nanoparticles CAR021 to CAR023 comprising pantothenic acid and panthenol-based ionized lipids 3-8, 3-9, and 3-10, comprising only the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid; lipid nanoparticles CAR024 to CAR026 comprising pantothenic acid and panthenol-based ionized lipids 3-8, 3-9, and 3-10, comprising only the oligo-gamma-glutamic acid derivative of Formula 1-2; and pantothenic acid and panthenol-based ionized lipids 3-8, 3-9, and 3-10 Lipid nanoparticles CAR027 to CAR029, which contain both the oligo-gammaglutamic acid derivative of Chemical Formula 1-2 and the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid, and the commercially available ionized lipid SM-102 were used to prepare lipid nanoparticles CAR-Con of a conventionally used composition. Lipid nanoparticles not containing the oligo-gammaglutamic acid derivative and the vitamin C-based lipid were prepared by replacing them with PEGylated lipids and DSPC helper lipids in the same component ratio. After transfecting NK cells with the prepared lipid nanoparticles, cell viability and CAR expression rates were confirmed, and the results are shown in Figure 11.

[0376] NK cell viability and CAR protein expression rates were verified through the following experiments. First, primary peripheral blood natural killer (PBNK) cells (1 M cell / 500 μL) dispersed in MACS medium were inoculated into a 24-well cell culture plate. Then, lipid nanoparticles encapsulated with CAR mRNA were added to each well at a concentration of 1 μg / 50 μL (based on RNA) and thoroughly mixed. The sequence of the CAR mRNA was denoted as SEQ ID NO. 1. After culturing for 3 hours, serum equivalent to 5% of the medium volume was added, and the cells were cultured for another 3 hours. After washing the cells with phosphate-buffered physiological saline, they were stained primarily by treating them with an antigen-biotin protein recognized by CAR and a Live / Dead dye for staining dead cells. Subsequently, streptavidin-APC was applied to the cells to finally stain the CAR expressed on the surface of the PBNK cells. Through flow cytometry, the red fluorescence labeling CAR expressed on the cell surface and the far-infrared fluorescence intensity stained on dead cells were measured.

[0377] While all lipid nanoparticles exhibited a cell viability of over 80%, CAR024 to CAR029, containing oligo-gammaglutamic acid derivatives, showed an excellent cell viability of over 93%. In contrast, lipid nanoparticles CAR018 to CAR020, which did not contain either oligo-gammaglutamic acid derivatives or the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid, and lipid nanoparticles CAR021 to CAR023, which contained only the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid, showed relatively low cell viability. Lipid nanoparticles CAR021 and CAR022, and CAR027 to CAR029, which contained the vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid in pantothenic acid and panthenol-based ionized lipids with chemical formulas 3-8, 3-9, and 3-10, showed a CAR expression efficiency of over 50%. In particular, lipid nanoparticles CAR027 to CAR029, which contain novel ionized lipids 3-8, 3-9, and 3-10 based on pantothenic acid and panthenol, as well as an oligo-gamma-glutamic acid derivative of chemical formula 1-2 and a vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid, showed an excellent cell viability of over 93% while exhibiting a CAR expression efficiency of over 50%.

[0378] In other words, it was confirmed that an ensemble of three types of lipids—pantothenic acid and panthenol-based ionized lipids (chemical formulas 3-8, 3-9, and 3-10) and vitamin C-based lipids (2,6-dipalmitoyl ascorbic acid) and oligo-gamma-glutamic acid derivatives (chemical formula 1-2)—is very important for the production of CAR-NK cells. Furthermore, as confirmed by the correlation between cell viability and expression efficiency, CAR027 and CAR029, which exhibited the most excellent cell viability and CAR expression efficiency, were additionally selected as lipid nanoparticles for CAR-NK production.

[0379] The additionally selected lipid nanoparticle CAR029 has the same composition as the previously selected lipid nanoparticle CAR017, but the component ratios have changed. The molar ratios of lipids based on oligo-gamma-glutamic acid derivatives, vitamin C-based helper lipids, ionized lipids, and cholesterol, excluding nucleic acids, were 1.5%, 15%, 45%, and 38.5% for CAR017, and 3.0%, 14.8%, 37.9%, and 44.3% for CAR029. Cell viability and CAR expression efficiency were confirmed using a flow cytometer after treatment for NK cell transformation, and the results are shown in Figure 12. Although CAR expression efficiency increases with a higher amount of treated CAR mRNA, it was confirmed that CAR029 showed a CAR expression efficiency improvement of more than 10% compared to CAR017, and cell viability was high at approximately 95%.

[0380] Additionally, by treating human peripheral blood mononuclear cells (PBMCs) with the lipid nanoparticles CAR027 and CAR029 for CAR-NK synthesis, it was confirmed that CAR expression is possible in CD3-labeled T cells, and the results are shown in Fig. 13. CAR027 and CAR029 [represented] 86.4% of the CD3-labeled T cells in the PBMCs used in the experiment + It was confirmed that these lipid nanoparticles can be transfected into T cells as well as NK cells by expressing CAR proteins in T cells at 63.2% and 82.1%, respectively.

[0381] In addition, lipid nanoparticles CAR030 to CAR034 were prepared using resveratrol as a cholesterol substitute, which is a natural product capable of activating NK cells. Furthermore, based on the observation that transfection within NK cells improved when the ratio of oligo-gamma-glutamic acid was increased from 1.5 mol% (CAR017) to 3 mol% (CAR029), lipid nanoparticles were prepared with the molar ratio of oligo-gamma-glutamic acid 1-2 to 1-3 increased to 10%. After transfecting the prepared lipid nanoparticles into NK cells, cell viability and CAR expression rates were confirmed, and the results are shown in Fig. 14.

[0382] Looking at Figure 14, when compounds of chemical formulas 3-8 to 3-14 were used as ionized lipids, and vitamin C-based lipids 2,6-dipalmitoyl ascorbic acid and resveratrol were used, the survival rate of NK cells decreased slightly to around 80%, and it was confirmed that lipid nanoparticles CAR 030 and CAR 033, in which the ionized lipids were chemical formulas 3-8 and 3-13, showed a CAR expression efficiency of more than 60%. In addition, when compounds of formulas 3-10 to 3-14 were used as ionized lipids, and vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid and oligo-gammaglutamic acid of formulas 1-2 to 1-3 were used, cell viability of over 90% was maintained, and in particular, lipid nanoparticles CAR035 and CAR036 using formula 3-10 as ionized lipids and oligo-gammaglutamic acid of formulas 1-2 and 1-3, and CAR039 and CAR040 using formulas 3-13 and 3-14 as ionized lipids and oligo-gammaglutamic acid of formula 1-2, showed excellent CAR expression rates of over 95%. In particular, it was confirmed that as the ratio of oligo-gamma-glutamic acid increased to 1.5 mol% (CAR017), 3 mol% (CAR029), and 10 mol% (CAR036), the CAR expression rate increased sequentially without affecting cell viability.

[0383] As a result, a total of 10 lipid nanoparticles suitable for CAR-NK cell production were selected as optimal lipid nanoparticles for CAR-NK production, including lipid nanoparticles CAR010 and CAR012 containing vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid and oligo-gamma-glutamic acid derivative formula 1-1 and pantothenic acid-based ionized lipid formulas 3-4 and 3-8, lipid nanoparticles CAR015 and CAR017 containing vitamin C-based lipid 2,6-dipalmitoyl ascorbic acid and oligo-gamma-glutamic acid derivative formulas 1-2 to 1-3 and pantothenic acid and panthenol-based ionized lipid formulas 3-8, 3-10, 3-13, and 3-14, and CAR027, CAR029, CAR035, CAR036, CAR039, and CAR040 with optimized component ratios.

[0384] Although the embodiments have been described above with reference to the limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, suitable results may be achieved even if the described techniques are performed in a different order than described, and / or if the components of the described system, structure, device, circuit, etc. are combined or assembled in a form different from described, or replaced or substituted by other components or equivalents.

[0385] Therefore, other embodiments, other manufacturing examples, and equivalents to the claims also fall within the scope of the claims set forth below.

[0386] The present invention was completed with the support of the National Research and Development Project No. 1711186495 and No. 2021M3E5E3080563, the National Research and Development Project No. 1711195963 and No. 00229101, and the institution's own project, Future Source Brain Science and Technology Development Project Artificial Brain Convergence Research (Brain Science) (2E32230:20230101~20231231:).

Claims

1. An LNP composition for immune cell transformation characterized by comprising: a nucleic acid; a lipid based on an oligo-gamma-glutamic acid derivative; a helper lipid based on vitamin C or gallic acid; an ionized lipid represented by the following chemical formula 3; and a structure-maintaining lipid; In the above chemical formula 3, R 1 to R 4 They may be the same or different from each other, and each C1 to C 12 It is an alkyl of, and R 5 and R 6 They may be the same or different from each other, and each is a C1 to C6 alkyl or combines with each other to form a C3 to C6 cycloalkyl or heterocycloalkyl, and A is any one selected from the group consisting of -C(=O)-NH-, -OC(=O)-, -C(=O)-O- and -OC(=O)-O-, and n and m are integers from 1 to 6, respectively.

2. In Paragraph 1, In the above chemical formula 3, R 1 to R 4 They may be the same or different from each other, and each C6 to C 12 It is a straight-chain alkyl, and R 5 and R 6 Each is methyl or ethyl, or combines with each other to form morpholine or oxazepane, and An LNP composition for immune cell transformation, wherein n and m are integers from 1 to 3, respectively.

3. In Paragraph 1, The above nucleic acid is an LNP composition for immune cell transformation, which is mRNA expressing CAR (Chimeric antigen receptors).

4. In Paragraph 1, An LNP composition for immune cell transformation, wherein the ionized lipid represented by [Chemical Formula 3] above is selected from the group consisting of:

5. In Paragraph 1, An LNP composition for immune cell transformation, selected from the group consisting of the following when the above helper lipid is vitamin C-based: and .

6. An LNP composition for immune cell transformation according to claim 1, wherein the helper lipid is gallic acid-based and is selected from the group consisting of the following:

7. In Paragraph 1, The above-mentioned oligo-gamma-glutamic acid derivative-based lipid is any one selected from the group consisting of the following: an LNP composition for immune cell transformation and 8. In Paragraph 1, An LNP composition for immune cell transformation, wherein the molar ratios of lipids based on oligo-gamma-glutamic acid derivatives excluding nucleic acids, vitamin C-based helper lipids, ionized lipids, and structure-maintaining lipids are 1.5 to 10%, 10 to 15%, 44 to 50%, and 30 to 38.5%, respectively.

9. An LNP composition for immune cell transformation according to claim 8, wherein the structural maintenance lipid is selected from either cholesterol or resveratrol.

10. An LNP composition for immune cell transformation according to claims 1 to 9, wherein the immune cell is either an NK cell or a T cell.

11. A method for transforming NK cells or T cells, comprising the step of treating NK cells or T cells with the composition of claim 10.

12. A pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, NK cells or T cells expressing a CAR on their surface, prepared by the method of claim 11.

13. In Paragraph 12, A pharmaceutical composition in which the above cancer is selected from the group consisting of pancreatic cancer, breast cancer, ovarian cancer, glioma, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, liver cancer, lung cancer, colorectal cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, blood cancer, and melanoma.

14. In Paragraph 12, The above pharmaceutical composition is a pharmaceutical composition that further comprises another anticancer agent.

15. In Paragraph 12, The above pharmaceutical composition is a pharmaceutical composition that inhibits the proliferation, survival, metastasis, recurrence, or anticancer drug resistance of cancer.

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