Lipid compound having asymmetric structure and lipid nanoparticle composition comprising same

Abstract

In some embodiments, a lipid compound has aliphatic chain-containing tail moieties that have an asymmetric structure. The lipid compound can be used as an ionizable lipid compound and stabilizes a pharmaceutically active substance, such as a nucleic acid-based therapeutic agent or vaccine. The expression efficiency of a target molecule encoded in a nucleic acid molecule is improved by using the ionizable lipid compound. The lipid compound can be applied to nucleic acid-based therapeutic agents or vaccines.

Description

Lipid compound having an asymmetric structure and lipid nanoparticle composition comprising the same

[0001] This patent application is related to the results of the research project for the development of toxicity evaluation technology for mRNA vaccines, etc. (led by the Catholic University of Korea Industry-Academic Cooperation Foundation) as part of the Infectious Disease Response Innovation Technology Support Research Project of the Ministry of Food and Drug Safety of the Republic of Korea, and the research project for the development of technology for evaluating the efficacy and quality of mRNA vaccines, etc. (led by the Catholic University of Korea Industry-Academic Cooperation Foundation) as part of the Ministry of Food and Drug Safety's mRNA vaccines, etc. efficacy and quality evaluation technology development project, regarding the results of the research project for the development of technology for evaluating the efficacy of mRNA vaccines for treating HPV-induced cancer (led by the Catholic University of Korea Industry-Academic Cooperation Foundation).

[0002] The present disclosure relates to lipid compounds, and more particularly, to lipid compounds capable of stabilizing nucleic acid molecules and lipid nanoparticle compositions comprising the same.

[0003] Since the development of genetic recombination technology in the 1970s, nucleic acid-based materials have been utilized as pharmaceuticals, such as therapeutics and vaccines. For example, nucleic acid-based vaccines are being developed, in which genes encoding therapeutic proteins or antigens are injected in nucleic acid form. With the rapid development of mRNA-based vaccines for COVID-19, which caused a global pandemic in 2019, nucleic acid-based medicines and vaccines are attracting attention.

[0004] However, nucleic acid-based drugs are easily degraded in the body by nucleases. Furthermore, because nucleic acid molecules are inherently negatively charged, they have a low intracellular delivery efficiency. Materials such as lipids, polymers, and dendrimers have been developed to facilitate the delivery of nucleic acid-based substances, the active ingredients of pharmaceuticals or vaccines. Among these, drugs using lipid nanoparticles are now being released on the market.

[0005] Currently, lipid nanoparticles are commonly used in a form containing a fixed ratio of four components: ionized lipids, phospholipids (helper lipids), cholesterol (structural maintenance lipids), and PEG-lipids. There is a need to develop lipid compounds capable of efficiently delivering nucleic acid-based pharmaceuticals into the body, and lipid nanoparticle compositions containing these lipid compounds.

[0006] The purpose of the present disclosure is to provide an ionizable lipid compound capable of improving the expression of a target molecule.

[0007] Another object of the present disclosure is to provide a lipid nanoparticle composition comprising an ionizable lipid compound.

[0008] In one aspect, the present disclosure provides a lipid compound, which is a compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0009] [Chemical Formula 1]

[0010]

[0011] In chemical formula 1,

[0012] R 1 and R 2 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 Alkynyl group; L 1 is linear or branched C1-C 40 Alkylene group, linear or branched C2-C 40 Alkenylene group or linear or branched C2-C 40 It is a divalent aliphatic linking group which is an alkynylene group, and the aliphatic linking group is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NRa -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a Can have C(=O)O-; R 3 Inland R 5 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle connected to the carbon atom. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a It can have C(=O)O-, and R 3 Inland R 5 At least two of the above are aliphatic substituents, one of the above aliphatic substituents has -SS- in the middle, and the other of the above aliphatic substituents does not have -SS- in the middle; R a is a hydrogen atom or C1-C 15 An alkyl group; k is an integer from 1 to 3.

[0013] In an exemplary embodiment, the lipid compound may comprise a compound represented by the following formula 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0014] [Chemical Formula 2]

[0015]

[0016] In chemical formula 2,

[0017] R 1 , R2 and L 1 are each the same as defined in chemical formula 1; R 6 and R 7 are each independently linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle connected to the carbon atom. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a It can have C(=O)O-, and R 6 and R 7 One of them has -SS- in the middle, R 6 and R 7 One of the others does not have -SS-; R a and k are each the same as defined in chemical formula 1.

[0018] In another embodiment, the lipid compound may include a compound represented by the following chemical formula 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0019] [Chemical Formula 3]

[0020]

[0021] In chemical formula 3,

[0022] R 11 and R 12 are each independently a hydrogen atom, linear or branched linear or branched C1-C 20 Alkyl group, linear or branched C2-C 20Alkenyl group or linear or branched C2-C 20 Alkynyl group; L 11 and L 12 are each independently linear or branched C1-C 20 Alkylene group, linear or branched C2-C 20 Alkenylene group or linear or branched C2-C 20 It is a divalent aliphatic linking group, which is an alkynylene group; Y 11 -O(C=O)-, -(C=O)O-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -Im; R 13 Inland R 15 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is connected to a carbon atom at the terminal or middle thereof as -O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -NR a C(=O)- or -C(=O)NR a - can have, R 13 Inland R 15 At least two of the above are aliphatic substituents, one of the above aliphatic substituents has -SS- in the middle, and the other of the above aliphatic substituents does not have -SS- in the middle; R a is the same as defined in chemical formula 1.

[0023] In an exemplary embodiment, the lipid compound may include a compound represented by the following chemical formula 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0024] [Chemical Formula 4]

[0025]

[0026] In chemical formula 4,

[0027] R11 , R 12 , L 11 , L 12 and Y 11 are each the same as defined in chemical formula 3; R 16 and R 17 are each independently linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is connected to a carbon atom at the terminal or middle thereof as -O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -NR a C(=O)- or -C(=O)NR a - can have, R 16 and R 17 One of them has -SS- in the middle, R 6 and R 7 One of the others does not have -SS-; R a is the same as defined in chemical formula 1.

[0028] In another embodiment, the lipid compound may include a compound represented by the following chemical formula 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0029] [Chemical Formula 5]

[0030]

[0031] In chemical formula 5,

[0032] R 21 and R 22 are each independently a hydrogen atom, linear or branched linear or branched C1-C 20 Alkyl group; L 21 and L 22 are each independently linear or branched C1-C 20 Alkylene group; Y 21 is -O(C=O)- or -(C=O)O-; Y 22 and Y 23are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -Im; R 23 and R 24 are each independently linear or branched C1-C 40 Alkyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkenyl group, and the aliphatic substituent may have -O(C=O)-, -(C=O)O- or -SS- in the middle, and R 23 and R 24 One of them has -SS- in the middle, R 23 and R 24 One of the others does not have -SS- in the middle; R a is the same as defined in chemical formula 1.

[0033] In an exemplary embodiment, the lipid compound may include a compound represented by the following chemical formula 6, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0034] [Chemical Formula 6]

[0035]

[0036] In chemical formula 6,

[0037] R 21 , R 22 , L 21 , L 22 and Y 21 are each identical to those defined in chemical formula 5; Y 24 is -O(C=O)-, -(C=O)O-; Y 25 is -NR a C(=O)- or -C(=O)NR a -Im; L 23 is linear or branched C1-C 20 Alkylene group or linear or branched C2-C 20An aliphatic linking group which is an alkenylene group, and the aliphatic linking group may have -O(C=O)-, -(C=O)O- in the middle; R 25 is linear or branched C1-C 40 Alkyl group or linear or branched C2-C 40 An aliphatic substitution is an alkenyl group, and the aliphatic substitution group may have -O(C=O)-, -(C=O)O- in the middle; R 26 is linear or branched C1-C 20 Alkyl group or linear or branched C2-C 20 An aliphatic substitution is an alkenyl group, and the aliphatic substitution group may have -O(C=O)-, -(C=O)O- in the middle; R a is the same as defined in chemical formula 1.

[0038] For example, R in chemical formula 6 21 and R 22 are each independently linear or branched C1-C 10 is an alkyl group, and L 21 and L 22 are each independently linear or branched C1-C 10 It is an alkylene group, L 23 is linear or branched C1-C 20 It is an alkylene group, and the above C1-C 20 The alkylene group can have -O(C=O)-, -(C=O)O- in the middle, and R 26 is linear or branched C1-C 20 It may be an alkyl group.

[0039] In another aspect, the present disclosure provides a lipid nanoparticle composition comprising the aforementioned lipid compound.

[0040] For example, the lipid nanoparticle composition may further comprise at least one of a phospholipid, a structure-maintaining lipid, a polyethylene glycol (PEG)-lipid, and an additive.

[0041] The lipid nanoparticle composition may further comprise a target molecule, for example, a nucleic acid molecule.

[0042] In an exemplary embodiment, the nucleic acid molecule may comprise a coding region encoding a target molecule and at least one expression regulatory element operably linked to the coding region.

[0043] The coding region may comprise a nucleotide sequence encoding at least one of a reporter peptide, a marker peptide, a selection peptide, a peptide associated with disease treatment or prevention or a fragment thereof, and an immunogen or a fragment thereof.

[0044] Lipid compounds may have tail moieties that are asymmetrical in structure, possibly consisting of aliphatic chains. One of the two or more tail moieties has a disulfide (S-S) bond in the middle, and the other does not have a disulfide bond in the middle.

[0045] In lipid nanoparticle compositions, the lipid compound can stabilize the nucleic acid molecule, thereby enhancing the expression of the target molecule from the coding region encoded in the nucleic acid molecule.

[0046] For example, lipid compounds and lipid nanoparticle compositions comprising the same can be utilized to synthesize and manufacture pharmaceuticals such as nucleic acid-based therapeutic agents or vaccines.

[0047] FIG. 1 is a graph showing the amount of hEPO expression measured in a mouse after injecting a lipid nanoparticle composition comprising a lipid compound synthesized in an example of the present disclosure and an mRNA nucleic acid molecule encoding a target molecule into the mouse.

[0048] FIGS. 2 and 3 are graphs showing the expression levels of chemokines MCP-1 and IL-6 measured in mice after injecting a lipid nanoparticle composition comprising a lipid compound synthesized in an example of the present disclosure and an mRNA nucleic acid molecule encoding a target molecule into the mouse, respectively.

[0049] FIGS. 4 to 6 are graphs showing the secretion amount of antibodies IgG1 and IgG2a specific to the target molecule, the influenza virus surface antigen Hemagglutin (HA), in the serum of mice that were first immunized with a lipid nanoparticle composition containing a lipid compound synthesized in an example of the present disclosure, and an mRNA nucleic acid molecule encoding a target molecule, respectively.

[0050] FIGS. 7 to 9 are graphs showing the secretion amount of antibodies IgG1 and IgG2a specific to the target molecule, the influenza virus surface antigen Hemagglutin (HA), in the serum of mice immunized for the second time with a lipid nanoparticle composition containing a lipid compound synthesized in an example of the present disclosure, and an mRNA nucleic acid molecule encoding a target molecule, respectively.

[0051] FIG. 10 is a graph showing neutralizing antibody titers measured in mice immunized by injection of a lipid nanoparticle composition comprising a lipid compound synthesized in an example of the present disclosure and an mRNA nucleic acid molecule encoding a target molecule.

[0052] FIGS. 11 to 13 are graphs showing the results of measuring an increased specific response to an antigen, which is a target molecule, in mice immunized by injecting a lipid nanoparticle composition containing a lipid compound synthesized in an example of the present disclosure and an mRNA nucleic acid molecule encoding a target molecule, respectively.

[0053] FIGS. 14 and 15 are graphs showing the amount of hEPO expression measured in a mouse after injecting a lipid nanoparticle composition comprising a lipid compound synthesized in an example of the present disclosure and an mRNA nucleic acid molecule encoding a target molecule into the mouse, respectively.

[0054] Figures 16 to 19 are graphs showing the expression levels of chemokines MCP-1 and IL-6 measured in mice after injecting a lipid nanoparticle composition containing a lipid compound synthesized in an example of the present disclosure and an mRNA nucleic acid molecule encoding a target molecule into the mouse, respectively.

[0055] FIG. 20 is a graph showing the secretion amount of antibodies IgG1 and IgG2a specific to the spike protein, a surface antigen of the omicron virus, which is a target molecule, in the serum of a mouse that was first immunized with a lipid nanoparticle composition containing a lipid compound synthesized in an example of the present disclosure and an mRNA nucleic acid molecule encoding a target molecule.

[0056] FIGS. 21 to 31 are graphs showing the results of hematological and serological analyses of the toxicity of a target molecule in mice immunized with a lipid nanoparticle composition comprising a lipid compound synthesized in an embodiment of the present disclosure, and an mRNA nucleic acid molecule encoding the target molecule, respectively.

[0057] The present disclosure is described in detail with reference to the attached drawings, if necessary.

[0058] [lipid compounds]

[0059] The lipid compound may include a head moiety having an amino group, a tail moiety including an aliphatic chain connected to the head moiety, and a linking moiety between the head moiety and the tail moiety. The plurality of tail moieties constituting the lipid compound have an asymmetric structure. For example, one of the plurality of tail moieties includes a disulfide bond, while the other does not include a disulfide bond. The lipid compound having a tail moiety with an asymmetric structure includes a compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0060] [Chemical Formula 1]

[0061]

[0062] In chemical formula 1,

[0063] R 1 and R 2 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 Alkynyl group; L 1 is linear or branched C1-C 40 Alkylene group, linear or branched C2-C 40 Alkenylene group or linear or branched C2-C 40 It is a divalent aliphatic linking group which is an alkynylene group, and the aliphatic linking group is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a Can have C(=O)O-; R 3 Inland R 5 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle connected to the carbon atom. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NRa It can have C(=O)O-, and R 3 Inland R 5 At least two of the above are aliphatic substituents, one of the above aliphatic substituents has -SS- in the middle, and the other of the above aliphatic substituents does not have -SS- in the middle; R a is a hydrogen atom or C1-C 15 An alkyl group; k is an integer from 1 to 3.

[0064] In the present specification, the term 'hetero' used in 'heteroaromatic', 'heterocycloalkylene group', 'heteroarylene group', 'heteroaralkylene group', 'heteroaryl oxylene group', 'heterocycloalkyl group', 'heteroaryl group', 'heteroaralkyl group', 'heteroaryl oxyl group', 'heteroaryl amino group', etc. means that one or more of the carbon atoms constituting these aromatic or alicyclic rings, for example, 1 to 5 carbon atoms, are substituted with one or more heteroatoms selected from the group consisting of N, O, S, Si, Se, P, B, and combinations thereof, for example, N, O, S, and combinations thereof.

[0065] In this specification, an alkyl group, an alkenyl group, an alkynyl group, and an alkoxy group are saturated or unsaturated chain-like aliphatic substituents, including both linear and branched groups. In this specification, an alkylene group, an alkenylene group, and an alkynylene group may be a divalent linking group corresponding to these aliphatic hydrocarbon groups.

[0066] In this specification, a cycloalkyl group, a cycloalkenyl group, a heterocycloalkyl group, and a heterocycloalkenyl group are saturated or unsaturated cyclic aliphatic substituents. In this specification, a cycloalkylene group, a cycloalkenylene group, a heterocycloalkylene group, and a heterocycloalkenylene group may be a divalent linking group corresponding to these cyclic aliphatic substituents.

[0067] In the present specification, an aryl group and a heteroaryl group may each correspond to an aromatic substituent or a heteroaromatic substituent. In the present specification, an arylene group and a heteroarylene group may each be a divalent linking group corresponding to these aromatic or heteroaromatic substituents. For example, an arylene group may include a divalent linking group corresponding to benzene, naphthalene, indene, or anthracene, and a heteroarylene group may include a divalent linking group corresponding to pyridine, furan, thiophene, pyrrole, pyrimidine, pyran, triazine, thiazine, oxazine, or quinone, but is not limited thereto.

[0068] In an exemplary embodiment, the pharmaceutically acceptable salt may be an acid addition salt formed with a pharmaceutically acceptable free acid. The acid addition salt may be prepared by dissolving the compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, and / or acetonitrile. The free acid may be an organic acid or an inorganic acid. Inorganic acids include, but are not limited to, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid.

[0069] For example, pharmaceutically acceptable organic acids include, but are not limited to, acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, benzoic acid, ascorbic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid 2-hydroxyethanesulfonic acid, camphoric acid, oxalic acid, tartaric acid, mandelic acid, propionic acid, citric acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, and aspartic acid.

[0070] In an exemplary embodiment, R of formula 1 1 Inland R 2are each independently C1-C 40 Alkyl group (e.g., C1-C 30 Alkyl group or C1-C 20 alkyl group), C2-C 40 Alkenyl group (e.g., C2-C 30 Alkenyl group or C2-C 20 alkenyl group), C2-C 40 Alkynyl group (e.g., C2-C 30 Alkynyl group or C2-C 20 It may be an aliphatic substituent (alkenyl group).

[0071] In an exemplary embodiment, R of formula 1 3 Inland R 5 At least two of them are C1-C 40 Alkyl group (e.g., C1-C 30 Alkyl group or C1-C 20 alkyl group), C2-C 40 Alkenyl group (e.g., C2-C 30 Alkenyl group or C2-C 20 alkenyl group), C2-C 40 Alkynyl group (e.g., C2-C 30 Alkynyl group or C2-C 20 It can be an aliphatic substituent (alkenyl group). R 3 Inland R 5 The aliphatic substituents that can be present at the terminal or intermediate positions connected to the carbon atom are -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O). k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-, for example, -O(C=O)-, -(C=O)O-, -S(O) k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)- or -C(=O)NRa - can have R 3 Inland R 5 Among the possible aliphatic substituents, one contains a disulfide (-S-S-) bond in the middle, and the other does not contain a disulfide bond in the middle. Therefore, the lipid compound has an asymmetric tail moiety structure.

[0072] In an exemplary embodiment, L of formula 1 1 Silver C1-C 40 Alkylene group (e.g., C1-C 30 Alkylene group or C1-C 20 alkylene group), C2-C 40 Alkenylene group (e.g., C2-C 30 Alkenylene group or C2-C 20 alkenylene group) or C2-C 40 Alkynylene group (e.g., C2-C 30 Alkynylene group or C2-C 20 It is a divalent aliphatic linking group (alkenylene group), and the aliphatic linking group is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-, for example, -O(C=O)-, -(C=O)O-, -S(O) k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)- or -C(=O)NR a - can have.

[0073] The compound represented by Chemical Formula 1 may include a tail moiety comprising two aliphatic chains having an asymmetric structure. The aliphatic substituents constituting the tail moiety are -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a It may have C(=O)O-. One of the two aliphatic chains may have a disulfide bond in the middle, and the other may not have a disulfide bond. In an exemplary embodiment, the lipid compound may include a compound represented by the following formula 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0074] [Chemical Formula 2]

[0075]

[0076] In chemical formula 2,

[0077] R 1 , R 2 and L 1 are each the same as defined in chemical formula 1; R 6 and R 7 are each independently linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle connected to the carbon atom. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a-, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a It can have C(=O)O-, and R 6 and R 7 One of them has -SS- in the middle, R 6 and R 7 One of the others does not have -SS-; R a and k are each the same as defined in chemical formula 1.

[0078] In an exemplary embodiment, L in Formula 1 and Formula 2 1 In the middle is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a It can be a divalent aliphatic linker having C(=O)O-.

[0079] In another embodiment, an aliphatic linking group comprising at least one ester group, a carbonyl group, or an amide group may be positioned between a hydrophilic head moiety comprising an amino group and a tail moiety comprising an aliphatic chain. For example, the lipid compound may include a compound represented by the following formula 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0080] [Chemical Formula 3]

[0081]

[0082] In chemical formula 3,

[0083] R 11 and R 12 are each independently a hydrogen atom, linear or branched linear or branched C1-C20 Alkyl group, linear or branched C2-C 20 Alkenyl group or linear or branched C2-C 20 Alkynyl group; L 11 and L 12 are each independently linear or branched C1-C 20 Alkylene group, linear or branched C2-C 20 Alkenylene group or linear or branched C2-C 20 It is a divalent aliphatic linking group, which is an alkynylene group; Y 11 -O(C=O)-, -(C=O)O-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -Im; R 13 Inland R 15 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is connected to a carbon atom at the terminal or middle thereof as -O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -NR a C(=O)- or -C(=O)NR a - can have, R 13 Inland R 15 At least two of the above are aliphatic substituents, one of the above aliphatic substituents has -SS- in the middle, and the other of the above aliphatic substituents does not have -SS- in the middle; R a is the same as defined in chemical formula 1.

[0084] In an exemplary embodiment, the compound represented by formula 3 may include a tail moiety comprising two aliphatic chains having an asymmetric structure. The aliphatic substituents constituting the tail moiety are terminal or intermediate and include O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -NR aC(=O)- or -C(=O)NR a - may have. One of the two aliphatic chains may have a disulfide bond in the middle, and the other may not have a disulfide bond. For example, the lipid compound may include a compound represented by the following chemical formula 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0085] [Chemical Formula 4]

[0086]

[0087] In chemical formula 4,

[0088] R 11 , R 12 , L 11 , L 12 and Y 11 are each the same as defined in chemical formula 3; R 16 and R 17 are each independently linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is connected to a carbon atom at the terminal or middle thereof as -O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -NR a C(=O)- or -C(=O)NR a - can have, R 16 and R 17 One of them has -SS- in the middle, R 6 and R 7 One of the others does not have -SS-; R a is the same as defined in chemical formula 1.

[0089] In an exemplary embodiment, L in Formula 3 and Formula 4 11 and L 12 are each independently linear or branched C1-C 20 is an alkylene group, and Y 11can be -O(C=O)- or -(C=O)O-.

[0090] In another embodiment, each tail moiety having an asymmetric structure may be connected to a carbon atom constituting the linking group via an ester bond or an amide bond. For example, the lipid compound may include a compound represented by the following chemical formula 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0091] [Chemical Formula 5]

[0092]

[0093] In chemical formula 5,

[0094] R 21 and R 22 are each independently a hydrogen atom, linear or branched linear or branched C1-C 20 Alkyl group; L 21 and L 22 are each independently linear or branched C1-C 20 Alkylene group; Y 21 is -O(C=O)- or -(C=O)O-; Y 22 and Y 23 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -Im; R 23 and R 24 are each independently linear or branched C1-C 40 Alkyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkenyl group, and the aliphatic substituent may have -O(C=O)-, -(C=O)O- or -SS- in the middle, and R 23 and R 24 One of them has -SS- in the middle, R 23 and R 24 One of the others does not have -SS- in the middle; R a is the same as defined in chemical formula 1.

[0095] Among the tail moieties of the asymmetric structure of the compound represented by Chemical Formula 5, an aliphatic chain having a disulfide bond and an aliphatic chain not having a disulfide bond may be connected to a carbon atom through different groups. For example, among the tail moieties of the asymmetric structure, one tail moiety may be connected to a carbon atom through an ester bond, and the other tail moiety may be connected to a carbon atom through an amide bond. For example, the lipid compound may include a compound represented by Chemical Formula 6 below, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0096] [Chemical Formula 6]

[0097]

[0098] In chemical formula 6,

[0099] R 21 , R 22 , L 21 , L 22 and Y 21 are each identical to those defined in chemical formula 5; Y 24 is -O(C=O)-, -(C=O)O-; Y 25 is -NR a C(=O)- or -C(=O)NR a -Im; L 23 is linear or branched C1-C 20 Alkylene group or linear or branched C2-C 20 An aliphatic linking group which is an alkenylene group, and the aliphatic linking group may have -O(C=O)-, -(C=O)O- in the middle; R 25 is linear or branched C1-C 40 Alkyl group or linear or branched C2-C 40 An aliphatic substitution is an alkenyl group, and the aliphatic substitution group may have -O(C=O)-, -(C=O)O- in the middle; R 26 is linear or branched C1-C 20Alkyl group or linear or branched C2-C 20 An aliphatic substitution is an alkenyl group, and the aliphatic substitution group may have -O(C=O)-, -(C=O)O- in the middle; R 1 is the same as defined in chemical formula 1.

[0100] In an exemplary embodiment, R of formula 6 21 and R 22 are each independently linear or branched C1-C 10 is an alkyl group, and L 21 and L 22 are each independently linear or branched C1-C 10 It is an alkylene group, L 23 is linear or branched C1-C 20 It is an alkylene group, and the above C1-C 20 The alkylene group can have -O(C=O)-, -(C=O)O- in the middle, and R 26 is linear or branched C1-C 20 It may be an alkyl group, but is not limited thereto.

[0101] In another embodiment, the compound represented by Chemical Formula 1 is at least one of a compound represented by Chemical Formula 7 below, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or may be selected from a compound represented by Chemical Formula 7 below, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, but is not limited thereto.

[0102] [Chemical Formula 7]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] [Lipid nanoparticle composition]

[0115] The compounds represented by Chemical Formulae 1 to 7, stereoisomers thereof, or pharmaceutically acceptable salts thereof can stabilize pharmaceutically active ingredients. The present disclosure provides a lipid nanoparticle composition comprising the compounds represented by Chemical Formulae 1 to 7, stereoisomers thereof, or pharmaceutically acceptable salts thereof. For example, the pharmaceutically active ingredients in the lipid nanoparticle composition include, but are not limited to, nucleic acid molecules and / or peptides (proteins) expressed from nucleic acid molecules.

[0116] Lipid nanoparticle compositions comprise a plurality of lipid molecules physically associated with each other, including microspheres (including unilamellar and multilamellar vesicles, such as liposomes), dispersed phases in emulsions, micelles, or the internal phases of suspensions. Lipid nanoparticles included in lipid nanoparticle compositions can be used to deliver or encapsulate nucleic acid molecules or peptides (proteins) expressed from nucleic acid molecules.

[0117] In an exemplary embodiment, the lipid nanoparticle composition can include an ionizable lipid, a phospholipid (helper lipid), a structure-maintaining lipid (cholesterol lipid), a polyethylene glycol (PEG)-lipid, an additive, and / or a nucleic acid molecule. The compound represented by Formulae 1 to 7, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof can function as the ionizable lipid in the lipid nanoparticle composition. The ionizable lipid has a positive charge, which allows it to well encapsulate a nucleic acid molecule having a negative charge (e.g., an mRNA encoding a target molecule, or a siRNA molecule), and when administered into the blood, it can lose its positive charge due to the alkaline environment of the blood, thereby reducing toxicity in the body.

[0118] Phospholipids encapsulate and protect the core formed by the interaction of ionized lipids and active ingredients within lipid nanoparticles. Furthermore, phospholipids bind to the phospholipid bilayer of target cells, facilitating the intracellular delivery of the active ingredient through the cell membrane and endosome escape. Phospholipids can include neutral, uncharged, or cationic phospholipids.

[0119] For example, phospholipids include 5-heptadecylbenzene-1,3-diol (resorcinol), phosphatidylcholine (PLPC), lysophosphatidylcholine, phosphatidylethanolamine (PE), phosphatidylcholine (EPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, dilauroylphosphatidylcholine (DLPC), 1,2-Diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine](DOPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], and combinations thereof, but is not limited thereto. For example, lipid nanoparticles comprising DOPE or DSPC may be effective in mRNA delivery (excellent drug delivery efficiency for mRNA), and lipid nanoparticles comprising DSPE may be effective in siRNA delivery (excellent drug delivery efficiency for siRNA).

[0120] The structural maintenance lipid (cholesterol lipid) provides morphological rigidity according to lipid loading within the lipid nanoparticle (LNP), and is dispersed in the core and surface of the nanoparticle, thereby enhancing the stability of the nanoparticle. In addition, the structural maintenance lipid may enhance the transfection of nucleic acid molecules into living cells and / or enhance membrane fusogenicity. The structural maintenance lipid may include a steroid, a sterol, and an alkyl resorcinol. Alternatively or additionally, the structural maintenance lipid may be selected from the group consisting of, but not limited to, cholesterol, 5-heptadecyl resorcinol, cholesterol hemisuccinate, and combinations thereof.

[0121] PEG-lipids contribute to the stability of lipid nanoparticles in serum and play a role in preventing aggregation between nanoparticles. For example, PEG-lipids can enhance the stability of nucleic acid molecules in the body by protecting nucleic acid molecules from degrading enzymes during in vivo delivery of nucleic acid molecules, and can increase the half-life of drugs encapsulated in lipid nanoparticles. In exemplary embodiments, PEG-lipids can include, but are not limited to, polymers having a hydrophilic head, such as PEG (polyethylene glycol or polyethylene oxide), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and poly N-(2-hydroxypropyl)methacrylamide.

[0122] For example, the PEG-lipid can be selected from the group consisting of, but is not limited to, PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG, DMG-PEG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-distearoylglycamide, PEG-cholesterol (1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and combinations thereof.

[0123] In an exemplary embodiment, the lipid nanoparticle composition may comprise, but is not limited to, ionizable lipids according to the present disclosure in a proportion of 20 to 60 mol%, for example, 25 to 50 mol%, phospholipids in a proportion of 5 to 40 mol%, for example, 10 to 40 mol%, structure-maintaining lipids in a proportion of 15 to 70 mol%, for example, 20 to 65 mol%, PEG-lipids in a proportion of 0.5 to 5 mol%, for example, 1 to 3 mol%, and additives in a proportion of 0 to 25 mol%.

[0124] If desired, the lipid nanoparticle composition may further comprise a nucleic acid molecule. For example, the nucleic acid molecule may be si-RNA or sh-RNA. In another embodiment, the nucleic acid molecule may comprise a coding region encoding a target molecule that can be utilized as a label or marker, and at least one expression regulatory element.

[0125] For example, the coding region may encode a reporter peptide (reporter protein) and / or a marker or selection peptide. For example, the reporter peptide may include, but is not limited to, luciferase, green fluorescent protein (GFP), enhanced GFP (EGFP), beta-galactosidase, and combinations thereof. Optionally, the marker or selection peptide may include, but is not limited to, alpha-globin, galactokinase, xanthine, and combinations thereof.

[0126] Optionally, the nucleic acid molecule may include a coding region encoding a therapeutically or prophylactically active pharmaceutical ingredient. The coding region may consist of an open reading frame (ORF) encoding a peptide and / or fragment thereof effective in treating or preventing a disease. For example, the peptide effective in treating or preventing a disease may be a cytokine, lymphokine, monokines, growth factors, receptors, signaling molecules, transcription factors, or apoptotic factors. The peptide encoded in the coding region and effective in treating or preventing a disease may be an adjuvant peptide.

[0127] In another embodiment, the coding region may consist of a nucleotide sequence encoding an immunogen or a fragment thereof. For example, the immunogen encoded in the coding region may include an antigen, an epitope of an antigen, a fragment of an antigen, or a peptide of an antigen. For example, the immunogen may be a protein or peptide antigen, such as a tumor antigen, an allergen or allergen, an autoimmune autoantigen, or a pathogenic antigen. The pathogenic antigen encoded in the coding region may be an antigen derived from a viral antigen, a bacterial antigen, a fungal antigen, a protozoan antigen, an animal antigen, and / or an allergenic antigen.

[0128] The coding region may consist of an open reading frame (ORF) encoding a protein or peptide that is a tumor antigen or a fragment thereof, a variant or derivative thereof. The tumor antigen may be a melanocyte-specific antigen, a testicular cancer antigen or a tumor-specific antigen, for example, a CT-X antigen, a non-X CT-antigen, a binding partner to a CT-X antigen or a binding partner to a non-X CT-antigen or a tumor-specific antigen, or a tumor-specific antigen, a variant or derivative of a tumor antigen.

[0129] For example, the coding region may consist of nucleotides encoding an antibody or a fragment thereof. For example, the antibody encoded in the coding region may be a naturally occurring or recombinantly produced antibody. In particular, it may be an antibody suitable for therapeutic and / or diagnostic purposes. For example, antibodies include monoclonal antibodies, polyclonal antibodies, and antibodies with multiple epitope specificities, as well as chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, endomers, fragments and variants thereof, naturally occurring antibodies, and antibodies produced in a host by immunization. The coding region may consist of an open reading frame (ORF) encoding a therapeutic or prophylactic protein / peptide, or a protein / peptide that is a fragment, variant, or derivative thereof.

[0130] The nucleic acid molecule may further comprise one or more transcriptional regulatory elements (e.g., promoter, enhancer), translational regulatory elements (e.g., 5'UTR and / or 3'UTR such as IRES, 5'-cap), expression regulatory sequences such as poly A and / or poly A recognition sequences, and / or multiple cloning sites (MCS) to promote expression of a target molecule encoded in the coding region. The coding region within the nucleic acid molecule may be operably linked to the expression regulatory sequences. The nucleic acid molecule may be of DNA type or RNA type. For example, the nucleic acid molecule may have an RNA platform structure.

[0131] In an exemplary embodiment, the lipid component and the nucleic acid molecule may be added to the composition in a weight ratio of from 10:1 to 60:1. Optionally, the N / P ratio of the nucleic acid molecule may be from about 2:1 to about 30:1. The N / P ratio is the number of ionizable nitrogens of the ionizable lipid divided by the number of phosphate groups of the nucleic acid molecule.

[0132] Hereinafter, the present disclosure will be described through exemplary embodiments, but the present disclosure is not limited to the technical ideas described in the following embodiments.

[0133] Synthesis Example 1: Synthesis of disulfide-bonded tail precursor 2-(tetradecylthio)ethan-1-ol (2)

[0134]

[0135] 2-Mercaptoethanol 1 (2.2 mL, 1 equiv.) and 1-tetradecanethiol (8.7 mL, 1 equiv.) were added to a reaction vessel and dissolved in CH2Cl2: MeOH = 3:1 (total 100 mL, 0.3 M). Then, I2 (8.1 g, 1 equiv.) and pyridine (5.2 mL, 2 equiv.) were added and stirred at room temperature for 12 h. The reaction mixture was distilled under reduced pressure to remove the solvent, and then column chromatography (SiO 2, The precursor compound 2 (5.1 g, 51%) was obtained as a white solid by purification with EtOAc / hexane 1:20 → 1:8.

[0136] Synthesis Example 2: Synthesis of an aliphatic chain tail precursor

[0137] (1) Synthesis of precursor Oxepane-2,7-dione (4b)

[0138]

[0139] Adipic acid 3a (2.5 g, 1 equiv.) and (CH 3 CO ) 2 O (6.5 mL, 4 equiv.) were added to a reaction vessel and heated under reflux for 7 hours. The reaction mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure. The precursor compound 4b was obtained as a white solid through a drying process using a vacuum pump without further purification.

[0140] (2) Synthesis of precursor Oxocane-2,8-dione (4c)

[0141]

[0142] Pimelic acid 3b (2.5 g, 1 equiv.) and (CH3CO)2O (6.5 mL, 4 equiv.) were added to a reaction vessel and heated under reflux for 7 h. The reaction mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure. The precursor compound 4c was obtained as a white solid through a drying process using a vacuum pump without further purification.

[0143] (3) Synthesis of precursor Oxocane-2,9-dione (4d)

[0144]

[0145] Suberic acid 3c (2.5 g, 1 equiv.) and (CH 3 CO ) 2 O (6.5 mL, 4 equiv.) were added to a reaction vessel and heated under reflux for 7 hours. The reaction mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure. The precursor compound 4d was obtained as a white solid through a drying process using a vacuum pump without further purification.

[0146] (4) Synthesis of precursor 5-((2-hexyldecyl)oxy)-5-oxopentanoic acid (5a)

[0147]

[0148] Glutaric anhydride4a (1.88 g, 2 equiv.) and 2-hexyl-1-decanol (2 g, 1 equiv.) were added to a reaction vessel and dissolved in CH2Cl2 (100 mL, 0.08 M). DMAP (2.6 g, 2.5 equiv.) was added and stirred at room temperature for 9 hours. Water (50 mL) was added to the reaction mixture, the temperature was lowered to 4°C, and 6 M HCl aqueous solution was added to acidify. The reaction mixture was then placed in a separatory funnel and extracted with CH2Cl2 (30 mL x 2). The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was then distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:20 → 1:8) to obtain precursor compound 5a (2.2 g, 77%) as a clear oil.

[0149] (5) Synthesis of precursor 5-((2-octyldodecyl)oxy)-5-oxopentanoic acid (5b)

[0150]

[0151] Glutaric anhydride 4a (1.5 g, 2 equiv.) and 2-octyl-1-dodecanol (2 g, 1 equiv.) were added to a reaction vessel and dissolved in CH2Cl2 (84 mL, 0.08 M). DMAP (2 g, 2.5 equiv.) was added and stirred at room temperature for 9 hours. Water (50 mL) was added to the reaction mixture, the temperature was lowered to 4°C, and 6 M HCl aqueous solution was added to acidify. The reaction mixture was then placed in a separatory funnel and extracted with CH2Cl2 (30 mL x 2). The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was then distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:20 → 1:4) to obtain precursor compound 5b (2.0 g, 75%) as a clear oil.

[0152] (6) Synthesis of precursor 6-((2-hexyldecyl)oxy)-6-oxohexanoic acid (5c)

[0153]

[0154] In a reaction vessel, precursor compound 4b (1.4 g, 1.3 equiv.) and 2-hexyl-1-decanol (2 g, 1 equiv.) were added and dissolved in CH2Cl2 (16 mL, 0.5 M). DMAP (2.6 g, 2.5 equiv.) was added and stirred at room temperature for 9 hours. Water (50 mL) was added to the reaction mixture, the temperature was lowered to 4°C, and 6 M HCl aqueous solution was added to acidify. The reaction mixture was then placed in a separatory funnel and extracted with CH2Cl2 (30 mL x 2). The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was then distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:20 → 1:8) to obtain precursor compound 3c (2 g, 66%) as a clear oil.

[0155] (7) Synthesis of precursor 6-((2-octyldodecyl)oxy)-6-oxohexanoic acid (5d)

[0156]

[0157] Precursor compound 4b (560 mg, 1.3 equiv.) and 2-octyl-1-dodecanol (1 g, 1 equiv.) were added to a reaction vessel and dissolved in CH2Cl2 (7 mL, 0.5 M). DMAP (1 g, 2.5 equiv.) was added and stirred at room temperature for 9 hours. Water (20 mL) was added to the reaction mixture, the temperature was lowered to 4°C, and 6 M HCl aqueous solution was added to acidify. The reaction mixture was then placed in a separatory funnel and extracted with CH2Cl2 (20 mL x 2). The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was then distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:20 → 1:8) to obtain precursor compound 5d (980 mg, 69%) as a clear oil.

[0158] (8) Synthesis of precursor 7-((2-hexyldecyl)oxy)-7-oxoheptanoic acid (5e)

[0159]

[0160] In a reaction vessel, precursor compound 4c (760 mg, 1.3 equiv.) and 2-hexyl-1-decanol (1 g, 1 equiv.) were added and dissolved in CH2Cl2 (41 mL, 0.5 M). DMAP (1.3 g, 2.5 equiv.) was added and stirred at room temperature for 9 hours. Water (20 mL) was added to the reaction mixture, the temperature was lowered to 4°C, and 6 M HCl aqueous solution was added to acidify. The reaction mixture was then placed in a separatory funnel and extracted with CH2Cl2 (20 mL x 2). The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was then distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:20 → 1:8) to obtain precursor compound 5e (1.1 g, 71%) as a clear oil.

[0161] (9) Synthesis of precursor 7-((2-octyldodecyl)oxy)-7-oxoheptanoic acid (5f)

[0162]

[0163] Precursor compound 4c (1.3 g, 1.3 equiv.) and 2-octyl-1-dodecanol (2 g, 1 equiv.) were added to a reaction vessel and dissolved in CH2Cl2 (13 mL, 0.5 M). DMAP (2 g, 2.5 equiv.) was added and stirred at room temperature for 9 hours. Water (30 mL) was added to the reaction mixture, the temperature was lowered to 4°C, and 6 M HCl aqueous solution was added to acidify. The reaction mixture was then placed in a separatory funnel and extracted with CH2Cl2 (30 mL x 2). The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was then distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:20 → 1:8) to obtain precursor compound 5f (2.2 g, 67%) as a clear oil.

[0164] (10) Synthesis of precursor 8-oxo-9-(pentadecan-7-yloxy)nonanoic acid (5g)

[0165]

[0166] Precursor compound 4d (1.8 g, 1.5 equiv.) and 2-hexyl-1-decanol (2 g, 1 equiv.) were added to a reaction vessel and dissolved in CH2Cl2 (13 mL, 0.6 M), then DMAP (2.6 g, 2.5 equiv.) was added and stirred at room temperature for 9 hours. Water (20 mL) was added to the reaction mixture, and the temperature was lowered to 4°C. 6 M HCl aqueous solution was added to acidify the mixture. The reaction mixture was then poured into a separatory funnel and poured into EtOAc. (30 mL x 2) was extracted, the separated organic layer was dried over anhydrous Na2SO4, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:20 → 1:10) to obtain 5 g (2.1 g, 70%) of the precursor compound as a clear oil.

[0167] (11) Synthesis of precursor 9-(nonadecan-9-yloxy)-8-oxononanoic acid (5h)

[0168]

[0169] Precursor compound 4d (2.3 g, 1.5 equiv.) and 2-octyl-1-dodecanol (3 g, 1 equiv.) were added to a reaction vessel and dissolved in CH2Cl2 (33 mL, 0.5 M). DMAP (2.9 g, 2.5 equiv.) was added and stirred at room temperature for 9 hours. Water (50 mL) was added to the reaction mixture, the temperature was lowered to 4°C, and 6 M HCl aqueous solution was added to acidify. The reaction mixture was then placed in a separatory funnel and extracted with CH2Cl2 (30 mL x 2). The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:20 → 1:8) to obtain precursor compound 5h (2.2 g, 51%) as a clear oil.

[0170] Synthesis Example 3: Synthesis of Asymmetric Ionized Lipid Precursor

[0171]

[0172] (1) Synthesis of intermediate compound (R)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propanoic acid (7)

[0173] D-pantothenic acid hemicalcium salt6 (5.0 g, 1 equiv.) and (MeO)2C(CH3)3 (42.0 mL, 0.5 mL) were placed in a reaction vessel, and p-TsOH·H2O (4.4 g, 1.1 equiv.) was added while stirring. After the reaction mixture was stirred at room temperature for 17 h, the produced white solid was filtered through celite using hexane as a washing liquid. The filtrate was distilled under reduced pressure and dried using a vacuum pump without additional purification to obtain intermediate compound 7 as a yellow solid.

[0174] (2) Synthesis of intermediate compound 2-(tetradecyldisulfaneyl)ethyl (R)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propanoate (8)

[0175] Compound 7 (2.3 g, 1.3 equiv.), EDC·HCl (1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride, 2.0 g, 1.5 equiv.), and 4-dimethylaminopyridine (DMAP) (167 mg, 20 mol%) were added to a reaction vessel, and the temperature of the solution dissolved in CH2Cl2 (17 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. Compound 2 (2.1 g, 1 equiv.) was added to another reaction vessel, dissolved in CH2Cl2 (10 mL), and slowly added dropwise to the mixture of intermediate compound 7, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (20 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (20 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:8 → 1:2) to obtain intermediate compound 8 (3.5 g, 92%) as a clear oil.

[0176] (3) Synthesis of intermediate compound 2-(tetradecyldisulfaneyl)ethyl (R)-3-(2,4-dihydroxy-3,3-dimethylbutanamido)propanoate (9)

[0177] Intermediate compound 8 (3.4 g, 1 equiv.) was added to a reaction vessel, and the temperature of the solution dissolved in MeCN:H2O = 1:1 (total 10 mL, 0.62 M) was lowered to 4°C, 2,2,2-trifluoroacetic acid (TFA, 5 mL, 0.81 M) was added dropwise, and then stirred at room temperature for 30 minutes. The temperature of the reaction mixture was adjusted to 4 °C by adding compound 7 (2.3 g, 1.3 equiv.), EDC·HCl (1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride, 2.0 g, 1.5 equiv.), and 4-dimethylaminopyridine (DMAP) (167 mg, 20 mol%) to a reaction vessel, dissolving the solution in CH2Cl2 (17 mL, 0.4 M), lowering the temperature to 4 °C, and stirring vigorously at room temperature for 30 minutes under nitrogen gas. In another reaction vessel, add compound 2 (2.1 g, 1 equiv.), dissolve it in CH2Cl2 (10 mL), and slowly add it dropwise to the mixture of intermediate compound 7, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (20 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (20 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:8 → 1:2) to obtain intermediate compound 8 (3.5 g, 92%) as a clear oil.

[0178] (3) Synthesis of intermediate compound 2-(tetradecyldisulfaneyl)ethyl (R)-3-(2,4-dihydroxy-3,3-dimethylbutanamido)propanoate (9)

[0179] Intermediate compound 8 (3.4 g, 1 equiv.) was added to a reaction vessel, and the temperature of the solution dissolved in MeCN:H2O = 1:1 (total 10 mL, 0.62 M) was lowered to 4℃, 2,2,2-trifluoroacetic acid (TFA, 5 mL, 0.81 M) was added dropwise, and the mixture was stirred at room temperature for 30 minutes. After lowering the temperature of the reaction mixture to 4℃, saturated aqueous NaHCO3 solution was added to make it alkaline. After that, the reaction mixture was placed in a separatory funnel and extracted with CH2Cl2 (50 mL x 2), the separated organic layer was dried over anhydrous Na2SO4, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, EtOAc / hexane 1:1 → 8:1) to obtain intermediate compound 9 (2.7 g, 86%) as a clear oil.

[0180] (4) Synthesis of precursor (R)-3-hydroxy-2,2-dimethyl-4-oxo-4-((3-oxo-3-(2-(tetradecyldisulfaneyl)ethoxy)propyl)amino)butyl 3-(dimethylamino)propanoate (11)

[0181] In a reaction vessel, compound 3-(dimethylamino)propanoic acid 10 (743 mg, 1.2 equiv.), EDC·HCl (1.2 g, 1.5 equiv.), and 4-dimethylaminopyridine (DMAP) (99 mg, 20 mol%) were added, and the temperature of the solution dissolved in CH2Cl2 (10 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature for 30 minutes under nitrogen gas. In another reaction vessel, compound 9 (2.05 g, 1 equiv.) was added, dissolved in CH2Cl2 (5 mL), and slowly added dropwise to the mixture of compound 10, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (10 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (10 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, MeOH / CH2Cl2 1:100 → 1:20) to obtain precursor compound 11 (1.9 g, 77%) as a clear oil.

[0182] Example 1: Synthesis of the asymmetric ionizable lipid compound 2-hexyldecyl ((R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl) glutarate (12)

[0183]

[0184] In a reaction vessel, precursor compound 5a (85 mg, 1.5 equiv.), EDCHCl (61 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (4 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.4 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (96 mg, 1 equiv.) was added, dissolved in CH2Cl2 (1 mL), and slowly added dropwise to the mixed solution of precursor compound 5a, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture solution was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture solution was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 12 (131 mg, 88%) as a clear oil.

[0185] 1 H NMR (CDCl3, 300 MHz): δ 7.54 (t,J= 6.0 Hz, 1H), 4.80 (s, 1H), 4.32 (t,J= 6.7 Hz, 2H), 3.99 (d,J= 11.2 Hz, 1H), 3.95 (d,J= 5.8 Hz, 2H), 3.74 (d,J= 11.2 Hz, 1H), 3.51-3.63 (m, 1H), 3.27-3.39 (m, 1H), 2.86 (t,J= 6.7 Hz, 2H), 2.63-2.69 (m, 3H), 2.45-2.59 (m, 7H), 2.37 (t,J= 7.3 Hz, 2H), 2.26 (s, 6H), 1.94 (p,J= 7.2 Hz, 2H), 1.58-1.69 (m, 3H), 1.20-1.34 (m, 46H), 1.03 (d,J= 7.1 Hz, 6H), 0.85 (t,J= 6.6 Hz, 9H).

[0186] Example 2: Synthesis of the asymmetric ionizable lipid compound 2-octyldodecyl ((R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl) glutarate (13)

[0187]

[0188] In a reaction vessel, precursor compound 5b (98 mg, 1.5 equiv.), EDCHCl (61 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (4 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.4 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (96 mg, 1 equiv.) was added, dissolved in CH2Cl2 (1 mL), and slowly added dropwise to the mixed solution of compound 5b, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated aqueous NaHCO3 solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 13 (126 mg, 80%) as a clear oil.

[0189] 1H NMR (CDCl3, 300 MHz): δ 7.54 (t,J= 6.1 Hz, 1H), 4.81 (s, 1H), 4.32 (t,J= 6.7 Hz, 2H), 3.99 (d,J= 11.2 Hz, 1H), 3.95 (d,J= 5.8 Hz, 2H), 3.74 (d,J= 11.2 Hz, 1H), 3.48-3.64 (m, 1H), 3.28-3.39 (m, 1H), 2.86 (t,J= 6.7 Hz, 2H), 2.62-2.73 (m, 3H), 2.43-2.60 (m, 7H), 2.37 (t,J= 7.3 Hz, 2H), 2.26 (s, 6H), 1.94 (p,J= 7.2 Hz, 2H), 1.59-1.69 (m, 3H), 1.20-1.34 (m, 54H), 1.03 (d,J= 7.0 Hz, 6H), 0.85 (t,J= 6.7 Hz, 9H).

[0190] Example 3: Synthesis of the asymmetric ionizable lipid compound 2-hexyldecyl ((R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl) adipate (14)

[0191]

[0192] In a reaction vessel, precursor compound 5c (88 mg, 1.5 equiv.), EDCHCl (61 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (4 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.4 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (96 mg, 1 equiv.) was added, dissolved in CH2Cl2 (1 mL), and slowly added dropwise to the mixed solution of compound 5c, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 14 (121 mg, 80%) as a clear oil.

[0193] 1 H NMR (CDCl3, 300 MHz): δ 7.50 (t,J= 5.9 Hz, 1H), 4.80 (s, 1H), 4.32 (t,J= 6.7 Hz, 2H), 3.99 (d,J= 11.2 Hz, 1H), 3.93 (d,J= 5.8 Hz, 2H), 3.73 (d,J= 11.2 Hz, 1H), 3.51-3.63 (m, 1H), 3.27-3.39 (m, 1H), 2.86 (t,J= 6.7 Hz, 2H), 2.62-2.73 (m, 3H), 2.46-2.59 (m, 5H), 2.39 (dd,J= 8.1, 6.7 Hz, 2H), 2.29 (d,J= 7.4 Hz, 2H), 2.26 (s, 6H), 1.63 (qd,J= 7.5, 3.7 Hz, 7H), 1.22-1.40 (m, 46H), 1.03 (d,J= 6.8 Hz, 6H), 0.85 (t,J= 6.6 Hz, 9H).

[0194] Example 4: Synthesis of the asymmetric ionizable lipid compound 2-octyldodecyl ((R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl) adipate (15)

[0195]

[0196] In a reaction vessel, precursor compound 5d (101 mg, 1.5 equiv.), EDCHCl (61 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (4 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.4 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (96 mg, 1 equiv.) was added, dissolved in CH2Cl2 (1 mL), and slowly added dropwise to the mixed solution of compound 5d, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 15 (141 mg, 88%) as a clear oil.

[0197] 1H NMR (CDCl3, 300 MHz): δ 7.50 (t,J= 5.9 Hz, 1H), 4.80 (s, 1H), 4.31 (t,J= 6.7 Hz, 2H), 3.98 (d,J= 11.2 Hz, 1H), 3.93 (d,J= 5.8 Hz, 2H), 3.73 (d,J= 11.2 Hz, 1H), 3.51-3.63 (m, 1H), 3.27-3.38 (m, 1H), 2.85 (t,J= 6.7 Hz, 2H), 2.61-2.72 (m, 3H), 2.45-2.58 (m, 5H), 2.38 (dd,J= 8.1, 6.7 Hz, 2H), 2.29 (d,J= 7.4 Hz, 2H), 2.25 (s, 6H), 1.56-1.69 (m, 7H), 1.19-1.40 (m, 54H), 1.03 (d,J= 6.9 Hz, 6H), 0.84 (t,J= 6.7 Hz, 9H).

[0198] Example 5: Synthesis of the asymmetric ionizable lipid compound 1-(2-hexyldecyl) 7-((R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl) heptanedioate (16)

[0199]

[0200] Precursor compound 5e (107 mg, 1.5 equiv.), EDC·HCl (72 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (5 mg, 20 mol%) were added to a reaction vessel, and the temperature of the mixed solution dissolved in CH2Cl2 (0.5 mL, 0.4 M) was lowered to 4°C and stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (113 mg, 1 equiv.) was added, dissolved in CH2Cl2 (1 mL), and slowly added dropwise to the mixed solution of compound 5e, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 16 (158 mg, 87%) as a clear oil.

[0201] 1 H NMR (CDCl3, 300 MHz): δ 7.49 (t,J= 5.9 Hz, 1H), 4.81 (s, 1H), 4.32 (t,J= 6.7 Hz, 2H), 3.99 (d,J= 11.2 Hz, 1H), 3.94 (d,J= 5.8 Hz, 2H), 3.74 (d,J= 11.2 Hz, 1H), 3.52-3.64 (m, 1H), 3.27-3.39 (m, 1H), 2.86 (t,J= 6.7 Hz, 2H), 2.64-2.73 (m, 3H), 2.46-2.59 (m, 5H), 2.39 (dd,J= 8.1, 6.8 Hz, 2H), 2.29 (d,J= 7.4 Hz, 2H), 2.26 (s, 6H), 1.57-1.67 (m, 7H), 1.23-1.41 (m, 48H), 1.04 (d,J= 6.6 Hz, 6H), 0.85 (t,J= 6.6 Hz, 9H).

[0202] Example 6: Synthesis of the asymmetric ionizable lipid compound 1-(2-octyldodecyl) 7-((R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl) heptanedioate (17)

[0203]

[0204] In a reaction vessel, precursor compound 5f (123 mg, 1.5 equiv.), EDCHCl (72 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (5 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.5 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (113 mg, 1 equiv.) was added, dissolved in CH2Cl2 (1 mL), and slowly added dropwise to the mixed solution of compound 5f, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 17 (151 mg, 79%) as a clear oil.

[0205] 1H NMR (CDCl3, 300 MHz): δ 7.49 (t,J= 5.9 Hz, 1H), 4.80 (s, 1H), 4.32 (t,J= 6.7 Hz, 2H), 3.99 (d,J= 11.2 Hz, 1H), 3.93 (d,J= 5.8 Hz, 2H), 3.73 (d,J= 11.2 Hz, 1H), 3.51-3.63 (m, 1H), 3.27-3.39 (m, 1H), 2.85 (t,J= 6.7 Hz, 2H), 2.61-2.73 (m, 3H), 2.46-2.59 (m, 5H), 2.38 (t,J= 7.5 Hz, 2H), 2.28 (d,J= 7.4 Hz, 2H), 2.26 (s, 6H), 1.57-1.69 (m, 7H), 1.20-1.40 (m, 56H), 1.03 (d,J= 6.6 Hz, 6H), 0.85 (t,J= 6.7 Hz, 9H).

[0206] Example 7: Synthesis of the asymmetric ionizable lipid compound 1-(2-hexyldecyl) 8-((R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl) octanedioate (18)

[0207]

[0208] In a reaction vessel, 5 g of precursor compound (111 mg, 1.5 equiv.), EDCHCl (72 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (5 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.5 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (113 mg, 1 equiv.) was added, dissolved in CH2Cl2 (1 mL), and slowly added dropwise to the mixed solution of 5 g of compound, and the mixture was stirred at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 18 (171 mg, 95%) as a clear oil.

[0209] 1 H NMR (CDCl3, 300 MHz): δ 7.47 (t,J= 5.9 Hz, 1H), 4.80 (s, 1H), 4.31 (t,J= 6.7 Hz, 2H), 3.98 (d,J= 11.2 Hz, 1H), 3.92 (d,J= 5.8 Hz, 2H), 3.72 (d,J= 11.2 Hz, 1H), 3.50-3.62 (m, 1H), 3.26-3.38 (m, 1H), 2.85 (t,J= 6.7 Hz, 2H), 2.60-2.72 (m, 3H), 2.45-2.58 (m, 5H), 2.39 (t,J= 7.5 Hz, 2H), 2.26 (d,J= 7.4 Hz, 2H), 2.25 (s, 6H), 1.56-1.68 (m, 7H), 1.21-1.34 (m, 50H), 1.02 (d,J= 6.3 Hz, 6H), 0.85 (t,J= 6.1 Hz, 9H).

[0210] Example 8: Synthesis of the asymmetric ionizable lipid compound 1-(2-octyldodecyl) 8-((R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl) octanedioate (19)

[0211]

[0212] In a reaction vessel, precursor compound 5h (127 mg, 1.5 equiv.), EDCHCl (72 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (5 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.5 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (113 mg, 1 equiv.) was added, dissolved in CH2Cl2 (1 mL), and slowly added dropwise to the mixed solution of compound 5h, followed by stirring at room temperature for 7 hours. The reaction was terminated by adding saturated NaHCO3 aqueous solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 19 (171 mg, 95%) as a clear oil.

[0213] 1H NMR (CDCl3, 300 MHz): δ 7.42 (t,J= 5.9 Hz, 1H), 4.81 (s, 1H), 4.31 (t,J= 6.7 Hz, 2H), 3.97 (d,J= 11.2 Hz, 1H), 3.93 (d,J= 5.8 Hz, 2H), 3.75 (d,J= 11.2 Hz, 1H), 3.50-3.60 (m, 1H), 3.28-3.40 (m, 1H), 2.85 (t,J= 6.7 Hz, 2H), 2.61-2.74 (m, 3H), 2.47-2.59 (m, 5H), 2.37 (t,J= 7.6 Hz, 2H), 2.26 (d,J= 7.3 Hz, 2H), 2.26 (s, 6H), 1.56-1.69 (m, 7H), 1.22-1.33 (m, 58H), 1.03 (d,J= 4.9 Hz, 6H), 0.84 (t,J= 6.7 Hz, 9H).

[0214] Example 9: Synthesis of asymmetric ionizable lipid compound (R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl 2-hexyldecanoate (20)

[0215]

[0216] In a reaction vessel, compounds 2-hexyldecanoic acid (122 mg, 1.5 equiv.), EDCHCl (121 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (8 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.8 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (192 mg, 1 equiv.) was added, dissolved in CH2Cl2 (2 mL), and slowly added dropwise to the mixed solution of compound 2-hexyldecanoic acid, and then stirred at room temperature for 7 hours. The reaction was terminated by adding saturated aqueous NaHCO3 solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 1:1) to obtain compound 20 (228 mg, 85%) as a clear oil.

[0217] 1H NMR (CDCl3, 300 MHz): δ 7.42 (t,J= 5.9 Hz, 1H), 4.78 (s, 1H), 4.31 (t,J= 6.8 Hz, 2H), 4.03 (d,J= 11.2 Hz, 1H), 3.69 (d,J= 11.2 Hz, 1H), 3.51 (dq,J= 12.7, 6.4 Hz, 1H), 3.37 (dq,J= 12.6, 6.2 Hz, 1H), 2.85 (t,J= 6.8 Hz, 2H), 2.61-2.73 (m, 3H), 2.46-2.59 (m, 5H), 2.33-2.44 (m, 1H), 2.26 (s, 6H), 1.56-1.66 (m, 4H), 1.40-1.47 (m, 2H), 1.11-1.36 (m, 42H), 1.03 (d,J= 10.8 Hz, 6H), 0.81-0.86 (m, 9H).

[0218] Example 10: Synthesis of the asymmetric ionizable lipid compound (R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl oleate (21)

[0219]

[0220] In a reaction vessel, compounds Oleic acid (134 mg, 1.5 equiv.), EDCHCl (121 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (8 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.8 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (192 mg, 1 equiv.) was added, dissolved in CH2Cl2 (2 mL), and slowly added dropwise to the mixed solution of compound Oleic acid, and then stirred at room temperature for 7 hours. The reaction was terminated by adding saturated aqueous NaHCO3 solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 21 (211 mg, 77%) as a clear oil.

[0221] 1H NMR (CDCl3, 300 MHz): δ 7.45 (t,J= 5.9 Hz, 1H), 5.25-5.37 (m, 2H), 4.82 (s, 1H), 4.32 (t,J= 6.7 Hz, 2H), 3.99 (d,J= 11.2 Hz, 1H), 3.75 (d,J= 11.2 Hz, 1H), 3.57 (dq,J= 12.6, 6.3 Hz, 1H), 3.34 (dq,J= 12.4, 6.3 Hz, 1H), 2.86 (t,J= 6.7 Hz, 2H), 2.61-2.73 (m, 3H), 2.46-2.59 (m, 5H), 2.37 (t,J= 7.6 Hz, 2H), 2.26 (s, 6H), 1.94-2.04 (m, 4H), 1.59-1.69 (m, 4H), 1.23-1.28 (m, 42H), 1.04 (d,J= 4.8 Hz, 6H), 0.85 (d,J=6.7 Hz, 6H).

[0222] Example 11: Synthesis of the asymmetric ionizable lipid compound (R)-2,8,8-trimethyl-5,10,14-trioxo-6,15-dioxa-18,19-dithia-2,11-diazatritriacontan-9-yl (9Z,12Z)-octadeca-9,12-dienoate (22)

[0223]

[0224] In a reaction vessel, compounds Linoleic acid (133 mg, 1.5 equiv.), EDCHCl (121 mg, 2 equiv.), and 4-dimethylaminopyridine (DMAP) (8 mg, 20 mol%) were added, and the temperature of the mixed solution dissolved in CH2Cl2 (0.8 mL, 0.4 M) was lowered to 4°C, and the mixture was stirred vigorously at room temperature under nitrogen gas for 30 minutes. In another reaction vessel, precursor compound 11 (192 mg, 1 equiv.) was added, dissolved in CH2Cl2 (2 mL), and slowly added dropwise to the mixed solution of compound Oleic acid, and the mixture was stirred at room temperature for 7 hours. The reaction was terminated by adding saturated aqueous NaHCO3 solution (5 mL) to the reaction mixture. Afterwards, the reaction mixture was placed in a separatory funnel, extracted with CH2Cl2 (5 mL x 3), and the separated organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by column chromatography (SiO2, 2% Et3N, EtOAc / hexane 1:4 → 2:1) to obtain compound 22 (220 mg, 80%) as a clear oil.

[0225] 1H NMR (CDCl3, 300 MHz): δ 7.45 (t,J= 6.0 Hz, 1H), 5.22-5.38 (m, 4H), 4.79 (s, 1H), 4.30 (t,J= 6.7 Hz, 2H), 3.97 (d,J= 11.2 Hz, 1H), 3.72 (d,J= 11.2 Hz, 1H), 3.57 (dq,J= 12.6, 6.3 Hz, 1H), 3.34 (dq,J= 12.4, 6.3 Hz, 1H), 2.84 (t,J= 6.7 Hz, 2H), 2.72 (t,J= 5.8 Hz, 2H), 2.61-2.68 (m, 3H), 2.44-2.57 (m, 5H), 2.35 (t,J= 7.6 Hz, 2H), 2.24 (s, 6H), 2.00 (q,J= 6.6 Hz, 4H), 1.56-1.67 (m, 4H), 1.15-1.33 (m, 36H), 1.02 (d,J= 5.4 Hz, 6H), 0.80-0.87 (m, 6H).

[0226] Example 12: Production of RNA Platform Nucleic Acid Molecules

[0227] A nucleotide (SEQ ID NO: 8) encoding a therapeutic peptide, human EPO (hEPO) peptide (SEQ ID NO: 7), or a nucleotide (SEQ ID NO: 10) encoding an immunogen, influenza virus Hemagglutinin (SEQ ID NO: 9), was inserted into the multi-cloning site (MCS, SEQ ID NO: 3) of the template DNA having the nucleotide sequence shown below to produce a template DNA into which a target gene was inserted. The template DNA into which each target gene was inserted and modified was cloned into a pGH vector, and RNA platform nucleic acid molecules were produced through the IVT process.

[0228] 5' - KpnⅠ recognition sequence (GGTACC) - T7 promoter (SEQ ID NO: 1) - Upstream translational regulatory element derived from human troponin T1 (TNNT1) (SEQ ID NO: 2) - PacⅠ recognition sequence (TTAATTAA) - Kozak sequence (GCCACC) - MCS (SEQ ID NO: 3) - Coding region (SEQ ID NO: 8 encoding the peptide of SEQ ID NO: 7 or SEQ ID NO: 10 encoding the peptide of SEQ ID NO: 9) - Downstream translational regulatory element derived from human Ribosomal protein (RPS) S27 (SEQ ID NO: 4) - Downstream translational regulatory element derived from human Ferrting light chain (FTL) (SEQ ID NO: 5) - EcoRI recognition sequence (GAATTC) - Poly A tail having a complementary sequence having a NotⅠ recognition sequence (GCGGCCGC) at the 3' end (SEQ ID NO: 6) - 3'

[0229] Hereinafter, the mRNA platform nucleic acid molecule prepared from a template DNA having a target gene (gene of interest) of SEQ ID NO: 8 inserted into the coding region is abbreviated as an hEPO nucleic acid molecule, and the mRNA nucleic acid molecule prepared from a template DNA having a target gene (gene of interest) of SEQ ID NO: 10 inserted is abbreviated as an HA nucleic acid molecule.

[0230] Experimental Example 1: Evaluation of nucleic acid molecule delivery efficiency of a composition including lipid nanoparticles.

[0231] (1) Preparation of a composition including lipid nanoparticles

[0232] Using a laboratory mixer and emulsifier (Enparticle, Encell, Inc), the RNA (hEPO nucleic acid molecule, HA nucleic acid molecule) containing solution (0.625 mg / mL RNA solution, 50 mM sodium citrate buffer, 110 mM NaCl, pH=4.0) prepared in Example 12 and the lipid mixture solution (ethanol solvent) were mixed to prepare lipid nanoparticles. The prepared lipid nanoparticles were solvent-changed to saline or DPBS using a centrifugal filter tube (UFC5010, Amicon), thereby preparing a lipid nanoparticle composition. A lipid nanoparticle composition including lipid nanoparticles and the RNA synthesized in Example 12 was prepared with the contents as shown in Table 1 below.

[0233] Composition of lipid nanoparticle compositions Sample N / Ratio Ionized lipid (mol%) Cholesterol lipid (mol%) Phospholipid (mol%) PEG-lipid (mol%) LNP 16 Compounds 12 (50) Cholesterol (38.5) DSPC (10) DMG-PEG 2K (1.5) LNP 26 Compounds 13 (50) Cholesterol (38.5) DSPC (10) DMG-PEG 2K (1.5) LNP 36 Compounds 14 (50) Cholesterol (38.5) DSPC (10) DMG-PEG 2K (1.5) LNP 46 Compounds 15 (50) Cholesterol (38.5) DSPC (10) DMG-PEG 2K (1.5) LNP 56 Compounds 16(50) Cholesterol(38.5)DSPC(10)DMG-PEG 2K(1.5)LNP 66 compounds 17(50) Cholesterol(38.5)DSPC(10)DMG-PEG 2K(1.5)LNP 76 compounds 18(50) Cholesterol(38.5)DSPC(10)DMG-PEG 2K(1.5)LNP 86 compounds 19(50) Cholesterol(38.5)DSPC(10)DMG-PEG 2K(1.5)LNP 96 compounds 20(50) Cholesterol(38.5)DSPC(10)DMG-PEG 2K(1.5)LNP 106 compounds 21(50) Cholesterol(38.5)DSPC(10)DMG-PEG 2K(1.5)LNP 116 Compound 22(50) Cholesterol(38.5) DSPC(10) DMG-PEG 2K(1.5)

[0234] (2) Evaluation of nucleic acid molecule delivery efficiency

[0235] hEPO mRNA-LNP was injected intramuscularly into the leg muscles of ICR model mice at a concentration of 10 μg / 40 μl (based on RNA), and hEPO expression levels were analyzed. Each experimental group was prepared at a dose of 10 μg / 40 μl (based on RNA) and injected individually into the left leg muscles. The expression levels were then determined considering the optimal expression time of the target protein. Saline injections were used as negative controls (NC). A nucleic acid molecule formulated using Moderna's LNP composition (SM-102 (50 mol%), cholesterol (38.5 mol%), DSPC (10 mol%), DMG-PEG 2k (1.5 mol%)) was used as positive control 1 (PC1). In addition, a nucleic acid molecule formulated in an LNP composition consisting of pantothene-based ionizable lipid (50 mol%), cholesterol (38.5 mol%), DSPC (10 mol%), DMG-PEG 2k (1.5 mol) was used as positive control 2 (PC2).

[0236] For the hEPO gene in the experiment, expression was confirmed at a time point of 6 hours after injection. The specific experimental procedure is as follows. After calibrating the mice, the appropriate drug for the experimental conditions was administered via an insulin syringe. After euthanizing them using CO2 at the appropriate time points, blood was collected via the iliac vein to collect serum. The collected serum was analyzed for hEPO expression using the human EPO ELISA from R&D. The measurement results are shown in Figure 1.

[0237] Experimental Example 2: Measurement of Chemokine Expression in Compositions Containing Ionized Lipids

[0238] Using the Cytokine ELISA kit from Invitrogen, the expression levels of MCP-1 (Monocyte Chemoattractant Protein-1) chemokine and IL-6 (Interleukin-6) cytokine in mouse serum were measured based on serum obtained according to the same procedure as Experimental Example 1. The measurement results are shown in Fig. 2 (MCP-1) and Fig. 3 (IL-6).

[0239] Experimental Example 3: Increased HA-specific antibody immune response by mRNA-LNP

[0240] To investigate the effect of LNP on antibody production, mRNA encoding HA, the surface antigen of influenza virus, was encapsulated into LNP, and 20 μg of mRNA was injected intramuscularly into 6-week-old mice (BALB / c). Two weeks after the first or second immunization, the antibody response was observed through the levels of IgG1 and IgG2a antibodies to HA-specific antibodies in the blood using an ELISA method. The appropriate drug for the experimental conditions was injected via an insulin syringe. Afterwards, blood was collected using a respiratory anesthesia machine on each corresponding date. Serum was separated from the obtained blood and then the experiment was performed.

[0241] Figures 4 to 6 show the results of measuring the amount of HA-specific immunoglobulin secreted in mouse serum two weeks after the first immunization by injecting HA nucleic acid molecules formulated with an LNP composition into mice, respectively. Figures 7 to 9 show the results of measuring the amount of HA-specific immunoglobulin secreted in mouse serum two weeks after the second immunization, respectively.

[0242] Experimental Example 4: Measurement of increased HA-specific neutralizing antibodies by mRNA-LNP

[0243] To investigate the immune response to LNP, we measured the levels of neutralizing antibodies capable of directly neutralizing influenza virus infection. Neutralizing antibody levels were determined using serum collected two weeks after the second immunization, using the same procedure as in Experimental Example 3.

[0244] Specifically, an appropriate amount of Madin-Darby canine kidney (MDCK) cells were cultured in a 96-well plate and infected with a virus (A / Puerto Rico / 8 / 1934 (H1N1)) with the same influenza virus antigen (HA) at a serial dilution of 1 / 10. After 1 to 2 days, when CPE (cytopathic effect) was observed, the cells were fixed with 4% formaldehyde and stained with Crystal violet to observe the cells. The TCID 50 (Tissue Culture Infective Dose 50%) was calculated using the Reed & Muench method, and the obtained serum was inactivated at 56℃ for 30 minutes. Afterwards, 50 ul of serum serially diluted with an appropriate dilution factor and 50 ul of virus (100 TCID 50) were mixed appropriately and reacted at 37℃ for 1 hour, and then infected into a 96-well plate containing an appropriate amount of MDCK cells. After culturing for the same period of time with TCID50, the cells were fixed with 4% formaldehyde and stained with Crystal violet to observe the cells and confirm the neutralizing antibody titer. The measurement results are shown in Figure 10.

[0245] Experimental Example 5: Confirmation of increased HA-specific cytokine expression in spleen cells by mRNA-LNPs against viral antigens through hemagglutination inhibition tests.

[0246] Spleen cells were collected from mice 2 weeks after the second immunization using the same process as in Experimental Example 3, and the expression levels of IFN-γ and TNF-α, cytokines related to the Th1 immune response, and IL-2, cytokine related to the Th2 immune response, were confirmed. 5x10 5The cells were cultured at 37°C for 72 hours with 5 μg of a mixture of eight HA-specific T cell epitope peptides (IYSTVASSL, LYEKVKSQL, DYEELREQL, SFERFEIFPKE, HNTNGVTAACSH, KLKNSYVNKKGK, NAYVSVVTSNYNRRF, CPKYVRSAKLRM). The obtained supernatant was then used to confirm the expression levels of IFN-γ, TNF-α, and IL-2 using a Cytokine ELISA kit from Invitrogen. The measurement results are shown in Figures 11 to 13.

[0247] In summary, when a lipid nanoparticle composition containing an mRNA encoding a target molecule and a lipid compound was used, it was confirmed that the lipid compound stabilized the nucleic acid molecule, thereby stably expressing the target molecule inserted into the nucleic acid molecule. In addition, compared to the positive control, the results of Experimental Example 2, which indicates reactogenicity, showed that IL-6 was measured relatively low, confirming that overall toxicity was reduced and safety was secured. In addition, it was confirmed that both the Th1 immune response, which is a cellular immune response, and the Th2 immune response, which is a humoral immune response, were induced.

[0248] Experimental Example 6: Evaluation of nucleic acid molecule delivery efficiency of a composition containing lipid nanoparticles

[0249] (1) Preparation of a composition including lipid nanoparticles

[0250] Using the same method as Experimental Example 1, a lipid nanoparticle composition was prepared, including lipid nanoparticles in which each lipid content was modified as shown in Table 2 below and the RNA synthesized in Example 12.

[0251] Composition of lipid nanoparticle composition Sample N / Ratio Ionized lipid (mol%) Cholesterol lipid (mol%) Phospholipid (mol%) PEG-lipid (mol%) OPT 16 Compound 12 (25) Cholesterol (62.5) DSPC or DOPE (11) DMG-PEG 2K (1.5) OPT 26 Compound 12 (25) Cholesterol (58.5) DSPC or DOPE (15) DMG-PEG 2K (1.5) OPT 36 Compound 12 (25) Cholesterol (48.5) DSPC or DOPE (25) DMG-PEG 2K (1.5) OPT 46 Compound 12 (25) Cholesterol (40.5) DSPC or DOPE (33) DMG-PEG 2K (1.5) OPT 56 Compound 12(25) Cholesterol(29.5)DSPC or DOPE (44)DMG-PEG 2K(1.5)OPT 66 compounds 12(45) Cholesterol(42.5)DSPC or DOPE (11)DMG-PEG 2K(1.5)OPT 76 compounds 12(45) Cholesterol(40.5)DSPC or DOPE (13)DMG-PEG 2K(1.5)OPT 86 compounds 12(45) Cholesterol(29.5)DSPC or DOPE (24)DMG-PEG 2K(1.5)OPT 96 compounds 12(45) Cholesterol(25)DSPC or DOPE (28.5)DMG-PEG 2K(1.5)OPT 106 compounds 12(45) Cholesterol(20) DSPC or DOPE (33.5) DMG-PEG 2K(1.5)

[0252] (2) Evaluation of nucleic acid molecule delivery efficiency

[0253] The hEPO mRNA-LNP having the composition shown in Table 2 was injected intramuscularly into the leg muscles of ICR model mice using the same method as in Experimental Example 1, and the expression level of hEPO was analyzed. The LNP composition consisting of pantothene-based ionizable lipid (50 mol%), cholesterol (38.5 mol%), DSPC (10 mol%), and DMG-PEG 2k (1.5 mol) was used as a positive control (PC). All measurement results were evaluated as relative values ​​to the positive control (PC). The measurement results are shown in Figs. 14 and 15. When the content of ionizable lipid was 45 mol% with respect to the nucleic acid molecule, the expression of the target molecule was relatively increased.

[0254] Experimental Example 7: Measurement of Chemokine Expression in Compositions Containing Ionized Lipids

[0255] After injecting lipid nanoparticles and mRNA nucleic acid molecules having the compositions shown in Table 2 into mice, the expression levels of MCP-1 chemokine and IL-6 cytokine in mouse serum were measured using the same method as in Experimental Example 2. All measurement results were evaluated as relative values ​​to the positive control (PC). The measurement results are shown in Figures 16 to 19. When the content of compound 12 added as an ionized lipid was 45 mol%, it was confirmed that DSPC induced a relatively stable initial immune response compared to DOPE.

[0256] Experimental Example 8: Increased S-Omicron-specific antibody immune response by mRNA-LNP

[0257] To investigate the effect of LNPs on antibody production, a nucleic acid molecule was synthesized in which a nucleotide sequence (SEQ ID NO: 12) encoding the spike protein (SEQ ID NO: 11), a surface antigen of SARS-CoV2 (Severe acute respiratory syndrome coronavirus 2), was inserted into the target gene (gene of interest) in the coding region. LNPs having a composition of OPT 7 to OPT 9 among the compositions in Table 2 and a nucleic acid molecule encoding the surface antigen of the SARS-CoV2 virus as a target molecule were immunized by intramuscular injection at a dose of 20 μg on an mRNA basis into 6-week-old mice (BALB / c). A nucleic acid molecule formulated in an LNP composition consisting of pantothene-based ionizable lipid (50 mol%), cholesterol (38.5 mol%), DSPC (10 mol%), and DMG-PEG 2k (1.5 mol) was used as a positive control (PC).

[0258] Next, an antibody response was induced using the same method as in Experimental Example 3. The measurement results are shown in Figure 20.

[0259] Experimental Example 9: Hematological / Serological Analysis Induced by mRNA-LNP

[0260] To investigate the effect of LNPs on antibody production, mRNA encoding the Spike protein, a surface antigen of the SARS-CoV-2 virus, was encapsulated in LNPs, and 50 μg of mRNA was injected intramuscularly into 6-week-old ICR mice. Blood was collected 2 days after the first or second immunization. Serum was isolated from the obtained blood or blood was used for experiments. A positive control was used in the same manner as in Experimental Example 8. Figures 21 to 31 are graphs showing the results of blood / serological analysis on the toxicity of the target molecule, the Spike protein, respectively. Compared to the positive control, it was confirmed that toxicity was relatively reduced in the serum of mice immunized with the lipid nanoparticle composition and nucleic acid molecules according to the present invention.

[0261] While the present disclosure has been described above based on exemplary embodiments and examples of the present disclosure, the present disclosure is not limited to the technical concepts described in the embodiments and examples. Rather, those skilled in the art to which the present disclosure pertains can readily devise various modifications and variations based on the aforementioned embodiments and examples. However, it is clear from the appended claims that all such modifications and variations fall within the scope of the present disclosure.

Claims

1. A lipid compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 1] In chemical formula 1, R 1 and R 2 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 alkynyl group; L 1 is linear or branched C1-C 40 Alkylene group, linear or branched C2-C 40 Alkenylene group or linear or branched C2-C 40 It is a divalent aliphatic linking group which is an alkynylene group, and the aliphatic linking group is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a Can have C(=O)O-; R 3 Inland R 5 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle connected to the carbon atom. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a It can have C(=O)O-, and R 3 Inland R 5 At least two of the above are aliphatic substituents, one of the above aliphatic substituents has -SS- in the middle, and the other of the above aliphatic substituents does not have -SS- in the middle. R a is a hydrogen atom or C1-C 15 Alkyl group; k is an integer from 1 to 3.

2. In paragraph 1, The lipid compound is a lipid compound comprising a compound represented by the following chemical formula 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 2] In chemical formula 2, R 1 , R 2 and L 1 are each identical to those defined in Chemical Formula 1; R 6 and R 7 are each independently linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle connected to the carbon atom. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a It can have C(=O)O-, and R 6 and R 7 One of them has -SS- in the middle, R 6 and R 7 One of the others does not have -SS-; R a and k are each the same as defined in chemical formula 1.

3. In paragraph 1, The lipid compound is a lipid compound comprising a compound represented by the following chemical formula 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 3] In chemical formula 3, R 11 and R 12 are each independently a hydrogen atom, linear or branched linear or branched C1-C 20 Alkyl group, linear or branched C2-C 20 Alkenyl group or linear or branched C2-C 20 alkynyl group; L 11 and L 12 are each independently linear or branched C1-C 20 Alkylene group, linear or branched C2-C 20 Alkenylene group or linear or branched C2-C 20 It is a divalent aliphatic linking group, which is an alkynylene group; Y 11 -O(C=O)-, -(C=O)O-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -lim; R 13 Inland R 15 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is connected to a carbon atom at the terminal or middle thereof as -O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -NR a C(=O)- or -C(=O)NR a - can have, R 13 Inland R 15 At least two of the above are aliphatic substituents, one of the above aliphatic substituents has -SS- in the middle, and the other of the above aliphatic substituents does not have -SS- in the middle; R a is the same as defined in chemical formula 1.

4. In paragraph 3, The lipid compound is a lipid compound comprising a compound represented by the following chemical formula 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 4] In chemical formula 4, R 11 , R 12 , L 11 , L 12 and Y 11 are each identical to those defined in chemical formula 3; R 16 and R 17 are each independently linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is connected to a carbon atom at the terminal or middle thereof as -O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -NR a C(=O)- or -C(=O)NR a - can have, R 16 and R 17 One of them has -SS- in the middle, R 6 and R 7 One of the others does not have -SS-; R a is the same as defined in chemical formula 1.

5. In paragraph 1, The lipid compound is a lipid compound comprising a compound represented by the following chemical formula 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 5] In chemical formula 5, R 21 and R 22 are each independently a hydrogen atom, linear or branched linear or branched C1-C 20 Alkyl group; L 21 and L 22 are each independently linear or branched C1-C 20 Alkylene group; Y 21 is -O(C=O)- or -(C=O)O-; Y 22 and Y 23 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -lim; R 23 and R 24 are each independently linear or branched C1-C 40 Alkyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkenyl group, and the aliphatic substituent may have -O(C=O)-, -(C=O)O- or -SS- in the middle, and R 23 and R 24 One of them has -SS- in the middle, R 23 and R 24 One of the others does not have -SS- in the middle; R a is the same as defined in chemical formula 1.

6. In paragraph 5, The lipid compound is a lipid compound comprising a compound represented by the following chemical formula 6, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 6] In chemical formula 6, R 21 , R 22 , L 21 , L 22 and Y 21 are each identical to those defined in chemical formula 5; Y 24 is -O(C=O)-, -(C=O)O-; Y 25 is -NR a C(=O)- or -C(=O)NR a -lim; L 23 is linear or branched C1-C 20 Alkylene group or linear or branched C2-C 20 An aliphatic linking group which is an alkenylene group, and the aliphatic linking group may have -O(C=O)-, -(C=O)O- in the middle; R 25 is linear or branched C1-C 40 Alkyl group or linear or branched C2-C 40 An aliphatic substitution is an alkenyl group, and the aliphatic substitution group may have -O(C=O)-, -(C=O)O- in the middle; R 26 is linear or branched C1-C 20 Alkyl group or linear or branched C2-C 20 An aliphatic substitution is an alkenyl group, and the aliphatic substitution group may have -O(C=O)-, -(C=O)O- in the middle; R a is the same as defined in chemical formula 1.

7. In paragraph 6, R of the above chemical formula 6 21 and R 22 are each independently linear or branched C1-C 10 It is an alkyl group, L 21 and L 22 are each independently linear or branched C1-C 10 It is an alkylene group, L 23 is linear or branched C1-C 20 It is an alkylene group, and the above C1-C 20 The alkylene group can have -O(C=O)-, -(C=O)O- in the middle, R 26 is linear or branched C1-C 20 Alkyl group lipid compound.

8. In paragraph 1, The lipid compound is at least one of a compound represented by the following chemical formula 7, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 7] 9. A lipid nanoparticle composition comprising a lipid compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 1] In chemical formula 1, R 1 and R 2 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 alkynyl group; L 1 is linear or branched C1-C 40 Alkylene group, linear or branched C2-C 40 Alkenylene group or linear or branched C2-C 40 It is a divalent aliphatic linking group which is an alkynylene group, and the aliphatic linking group is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a Can have C(=O)O-; R 3 Inland R 5 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle connected to the carbon atom. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a It can have C(=O)O-, and R 3 Inland R 5 At least two of the above are aliphatic substituents, one of the above aliphatic substituents has -SS- in the middle, and the other of the above aliphatic substituents does not have -SS- in the middle. R a is a hydrogen atom or C1-C 15 Alkyl group; k is an integer from 1 to 3.

10. In paragraph 9, The lipid compound is a lipid nanoparticle composition comprising a compound represented by the following chemical formula 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 2] In chemical formula 2, R 1 , R 2 and L 1 are each identical to those defined in Chemical Formula 1; R 6 and R 7 are each independently linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) at the terminal or middle connected to the carbon atom. k -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - or -NR a It can have C(=O)O-, and R 6 and R 7 One of them has -SS- in the middle, R 6 and R 7 One of the others does not have -SS-; R a and k are each the same as defined in chemical formula 1.

11. In paragraph 9, A lipid nanoparticle composition comprising a compound represented by the following chemical formula 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 3] In chemical formula 3, R 11 and R 12 are each independently a hydrogen atom, linear or branched linear or branched C1-C 20 Alkyl group, linear or branched C2-C 20 Alkenyl group or linear or branched C2-C 20 alkynyl group; L 11 and L 12 are each independently linear or branched C1-C 20 Alkylene group, linear or branched C2-C 20 Alkenylene group or linear or branched C2-C 20 It is a divalent aliphatic linking group, which is an alkynylene group; Y 11 -O(C=O)-, -(C=O)O-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -lim; R 13 Inland R 15 are each independently a hydrogen atom, linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is connected to a carbon atom at the terminal or middle thereof as -O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -NR a C(=O)- or -C(=O)NR a - can have, R 13 Inland R 15 At least two of the above are aliphatic substituents, one of the above aliphatic substituents has -SS- in the middle, and the other of the above aliphatic substituents does not have -SS- in the middle; R a is the same as defined in chemical formula 1.

12. In paragraph 11, A lipid nanoparticle composition comprising a compound represented by the following chemical formula 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 4] In chemical formula 4, R 11 , R 12 , L 11 , L 12 and Y 11 are each identical to those defined in chemical formula 3; R 16 and R 17 are each independently linear or branched C1-C 40 Alkyl group, linear or branched C2-C 40 Alkenyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkynyl group, and the aliphatic substituent is connected to a carbon atom at the terminal or middle thereof as -O(C=O)-, -(C=O)O-, -C(=O)-, -SS-, -NR a C(=O)- or -C(=O)NR a - can have, R 16 and R 17 One of them has -SS- in the middle, R 6 and R 7 One of the others does not have -SS-; R a is the same as defined in chemical formula 1.

13. In paragraph 9, A lipid nanoparticle composition comprising a compound represented by the following chemical formula 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 5] In chemical formula 5, R 21 and R 22 are each independently a hydrogen atom, linear or branched linear or branched C1-C 20 Alkyl group; L 21 and L 22 are each independently linear or branched C1-C 20 Alkylene group; Y 21 is -O(C=O)- or -(C=O)O-; Y 22 and Y 23 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -NR a C(=O)- or -C(=O)NR a -lim; R 23 and R 24 are each independently linear or branched C1-C 40 Alkyl group or linear or branched C2-C 40 An aliphatic substituent which is an alkenyl group, and the aliphatic substituent may have -O(C=O)-, -(C=O)O- or -SS- in the middle, and R 23 and R 24 One of them has -SS- in the middle, R 23 and R 24 One of the others does not have -SS- in the middle; R a is the same as defined in chemical formula 1.

14. In paragraph 13, A lipid nanoparticle composition comprising a compound represented by the following chemical formula 6, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 6] In chemical formula 6, R 21 , R 22 , L 21 , L 22 and Y 21 are each identical to those defined in chemical formula 5; Y 24 is -O(C=O)-, -(C=O)O-; Y 25 is -NR a C(=O)- or -C(=O)NR a -lim; L 23 is linear or branched C1-C 20 Alkylene group or linear or branched C2-C 20 An aliphatic linking group which is an alkenylene group, and the aliphatic linking group may have -O(C=O)-, -(C=O)O- in the middle; R 25 is linear or branched C1-C 40 Alkyl group or linear or branched C2-C 40 An aliphatic substitution is an alkenyl group, and the aliphatic substitution group may have -O(C=O)-, -(C=O)O- in the middle; R 26 is linear or branched C1-C 20 Alkyl group or linear or branched C2-C 20 An aliphatic substitution is an alkenyl group, and the aliphatic substitution group may have -O(C=O)-, -(C=O)O- in the middle; R a is the same as defined in chemical formula 1.

15. In paragraph 14, R of the above chemical formula 6 21 and R 22 are each independently linear or branched C1-C 10 It is an alkyl group, L 21 and L 22 are each independently linear or branched C1-C 10 It is an alkylene group, L 23 is linear or branched C1-C 20 It is an alkylene group, and the above C1-C 20 The alkylene group can have -O(C=O)-, -(C=O)O- in the middle, R 26 is linear or branched C1-C 20 A composition of lipid nanoparticles containing an alkyl group.

16. In paragraph 9, A lipid nanoparticle composition wherein the lipid compound is at least one of a compound represented by the following chemical formula 7, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 7] 17. In paragraph 9, The lipid nanoparticle composition further comprises at least one of a phospholipid, a structure-maintaining lipid, a polyethylene glycol (PEG)-lipid, and an additive.

18. In paragraph 17, The lipid nanoparticle composition further comprises a nucleic acid molecule.

19. In paragraph 18, The nucleic acid molecule comprises a coding region encoding a target molecule, A lipid nanoparticle composition comprising at least one expression regulatory element operably linked to the coding region.

20. In paragraph 19, A lipid nanoparticle composition, wherein the coding region comprises a nucleotide sequence encoding at least one of a reporter peptide, a marker peptide, a selection peptide, a peptide associated with disease treatment or prevention or a fragment thereof, and an immunogen or a fragment thereof.

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