Cationic lipids, lipid nanoparticles comprising the same and methods of delivering nucleic acids

Abstract

A cationic lipid, a lipid nanoparticle containing the cationic lipid, and a method of delivering a nucleic acid encapsulated in the lipid nanoparticle to a cell or a subject.

Description

NDJP.023WO2 PATENT CATIONIC LIPIDS, LIPID NANOPARTICLES COMPRISING THE SAME AND METHODS OF DELIVERING NUCLEIC ACIDS FIELD

[0001] The present disclosure relates to a cationic lipid, a lipid nanoparticle comprising the cationic lipid, and a method of delivering a nucleic acid encapsulated in the lipid nanoparticle to a cell or a subject. BACKGROUND

[0002] Lipid nanoparticles (LNPs) are used as carriers to encapsulate lipophilic drugs and nucleic acids such as siRNA (short interfering RNA) and mRNA to deliver to target cells and organs. For example, lipid nanoparticles comprising cationic lipids as constituent lipids are reported as lipid nanoparticles which serve as carriers to efficiently deliver nucleic acids such as siRNA into target cells.

[0003] U.S. Publication No. U.S. 2013 / 0022665 discloses cationic lipids, such as HEDC, as components of lipid nanoparticles which are useful for delivering nucleic acids to target cells and organs.

[0004] PCT publication No. WO 2013 / 185116 discloses cationic lipids, such as HEDC-M1, as components of lipid nanoparticles which are useful for delivering nucleic acids to target cells and organs.

[0005] There is a continuing need for lipid molecules for efficient delivery of nucleic acids and other agents to target cells and organs. 1NDJP.023WO2 PATENT SUMMARY

[0006] The present disclosure relates to a cationic lipid that is useful for preparing nanoparticles that have favorable transfection property to target cells.

[0007] Specifically, the present disclosure includes following Embodiments [1] to

[0013] .

[0008] Embodiment [1]. A compound of Formula I or pharmaceutically acceptable salt thereof:Formula I wherein: R1and R2are each independently selected from the group consisting of a C10-C20 alkyl and a C10-C20 alkenyl, A1is selected from the group consisting of —NR20C(O)—, —C(O)NR20—, — C(O)O—, —NR20C(O)NR21—, —NR20C(O)O—, and —OC(O)O—, n1 and n2 are each independently 2, 3, 4, or 5, n3 is 1, 2, 3, 4, or 5, B1is selected from the group consisting of:wherein the arrow indicates a bond to A1, wherein n4 is 1, 2, 3, 4, or 5,NDJP.023WO2 PATENT wherein n5 is 0, 1, 2, 3, 4, or 5, wherein B2is a 5- or 6-membered nitrogen-containing saturated aliphatic heterocyclyl or a 5- or 6-membered nitrogen-containing heteroaryl, that is optionally substituted by one or two of a hydroxy or a C1 to C4 alkyl, wherein R11, R12, and R13are each independently a C1 to C4 alkyl, wherein R20and R21are each independently a hydrogen or a C1 to C4 alkyl, and wherein X- is a pharmaceutically acceptable counter anion.

[0009] Embodiment [2]. The compound of Embodiment [1] or pharmaceutically acceptable salt thereof, wherein n3 is 1, 2, or 3.

[0010] Embodiment [3]. The compound of Embodiment [1] or [2] or pharmaceutically acceptable salt thereof, wherein R1and R2are each independently a C12 to C18 alkyl or a C12 to C18 alkenyl.

[0011] Embodiment [4]. The compound of any one of Embodiments [1] to [3] or pharmaceutically acceptable salt thereof, wherein R1and R2are each independently selected from the group consisting of:, wherein the arrow indicates a bond to C(O).

[0012] Embodiment [5]. The compound of any one of Embodiments [1] to [4] or pharmaceutically acceptable salt thereof, wherein B2is selected from the group consisting of:wherein the arrow indicates a bond to the adjacent carbon atom or A1, wherein R14and R15are each independently a hydrogen or a hydroxy, and wherein R16is a C1 to C4 alkyl.NDJP.023WO2 PATENT

[0013] Embodiment [6]. The compound of any one of Embodiments [1] to [5] or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of Compound Nos.1 to 29 shown in the following Table 1-1: Table 1-1NDJP.023WO2 PATENTNDJP.023WO2 PATENTNDJP.023WO2 PATENTNDJP.023WO2 PATENTNDJP.023WO2 PATENTNDJP.023WO2 PATENT

[0014] Embodiment [7]. A lipid nanoparticle comprising the compound of any one of Embodiments [1] to [6] or pharmaceutically acceptable salt thereof.

[0015] Embodiment [8]. The lipid nanoparticle of Embodiment [7], further comprising a sterol, a phospholipid, and a polyalkylene glycol-modified lipid.

[0016] Embodiment [9]. The lipid nanoparticle of Embodiment [8], wherein the phospholipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and wherein the polyalkylene glycol-modified lipid is 1,2-dimyristoyl-rac-glycero- 3-methoxypolyethylene glycol (DMG-PEG).

[0017] Embodiment

[0010] . The lipid nanoparticle of Embodiment [8] or [9], wherein the lipid nanoparticle comprises: 40-60 mol% of the compound of any one of Embodiments [1] to [6] or pharmaceutically acceptable salt thereof to the total lipid amount of the lipid nanoparticle; 30-50 mol% of the sterol to the total lipid amount of the lipid nanoparticle; 5-15 mol% of the phospholipid selected from the group consisting of DSPC, DOPC, and DOPE to the total lipid amount of the lipid nanoparticle; and 1-5 mol% of the polyalkylene glycol-modified lipid that is DMG-PEG to the total lipid amount of the lipid nanoparticle.

[0018] Embodiment

[0011] . The lipid nanoparticle of Embodiment [8], wherein the phospholipid is selected from the group consisting of 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3- phosphorylethanolamine (DSPE), and 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), wherein the polyalkylene glycol-modified lipid is selected from the group consisting of N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE-PEG), N-[carbonyl-methoxypolyethylene glycol]-1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG), and N-[carbonyl- methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), andNDJP.023WO2 PATENT wherein the lipid nanoparticle further comprises a compound of Formula II.

[0019] Embodiment

[0012] . The lipid nanoparticle of Embodiment [8] or

[0011] , wherein the lipid nanoparticle comprises: 20-60 mol% of the compound of Embodiment [1] or pharmaceutically acceptable salt thereof to the total lipid amount of the lipid nanoparticle; 15-50 mol% of the sterol to the total lipid amount of the lipid nanoparticle; 5-40 mol% of the phospholipid selected from the group consisting of DOPE, DSPE, and DPPE to the total lipid amount of the lipid nanoparticle; 0.5-10 mol% of the polyalkylene glycol-modified lipid selected from the group consisting of DPPE-PEG, DMPE-PEG, and DSPE-PEG to the total lipid amount of the lipid nanoparticle, and 0.5-10 mol% of the compound of Formula II to the total lipid amount of the lipid nanoparticle .NDJP.023WO2 PATENT

[0020] Embodiment

[0013] . The lipid nanoparticle of any one of Embodiments [7] to

[0012] , wherein a nucleic acid is encapsulated in the lipid nanoparticle. Embodiment

[0014] . A method of delivering a nucleic acid to a cell, comprising contacting the cell with the lipid nanoparticle of Embodiment

[0013] . Embodiment

[0015] . A method of delivering a nucleic acid to a subject in need thereof, comprising administering to the subject the lipid nanoparticle of Embodiment

[0013] .

[0021] Embodiment

[0016] . A lipid nanoparticle comprising a cationic lipid, a sterol, a phospholipid, and a polyalkylene glycol-modified lipid, wherein the lipid nanoparticle comprises: 20 to 60 mol% of the cationic lipid based on the total lipid content of the lipid nanoparticle; 15 to 50 mol% of the sterol based on the total lipid content of the lipid nanoparticle; 5 to 40 mol% of the phospholipid selected from the group consisting of 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3- phosphorylethanolamine (DSPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), based on the total lipid content of the lipid nanoparticle; 0.5 to 10 mol% of the polyalkylene glycol-modified lipid selected from the group consisting of N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE-PEG), N-[carbonyl-methoxypolyethylene glycol]-1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG), and N-[carbonyl- methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE- PEG), based on the total lipid content of the lipid nanoparticle; and 0.5 to 10 mol% of a compound represented by Formula II, based on the total lipid content of the lipid nanoparticle,NDJP.023WO2 PATENT. Embodiment

[0017] . The lipid nanoparticle of Embodiment

[0016] , wherein the cationic lipid is selected from the group consisting of Compound Nos. 30 to 38 having the structures shown in the following Table 1-2: Table 1-2NDJP.023WO2 PATENTNDJP.023WO2 PATENT.

[0022] Embodiment

[0018] . The lipid nanoparticle of Embodiment

[0016] or

[0017] , wherein the sterol is cholesterol. Embodiment

[0019] . The lipid nanoparticle of any one of Embodiments

[0016] to

[0018] , wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Embodiment

[0020] . The lipid nanoparticle of any one of Embodiments

[0016] to

[0019] , wherein the polyalkylene glycol-modified lipid is N-[carbonyl-methoxypolyethylene glycol]- 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG). Embodiment

[0021] . The lipid nanoparticle of any one of Embodiments

[0016] to

[0020] , wherein the lipid nanoparticle comprises the cationic lipid, cholesterol, DOPE, DPPE- PEG2000, and the compound represented by Formula II in a ratio of 30:30:30:5:5 mol%. Embodiment

[0022] . The lipid nanoparticle of any one of Embodiments

[0016] to

[0021] , wherein a nucleic acid is encapsulated in the lipid nanoparticle. Embodiment

[0023] . A method of delivering a nucleic acid to a cell, comprising contacting the cell with the lipid nanoparticle of Embodiment

[0021] . Embodiment

[0024] . A method of delivering a nucleic acid to a subject in need thereof, comprising administering to the subject the lipid nanoparticle of Embodiment

[0021] . Embodiment

[0025] . A pharmaceutical composition comprising the lipid nanoparticle of any one of Embodiments

[0016] to

[0022] , and a pharmaceutically acceptable carrier.NDJP.023WO2 PATENT Embodiment

[0026] . A cationic lipid selected from the group consisting of Compound Nos.31 to 36 and 38 having the structures shown in Table 1-2. DETAILED DESCRIPTION Compound

[0023] In one aspect, the present disclosure relates to a cationic lipid that is a compound represented by the following general formula I or pharmaceutically acceptable salt thereof (hereinafter may be referred to as “cationic lipids of the present disclosure”):Formula I wherein: R1and R2are each independently selected from the group consisting of a C10-C20 alkyl and a C10-C20 alkenyl, A1is selected from the group consisting of —NR20C(O)—, —C(O)NR20—, — C(O)O—, —NR20C(O)NR21—, —NR20C(O)O—, and —OC(O)O—, n1 and n2 are each independently 2, 3, 4, or 5, n3 is 1, 2, 3, 4, or 5, B1is selected from the group consisting of:wherein the arrow indicates a bond to A1, wherein n4 is 1, 2, 3, 4, or 5,NDJP.023WO2 PATENT wherein n5 is 0, 1, 2, 3, 4, or 5, wherein B2is a 5- or 6-membered nitrogen-containing saturated aliphatic heterocyclyl or a 5- or 6-membered nitrogen-containing heteroaryl, that is optionally substituted by one or two of a hydroxy or a C1 to C4 alkyl, wherein R11, R12, and R13are each independently a C1 to C4 alkyl, wherein R20and R21are each independently a hydrogen or a C1 to C4 alkyl, and wherein X- is a pharmaceutically acceptable counter anion.

[0024] The cationic lipid of the present disclosure comprises, but is not limited to, a compound disclosed in Japanese Patent Application No. 2024-171245 and PCT / JP2025 / 008116, the entire disclosures of which are incorporated herein by reference. As used herein, the term “cationic lipid” includes an ionizable cationic lipid and permanently cationic lipid. As used herein, the term “ionizable cationic lipid” refers to a lipid that comprises an ionizable moiety capable of bearing a positive charge under certain conditions (e.g., at a certain pH range, e.g., under physiological conditions). The ionizable moiety may comprise an amine. In addition to the ionizable moiety, the ionizable cationic lipid may contain an alkyl or alkenyl group. As used herein, the term “permanently cationic lipid” refers to a lipid that comprises a cationic moiety that is positively charged at any pH range. The permanently cationic moiety may comprise a quaternary amine. In addition to the cationic moiety, the permanently cationic lipid may contain an alkyl or alkenyl group.

[0025] As used herein, the term “alkyl” refers to a linear or a branched hydrocarbyl radical of a saturated aliphatic group, which can be of any length unless otherwise specified. The term “C1-C4 alkyl” includes methyl, ethyl, n-propyl, n-butyl, isopropyl, tert-butyl and isobutyl. The term “C10-C20 alkyl” includes decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, ocatdecyl, nonadecyl, icocyl and the branched isomers thereof.

[0026] As used herein, the term “alkenyl” refers to a linear or a branched hydrocarbyl radical having at least one carbon-carbon double bond, which can be of any length unless otherwise specified. The term “C10-C20 alkenyl” includes, but not limited to, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, decadienyl, undecadienyl, dodecadienyl, tridecadienyl, tetradecadienyl, pentadecadienyl, hexadecadienyl, heptadecadiynyl, octadecadienyl, 17NDJP.023WO2 PATENT nonadecadienyl, icosadienyl, decatrienyl, undecatrienyl, dodecatrienyl, tridecatrienyl, tetradecatrienyl, pentadecatrienyl, hexadecatrienyl, heptadecatrienyl, octadecatrienyl, nonadecatrienyl, icosatrienyl and the branched isomers thereof.

[0027] In some embodiments, R1and R2are each independently a C10 to C19 alkyl, C10 to C18 alkyl, C10 to C17 alkyl, C11 to C19 alkyl, C11 to C18 alkyl, C11 to C17 alkyl, C12 to C19 alkyl, C12 to C18 alkyl, C12 to C17 alkyl, C13 to C19 alkyl, C13 to C18 alkyl, C13 to C17 alkyl, C10 to C19 alkenyl, C10 to C18 alkenyl, C10 to C17 alkenyl, C11 to C19 alkenyl, C11 to C18 alkenyl, C11 to C17 alkenyl, C12 to C19 alkenyl, C12 to C18 alkenyl, C12 to C17 alkenyl, C13 to C19 alkenyl, C13 to C18 alkenyl, or C13 to C17 alkenyl.

[0028] In some embodiments, R1and R2are each independently selected from the group consisting of:, wherein the arrow indicates a bond to C(O).

[0029] In one embodiment, R1is the same as R2. In another embodiment, R1is different from R2.

[0030] In some embodiments, A1is—NR20C(O)— or —C(O)NR20— wherein R20is a hydrogen or a C1 to C4 alkyl. In some embodiments, R20is methyl, ethyl, n-propyl or isopropyl. In some embodiments, R20is methyl or ethyl. In some embodiments, R20is methyl.

[0031] In some embodiments, n1 and n2 are each independently 2, 3, or 4.

[0032] In one embodiment, n1 is the same as n2. In another embodiment, n1 is different from n2.

[0033] In some embodiments, n3 is 1, 2, 3, or 4. In some embodiments, n3 is 1, 2, or 3.

[0034] In some embodiments, n4 is 1, 2, 3, or 4.

[0035] In some embodiments, n5 is 0, 1, 2, 3, or 4. In some embodiments, n5 is 0, 1, 2, or 3. In some embodiments, n5 is 0, 1, or 2.NDJP.023WO2 PATENT

[0036] In some embodiments, B2is a 5- or 6-membered nitrogen-containing saturated aliphatic heterocyclyl that is optionally substituted by one or two of a hydroxy or a C1 to C4 alkyl. As used herein, the term “5- or 6-membered nitrogen-containing saturated aliphatic heterocyclyl” refers to 5- or 6-membered saturated aliphatic heterocyclyl having at least one nitrogen atom. The 5- or 6-membered nitrogen-containing saturated aliphatic heterocyclyl includes, but not limited to, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 4-morpholinyl, 4-thiomorpholinyl, and 1-piperazinyl. In some embodiments, B2is a 5- or 6-membered nitrogen-containing saturated aliphatic heterocyclyl selected from the group consisting of:wherein the arrow indicates a bond to the adjacent carbon atom or A1, wherein R14and R15are each independently a hydrogen or a hydroxy, wherein R16and R17are each independently a C1 to C4 alkyl, and wherein X- is a pharmaceutically acceptable counter anion.

[0037] In some embodiments, B2is selected from the group consisting of:wherein the arrow indicates a bond to the adjacent carbon atom or A1, wherein R14and R15are each independently a hydrogen or a hydroxy, and wherein R16is a C1 to C4 alkyl.

[0038] In some embodiments, B2is a 5- or 6-membered nitrogen-containing heteroaryl that is optionally substituted by one or two of a hydroxy or a C1 to C4 alkyl. As used herein, the term “5- or 6-membered nitrogen-containing heteroaryl” refers to 5- or 6- membered aryl having at least one nitrogen atom. The 5- or 6-membered nitrogen-containing heteroaryl includes, but not limited to, pyrrolyl, thiazolyl, oxazolyl, imidazolyl, pyrazolyl,NDJP.023WO2 PATENT isoxazolyl, triazolyl and isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. In some embodiments, B2is, wherein the arrow indicates a bond to the adjacent carbon atom or A1.

[0039] As used herein, the term “pharmaceutically acceptable” refers to being compatible with use in subjects, for example, mammals such as human.

[0040] The pharmaceutically acceptable counter anion of the present disclosure includes, but is not limited to, a chloride, bromide, fluoride, iodide, nitrate, sulfate, methyl sulfate, phosphate, acetate, benzoate, citrate, glutamate, and lactate.

[0041] The pharmaceutically acceptable salt of the present disclosure includes, but not limited to, salts containing a chloride, bromide, fluoride, iodide, nitrate, sulfate, methyl sulfate, phosphate, acetate, benzoate, citrate, glutamate, and / or lactate. The pharmaceutically acceptable salt of the compound of Formula I of the present disclosure can be synthesized by conventional chemical methods. For example, the salt of the compound is prepared either by ion exchange chromatography or by reacting the free base in the compound with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.

[0042] In general, a compound may contain one or more chiral centers. Compounds containing one or more chiral centers may include those described as an “isomer”, a “stereoisomer”, a “diastereomer”, an “enantiomer”, an “optical isomer”, or a “racemic mixture”. Conventions for stereochemical nomenclature, for example the stereoisomer naming rules of Cahn, Ingold and Prelog, as well as methods for the determination of stereochemistry and the separation of stereoisomers are known in the art. See, for example, Michael B. Smith and Jerry March, March's Advanced Organic Chemistry, 5th edition, 2001. The compounds and structures of the present disclosure, including chemical drawings, are meant to encompass all possible isomers, chemically reasonable positional isomers, stereoisomers, diastereomers, enantiomers, and / or optical isomers that would be understood to exist for the specified compound or structure, including any mixture, racemic or otherwise, thereof.

[0043] The lipids represented by general formula I can be easily produced, for example, by the methods specifically shown in Examples herein. By referring to theseNDJP.023WO2 PATENT production methods and appropriately selecting raw material compounds, reagents, and reaction conditions, one skilled in the art can easily produce any lipids included in the range of general formula I. Lipid nanoparticle

[0044] In one aspect, the present disclosure relates to a lipid nanoparticle comprising the cationic lipids of the present disclosure (hereinafter may be referred to as “lipid nanoparticles according to the present disclosure”).

[0045] In the constituent lipids of the lipid nanoparticles according to the present disclosure, lipids which are generally used to form liposomes can generally be used as lipids other than the cationic lipids of the present disclosure. Such lipids include, for example, phospholipids, sterols, glycolipids, or saturated or unsaturated fatty acids, etc. These can be used in one type or a combination of two or more types. In some embodiments, the lipid nanoparticle comprises the cationic lipid of the present disclosure, a phospholipid, a sterol, and a polyalkylene glycol-modified lipid.

[0046] The phospholipids can include glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphorylcholine, cardiolipin, plasmalogen, ceramide phosphorylglycerol phosphate, phosphatidic acid; sphingophospholipids such as sphingomyelin, ceramide phosphorylglycerol, ceramide phosphoryl ethanolamine; etc. In addition, phospholipids derived from natural products such as egg yolk lecithin and soy lecithin can also be used. Fatty acid residues in glycerophospholipids and sphingophospholipids are not particularly limited, but can include, for example, saturated or unsaturated fatty acid residues having carbon number of 12-24, saturated or unsaturated fatty acid residues having carbon number of 14-20 are preferable. Specifically, acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, and lignoceric acid can be included. When these glycerolipids or sphingolipids have two or more fatty acid residues, all fatty acid residues may be the same group or may be different group from each other. Examples of the phospholipids includes diphytanoyl phosphatidyl ethanolamine (DPhPE) and 1,2-Diphytanoyl-sn-Glycero-3- Phosphocholine (DPhPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine 21NDJP.023WO2 PATENT (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn- glycero-3-phosphorylethanolamine (DSPE), and 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE).

[0047] Sterols include, for example, animal-derived sterols such as cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol;plant- -sitosterol, campesterol,brassicasterol; microorganism-derived sterols such as zymosterol and ergosterol, etc. Glycolipids include, for example, glyceroglycolipids such as sulfoxyribosylglyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride; sphingoglycolipids such as galactosylcerebroside, lactosylcerebroside, ganglioside; etc. Saturated or unsaturated fatty acids include, for example, saturated or unsaturated fatty acids having carbon number of 12-20 such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

[0048] The constituent lipids of the lipid nanoparticles according to the present disclosure, in addition to the cationic lipids of the present disclosure, preferably comprise neutral lipid, more preferably comprise phospholipid or sterol, further preferably comprise sterol, and more further preferably comprise cholesterol.

[0049] The lipid nanoparticles according to the present disclosure preferably comprise polyalkylene glycol-modified lipids as a lipid component. Polyalkylene glycol is a hydrophilic polymer, and, by constructing lipid nanoparticles using polyalkylene glycol- modified lipids as lipid membrane constituent lipids, surface of the lipid nanoparticles can be modified with polyalkylene glycol. Surface modification with polyalkylene glycol may be able to enhance the stability such as blood retention of lipid nanoparticles.

[0050] As polyalkylene glycol, for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, etc. can be used. The average molecular weight of polyalkylene glycol is, for example, approximately between 200 and 10,000, preferably approximately between 500 and 10,000, further preferably approximately between 1,000 and 5,000. In some embodiments, the molecular weight of polyalkylene glycol is about 200, 300, 350, 400, 500, 550, 750, 1000, 1500, 2000, 3000, 3500, 4000, 5000 or 10,000 Da. 22NDJP.023WO2 PATENT

[0051] For example, stearylated polyethylene glycol (e.g., PEG-45 stearate (STR- PEG45), etc.) can be used for modification of lipids by polyethylene glycol. Additionally, polyethylene glycol derivatives such as N-[carbonyl-methoxypolyethylene glycol]-1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), N-[carbonyl- methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE- PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), N-[carbonyl- methoxypolyethylene glycol]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE- PEG) can be used. For example, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG2000), n-[carbonyl- methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero- 3-phosphoethanolamine (DSPE-PEG750), N-[carbonyl-methoxypolyethylene glycol-2000]- 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000), N-[carbonyl- methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine(DSPE- PEG5000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-750 (DMG- PEG750),1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DMG- PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE-PEG750), N-[carbonyl-methoxypolyethylene glycol2000]-1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2000), N-[carbonyl- methoxypolyethylene glycol5000]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG5000) etc. can be used.

[0052] In some embodiments, the lipid nanoparticles according to the present disclosure comprises any one of DSPC, DOPC, and DOPE as the phospholipid and DMG-PEG as the polyalkylene glycol-modified lipid. For example, some embodiments of the lipid nanoparticles according to the present disclosure comprise 40-60 (such as 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5 or 60) mol% of the cationic lipids of the present disclosure to the total lipid amount of the lipid nanoparticle; 30- 50 (such as 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48,NDJP.023WO2 PATENT 48.5, 49, 49.5 or 50) mol% of the sterol (such as cholesterol) to the total lipid amount of the lipid nanoparticle; 5-15 (such as 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15) mol% of any one of DSPC, DOPC, and DOPE to the total lipid amount of the lipid nanoparticle; and 1-5 (such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5) mol% of DMG-PEG (such as DMG-PEG2000) to the total lipid amount of the lipid nanoparticle. The molar ratio of the cationic lipids of the present disclosure, the sterol, the phospholipid selected from the group consisting of DSPC, DOPC, and DOPE, and DMG-PEG in the lipid nanoparticle according to the present disclosure (cationic lipid / sterol / phospholipid / DMG-PEG) includes, but not limited to, (60 / 31 / 8 / 1), (60 / 31 / 7.5 / 1.5), (60 / 31 / 7 / 2), (60 / 31 / 6.5 / 2.5), (60 / 31 / 6 / 3), (60 / 31 / 5.5 / 3.5), (60 / 31 / 5 / 4), (60 / 31 / 4.5 / 4.5), (60 / 31 / 4 / 5), (60 / 31.5 / 7.5 / 1), (60 / 30.5 / 7.5 / 2), (60 / 30 / 7.5 / 2.5), (60 / 29.5 / 7.5 / 3), (60 / 29 / 7.5 / 3.5), (60 / 28.5 / 7.5 / 4), (60 / 28 / 7.5 / 4.5), (60 / 27.5 / 7.5 / 5), (59 / 32 / 7.5 / 1.5), (58 / 33 / 7.5 / 1.5), (57 / 34 / 7.5 / 1.5), (56 / 35 / 7.5 / 1.5), (55 / 36 / 7.5 / 1.5), (60 / 29 / 10 / 1), (60 / 28.5 / 10 / 1.5), (60 / 28 / 10 / 2), (60 / 27.5 / 10 / 2.5), (60 / 27 / 10 / 3), (60 / 26.5 / 10 / 3.5), (60 / 26 / 10 / 4), (60 / 25.5 / 10 / 4.5), (60 / 25 / 10 / 5), (60 / 27 / 12 / 1), (60 / 27 / 11.5 / 1.5), (60 / 27 / 11 / 2), (60 / 27 / 10.5 / 2.5), (60 / 27 / 9.5 / 3.5), (60 / 27 / 9 / 4), (60 / 27 / 8.5 / 4.5), (60 / 27 / 8 / 5), (59 / 28 / 10 / 3), (58 / 29 / 10 / 3), (57 / 30 / 10 / 3), (56 / 31 / 10 / 3), (55 / 32 / 10 / 3), (50 / 39 / 10 / 1), (50 / 38.5 / 10 / 1.5), (50 / 38 / 10 / 2), (50 / 37.5 / 10 / 2.5), (50 / 37 / 10 / 3), (50 / 36.5 / 10 / 3.5), (50 / 36 / 10 / 4), (50 / 35.5 / 10 / 4.5), (50 / 35 / 10 / 5), (50 / 38.5 / 10.5 / 1), (50 / 38.5 / 9.5 / 2), (50 / 38.5 / 9 / 2.5), (50 / 38.5 / 8.5 / 3), (50 / 38.5 / 8 / 3.5), (50 / 38.5 / 7.5 / 4), (50 / 38.5 / 7 / 4.5), (50 / 38.5 / 6.5 / 5), (51 / 37.5 / 10 / 1.5), (52 / 36.5 / 10 / 1.5), (53 / 35.5 / 10 / 1.5), (54 / 34.5 / 10 / 1.5), (55 / 33.5 / 10 / 1.5), (49 / 39.5 / 10 / 1.5), (48 / 40.5 / 10 / 1.5), (47 / 41.5 / 10 / 1.5), (46 / 42.5 / 10 / 1.5), (45 / 43.5 / 10 / 1.5), (51 / 34 / 10 / 5), (52 / 33 / 10 / 5), (53 / 32 / 10 / 5), (54 / 31 / 10 / 5), (55 / 30 / 10 / 5), (40 / 44 / 15 / 1), (40 / 43.5 / 15 / 1.5), (40 / 43 / 15 / 2), (40 / 42.5 / 15 / 2.5), (40 / 42 / 15 / 3), (40 / 41.5 / 15 / 3.5), (40 / 41 / 15 / 4), (40 / 40.5 / 15 / 4.5), (40 / 40 / 15 / 5), (41 / 39 / 15 / 5), (42 / 38 / 15 / 5), (43 / 37 / 15 / 5), (44 / 36 / 15 / 5), (45 / 35 / 15 / 5), (41 / 41 / 14 / 4), (42 / 42 / 13 / 3), (43 / 43 / 12 / 2), and (44 / 44 / 11 / 1).

[0053] In some embodiments, the lipid nanoparticles according to the present disclosure comprises a compound of Formula II. The compound of Formula II is also referred to as “Compound A” in the present disclosure. It corresponds to Compound T3 in WO 2019 / 090359, which is hereby expressly incorporated by reference in its entirety.NDJP.023WO2 PATENT

[0054] In some embodiments, the lipid nanoparticles according to the present disclosure comprises any one of DOPE, DSPE and DPPE as the phospholipid, any one of DPPE-PEG, DMPE-PEG and DSPE-PEG as the polyalkylene glycol-modified lipid, and Compound A. For example, some embodiments of the lipid nanoparticles according to the present disclosure comprise 20-60 (such as 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5 or 60) mol% of the cationic lipids of the present disclosure to the total lipid amount of the lipid nanoparticle; 15-50 (such as 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5 or 50) mol% of the sterol (such as cholesterol) to the total lipid amount of the lipid nanoparticle; 5-40 (such as 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40) mol% of any one of DOPE, DSPE and DPPE to the total lipid amount of the lipid nanoparticle; 0.5-10 (such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10) mol% of any one of DPPE-PEG (such as DPPE-PEG2000), DMPE-PEG(such as DMPE-PEG2000) andNDJP.023WO2 PATENT DSPE-PEG (such as DSPE-PEG2000) to the total lipid amount of the lipid nanoparticle; and 0.5-10 (such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10) mol% of Compound A. The molar ratio of the cationic lipids of the present disclosure, the sterol, the phospholipid selected from the group consisting of DOPE, DSPE and DPPE, the polyalkylene glycol-modified lipid selected from the group consisting of DPPE-PEG, DMPE-PEG and DSPE-PEG, and Compound A in the lipid nanoparticle according to the present disclosure (cationic lipid / sterol / phospholipid / glycol-modified lipid / Compound A) includes, but not limited to, (30 / 28 / 30 / 6 / 6), (30 / 29 / 30 / 5.5 / 5.5), (30 / 30 / 30 / 5 / 5), (30 / 31 / 30 / 4.5 / 4.5), (30 / 32 / 30 / 4 / 4), (30 / 33 / 30 / 3.5 / 3.5), (30 / 34 / 30 / 3 / 3), (30 / 35 / 30 / 2.5 / 2.5), (30 / 36 / 30 / 2 / 2), (30 / 37 / 30 / 1.5 / 1.5), (30 / 38 / 30 / 1 / 1), (30 / 30 / 31 / 4.5 / 4.5), (30 / 30 / 32 / 4 / 4), (30 / 30 / 33 / 3.5 / 3.5), (30 / 30 / 34 / 3 / 3), (30 / 30 / 35 / 2.5 / 2.5), (30 / 30 / 36 / 2 / 2), (30 / 30 / 37 / 1.5 / 1.5), (30 / 30 / 38 / 1 / 1), (35 / 25 / 30 / 5 / 5), (34 / 26 / 30 / 5 / 5), (33 / 27 / 30 / 5 / 5), (32 / 28 / 30 / 5 / 5), (31 / 29 / 30 / 5 / 5), (40 / 25 / 25 / 5 / 5), (38 / 26 / 26 / 5 / 5), (36 / 27 / 27 / 5 / 5), (34 / 28 / 28 / 5 / 5), (32 / 29 / 29 / 5 / 5), (50 / 20 / 20 / 5 / 5), (45 / 20 / 25 / 5 / 5), (40 / 25 / 25 / 5 / 5), (35 / 30 / 25 / 5 / 5), (30 / 35 / 25 / 5 / 5), (50 / 21 / 20 / 4.5 / 4.5), (50 / 22 / 20 / 4 / 4), (50 / 23 / 20 / 3.5 / 3.5), (50 / 24 / 20 / 3 / 3), (50 / 25 / 20 / 2.5 / 2.5), (50 / 26 / 20 / 2 / 2), (50 / 27 / 20 / 1.5 / 1.5), (50 / 28 / 20 / 1 / 1), (30 / 50 / 20 / 1 / 1), (30 / 48 / 20 / 2 / 2), (30 / 46 / 20 / 3 / 3), (30 / 44 / 20 / 4 / 4), (30 / 42 / 20 / 5 / 5), (25 / 40 / 25 / 5 / 5), (25 / 40 / 30 / 3 / 2), (25 / 35 / 35 / 3 / 2), (25 / 45 / 25 / 4 / 1), (23 / 35 / 35 / 5 / 2).

[0055] The lipid nanoparticles according to the present disclosure can be subjected to appropriate surface modification, as necessary. The lipid nanoparticles according to the present disclosure can be modified on the surface with hydrophilic polymers, etc. to enhance blood retention. Surface modification may be able to be achieved by using lipids modified with these modifying groups as constituent lipid of the lipid nanoparticles.

[0056] In the production of lipid nanoparticles according to the present disclosure, for example, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, and polyglycerol phospholipid derivatives, etc. can be used as lipid derivatives to enhance blood retention. In addition, dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin and carrageenan, etc., other than polyalkylene glycol, can be used for surface modification, as hydrophilic polymers to enhance blood retention.NDJP.023WO2 PATENT

[0057] In addition, in order to facilitate nuclear translocation of the lipid nanoparticles according to the present disclosure, for example, lipid nanoparticles can be surface-modified with oligosaccharide compounds with three or more saccharides. The type of oligosaccharide compounds with three or more saccharides is not particularly limited, but for example, oligosaccharide compounds in which approximately between 3 and 10 saccharide units are bound can be used, preferably oligosaccharide compounds in which approximately between 3 and 6 saccharide units are bound can be used. Among them, preferably, oligosaccharide compounds with trimer or hexamer of glucose can be used, and, further preferably, oligosaccharide compounds with trimer or tetramer of glucose can be used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, or maltohexaose can be preferably used, among which maltotriose, maltotetraose, maltopentaose, -4 bound glucose are further preferable. Particularly preferred are maltotriose or maltotetraose, and most preferred is maltotriose. Surface modification amount of lipid nanoparticles by oligosaccharide compound is not particularly limited, but, for example, it is approximately between 1 and 30 mol%, preferably approximately between 2 and 20 mol%, and more preferably approximately between 5 and 10 mol% to the total lipid amount.

[0058] The method for surface modifying lipid nanoparticles with oligosaccharide compound is not particularly limited, but, for example, liposomes in which lipid nanoparticles are surface modified with monosaccharides such as galactose and mannose (PCT publication No. WO 2007 / 102481) are known, so the method for the surface modification described in the publication can be employed. The surface modification method described in this publication can be adopted. All of the disclosures in above publication shall be included by reference as the disclosures in the specification of this application.

[0059] In addition, the lipid nanoparticles according to the present disclosure can also be imparted any one or more functions such as temperature change sensitive function, membrane permeability function, gene expression function, and pH-sensitive function. Adding these functions appropriately can improve the retention of lipid nanoparticles in the blood and allow the lipid nanoparticles to efficiently escape from endosomes after endocytosis in target cells.

[0060] The lipid nanoparticles according to the present disclosure may comprise one or more substances selected from the group consisting of anti-oxidizing agents such asNDJP.023WO2 PATENT tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charged substances, and membrane polypeptides, etc. Charged substances which impart positive charges can include, for example, saturated or unsaturated aliphatic amines such as stearylamine and oleylamine, and charged substances which impart negative charges can include, for example, dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, phosphatidic acid, etc. Membrane polypeptides include, for example, membrane extrinsic polypeptide or membrane intrinsic polypeptide, etc. The amount of these substances is not particularly limited and can be appropriately selected according to the purpose.

[0061] The average particle size of the lipid nanoparticles according to the present disclosure is, for example, 400 nm or less, 300 nm or less, 200 nm or less, or 150 nm or less. As used herein, the “average particle size of the lipid nanoparticles” means the number-average particle size measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be carried out by usual method using commercially available DLS equipment, etc.

[0062] The polydispersity index (PDI) of the lipid nanoparticles according to the present disclosure is, for example, approximately between 0.01 and 0.7, preferably approximately between 0.01 and 0.6, further preferably approximately between 0.03 and 0.3. The zeta potential at pH 7.4 can be in the range of -50 mV-5 mV, preferably -45 mV-5 mV.

[0063] The morphology of the lipid nanoparticles according to the present disclosure is not particularly limited, but can include, for example, unilamellar liposome, multilayer liposome, spherical micelle, or unshaped layered structure as morphology dispersed in aqueous solvent. The lipid nanoparticles according to the present disclosure are preferably unilamellar liposome, multilayer liposome.

[0064] The lipid nanoparticles according to the present disclosure preferably encapsulate components for the purpose of being delivered into the target cells inside the particle covered with lipid membranes. The components which the lipid nanoparticles according to the present disclosure encapsulate inside the particles are not limited as long as they are sized available to be encapsulated. The lipid nanoparticles according to the present disclosure can encapsulate any component such as nucleic acids, saccharides, peptides, lowNDJP.023WO2 PATENT molecular weight compounds, and metallic compounds. In some embodiment, the component is an active pharmaceutical ingredient.

[0065] The component encapsulated in the lipid nanoparticles according to the present disclosure is preferably nucleic acid. The nucleic acid may be DNA, or may be RNA, or also may be analogs or derivatives thereof (e.g., peptide nucleic acid (PNA) or phosphorothioate DNA, etc.). The nucleic acids to be encapsulated in the lipid nanoparticles according to the present disclosure may be single-stranded nucleic acids, may be double- stranded nucleic acids, also may be linear, or cyclic.

[0066] In some embodiments, the nucleic acids to be encapsulated in the lipid nanoparticles according to the present disclosure comprise a foreign gene to be expressed in the target cell, preferably they are nucleic acids which functions to express the foreign gene in the cell by being taken up into the cell. The foreign genes may be genes originally comprised in the genomic DNA of the target cells, or they may be genes not comprised in the genomic DNA. Such nucleic acids include gene expression vectors comprising nucleic acids consisting of base sequences encoding genes of interest to be expressed. The gene expression vectors may be present as extrachromosomal genes in the introduced cell, or it may be taken up into the genomic DNA by homologous recombination.

[0067] The gene expression vectors to be encapsulated in the lipid nanoparticles according to the present disclosure are not particularly limited, and vectors generally used in gene therapy, etc. can be used. The gene expression vectors to be encapsulated in the lipid nanoparticles according to the present disclosure are preferably nucleic acid vectors such as plasmid vectors. The plasmid vectors may remain in a circular form or may be encapsulated in the lipid nanoparticles according to the present disclosure in a in a pre-cut linear form. The gene expression vectors can be designed by usual method using commonly used molecular biological tools based on the base sequence information of the gene of the target to be expressed and can be produced by various known methods.

[0068] The nucleic acids to be encapsulated in the lipid nanoparticles according to the present disclosure are also preferably functional nucleic acids which control the expression of target genes present in the target cells. The functional nucleic acids include antisense oligonucleotide, antisense oligonucleotide (including antisense DNA and antisense RNA), siRNA, microRNA(miRNA), and mRNA, etc. Also, they may be plasmid DNA (pDNA)NDJP.023WO2 PATENT becoming siRNA expression vectors which express siRNA in the cells. The siRNA expression vectors can be prepared from commercially available siRNA expression vectors, also which may be appropriately modified. In one embodiment of the present disclosure, the lipid nanoparticles according to the present disclosure comprise cationic lipids of the present disclosure and mRNA.

[0069] As used herein, the “N / P ratio” is the ratio of the number of cationic nitrogen atoms (N) of the cationic lipids of the present disclosure to the number of phosphate residues (P) of the nucleic acids encapsulated in the lipid nanoparticles according to the present disclosure. When the nucleic acids are mRNAs, the N / P ratio may be, for example, in the range of 3.0 to 12.0.

[0070] The production method of lipid nanoparticles according to the present disclosure is not particularly limited, and any method available to those skilled in the art can be adopted. As an example, they can be produced by, after forming a lipid film by dissolving all lipid components in an organic solvent such as chloroform and then drying under reduced pressure by an evaporator or spray drying by a spray dryer, adding components to be encapsulated into the lipid nanoparticles (for example, aqueous solvent comprising nucleic acids, etc.) to dried above mixture, then emulsifying by emulsifier such as homogenizer, ultrasonic emulsifier, or high pressure jet spray emulsifier, etc. They can also be produced by a well-known method for producing liposomes, for example, reversed-phase evaporation method. If the size of the lipid nanoparticles is to be controlled, extrusion (extruding filtration) may be carried out under high pressure using membrane filter with uniform pore size, etc.

[0071] The composition of the aqueous solvents (dispersion media) is not particularly limited, but can include, for example, buffer solutions such as phosphate buffer solution, citrate buffer solution, and phosphate buffered physiological saline, physiological saline, and culture media for cell culture. These aqueous solvents (dispersion media) can stably disperse lipid nanoparticles, but they may furthermore be added saccharides (aqueous solution) such as: monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and meredinose; polysaccharides such as cyclodextrin; sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, maltitol; and polyalcohols (aqueous solution) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, 30NDJP.023WO2 PATENT ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol. In order to stably store the lipid nanoparticles dispersed in this aqueous solvent for a long time, it is desirable to eliminate electrolytes in the aqueous solvent as much as possible in terms of physical stability such as aggregation control, etc. In addition, in terms of chemical stability of the lipids, it is desirable to set the pH of the aqueous solvent between weak acidity and near neutral (approximately between pH 3.0 and 8.0) and / or to remove dissolved oxygen by nitrogen bubbling, etc.

[0072] The lipid nanoparticles according to the present disclosure also can be produced by alcohol dilution method using flow channel. The method is a method for producing lipid nanoparticles by introducing a solution in which lipid components are dissolved in alcohol solvent and a solution in which water-soluble components to be included in lipid nanoparticles are dissolved in aqueous solvent from different flow channels and merging them together. By using microchannel with built-in three-dimensional micromixer which can achieve instantaneous mixing of two liquids, lipid nanoparticles with a diameter of about 30 nm can be produced at high reproducibility (see Leung et al., Journal of Physical Chemistry C Nanomater Interfaces, 2012, vol.116(34), p.18440-18450).

[0073] When obtained aqueous dispersions of lipid nanoparticles is lyophilized or spray dried, the stability may be able to be improved using, for example, saccharide (aqueous solution) such as: monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and meredinose; polysaccharides such as cyclodextrin; sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, maltitol. In addition, when freezing above aqueous dispersions, the stability may be able to be improved using, for example, aforementioned saccharides and polyalcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol.

[0074] In one embodiment of the present disclosure, the lipid nanoparticles according to the present disclosure are lyophilized.NDJP.023WO2 PATENT

[0075] In some embodiments, the lipid nanoparticles according to the present disclosure are synthesized by injecting ethanol solution of lipids into a buffer solution including a nucleic acid in the same manner as described in U.S. Publication No. U.S. 2013 / 0022665, PCT publication No. WO 2019 / 090359, and PCT publication No. WO 2020 / 102668. In some embodiments, the lipid nanoparticles according to the present disclosure are synthesized by combining a lipid solution with a nucleic acid using a microfluidic mixing device such as NanoAssemblrTM(Precision Nano Systems).

[0076] In some embodiments, the lipid nanoparticles according to the present disclosure have excellent stability. The lipid nanoparticles of the invention are, for example, stable for at least 1 week when kept at -80°C.

[0077] An apparent pKa values of the lipid nanoparticles according to the present disclosure is not particularly limited, but can be selected, for example, in the range of approximately between 4.0 and 10.0, preferably approximately between 4.5 and 9.5, if it can be obtained. The pKa values are determined by using 2-(p-toluidino)-6-napthalene sulfonic acid (TNS) (for example, see PCT publication No. WO 2022 / 071582) Method of delivering a nucleic acid to a cell or a subject

[0078] In one aspect, the present disclosure relates to a method of delivering a nucleic acid to a cell, comprising contacting the lipid nanoparticle according to the present disclosure that encapsulates the nucleic acid with the cell. In certain embodiments, the cell is in vitro. In certain embodiments, the cell is in vivo. In certain embodiments, the cell is ex vivo.

[0079] In one aspect, the present disclosure relates to a method of delivering a nucleic acid to a subject in need thereof, comprising administering the lipid nanoparticle according to the present disclosure that encapsulates the nucleic acid to the subject. The subject may be human or non-human animals. The non-human animals include mammals such as cattle, pig, horse, sheep, goat, monkey, dog, cat, rabbit, mouse, rat, hamster, and guinea pig, and birds such as chicken, quail, and duck, etc. The lipid nanoparticle may be administered by any means known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, or airway (aerosol) administration.NDJP.023WO2 PATENT EXAMPLES AbbreviationsEDC·HCl: N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hydrochlorideDMAP: 4-Dimethylaminopyridine DCM: Dichloromethane DIEA: N,N-Diisopropylethylamine MSA: Methane sulfonic acid cis-DHP.HCl: cis-3,4-Dihydroxypyrrolidine hydrochloride HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate MPLC: Medium pressure liquid chromatography DI-H2O: Deionized water DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene RBF: round-bottom flask Example 1: Synthesis of cationic lipids Synthesis of Compounds 1, 2, 3, 6, 7, 8, 24, 25, 26, 27 of Table 1-1

[0080] Compound 1 was prepared according to Scheme 1.Scheme 1

[0081] Synthesis of Intermediate 1 in Scheme 1: A solution of N-Boc diethanol amime (6.4 g, 31.1 mmol) in anhydrous DCM (100 mL) was stirred at room temperature and treated with triethyl amine (17.4 mL, 124 mmol). After 10 min stirring, myristoyl chlorideNDJP.023WO2 PATENT (18.8 mL, 68.5 mmol) was slowly added to the solution. After 30 min, the reaction mixture was transferred to a separating funnel and washed with sodium bicarbonate solution (75 mL), water (50 mL), and brine (70 mL). The organic phase was separated, dried with Na2SO4, and filtered. Solvent was concentrated under reduced pressure, and the crude product was purified by ISCO / MPLC using a silica gel column (220 g). Column was eluted with (0-40)% hexane- ethyl acetate gradient. Fractions were combined and concentrated under reduced pressure using a rotary evaporator to yield Intermediate 1 (15.5 g, 79.5% yield). ESI MS (m / z): 626.77 (M+H)+.

[0082] Synthesis of Intermediate 2 in Scheme 1: Intermediate 1 (5 g, 7.9 mmol) was dissolved in DCM (50 ml) in a 250 ml round bottom flask outfitted with a magnetic stir bar. MSA (2.3 mL, 23.9 mmol) was added to the solution, and the resulting clear / colorless solution was stirred at ambient temperature under a steady stream of Nitrogen (N2) gas. IPC (LCMS) after overnight showed complete deprotection of the Boc protecting group. The reaction mixture was cooled by adding to an ice-bath and then proceeded to add DIEA (8.5 ml, 47.9 mmol) and succinic anhydride (0.99 g, 9.9 mmol). The reaction mixture was stirred overnight while slowly warming to ambient temperature. Next day, IPC (LCMS) showed complete conversion to Intermediate 2. Water (50 mL) was added to the reaction mixture, and pH was adjusted to 3-4 of aqueous layer with saturated citric acid solution. The reaction mixture was transferred to a separating funnel and separated to two layers. The organic layer was washed with saturated brine solution (50 ml). Back extracted aqueous washes with DCM (100 ml), dried with Na2SO4, filtered and concentrated via rotary evaporator to yield crude oil. Precipitation of product was obtained by addition of n-hexanes and placement of material in5 C cold room. The product was filtered via Buchner funnel and dried fully under high vacuumto yield white crystalline solid (4.5 g, 90%). ESI MS (m / z): 626.69 (M+H)+.

[0083] Synthesis of Compound 1: Intermediate 2 (1.00 g, 1.60 mmol), EDC·HCl (383 mg, 2.00 mmol), and DMAP (39 mg, 0.32 mmol) were dissolved in DCM (15 ml) in a scintillation vial (40 ml) equipped with a magnetic stir bar. The resulting solution was stirred at ambient temperature under a blanket of N2 gas. N, N-dimethylenediamine (176 mg, 2.00 mmol) was added, and the reaction continued overnight. Next day, IPC (LCMS) showed that the reaction was complete conversion. The reaction mixture was washed with DI-H2O (40 ml). The organic layer (DCM) was concentrated via rotary evaporator, and the solvent wasNDJP.023WO2 PATENTexchanged to n-hexanes (40-50 ml). The material was placed in 5 C cold room overnightwhere the product material precipitated out. The precipitate was filtered via Buchner funnel and dried fully under high vacuum to yield Compound 1 as solid (695 mg, 64 % yield). ESI MS (m / z): 697.70 (M+H)+.

[0084] Synthesis of Compound 2: Intermediate 2 in Scheme 1 (1.00 g, 1.60 mmol), EDC·HCl (383 mg, 2.00 mmol), and DMAP (39 mg, 0.32 mmol) were dissolved in DCM (15 ml) in a scintillation vial (40 ml) equipped with a magnetic stir bar. The resulting solution was stirred at ambient temperature under a blanket of N2gas. 1-(2-aminoethyl) piperidine (256 mg, 2.00 mmol) was added to the solution, and the reaction continued overnight. Next day, IPC (LCMS) showed that the reaction was complete conversion. The reaction mixture was washed with DI-H2O (40 ml). The organic layer (DCM) was concentrated via rotary evaporator, and the solvent was exchanged to n-hexanes (40-50 ml).The material was placed in 5 C cold room overnight where the product material precipitatedout. The precipitate was filtered via Buchner funnel and dried fully under high vacuum to yield Compound 2 as solid (902 mg, 76 % yield). ESI MS (m / z): 736.73 (M+H)+.

[0085] Intermediate 3 for the synthesis of Compound 3 was prepared according to Scheme 2.Scheme 2

[0086] cis-DHP.HCl (15.08 g, 108 mmol) was suspended in pyridine (150 ml) in a round bottom flask equipped with a magnetic stir bar. The mixture was then cooled with an ice-bath and TIPDSi-Cl2(36.3 ml, 113 mmol) was added. After the TIPDSi-Cl2addition, the ice bath was removed and the reaction was allowed to stir overnight with a blanket of N2gas. Next day, IPC (LCMS & TLC) showed complete transformation. Partitioned material between EtOAc (600 ml) and H2O (600 ml). The organics were collected, washed with sat. brine (600 ml), dried with Na2SO4, filtered and concentrated via rotary evaporator. The collected brown residue crude was then diluted with hexane (500 ml), cooled in ice-bath and filtered through a Buchner funnel, rinsing with cool hexane (25ml) to yield an off-white precipitate (~10 g). ForNDJP.023WO2 PATENT the remaining product material trapped in the mother liquor: concentrated via rotary evaporator to yield brown oil (33.4 g), purified by ISCO MPLC using multiple runs (120 g & 220 g Si columns, 65 g SLC, (0-15)% EtOAc / IPA gradients. Pooled fractions and concentrated via rotary evaporator to yield TIPDSi-cDHP as white solid (12 g). Dried fully under high vacuum and verified by LCMS.

[0087] TIPDSi-cDHP (2.31 g, 6.69 mmol) and N-Boc-2-bromoethanol-amine (1.50 g, 6.69 mmol) were dissolved in DCM (50 ml) and Et3N (1.17 ml, 8.37 mmol) was added. The reaction mixture was stirred overnight at ambient temperature under a blanket of N2gas. Next day, IPC (LCMS) showed that the reaction was complete. The organics were washed with H2O (75 ml), dried with Na2SO4, and concentrated via rotary evaporator to yield crude product. The crude product was purified by ISCO MPLC using 40 g Si-column, 25 g SLC, and (0-100)% Hex / EtOAc gradient. Fractions were pooled and concentrated via rotary evaporator, dried fully via high-vacuum to yield desired Boc-protected intermediate (1.59 g). Boc-removal achieved by dissolving intermediate in DCM (50 ml) and adding MSA (676 μl, 20.1 mmol). The resulting solution was stirred at ambient temperature under a blanket of N2gas for about 4 hours; then IPC (LCMS) showed that the reaction was complete. The reaction mixture was washed with sat. bicarb (100 ml), and pH of aqueous layer was checked; it was still basic. The reaction mixture was dried with Na2SO4, filtered, and concentrated via rotary evaporator to yield desired crude product (1.41 g, 54 % yield) that was carried forward without any further refinement.

[0088] Synthesis of Compound 3: Intermediate 2 in Scheme 1 (1.82 g, 2.90 mmol), EDC·HCl (834 mg, 4.35 mmol), DIEA (3.54 ml, 20.3 mmol), and DMAP (71 mg, 0.58 mmol) were dissolved in DCM (55 ml) in a round bottom flask equipped with a magnetic stir bar. The resulting solution was stirred at ambient temperature under a blanket of N2gas. Intermediate 3 (1.41 g, 3.62 mmol) was added, and the reaction continued overnight. Next day, IPC (LCMS) showed that the reaction was complete conversion. The reaction mixture was washed with DI-H2O (100 ml). The organic layer (DCM) was concentrated via rotary evaporator. The crude product was purified by ISCO MPLC using 80 g Si-column, 32 g SLC and (0-100)% Hex / EtOAc (0-10)% DCM / MeOH gradients. Fractions were pooled and concentrated via rotary evaporator to yield TIPDSi-protected product (1.16 g). Material was dissolved in EtOAc (75 ml) and TBAF (380 mg) added. IPC (LCMS) showed that the 36NDJP.023WO2 PATENT deprotection reaction was complete in 90 minutes. The reaction mixture was washed with H2O (75 ml), dried with Na2SO4, filtered, and concentrated via rotary evaporator to yield product crude material. The crude product was purified by ISCO MPLC using 80 g Si column, 32 g SLC and (0-100) % Hex / EtOAc (0-20)% DCM / MeOH gradients. Fractions were pooled and concentrated via rotary evaporator to yield Compound 3 (507 mg, 58 % yield). ESI MS (m / z): 753.59 (M+H)+.

[0089] Synthesis of Compound 6: Intermediate 2 in Scheme 1 (500 mg, 0.79 mmol, 1eq) was treated with N,N’-dipropyl amino ethyl amine (138 mg, 0.95 mmol, 1.2 eq) according to the same procedure as described in the synthesis of Compound 1 to yield Compound 6 as solid (417 mg, 69% yield). ESI MS (m / z): 752.75 (M+H)+.

[0090] Synthesis of Compound 7: Intermediate 2 in Scheme 1 (500 mg, 0.79 mmol, 1eq) was treated with N, N’-dimethyl amino propylamine (97 mg, 1.42 mmol, 1.2 eq) according to the same procedure as described in the synthesis of Compound 1 to yield Compound 7 as solid (154 mg, 27% yield). ESI MS (m / z): 710.68 (M+H)+.

[0091] Synthesis of Compound 8: Intermediate 2 in Scheme 1 (500 mg, 0.79 mmol, 1eq) was treated with N, N’-dimethyl amino butylamine (111 mg, 1.42 mmol, 1.2 eq) according to the same procedure as described in the synthesis of Compound 1 to yield Compound 8 as solid (178 mg, 30% yield). ESI MS (m / z): 724.71 (M+H)+.

[0092] Synthesis of Compound 24: Intermediate 2 in Scheme 1 (400 mg, 0.63 mmol, 1eq) was treated with N-methyl 4-aminopiperidine (109 mg, 0.95 mmol, 1.5 eq) according to the same procedure as described in the synthesis of Compound 1 to yield Compound 24 as solid (308 mg, 68% yield). ESI MS (m / z): 722.7 (M+H)+.

[0093] Synthesis of Compound 25: Intermediate 2 in Scheme 1 (400 mg, 0.63 mmol, 1eq) was treated with N-methyl 4-aminopiperidine (122 mg, 0.95 mmol, 1.5 eq) according to the same procedure as described in the synthesis of Compound 1 to yield Compound 25 as solid (315 mg, 67% yield). ESI MS (m / z): 736.7 (M+H)+.

[0094] Synthesis of Compound 26: Intermediate 2 in Scheme 1 (400 mg, 0.63 mmol, 1eq) was treated with N1, N1, N2-trimethylethane-1,2-diamine (97 mg, 0.95 mmol, 1.5 eq) according to the same procedure as described in the synthesis of Compound 1 to yield Compound 26 as solid (290 mg, 64% yield). ESI MS (m / z): 736.7 (M+H)+.NDJP.023WO2 PATENT

[0095] Synthesis of Compound 27: Intermediate 2 in Scheme 1 (400 mg, 0.63 mmol, 1eq) was treated with 4-hydroxy-1-methylpiperidine (109 mg, 0.95 mmol, 1.5 eq) according to the same procedure as described in the synthesis of Compound 1 to yield Compound 27 as solid (47 mg, 10% yield). ESI MS (m / z): 723.7 (M+H)+. Synthesis of Compound 4 of Table 1-1

[0096] Compound 4 was prepared according to Scheme 3.Scheme 3

[0097] Synthesis of Intermediate 2 in Scheme 3: Intermediate 1 in Scheme 3 was prepared according to the same procedure as described in PCT publication No. WO 2013 / 185116. Intermediate 2 in Scheme 3 was prepared according to the same procedure as described in the synthesis of Intermediate 2 in Scheme 1.

[0098] Synthesis of Compound 4: Compound 4 was prepared according to the same procedure as described in the synthesis of Compound 1 in Scheme 1, except for using 1- (2-aminoethyl) pyrrolidine instead of N, N-dimethylenediamine. Compound 4 was obtained as oil. ESI MS (m / z): 825.66 (M+H)+. Synthesis of Compounds 5, 13, 14 of Table 1-1

[0099] Compound 13 was prepared according to Scheme 4.NDJP.023WO2 PATENTScheme 4

[0100] Synthesis of Intermediate 2 in Scheme 4: Intermediate 1 in Scheme 4 was prepared according to the same procedure as described in PCT publication No. WO 2019 / 090359. Intermediate 2 in Scheme 4 was prepared according to the same procedure as described in the synthesis of Intermediate 2 in Scheme 1.

[0101] Synthesis of Compound 13: Intermediate 2 in Scheme 4 (400 mg, 0.58 mmol, 1 eq) was dissolved in anhydrous DCM (10 mL). N, N’-dimethyl amino ethyl amine (51 mg, 0.58 mmol, 1.0 eq), triethyl amine (163 , 1.17 mmol, 2 eq), and HATU (245 mg, 0.63 mmol, 1.1eq) were added to the reaction mixture. After overnight stirring at room temperature, LC / MS confirmed that the reaction was complete. The reaction mixture was diluted with DCM then washed with sat. aqueous sodium bicarbonate (15 mL), water (15 mL), and brine (15 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (40 g). Column was eluted with (0-100)% Hexane-Ethyl acetate and (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 13 as solid (280 mg, 64% yield). ESI MS (m / z): 752.83 (M+H)+.

[0102] Synthesis of Compound 14: Intermediate 2 in Scheme 4 (400 mg, 0.58 mmol, 1eq) was treated with N, N’-dimethyl amino propyl amine (84 mg, 0.58 mmol, 1.0 eq) according to the same procedure as described in the synthesis of Compound 13 to yield Compound 14 as solid (180 mg, 22% yield). ESI MS (m / z): 766.85 (M+H)+.

[0103] Synthesis of Compound 5: Compound 5 was prepared according to the same procedure as described in the synthesis of Compound 13 in Scheme 4, except for usingNDJP.023WO2 PATENT 1-(2-aminoethyl) pyrrolidine instead of N, N-dimethylenediamine. Compound 5 was obtained as colorless oil. ESI MS (m / z): 778.64 (M+H)+. Synthesis of Compounds 9 and 10 of Table 1-1

[0104] Compound 9 was prepared according to Scheme 5.Scheme 5

[0105] Synthesis of Intermediate 2 in Scheme 5: Intermediate 1 in Scheme 5 is the same as Intermediate 1 in Scheme 1. Deprotection of the Boc protecting group of Intermediate 1 was carried out using MSA according to the same procedure as described in the synthesis of Intermediate 2 in Scheme 1.

[0106] Triethyl amine (2 mL, 14.5 mmol) was added to a suspension of the MSA salt of the deprotected product (3g, 4.8 mmol, 1.0 eq) in DCM (40 ml), followed by addition of EDC·HCl (1.3g, 6.7 mmol), cat DMAP and N-Boc amino alanine (1.3g, 7.2 mmol) at room temperature. After overnight stirring, LC / MS confirmed formation of the product. The reaction mixture was washed with sat aqueous NaHCO3 (20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (80 g). Column was eluted with (0-60)% Hexane-Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 2 as solid (1.9g, 57% yield). ESI MS (m / z): 697.70 (M+H)+

[0107] Synthesis of Compound 9: MSA (93 , 1.42 mmol, 2.0 eq) was added to a solution of Intermediate 2 in Scheme 5 (500 mg, 0.71 mmol, 1eq) in DCM (15 ml) under N2 gas. The clear solution was allowed to stir overnight at room temperature. Next day, LC / MS confirmed complete cleavage of Boc group. The reaction flask was cooled with an ice-bath,NDJP.023WO2 PATENT then N, N’ dimethyl amino propionic acid (105 mg, 0.89, 1.25 eq), EDC·HCl (205 mg, 1.07 mmol, 1.5eq.), DIEA (0.63 mL, 3.5 mmol, 5eq.), and DMAP (17 mg) were sequentially added to the reaction mixture. After 15 min, the ice-bath was removed, and the reaction was stirred overnight. LC / MS confirmed formation of product. The reaction mixture was diluted with DCM (20 mL). The organic phase was washed with sat. aqueous NaHCO3 (20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (40 g). Column was eluted with (0-100)% Hexane-ethyl acetate and (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 9 as solid (200 mg, 40% Yield). ESI MS (m / z): 696.7 (M+H)+.

[0108] Synthesis of Compound 10: Intermediate 2 in Scheme 5 (500 mg, 0.71 mmol, 1eq) was treated with N, N’ dimethyl amino butanoic acid (117 mg, 0.89, 1.25 eq.) according to the same procedure as described in the synthesis of Compound 9 to yield Compound 10 as solid (158 mg, 31% yield). ESI MS (m / z): 710.72 (M+H)+Synthesis of Compounds 11 and 12 of Table 1-1

[0109] Compound 11 was prepared according to Scheme 6.Scheme 6

[0110] Synthesis of Intermediate 2 in Scheme 6: Intermediate 1 in Scheme 6 is the same as Intermediate 1 in Scheme 1. Deprotection of the Boc protecting group of Intermediate 1 was carried out using MSA according to the same procedure as described in the synthesis of Intermediate 2 in Scheme 1.

[0111] Triethyl amine (2 mL, 14.5 mmol) was added to a suspension of the MSA salt of the deprotected product (3g, 4.8 mmol, 1.0 eq) in DCM (40 ml), followed by addition of EDC·HCl (1.3g, 6.7 mmol), cat DMAP, and N-Boc amino butanoic acid (1.4g, 7.2 mmol)NDJP.023WO2 PATENT at room temperature. After overnight stirring, LC / MS confirmed formation of the product. The organic layer was washed with sat aqueous NaHCO3(20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (80 g). Column was eluted with (0-60)% Hexane-Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 2 as solid (2.1g, 62% yield). ESI MS (m / z): 711.72 (M+H)+.

[0112] Synthesis of Compound 11: Methane sulfonic acid (MSA, 91 , 1.4 mmol, 2.0 eq) was added to a solution of Intermediate 2 in Scheme 6 (500 mg, 0.71 mmol, 1eq) in DCM (10 ml) under N2gas. The clear solution was stirred overnight at room temperature. Next day, LC / MS confirmed complete cleavage of Boc group. The reaction flask was cooled with an ice-bath, then N,N’ dimethyl amino propionic acid (102 mg, 0.87, 1.25 eq.), EDC·HCl (201 mg, 1.05 mmol, 1.5eq.), DIEA (0.63 mL, 3.5 mmol, 5eq.), and DMAP (17 mg) were sequentially added to the reaction mixture. After 15 min, the ice-bath was removed, and the reaction was stirred overnight. LC / MS confirmed formation of product. The reaction mixture was diluted with DCM (20 mL), and washed with sat aqueous NaHCO3(20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (40 g). Column was eluted with (0-100)% Hexane-Ethyl acetate and (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 11 as solid (196 mg, 39% yield). ESI MS (m / z): 710.76 (M+H)+.

[0113] Synthesis of Compound 12: Intermediate 2 in Scheme 6 (500 mg, 0.71 mmol, 1eq) was treated with N, N’ dimethyl amino glycine HCl (120 mg, 0.87, 1.25 eq.) according to the same procedure as described in the synthesis of Compound 11 to yield Compound 12 as solid (60 mg, 12% yield). ESI MS (m / z): 696.70 (M+H)+.NDJP.023WO2 PATENT Synthesis of Compounds 15, 16, 17, and 18 of Table 1-1

[0114] Compound 15 was prepared according to Scheme 7.Intermediate 3 Scheme 7

[0115] Synthesis of Intermediate 1 in Scheme 7: Oleic acid (4.1 g, 20 mmol) was dissolved in anhydrous DCM (200 mL). EDC·HCl (7.85 g, 41 mmol) and DMAP (0.05 g, 0.038 mmol) were added. The reaction mixture was stirred at 0 °C for 30 minutes, then stirred at room temperature overnight. The next day, LC / MS showed that the reaction was complete. Solvent was evaporated under reduced pressure via rotary evaporator, and the resulting residue was suspended in ethyl acetate (150 mL). The organic layer was washed with saturated solution NaHCO3 (100 mL), water (100 mL), and brine solution (50 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated under reduced pressure using an evaporator, and crude product was purified by flash chromatography using a silica column (120 g). Column was eluted with a (0-10) % Hexane – ethyl acetate gradient. Fractions were concentrated and dried under high vacuum to yield Intermediate 1 (9.98 g, 68.0 yield).

[0116] Synthesis of Intermediate 2 in Scheme 7: To a cold solution of intermediate 1 in Scheme 7 (5.17 g, 7 mmol) in an anhydrous DCM at 0°C, methane sulfonic acid (2.48 g, 21 mmol) was added. The reaction mixture was stirred at 0°C for 1 hour. LC / MS showed that the reaction was completed. The reaction was quenched with saturated Na2CO3 (100 mL). The crude product was extracted with DCM three times (150 mL). The combined organic phase was dried with Na2SO4and filtered. Solvent was concentrated under reduced pressure using an evaporator to afford intermediate 2 crude product (4.44 g, 100% yield).

[0117] Synthesis of Intermediate 3 in Scheme 7: To a solution of Intermediate 2 in Scheme 7 (4.44 g, 7 mmol) in anhydrous DCM (75 mL), triethylamine (1.22 mL, 8.7 mmol)NDJP.023WO2 PATENT and succinic anhydride (1.04 g) were added. The reaction mixture was stirred at room temperature for four hours. LC / MS showed that the reaction was completed. The reaction was quenched with 5% citric acid (100 mL). Crude product was extracted with DCM three times (150 mL). The combined organic phase was dried with Na2SO4 and filtered. Solvent was concentrated under reduced pressure using an evaporator, and crude product was purified by flash chromatography using a silica column (120 g). Column was eluted with a (0-20) % hexane – ethyl acetate gradient. Fractions were concentrated and dried under high vacuum to yield Intermediate 3 (4.71 g, 64.2 % yield). ESI MS (m / z) 734.73 (M+ H)+.

[0118] Synthesis of Compound 15: Intermediate 3 in Scheme 7 (0.55 g, 0.75 mmol) was dissolved in anhydrous DCM (10 mL). EDC·HCl (0.15 g, 0.77 mmol) and DMAP (0.05 g, 0.038 mmol) were added to the solution. N, N-dimethylethylenediamine (0.068 g, 0.77 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. The next day, LC / MS showed that the reaction was complete. Solvent was evaporated under reduced pressure via rotary evaporator, and the resulting residue was suspended in ethyl acetate (60 mL). The organic layer was washed with water (50 mL) and brine solution (50 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated under reduced pressure using an evaporator, and crude product was purified by flash chromatography using a silica column (40 g). Column was eluted with a (0-5) % DCM – methanol gradient. Fractions were concentrated and dried under high vacuum to yield Compound 15 (0.485 g, 60.0 % yield). ESI MS (m / z) 804.86 (M+ H)+.

[0119] Synthesis of Compound 16: Intermediate 3 in Scheme 7 (0.48 g, 0.65 mmol) was dissolved in anhydrous DCM (10 mL). EDC·HCl (0.13 g, 0.68 mmol) and DMAP (0.04 g, 0.033 mmol) were added. N, N-dimethyl-1,3-propanediamine (0.066 g, 0.68 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. The next day, LC / MS showed that the reaction was complete. Solvent was evaporated under reduced pressure via rotary evaporator, and the resulting residue was suspended in ethyl acetate (50 mL). The organic layer was washed with water (30 mL) and brine solution (30 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated under reduced pressure using an evaporator, and crude product was purified by flash chromatography using a silica column (40 g). Column was eluted with a (0-5) % DCM –NDJP.023WO2 PATENT methanol gradient. Fractions were concentrated and dried under high vacuum to yield Compound 16 (0.30 g, 37.0 % yield). ESI MS (m / z) 818.91 (M+ H)+.

[0120] Synthesis of Compound 17: Intermediate 3 in Scheme 7 (0.48 g, 0.65 mmol) was dissolved in anhydrous DCM (10 mL). EDC·HCl (0.13 g, 0.68 mmol) and DMAP (0.04 g, 0.033 mmol) were added. N, N-dimethyl 1,4-butanediamine (0.079 g, 0.68 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. The next day, LC / MS showed that the reaction was complete. Solvent was evaporated under reduced pressure via rotary evaporator, and the resulting residue was suspended in ethyl acetate (50 mL). The organic layer was washed with water (30 mL) and brine solution (30 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated under reduced pressure using an evaporator, and crude product was purified by flash chromatography using a silica column (40 g). Column was eluted with a (0-5) % DCM – methanol gradient. Fractions were concentrated and dried under high vacuum to yield Compound 17 (0.30 g, 37.0 % yield). ESI MS (m / z) 832.93 (M+ H)+.

[0121] Synthesis of Compound 18: Intermediate 3 in Scheme 7 (158 mg, 0.25 mmol) was dissolved in anhydrous DCM (5 mL). EDC·HCl (50.2 g, 0.26 mmol) and DMAP (1.5 mg, 0.013 mmol) were added. N, N-dipropyl ethylene diamine (38 mg, 0.26 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. The next day, LC / MS showed that the reaction was complete. The reaction mixture was diluted with DCM (20 mL). The organic phase was washed with sat. aqueous NaHCO3 (20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (24 g). Column was eluted with (0-100)% Hexane-ethyl acetate and (0-20)% DCM- MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 18 (120 mg, 56 % yield). ESI MS (m / z) 860.98 (M+ H)+.NDJP.023WO2 PATENT Synthesis of Compound 19 of Table 1-1

[0122] Compound 19 was prepared according to Scheme 8.Scheme 8

[0123] Synthesis of Intermediate 2 in Scheme 8: Intermediate 1 in Scheme 8 is the same as Intermediate 1 in Scheme 3. To solution of Intermediate 1 (2g, 2.7 mmol) in anhydrous DCM (20 mL), methane sulfonic acid (0.35 mL, 5.4 mmol, 2eq) was added, and the reaction mixture was stirred at room temperature. Next day, LC / MS confirmed complete deprotection of Boc group. Triethyl amine (0.77 mL, 13.5 mmol, 5eq) was slowly added, followed by addition of glutaric anhydride (468 mg, 4.1 mmol, 5 eq) to the reaction mixture. The reaction mixture was stirred overnight. Next day, LC / MS confirmed that the reaction was complete. The reaction mixture was diluted with DCM (20 mL). The organic phase was washed with water (25 mL) and brine (25 mL). The organic phase was dried with Na2SO4. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (80 g). Column was eluted with (0-100)% Hexane-Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 2 as oil (900 mg, 45% yield). ESI MS (m / z): 744.71 (M+H)+.

[0124] Synthesis of Compound 19: To a solution of Intermediate 2 in Scheme 8 (300 mg, 0.4 mmol, 1eq) in DCM (5 ml), N, N’-dipropyl ethylene diamine (87 mg, 0.6 mmol, 1.5 eq), DIEA (143 L, 0.68 mmol, 2 eq), and HATU (229 mg, 0.51 mmol, 1.5eq) were added. The resulting solution was stirred at ambient temperature under a blanket of N2gas overnight. Next day, LCMS showed that the reaction was complete. The reaction mixture was diluted with DCM (20 mL). The organic phase was washed with sat aqueous NaHCO3 (10 mL), water (10 mL), and brine (10 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica 46NDJP.023WO2 PATENT column (40 g). Column was eluted with (0-100)% Hexane-Ethyl acetate and (0-20)% DCM- MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 19 as oil (183 mg, 52% yield). ESI MS (m / z): 870.97 (M+H)+. Synthesis of Compounds 20 and 21 of Table 1-1

[0125] Compound 20 was prepared according to Scheme 9.Scheme 9

[0126] Synthesis of Intermediate 1 in Scheme 9: To a mixture of NBoc-amino diethanol (2.9g, 14.1 mmol) and 6-hexyloctanoic acid (8.0g, 35.3 mmol, 2.5eq) in anhydrous DCM (50 mL), EDC·HCl (172 mg, 35.3 mmol) and DMAP (172 mg, 1.41 mmol, 0.1 eq) were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (30 mL). The organic phase was washed with sat aqueous NaHCO3 (50 mL), water (40 mL), and brine (40 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (80 g). Column was eluted with (0-40)% Hexane-ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 1 as solid (6g, 68% yield). ESI MS (m / z): 626.76 (M+H)+.

[0127] Synthesis of Intermediate 2 in Scheme 9: To a solution of Intermediate 1 in Scheme 9 (2.0g, 3.2 mmol) in DCM (50 ml), methane sulfonic acid (0.4 mL, 6.4 mmol, 2eq) was added. The reaction mixture was stirred at room temperature. Next day, LC / MS confirmedNDJP.023WO2 PATENT complete deprotection of Boc group. Triethylamine (2.3 mL, 16.4 mmol, 5eq) and succinic anhydride (0.47g, 4.9 mmol) were added to the reaction mixture at room temperature. The reaction mixture was stirred overnight. Next day, LC / MS confirmed that the reaction was complete. The reaction mixture was diluted with water (20 m). Saturated citric acid was added to the mixture until pH becomes 4-5. The reaction mixture was extracted with DCM (2x25 mL). The organic phase was washed with water (25 mL) and brine (25 mL). The organic phase was dried with Na2SO4. Solvent was concentrated under reduced pressure to provide Intermediate 2 as solid in quantitative yield. ESI MS (m / z): 626.72 (M+H)+.

[0128] Synthesis of Compound 20: Triethyl amine was added to a mixture of Intermediate 2 in Scheme 9 (300 mg, 0.47 mmol, 1eq) and N, N’-dimethyl ethylene diamine (84 mg, 0.94 mmol, 2 eq) in DCM (15 ml), followed by addition of HATU. The resulting solution was stirred at ambient temperature under a blanket of N2 gas overnight. Next day, LCMS showed that the reaction was completed. The reaction mixture was diluted with DCM (20 mL). The organic phase was washed with sat aqueous NaHCO3 (20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (40 g). Column was eluted with (0-100)% Hexane-Ethyl acetate and (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 20 as solid (100 mg, 30% yield). ESI MS (m / z): 696.8 (M+H)+.

[0129] Synthesis of Compound 21: Intermediate 2 in Scheme 9 (300 mg, 0.47 mmol, 1eq) was treated with N,N’-dipropyl ethylene diamine (97 mg, 94 mmol, 2 eq) according to the same procedure as described in the synthesis of Compound 20 to yield Compound 21 as solid (125 mg, 37% yield). ESI MS (m / z): 710.8 (M+H)+.NDJP.023WO2 PATENT Synthesis of Compound 22 of Table 1-1

[0130] Compound 22 was prepared according to Scheme 10.Scheme 10

[0131] Synthesis of Intermediate 3 in Scheme 10: Intermediates 1 and 2 in Scheme 10 are the same as Intermediates 1 and 2 in Scheme 7, respectively. To a solution of Intermediate 2 in Scheme 10 (4.44 g, 7 mmol) in anhydrous DCM (50 mL), triethylamine (1.17 mL, 8.4 mmol) and glutaric anhydride (0.96 g, 8.4 mmol) were added. The reaction mixture was stirred at 0 °C for 30 minutes, then stirred at room temperature for four hours. LC / MS showed the reaction completed. The reaction was quenched with 0.1 N HCl (100 mL). Crude product was extracted with DCM three times (50 mL). The combined organic phase was dried with Na2SO4and filtered. Solvent was concentrated under reduced pressure using an evaporator, and crude product was purified by flash chromatography using a silica column (120 g). Column was eluted with a (10-20) % hexane – ethyl acetate gradient. Fractions were concentrated and dried under high vacuum to yield intermediate 3 (2.35 g, 45 % yield).

[0132] Synthesis of Compound 22: Intermediate 3 in Scheme 10 (0.55 g, 0.75 mmol) was dissolved in anhydrous DCM (10 mL). EDC·HCl (0.15 g, 0.77 mmol) and DMAP (0.05 g, 0.038 mmol) were added to the solution. N, N-dimethylethylenediamine (0.068 g, 0.77 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. The next day, LC / MS showed that the reaction was complete. Solvent was evaporated under reduced pressure via rotary evaporator, and the resulting residue was suspended in ethyl acetate (60 mL). The organic layer was washed with water (50 mL) and brine solution (50 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated under reduced pressure using an evaporator, and crude product was purified by flash chromatography using a silica column (40 g). Column was eluted with a (0-5) % DCM – 49NDJP.023WO2 PATENT methanol gradient. Fractions were concentrated and dried under high vacuum to yield final product (0.485 g, 60.0 % yield). ESI MS (m / z) 818.95 (M+ H)+. Synthesis of Compound 23 of Table 1-1

[0133] Compound 23 was prepared according to Scheme 11.Scheme 11

[0134] Synthesis of Intermediate 2 in Scheme 11: Intermediate 1 in Scheme 11 is the same as Intermediate 1 in Scheme 3. To a suspension of Intermediate 1 in Scheme 11 (3g, 4.1 mmol, 1.0 eq) in DCM (50 ml), MSA (0.53 mL, 8.2 mmol, 2eq) was added. The reaction mixture was stirred at room temperature. Next day, LC / MS confirmed complete deprotection of Boc group. Triethyl amine (2.3 mL, 16.4 mmol, 4eq) was added to the reaction mixture, followed by addition of HATU (2.3g, 6.1 mmol, 1.5 mmol) and N-Boc glycine (1.3g, 6.1 mmol) at room temperature. After overnight stirring, LC / MS confirmed formation of the product. The organic phase was washed with sat aqueous NaHCO3 (20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (80 g). Column was eluted with (0-60)% Hexane-Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 2 as oil (3g, 93% yield). ESI MS (m / z): 787.76 (M+H)+.

[0135] Synthesis of Compound 23: To a solution of Intermediate 2 in Scheme 11 (260 mg, 0.33 mmol, 1eq) in DCM (5 ml), MSA (45 , 0.64 mmol, 2.0 eq) was added under N2 gas. The clear solution was stirred overnight at room temperature. Next day, LC / MS confirmed complete cleavage of Boc group. The reaction flask was cooled with an ice-bath, then N, N’ dimethyl glycine HCl (91 mg, 0.66 mmol, 2 eq), EDC·HCl (130 mg, 0.66 mmol, 2NDJP.023WO2 PATENT eq), and diisopropylethylamine (117 , 0.66 mmol, 2eq) were sequentially added. After 15 min, the ice-bath was removed, and the reaction was stirred overnight. LC / MS confirmed formation of the product. The reaction mixture was diluted with DCM (20 mL). The organic phase was washed with sat aqueous NaHCO3 (20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4 and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (24 g). Column was eluted with (0-100)% Hexane-Ethyl acetate and (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 23 as oil (100 mg, 39% yield). ESI MS (m / z): 772.88 (M+H)+. Synthesis of Compound 28 of Table 1-1

[0136] Compound 28 was prepared according to Scheme 12.Intermediate 2Scheme 12

[0137] Synthesis of Intermediate 1 in Scheme 12: To a solution of NBoc-amino diethanol (1.5 g, 5.7 mmol) in anhydrous DCM (30 mL), triethyl amine (3.2 mL, 22 mmol, 4 eq) was added, and followed by slow addition of myristoyl chloride (3.1 mL, 12.6 mmol, 2.2 eq) at 0oC. The reaction flask was warmed to room temperature. After 2h stirring, LC / MS confirmed completion of the reaction. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (20 mL). The organic phase was washed with sat aqueous NaHCO3 (30 mL), water (30 mL), and brine (30 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (80 g). Column was eluted with (0- 50)% Hexane-Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 1 as solid (3.4 g, 87% yield). ESI MS (m / z): 682.82 (M+H)+.NDJP.023WO2 PATENT

[0138] Synthesis of Intermediate 2 in Scheme 12: To a solution of Intermediate 1 (500 mg, 0.73 mmol) in DCM (15 ml), methane sulfonic acid (95 , 1.4 mmol, 2eq) was added, and the reaction mixture was stirred at room temperature. Next day, LC / MS confirmed complete deprotection of Boc group. Triethylamine (0.5 mL, 3.6 mmol, 5eq) and succinic anhydride (0.1 g, 1.1 mmol) were added to the reaction mixture at room temperature. The reaction mixture was stirred overnight. Next day, LC / MS confirmed that the reaction was completed. The reaction mixture was diluted with water (15 m). Saturated citric acid was added to the mixture until pH becomes 4-5. The reaction mixture was extracted with DCM (2x20 mL). The organic phase was washed with water (15 mL) and brine (15 mL). The organic phase was dried with Na2SO4. Solvent was concentrated under reduced pressure to provide Intermediate 2 as solid (375 mg, 70% yield).

[0139] Synthesis of Compound 28: was added to a mixture of Intermediate 2 in Scheme 12 (200 mg, 0.29 mmol, 1eq) and N, N’-dimethyl ethylene diamine (38 mg, 94 mmol, 2 eq) in DCM (15 ml),followed by addition of HATU (167 mg, 0.44 mmol, 1.5 eq). The resulting solution was stirred at ambient temperature under a N2gas overnight. Next day, LCMS showed that the reaction was completed. The reaction mixture was diluted with DCM (20 mL). The organic phase was washed with sat aqueous NaHCO3(20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (24 g). Column was eluted with (0-100)% Hexane-Ethyl acetate and (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 28 as solid (140 mg, 63% yield). ESI MS (m / z): 752.6 (M+H)+NDJP.023WO2 PATENT Synthesis of Compound 29 of Table 1-1

[0140] Compound 29 was prepared according to Scheme 13.Scheme 13

[0141] Synthesis of Intermediate 1 in Scheme 13: To a mixture of N-Boc amino dibutanol (1.5 g, 5.7 mmol, 1.0 eq) and 6-hexyloctanoic acid (3.1 g, 13.7 mmol, 2.4eq) in anhydrous DCM (25 mL), EDC·HCl (2.4 g, 13.7 mmol) and DMAP (70 mg, 0.57 mmol, 0.1 eq) were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (30 mL). The organic phase was washed with sat aqueous NaHCO3(25 mL), water (25 mL), and brine (25 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated, the crude product was purified by ISCO / MPLC system using a silica column (80 g). Column was eluted with (0-40)% Hexane-Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 1 as solid (1.1 g, 28% yield). ESI MS (m / z): 682.82 (M+H)+.

[0142] Synthesis of Intermediate 2 in Scheme 13: Methane sulfonic acid (0.13 mL, 2.0 mmol, 2eq) was added to a suspension of Intermediate 1 (700 mg, 1.0 mmol, 1.0 eq) in DCM (20 ml), and the reaction mixture was stirred at room temperature. Next day, LC / MS confirmed complete deprotection of Boc group. Diispropylethyl amine (0.73 mL, 4.1 mmol, 4eq) was added to the reaction mixture, followed by addition of HATU (580 mg, 1.5 mmol, 1.5eq) and N-Boc glycine (260 mg, 1.5 mmol, 1.5 eq) at room temperature. After 3h stirring, LC / MS confirmed formation of the product. The organic phase was washed with sat aqueousNDJP.023WO2 PATENT NaHCO3(20 mL), water (20 mL), and brine (20 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated, the crude product was purified by ISCO / MPLC system using a silica column (40 g). Column was eluted with (0-60)% Hexane-Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 2 as oil (420 mg, 55% yield). ESI MS (m / z): 739.50 (M+H)+.

[0143] Synthesis of Compound 29: was added to a solution of Intermediate 2 (140 mg, 0.19 mmol, 1eq) in DCM (5 ml) under N2gas. The clear solution was stirred overnight at room temperature. Next day, LC / MS confirmed complete cleavage of Boc group. The reaction flask was cooled with an ice-bath, then 4- (dimethylamino) butanoic acid HCl (47 mg, 0.28 mmol, 1.5 eq.), HATU (122 mg, 0.64 mmol, 1.5 eq), and DIEA (0.28 , 1.5 mmol, 5eq) were sequentially added. After 15 min, the ice- bath was removed, and the reaction was stirred for 4h. LC / MS confirmed formation of the product. The reaction mixture was diluted with DCM (15 mL). The organic phase was washed with sat aqueous NaHCO3 (10 mL), water (10 mL), and brine (10 mL). The organic phase was dried with Na2SO4and filtered. Solvent was concentrated, and the crude product was purified by ISCO / MPLC system using a silica column (24 g). Column was eluted with (0-100)% Hexane-ethyl acetate and (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 29 as solid (63 mg, 44% yield). ESI MS (m / z): 752.7 (M+H)+. Example 2: In vitro expression of Fluc mRNA

[0144] LNP formulations with Fluc mRNA were prepared with the following compositions: (Test compound / cholesterol / DSPC / DMG-PEG2000) = 50 / 38 / 10 / 2 (mol%)

[0145] The test compound is one selected from the group consisting of Compounds Nos.1 to 29, HEDC, and HEDC-M1. HEDC was prepared according to U.S. publication No. 2013 / 0022665, which is hereby expressly incorporated by reference in its entirely. HEDC-M1 was prepared according to PCT publication No. WO 2013 / 185116, which is hereby expressly incorporated by reference in its entirely. N / P ratio was fixed at 6.0 here.

[0146] LNP formulations were prepared by injecting ethanol solution of lipids into a Fluc mRNA (TriLink, 5moU) buffer solution, in the same manner as described in U.S. Publication No. U.S. 2013 / 0022665, PCT publication No. WO 2019 / 090359, and WO 54NDJP.023WO2 PATENT 2020 / 102668, which are hereby expressly incorporated by reference in its entirely. The average particle size (PS), the polydispersity index (PDI), the encapsulation efficiency for mRNA (%EE), and the yield of each LNP formulation are shown in the following Table 2. PS and PDI were obtained by using Malvern Zetasizer Nano-ZS ZEN 3600. %EE were obtained by the Ribogreen fluorescence assay following GenVoy-ILMTMUser Guide by Precision NanoSystems. Table 2NDJP.023WO2 PATENT

[0147] In vitro expression of Fluc mRNA were measured according to the following protocol 1 or 2: Protocol 1:

[0148] A549, Hep3B, and Panc-1 cell lines were cultured in media supplemented with 10% HI-FBS (Gibco Ref # 10082-147). F-12K media (ATCC Ref # 30-2004), EMEM media (ATCC Ref # 30-2003), and DMEM media (Gibco Ref # 11965-092) were used respectively. On day 0, cells were plated in white opaque 96-well TC-treated plates (Greiner were placed in a 37°C incubator with 5% CO2overnight to allow cell attachment. On day 1, the mRNA / LNP complex was equilibrated to room temperature, then diluted with DPBS (Gibco Ref # 14190-144) to create a dose- Plates were placed back in the 37°C incubator with 5% CO2 for 24 hours. On day 2, the Promega Luciferase Assay System (Ref # E1501) buffer and substrate were equilibrated to room temperature and combined, then added to the SpectraMax L Luminometer (Molecular Devices) injectors. Plates were prepared according to Promega kit guidelines by first removing reporter lysis buffer (Ref # E397A) to each well. Plates were placed in the Luminometer and were obtained. EC50 values were determined by fitting dose-response curve with 4-parameter logistic model using GraphPad / Prism. EC50 values of the LNP formulation including one of Compounds 1 to 22 and HEDC were determined according to Protocol 1. Protocol 2:

[0149] A549, Hep3B, and Panc-1 cell lines were cultured in media supplemented with 10% HI-FBS (Gibco Ref # 10082-147). F-12K media (ATCC Ref # 30-2004), EMEM media (ATCC Ref # 30-2003), and DMEM media (Gibco Ref # 11965-092) were used respectively. On day 0, cells were plated in white opaque 384-well TC-treated plates (USANDJP.023WO2 PATENT Scientific Ref # 5678-1080) at a density of 1500 cells / well using the Multidrop Combi+ were placed in a 37°C incubator with 5% CO2 overnight to allow cell attachment. On day 1, the mRNA / LNP complex was equilibrated to room temperature, then diluted with DPBS (Gibco Ref # 14190-144) tocreate a dose- were placedback in the 37°C incubator with 5% CO2 for 24 hours. On day 2, the ONE-Glo EX Luciferase Assay System (Promega Ref # E8130) was equilibrated to room temperature and combined. Plates were to each well. Plates were placed on a plate shaker for 3 minutes to assure cell lysis. Once lysis was complete, the plates were added to the Luminometer (Molecular Devices) and luminescence values were obtained. EC50 values were determined by fitting dose-response curve with 4-parameter logistic model using GraphPad / Prism. EC50 values of the LNP formulation including one of Compounds 23 to 29 and HEDC-M1were determined according to Protocol 2. Experimental results are shown in Table 3 below. Table 3NDJP.023WO2 PATENTND: No Data

[0150] All of LNP 1a to LNP 29a showed higher transfection property to A549 cells, HEP3B cells or Panc-1 cells than LNP R1a. Specifically, when LNP 1a, LNP 5a, LNP 6a, LNP 8a, LNP 9a, LNP 12a, LNP 20a, and LNP 23a were applied to A549 cells, the EC50value was about 1.1 to 30-fold lower than when LNP R1a was applied to the same cells. When LNP 3a, LNP 5a, LNP 9a, LNP 12a, and LNP 13a were applied to HEP3B cells, the EC50value was 1.5 to 7.5- fold lower than when LNP R1a was applied to the same cells. When LNP 1a, LNP 2a, LNP 4a, LNP 5a, LNP 7a, LNP 8a, LNP 9a, LNP 10a, LNP 11a, LNP 12a, LNP 14a, LNP 15a, LNP 16a, LNP 17a, LNP 18a, LNP 19a, LNP 20a, LNP 21a, LNP 22a, LNP 24a, LNP 25a, LNP 26a, LNP 27a, LNP 28a, and LNP 29a were applied to Panc-1 cells, the EC50 value was about 1.2 to 24 -fold lower than when LNP R1a was applied to the same cells.

[0151] In addition, when LNP 1a, LNP 2a, LNP 5a, LNP 6a, LNP 7a, LNP 8a, LNP 9a, LNP 11a, LNP 12a, LNP 15a, LNP 16a, LNP 19a, LNP 20a, LNP 21a, LNP 23a, LNP 24a, LNP 26a, and LNP 29a were applied to A549 cells, the EC50value was about 1.2 to 83-fold lower than when LNP R2a was applied to the same cells. When LNP 3a and LNP 5a were applied to HEP3B cells, the EC50 value was about 1.4 to 2.5-fold lower than when LNP R2a was applied to the same cells.NDJP.023WO2 PATENT Example 3: In vitro expression of Fluc mRNA

[0152] LNP formulations with Fluc mRNA were prepared with the following compositions: (Test compound / cholesterol / DOPE / DPPE-PEG2000 / Compound A) = 30 / 30 / 30 / 5 / 5 (mol%)

[0153] The test compound is one selected from the group consisting of Compounds Nos. 1 to 8, 10 to 19, 21 to 24, 26 to 29 and HEDC-M1. Compound A corresponds to Compound T3 in WO 2019 / 090359, which is hereby expressly incorporated by reference in its entirety.described in Example 2. The average particle size (PS), the polydispersity index (PDI), the encapsulation efficiency for mRNA (%EE), and the yield of each LNP formulation are shown in the following Table 4. PS and PDI were obtained by using Malvern Zetasizer Nano-ZS ZEN 3600. %EE were obtained by the Ribogreen fluorescence assay following GenVoy- ILMTMUser Guide by Precision NanoSystems. Table 459NDJP.023WO2 PATENT

[0155] In vitro expression of Fluc mRNA of LNP1b to LNP22b in Table 4 were measured according to Protocol 1 described in Example 2. In vitro expression of Fluc mRNA of LNP 23b to LNP29b and LNPR2b in Table 4 were measured according to Protocol 2 described in Example 2. Experimental results are shown in Table 5 below. Table 560NDJP.023WO2 PATENTND: No Data

[0156] All of LNP 1b to LNP 29b in Table 5 showed higher transfection property to A549 cells, HEP3B cells or Panc-1 cells than LNP R2b. Specifically, when LNP 3b, LNP5b, LNP7b, LNP11b, LNP12b, LNP13b, LNP14b, LNP15b, LNP16b, LNP17b, LNP18b, LNP22b, LNP23b, LNP27b or LNP29b were applied to A549 cells, the EC50value was about 1.1 to 17-fold lower than when LNP R2b was applied to the same cells. When LNP 1b, LNP2b, LNP 3b, LNP5b, LNP6b, LNP 8b, LNP11b, LNP12b, LNP13b, LNP14b, LNP15b, LNP16b, LNP17b, LNP18b, LNP 19b, LNP22b, LNP26b or LNP29b were applied to HEP3B cells, the EC50 value was about 1.2 to 29- fold lower than when LNP R2b was applied to the same cells. When LNP3b, LNP4b, LNP5b, LNP6b, LNP7b, LNP10b, LNP12b, LNP13b, LNP14b, LNP15b, LNP16b, LNP17b, LNP18b, LNP19b, LNP21b, LNP22b, LNP24b LNP27b,NDJP.023WO2 PATENT LNP28b or LNP29b were applied to Panc-1 cells, the EC50value was about 1.4 to 12 -fold lower than when LNP R2b was applied to the same cells.

[0157] Also, the following LNP formulations with Fluc mRNA were prepared with the following compositions: (Test compound / cholesterol / DOPE / DPPE-PEG2000 / Compound A) = 30 / 30 / 30 / 5 / 5 (mol%)

[0158] The test compound is one selected from the group consisting of Compounds Nos.30-38 having the structures shown in the following Table 6: Table 6NDJP.023WO2 PATENTNDJP.023WO2 PATENT

[0159] LNP formulations were prepared according to the same procedure as described in Example 2. The average particle size (PS), the polydispersity index (PDI), the encapsulation efficiency for mRNA (%EE), and the yield of each LNP formulation are shown in the following Table 7. PS and PDI were obtained by using Malvern Zetasizer Nano-ZS ZEN 3600. %EE were obtained by the Ribogreen fluorescence assay following GenVoy- ILMTMUser Guide by Precision NanoSystems. Table 7

[0160] In vitro expression of Fluc mRNA of LNP30b to LNP38b in Table 7 were measured according to Protocol 1 described in Example 2. Experimental results are shown in Table 8 below. Table 864NDJP.023WO2 PATENT

[0161] All of LNP 30b to LNP 38b in Table 8 showed higher transfection property to A549 cells, HEP3B cells or Panc-1 cells than LNP R2b. Specifically, when LNP 30b to LNP 38b were applied to A549 cells, the EC50 value was about 2.2 to 17.4-fold lower than when LNP R2b was applied to the same cells. When LNP 30b to LNP 38b were applied to HEP3B cells, the EC50value was about 2.9 to 29-fold lower than when LNP R2b was applied to the same cells. When LNP 30b to LNP 38b were applied to Panc-1 cells, the EC50value was about 3.1 to 109-fold lower than when LNP R2b was applied to the same cells. Synthesis of Compounds 30-38 of Table 1-2

[0162] Compound 31 was prepared according to Scheme 14.NDJP.023WO2 PATENTScheme 14

[0163] Synthesis of Intermediate 1 in Scheme 14 (Step 1): N-Boc-diethanolamine (32.8 g, 160 mmol, 1 eq), myristic acid (82.2 g, 360 mmol, 2.2 eq) and EDC.HCl were dissolved in DCM (1000 mL). To the above reaction solution, DMAP (7.81 g, 63.9 mmol, 0.4 eq) was added. The resulting clear / colorless solution was stirred at room temperature under N2gas. Next day, LC / MS showed product formation. The reaction solution was transferred to a 2000 mL separating-funnel and washed with saturated aqueous NaHCO3solution followed by water and brine solution. The organic layer was separated, dried over Na2SO4 and filtered. The solvent was concentrated under reduced pressure via a roto-evaporator and the crude material was purified on ISCO MPLC using 330 g silica column. Column was eluted with 0-15% Hexane / Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 1 as colorless oil that solidified at -20oC. (Yield: Quantitative)

[0164] Synthesis of Intermediate 2 in Scheme 14 (Step 2): Intermediate 1 (10 g, 15.9 mmol, 1eq) was dissolved in DCM (100 mL) in a RBF (250 mL) equipped with a magnetic stir bar. Methane sulfonic acid (MSA) (3.1 mL, 47.9 mmol, 3 eq) was slowly added to above solution at room temperature. The clear solution was stirred at room temperature overnight under N2 gas. Next day, LC / MS showed complete conversion to product. Reaction flask wasNDJP.023WO2 PATENT cooled to 0-5oC with ice bath. Chloroacetyl chloride (1.58 mL, 19.9 mmol, 1.25 eq) was added followed by drop-wise addition of triethyl amine (13.3 mL, 95.8 mmol, 6 eq) to the above reaction solution. After 15 min, ice-bath was removed, the reaction solution was warmed to room temperature and stirred for 3 hr. LC / MS showed reaction was complete. The reaction solution was diluted with DCM, then washed sequentially with water (2x) and brine solution. The organic layer was separated, dried over NaSO4 and filtered. The solvent was evaporated under reduced pressure using rotary evaporator. The crude product was purified by ISCO MPLC using 220 g silica column. Column was eluted with 0-50% Hexane-EtOAc gradient. Fractions were combined and concentrated to yield Intermediate 2 as oil that solidified at -20oC (9 g, 93.7%). MS ESI (m / z): 619.47 (M+NH4)+

[0165] Synthesis of Intermediate 3 in Scheme 14 (Step 3): Intermediate 2 (4.5 g, 7.9 mmol, 1eq) and (R) pyrrolidine 3-ol-HCl (2 eq) were suspended in LC / MS grade acetonitrile (80 mL) in a RBF (200 mL) equipped with a magnetic stir bar. DBU (2.3 mL, 15.9 mmol, 2 eq) was added and the reaction flask was heated at 45oC and stirred overnight under N2 gas. Next day, LC / MS showed complete conversion to product. The heating was turned off and the reaction flask was cooled to room temperature. The solvent was concentrated under reduced pressure using a rotary evaporator. The reaction mixture was diluted with DCM then washed sequentially with water (40 mL) and brine solution (40 mL). The organic layer was separated, dried over NaSO4and filtered. The crude product was purified by MPLC / ISCO using 120 g silica column. Column was eluted with 0-100% Hexane-EtOAc and 0-20% DCM-MeOH gradient. Fractions were combined and concentrated to yield Intermediate 3 as a solid (4.2 g, 80%). ESI MS (m / z): 653.62 (M+H)+

[0166] Synthesis of Compound 31 in Scheme 14 (Step 4): Intermediate 3 (4.2 g, 6.4 mmol, 1eq) was taken in 200 mL round bottle flask and bromo ethanol (2.3 mL, 32.1 mmol) was added. The reaction flask was heated at 70oC and stirred overnight under N2gas. Next day, LC / MS showed starting material was completely converted to methyl iodide salt. The solvent was evaporated and the residue was purified by ISCO MPLC using 120 g silica column. Column was eluted with (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated to yield Intermediate as bromide counter ion. This intermediate was dissolved to a minimum volume of DCM and passed through a column of Amberlyst A26 (activated with HCl). Column was eluted with 100% DCM and all fractions obtained were concentrated. TheNDJP.023WO2 PATENT resulting residue was dissolved again to a minimum volume of DCM and passed through above column. Column was eluted again with DCM and all fractions were concentrated. The product was dried under high vacuum to give desired product Compound 31 as chloride counter ion (1.4 g, 28% yield). MS ESI (m / z): 697.73 (M-Cl)+

[0167] Compound 32 was prepared according to Scheme 15.Scheme 15

[0168] Synthesis of Intermediate 1 in Scheme 15 (Step 1): N-Boc-diethanolamine (32.8 g, 160 mmol, 1 eq), Myristic acid (82.2 g, 360 mmol, 2.2 eq) and EDC.HCl were dissolved in DCM (1000 mL). To the above reaction solution, DMAP (7.81 g, 63.9 mmol, 0.4 eq) was added. The resulting clear / colorless solution was stirred at room temperature under N2gas. Next day, LC / MS showed product formation. The reaction solution was transferred to a 2000 mL separating-funnel and washed with saturated aqueous NaHCO3solution followed by water and brine solution. The organic layer was separated, dried over Na2SO4 and filtered. The solvent was concentrated under reduced pressure via a roto-evaporator and the resulting crude material was purified by ISCO MPLC using 330 g silica column. Column was eluted with 0- 15% Hexane / Ethyl acetate gradient. Fractions were combined and concentrated via rotaryNDJP.023WO2 PATENT evaporator to yield Intermediate 1 as colorless oil that solidified at -20oC. (Yield: Quantitative)

[0169] Synthesis of Intermediate 2 in Scheme 15 (Step 2): Intermediate 1 (10 g, 15.9 mmol, 1eq) was dissolved in DCM (100 mL) in a RBF (250 mL) equipped with a magnetic stir bar. Methane sulfonic acid (MSA) (3.1 mL, 47.9 mmol, 3 eq) was slowly added to the above solution at room temperature. The clear solution was stirred at room temperature overnight under N2gas. Next day, LC / MS showed complete conversion to the product. The reaction flask was cooled to 0-5oC with ice bath. Chloroacetyl chloride (1.58 mL, 19.9 mmol, 1.25 eq) was added followed by drop-wise addition of triethyl amine (13.3 mL, 95.8 mmol, 6 eq) to the above reaction solution. After 15 min, ice-bath was removed, the reaction was warmed to room temperature and stirred for 3 hr. LC / MS showed reaction was complete. The obtained mixture was diluted with DCM then washed sequentially with water (2x) and brine solution. The organic layer was separated, dried over NaSO4 and filtered. The solvent was evaporated under reduced pressure using rotary evaporator. The crude product was purified by ISCO MPLC sing 220 g silica column. Column was eluted with 0-50% Hexane-EtOAc gradient. Fractions were combined and concentrated to yield Intermediate 2 as oil that solidified at -20oC (9 g, 93.7%). MS ESI (m / z): 619.47 (M+NH4)+

[0170] Synthesis of Intermediate 3 in Scheme 15 (Step 3): This is a general procedure to get Intermediate 3 or Ionizable lipid by treating Intermediate 2 with various substituted amines. Intermediate 2 (800 mg, 1.32 mmol, 1eq) and amine (2 eq) were suspended in LC / MS grade acetonitrile (40 mL) in a RBF (100 mL) equipped with a magnetic stir bar. DBU (0.39 mL, 2.64 mmol, 2 eq) was added and the reaction flask was heated at 45oC and stirred overnight under N2 gas. Next day, LC / MS showed complete conversion to the product. The heating was turned off and the reaction flask was cooled to room temperature. Solvent was concentrated under reduced pressure using a rotary evaporator and the crude product was purified by MPLC / ISCO using 40 g silica column. Column was eluted with 0-100% Hexane- EtOAc and 0-20% DCM-MeOH gradient. Fractions were combined and concentrated to yield the corresponding lipid.

[0171] Synthesis of Compound 32 in Scheme 15 (Step 4): This is a general procedure to get cationic version and analogs by treating Intermediate 3 and its analogs with MeI followed by ion exchange with Amberlite A26 resin (HCl form). Intermediate 3 (500 mg, 0.74 mmol, 69NDJP.023WO2 PATENT 1eq) was taken in 15 mL scintillation vial and MeI (2.5 mL, excess) was added. The resulting clear solution was stirred at room temperature overnight. Next day, LC / MS showed that the starting material was completely converted to methyl iodide salt. Excess MeI was evaporated and the residue was purified by ISCO MPLC using 80 g silica column. The obtained mixture was eluted with (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated to yield Intermediate as iodide counter ion. This intermediate was dissolved to a minimum volume of DCM and passed through a column of Amberlyst A26 (activated with HCl). Column was eluted with 100% DCM and all fractions were combined and concentrated. The resulting residue was dissolved again to a minimum volume of DCM and passed through above column. The mixture was eluted again with DCM and all fractions were combined and concentrated. The product was dried under high vacuum to give desired product Compound 32 as chloride counter ion (376 mg, 70% yield). MS ESI (m / z): 683.57 (M-Cl)+

[0172] Compound 34 was prepared according to Scheme 16. Compound 34: Intermediate 2 (700 mg, 1.16 mmol, 1eq) was reacted with piperidine 4-yl-methanol (267 mg, 2.32 mmol, 2 eq) by the general procedure described in Step 3 of Scheme 15, yielding Compound 34 as white solid. (507 mg, 63.7%) ESI MS (m / z): 681.59 (M+H)+Scheme 16

[0173] Compound 33 was prepared according to Scheme 17.NDJP.023WO2 PATENTScheme 17

[0174] Synthesis of Intermediate 1 in Scheme 17 (Step 1): N-Boc-diethanolamine (10g, 48.7 mmol, 1 eq) and Linoleic acid (30.7 g, 109.6 mmol, 2.2 eq) were dissolved in DCM (300 mL) in a RBF (500 mL) equipped with a magnetic stir bar. To the above reaction solution, EDC.HCl (23.3 g, 121.8 mmol, 2.5 eq) and DMAP (2.4 g, 195 mmol, 04 eq) were added simultaneously. The resulting clear solution was stirred at room temperature under N2gas. Next day, LC / MS showed product formation. The reaction solution was transferred to a 500 mL separating-funnel and washed with saturated aqueous NaHCO3 solution followed by water and brine solution. The organic layer was separated, dried over Na2SO4 and filtered. The solvent was concentrated under reduced pressure via a roto-evaporator and the resulting crude material was purified on ISCO MPLC using 220 g silica column. The column was eluted with 0-20% Hexane / Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to give Intermediate 1 as colorless oil. (18.9 g, 55%). MS ESI (m / z): 630.73 (M+H)+

[0175] Synthesis of Intermediate 2 in Scheme 17 (Step 2): Intermediate 1 (14.1 g, 19.3 mmol, 1eq) was dissolved in DCM (95 mL) in a RBF (250 mL) equipped with a magnetic stir bar. Methane sulfonic acid (MSA) (3.7 mL, 57.9 mmol, 3 eq) was slowly added to aboveNDJP.023WO2 PATENT solution at room temperature. The clear solution was stirred at room temperature overnight under N2gas. Next day, LC / MS showed complete conversion to the product. The reaction flask was cooled to 0-5oC with ice bath. Chloroacetyl chloride (1.9 mL, 24.1 mmol, 1.25 eq) was added followed by drop-wise addition of triethyl amine (16.1 mL, 115 mmol, 6 eq) to the above reaction solution. After 15 min, ice-bath was removed, and the reaction solution was warmed to room temperature and stirred for 3 hr. LC / MS showed that the reaction was complete. The reaction mixture was diluted with DCM then washed sequentially with saturated aqueous NaHCO3solution, water and brine solution. The organic layer was separated, dried over NaSO4and filtered. The solvent was evaporated under reduced pressure using rotary evaporator. The crude product was purified by ISCO MPLC sing 220 g silica column. Column was eluted with 0-50% Hexane-EtOAc gradient. Fractions were combined and concentrated to yield Intermediate 2 as oil (12.0 g, 85.7%). MS ESI (m / z): 723.59 (M+Na)+

[0176] Synthesis of Compound 30 and analogs in Scheme 17 (Step 3): This is a general procedure to prepare Compound 30 and analogs with different head groups by treating Intermediate 2 with various substituted amines. Intermediate 2 (800 mg, 1.13 mmol, 1eq) and amine (2-3 eq) were suspended in LC / MS grade acetonitrile (40 mL) in a RBF (100 mL) equipped with a magnetic stir bar. DBU (0.33 mL, 2.26 mmol, 2 eq) was added and the reaction flask was heated at 45oC and stirred overnight under N2gas. Next day, LC / MS showed complete conversion to the product. The heating was turned off and the reaction flask was cooled to room temperature. The solvent was concentrated under reduced pressure using rotary evaporator and the crude product was purified by MPLC / ISCO using 40 g silica column. Column was eluted with 0-100% Hexane-EtOAc and 0-20% DCM-MeOH gradient. Fractions were combined and concentrated to yield the corresponding lipid.

[0177] Synthesis of Compound 33 in Scheme 17 (Step 4): This is a general procedure to get cationic version of Compound 33 by treating Compound 30 or its analogs with MeI followed by ion exchange with Amberlite A26 resin (HCl form). Compound 30 (2 g, 2.5 mmol, 1eq) was taken in 15 mL scintillation vial and MeI (3 mL, excess) was added. The resulting clear solution was stirred at room temperature for overnight. Next day, LC / MS showed that the starting material was completely converted to methyl iodide salt. The excess MeI was evaporated and the residue was suspended in DCM (40 mL). The organic layer was sequentially washed with saturated aqueous NaHCO3 solution (25 mL) and brine solution (25NDJP.023WO2 PATENT mL). The organic phase was dried over Na4SO4and filtered. The solvent was concentrated and the intermediate was purified on ISCO MPLC using 80 g silica column. Column was eluted with (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated to yield Intermediate as iodide counter ion. This intermediate was dissolved to a minimum volume of DCM and passed through a column of Amberlyst A26 (activated with HCl). Column was eluted with 100% DCM and all fractions were combined and concentrated. The resulting residue was dissolved again to a minimum volume of DCM and passed through above column. The column was eluted again with DCM and all fractions were combined and concentrated. The obtained product was dried under high vacuum to give the desired product Compound 33 as chloride counter ion (1.19, 58% yield). MS ESI (m / z): 787.6 (M-Cl)+

[0178] Compound 35 was prepared according to Scheme 18.NDJP.023WO2 PATENT Scheme 18

[0179] Synthesis of Intermediate 1 in Scheme 18 (Step 1): N-Boc-diethanolamine (30.0 g, 146 mmol, 4.0 eq.) and EDC HCl salt (6.44 g, 33.6 mmol, 1.15 eq.) was dissolved in DCM (250 ml) in a RBF (1000 ml) equipped with a magnetic stir bar. Linoleic acid (8.2 g, 29.2 mmol, 1.0 eq.) and DMAP (713 mg, 5.84 mmol, 0.2 eq.) were added and the clear / colorless solution was stirred at ambient temperature under Nitrogen gas. Approximately 5 hours later, IPC (LCMS) showed complete conversion to the product. The obtained mixture was washed with H2O (150 ml), saturated NaHCO3solution (150 ml) and saturated brine (150 ml). The extracted aqueous solution was washed with DCM (~50 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (31.02 g). The crude oil was purified by ISCO MPLC using a Si column (330 g), Solid Load Cartridge (SLC, 65 g) and a (0-40)% Hexane / Ethyl Acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 1 as clear colorless oil (10.47 g, 76.4 % Yield).

[0180] Synthesis of Intermediate 2 in Scheme 18 (Step 2): Intermediate 1 (10.47 g, 22.4 mmol, 1.0 eq), EDC HCl salt (5.37 g, 28.0 mmol, 1.25 eq.) and Myristic acid (5.89 g, 25.8 mmol, 1.15 eq.) were dissolved in DCM (250 ml) in a RBF (1000 ml) equipped with a magnetic stir bar. DMAP (547 mg, 4.48 mmol, 0.2 eq.) was added and the clear / colorless solution was stirred at ambient temperature under Nitrogen gas. The next day, IPC (LCMS) showed complete conversion to the product. The obtained mixture was washed with H2O (150 ml) and saturated brine (150 ml). The extracted aqueous solution was washed with DCM (~50 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (16 g). The crude oil was purified by ISCO MPLC using a Si column (330 g), SLC (65 g) and a (0-10-25)% Hexane / Ethyl Acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 2 as clear colorless oil (14.41 g, 93.0 % Yield).

[0181] Intermediate 2 (14.4 g, 21.2 mmol, 1.0 eq) was dissolved in DCM (200 ml) and methane sulfonic acid (MSA, 4.1 ml, 63.6 mmol, 3.0 eq) was added in a RBF (1000 ml) equipped with a magnetic stir bar. The clear / colorless solution was allowed to stir overnight at ambient temperature under N2 gas. Next day, IPC (LCMS) confirmed that the reaction was 74NDJP.023WO2 PATENT complete. The RBF was placed into an ice-bath where chloroacetyl-chloride (2.1 ml, 26.5 mmol, 1.25 eq.) was then added followed by dropwise addition of triethylamine (Et3N, 17.8 ml, 127 mmol, 6.00 eq.). When the addition was complete, the ice bath was removed and the reaction mixture was allowed to warm to ambient temperature. Approximately 2 hours later, IPC (LCMS) confirmed that the reaction was complete. The obtained mixture was washed with H2O (150 ml) and saturated brine (150 ml). The extracted aqueous solution was washed with DCM (~50 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (14.09 g). The crude oil was purified by ISCO MPLC using a Si column (330 g), SLC (32 g) and a (0-25) % Hexane / Ethyl Acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 3 as clear colorless oil (12.47 g, 89.7 % Yield).

[0182] Synthesis of Compound 36 in Scheme 18 (Step 3): Intermediate 3 (3.47 g, 5.30 mmol, 1.0 eq.) was dissolved in ACN (50 ml) and cis-dihydroxypyrollidine HCl salt (cDHP, 1.48 g, 10.6 mmol, 2.0 eq) was added in a RBF (250 ml) equipped with a magnetic stir bar. DBU (1.66 ml, 11.1 mmol, 2.1 eq.) was added and the resulting solution was then heated to 45 °C and allowed to stir under N2gas. Next day, IPC (LCMS) confirmed that the reaction was complete. Material was then concentrated under reduced pressure using a rotary evaporator and purified by ISCO MPLC using a Si column (220 g), SLC (32 g) and a (0-100) % Hexane / Ethyl Acetate followed by (0-25) % Ethyl Acetate / Ethanol gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 36 as an oil (3.35 g, 87.7 % Yield).

[0183] Synthesis of Compound 35 in Scheme 18 (Step 4): Compound 36 (1.36 g, 1.89 mmol, 1.0 eq) was dissolved in ACN (25 ml) and iodomethane (MeI, 235 μl, 3.77 mmol, 2.0 eq) was added in a RBF (50 ml) equipped with a magnetic stir bar. The resulting solution was then heated to 45 °C and allowed to stir under N2gas. Next day, IPC (LCMS) confirmed that the reaction was complete. Material was then concentrated under reduced pressure using a rotary evaporator and purified by ISCO MPLC using a Si column (40 g), SLC (5 g) and a (0- 100) % Hexane / Ethyl Acetate followed by (0-15) % DCM / MeOH gradient. Fractions were combined and concentrated via rotary evaporator. The residue was diluted with DCM and passed through short column packed with Activated Amberlyst A21 resin, rinsing with moreNDJP.023WO2 PATENT DCM. The obtained product was concentrated via rotary evaporator and dried fully under high vacuum to yield Compound 35 (1177 mg, 80.6 % Yield).

[0184] Compound 36 was prepared according to Scheme 19.Scheme 19

[0185] Synthesis of Intermediate 1 in Scheme 19 (Step 1): N-Boc-diethanolamine (30.0 g, 146 mmol, 4.0 eq.) and EDC HCl salt (6.44 g, 33.6 mmol, 1.15 eq.) were dissolved in DCM (250 ml) in a RBF (1000 ml) equipped with a magnetic stir bar. Linoleic acid (8.2 g, 29.2 mmol, 1.0 eq.) and DMAP (713 mg, 5.84 mmol, 0.2 eq.) were added and the clear / colorless solution was stirred at ambient temperature under Nitrogen gas. Approximately 5 hours later, IPC (LCMS) showed complete conversion to the product. The reaction mixture was washed with H2O (150 ml), saturated NaHCO3solution (150 ml) and saturated brine (150 ml). The extracted aqueous solution was washed with DCM (~50 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (31.02 g). The crude oil was purified by ISCO MPLC using a Si column (330 g), Solid Load Cartridge (SLC, 65 g) and a (0-40)% Hexane / Ethyl AcetateNDJP.023WO2 PATENT gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 1 as clear colorless oil (10.47 g, 76.4 % Yield).

[0186] Synthesis of Intermediate 2 in Scheme 19 (Step 2): Intermediate 1 (10.47 g, 22.4 mmol, 1.0 eq), EDC HCl salt (5.37 g, 28.0 mmol, 1.25 eq.) and myristic acid (5.89 g, 25.8 mmol, 1.15 eq.) were dissolved in DCM (250 ml) in a RBF (1000 ml) equipped with a magnetic stir bar. DMAP (547 mg, 4.48 mmol, 0.2 eq.) was added and the clear / colorless solution was stirred at ambient temperature under Nitrogen gas. The next day, IPC (LCMS) showed complete conversion to the product. The reaction mixture was washed with H2O (150 ml) and saturated brine (150 ml). The extracted aqueous solution was washed with DCM (~50 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (16 g). The crude oil was purified by ISCO MPLC using a Si column (330 g), SLC (65 g) and a (0-10-25)% Hexane / Ethyl Acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 2 as clear colorless oil (14.41 g, 93.0 % Yield).

[0187] Synthesis of Intermediate 3 in Scheme 19 (Step 3): Intermediate 2 (14.4 g, 21.2 mmol, 1.0 eq) was dissolved in DCM (200 ml) and methane sulfonic acid (MSA, 4.1 ml, 63.6 mmol, 3.0 eq) was added in a RBF (1000 ml) equipped with a magnetic stir bar. The clear / colorless solution was allowed to stir overnight at ambient temperature under N2gas. Next day, IPC (LCMS) confirmed that the reaction was complete. The RBF was placed into an ice-bath where chloroacetyl-chloride (2.1 ml, 26.5 mmol, 1.25 eq.) was then added followed by dropwise addition of triethylamine (Et3N, 17.8 ml, 127 mmol, 6.00 eq.). When the addition was complete, the ice bath was removed and the reaction mixture was allowed to warm to ambient temperature. Approximately 2 hours later, IPC (LCMS) confirmed that the reaction was complete. The reaction mixture was washed with H2O (150 ml) and saturated brine (150 ml). The extracted aqueous solution was washed with DCM (~50 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (14.09 g). The crude oil was purified by ISCO MPLC using a Si column (330 g), SLC (32 g) and a (0-25) % Hexane / Ethyl Acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 3 as clear colorless oil (12.47 g, 89.7 % Yield).NDJP.023WO2 PATENT

[0188] Synthesis of Compound 36 in Scheme 19 (Step 4): Intermediate 3 (3.47 g,5.30 mmol, 1.0 eq.) was dissolved in ACN (50 ml) and cis-dihydroxypyrollidine HCl salt (cDHP, 1.48 g, 10.6 mmol, 2.0 eq) was added in a RBF (250 ml) equipped with a magnetic stir bar. DBU (1.66 ml, 11.1 mmol, 2.1 eq.) was added and the resulting solution was then heated to 45 °C and allowed to stir under N2 gas. Next day, IPC (LCMS) confirmed that the reaction was complete. Material was then concentrated under reduced pressure using a rotary evaporator and purified by ISCO MPLC using a Si column (220 g), SLC (32 g) and a (0-100) % Hexane / Ethyl Acetate followed by (0-25) % Ethyl Acetate / Ethanol gradient. Fractions werecombined and concentrated via rotary evaporator to yield Compound 36 as an oil (3.35 g, 87.7% Yield).

[0189] Compound 37 was prepared according to Scheme 20.Intermediate 137Scheme 20

[0190] Synthesis of Compound 37 in Scheme 20: Intermediate 1 (200 mg, 0.27mmol) and trans-3,4-pyrrolidinediol (28 mg, 0.27 mmol) in an oven-dried vial (40 mL) with amagnetic bar were added into anhydrous DCM (10 mL) and DMSO (1 mL). The mixture was stirred at ambient temperature for 2 minutes to a clear solution. Triethylamine (50 mL, 0.32 mmol) was then added to the mixture and the mixture was stirred at ambient temperature overnight (17 hours). The reaction was quenched with 10% K2CO3solution (50 mL) and extracted with DCM (2×50 mL). The organic layers were then combined, dried over MgSO4(5 g), filtered, and concentrated under reduced pressure. The crude was dissolved in 5 mL DCM and purified with a 12 g silica column using a gradient of EtOAc for 5 min and 0-30% MeOH / DCM for 30 min under the flow rate at 20 mL / min. The product fractions werecollected and concentrated to yield Compound 37 (389 mg, 78% yield) as a colorless liquid.

[0191] Compound 38 was prepared according to Scheme 21.NDJP.023WO2 PATENTScheme 21

[0192] Synthesis of Intermediate 1 in Scheme 21 (Step 1): N-Boc-diethanolamine (50.0 g, 244 mmol, 4.0 eq.) and EDC HCl salt (13.4 g, 70.0 mmol, 1.15 eq.) were dissolved in DCM (300 ml) in a RBF (1000 ml) equipped with a magnetic stir bar. Linoleic acid (17.1 g, 60.9 mmol, 1.0 eq.) and DMAP (1.49 g, 12.2 mmol, 0.2 eq.) were added and the clear / colorless solution was stirred at ambient temperature under Nitrogen gas. Next day, IPC (LCMS) showed complete conversion to the product. The reaction mixture was washed with H2O (150 ml), saturated NaHCO3 solution (150 ml) and saturated brine (150 ml). The extracted aqueous solution was washed with DCM (~50 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (~58 g). The crude oil was purified by ISCO MPLC using a Si column (330 g), SLC (65 g) and a (0-40) % Hexane / Ethyl Acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 1 as clear colorless oil (23.92 g, 83.4 % Yield).

[0193] Synthesis of Intermediate 2 in Scheme 21 (Step 2): Intermediate 1 (5.00 g, 10.7 mmol, 1.0 eq), EDC HCl salt (2.57 g, 13.4 mmol, 1.25 eq.) and stearic acid (3.50 g, 12.3 mmol, 1.15 eq.) were dissolved in DCM (50 ml) in a RBF (200 ml) equipped with a magnetic stir bar. DMAP (261 mg, 2.14 mmol, 0.2 eq.) was added and the clear / colorless solution wasNDJP.023WO2 PATENT stirred at ambient temperature under Nitrogen gas. Next day, IPC (LCMS) showed complete conversion to the product. The reaction mixture was washed with H2O (100 ml) and saturated brine (100 ml). The extracted aqueous solution was washed with DCM (~50 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil (~8 g). The crude oil was purified by ISCO MPLC using a Si column (80 g), SLC (25 g) and a (0-10-25) % Hexane / Ethyl Acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 2-C18 / C18:2 as clear colorless oil (7.41 g, 94.3 % Yield).

[0194] Synthesis of Intermediate 3 in Scheme 21 (Step 3): Intermediate 2- C18 / C18:2 1.00 g, 1.36 mmol, 1.0 eq) was dissolved in DCM (10 ml) and methane sulfonic acid (MSA, 265 μl, 4.09 mmol, 3.0 eq) was added in a scintillation vial (20 ml) equipped with a magnetic stir bar. The clear / colorless solution was allowed to stir overnight at ambient temperature under N2 gas. Next day, IPC (LCMS) confirmed that the reaction was complete and Intermediate 3 (assumed quantitative yield) was carried forward without any further refinement.

[0195] Synthesis of Compound 38 in Scheme 21 (Step 4): The scintillation vial, containing Intermediate 3 in DCM (10 ml), was placed into an ice-bath where N,N- Dimethylglycine HCl salt (237 mg, 1.70 mmol, 1.25 eq) and EDC HCl salt (391 mg, 2.04 mmol, 1.5 eq) was added followed by dropwise addition of Diisopropylethylamine (DIEA, 726 μl, 4.08 mmol, 3.00 eq.). DMAP (33 mg, 0.272 mmol, 0.2 eq) was also added. When the addition was complete, the ice bath was removed, and the reaction mixture was allowed to warm to ambient temperature. Next day, IPC (LCMS) confirmed that the reaction was complete. The reaction mixture was washed with H2O (20 ml) and saturated brine (20 ml). The extracted aqueous solution was washed with DCM (~30 ml). The organics were combined, dried with Na2SO4, filtered and concentrated under reduced pressure using a rotary evaporator to yield crude oil. The crude oil was purified by ISCO MPLC using a Si column (40 g), SLC (12 g) and a (0-100) % Hexane / Ethyl Acetate gradient followed by (0-25) % DCM / MeOH gradient. Fractions were combined and concentrated via rotary evaporator to yield Compound 38 as clear colorless oil (394 mg, 40.2 % Yield).NDJP.023WO2 PATENT

[0196] The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0197] The above description discloses several methods and materials of the present disclosure. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it covers all modifications and alternatives coming within the true scope and spirit of the invention.

[0198] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

Claims

NDJP.023WO2 PATENT WHAT IS CLAIMED IS:

1. A lipid nanoparticle comprising a cationic lipid, a sterol, a phospholipid, and a polyalkylene glycol-modified lipid, wherein the lipid nanoparticle comprises: 20 to 60 mol% of the cationic lipid based on the total lipid content of the lipid nanoparticle; 15 to 50 mol% of the sterol based on the total lipid content of the lipid nanoparticle; 5 to 40 mol% of the phospholipid selected from the group consisting of 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3- phosphorylethanolamine (DSPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), based on the total lipid content of the lipid nanoparticle; 0.5 to 10 mol% of the polyalkylene glycol-modified lipid selected from the group consisting of N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE-PEG), N-[carbonyl-methoxypolyethylene glycol]-1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG), and N-[carbonyl- methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE- PEG), based on the total lipid content of the lipid nanoparticle; and 0.5 to 10 mol% of a compound represented by Formula II, based on the total lipid content of the lipid nanoparticle,NDJP.023WO2 PATENT 2. The lipid nanoparticle of claim 1, wherein the cationic lipid is selected from the group consisting of Compound Nos. 30 to 38 having the structures shown in the following Table: Table83NDJP.023WO2 PATENTNDJP.023WO2 PATENT 3. The lipid nanoparticle of claim 1 or 2, wherein the sterol is cholesterol.

4. The lipid nanoparticle of any one of claims 1-3, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

5. The lipid nanoparticle of any one of claims 1-4, wherein the polyalkylene glycol-modified lipid is N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE-PEG).

6. The lipid nanoparticle of any one of claims 1-5, wherein the lipid nanoparticle comprises: 20 to 40 mol% of the cationic lipid based on the total lipid content of the lipid nanoparticle; 20 to 40 mol% of cholesterol based on the total lipid content of the lipid nanoparticle; 20 to 40 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) based on the total lipid content of the lipid nanoparticle; 0.5 to 10 mol% of N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE-PEG) based on the total lipid content of the lipid nanoparticle; and 0.5 to 10 mol% of the compound represented by Formula II based on the total lipid content of the lipid nanoparticle.

7. The lipid nanoparticle of any one of claims 1-5, wherein the lipid nanoparticle comprises: 25 to 35 mol% of the cationic lipid based on the total lipid content of the lipid nanoparticle; 25 to 35 mol% of cholesterol based on the total lipid content of the lipid nanoparticle; 25 to 35 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) based on the total lipid content of the lipid nanoparticle;NDJP.023WO2 PATENT 2.5 to 7.5 mol% of N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE-PEG) based on the total lipid content of the lipid nanoparticle; and 2.5 to 7.5 mol% of the compound represented by Formula II based on the total lipid content of the lipid nanoparticle.

8. The lipid nanoparticle of any one of claims 1-5, wherein the lipid nanoparticle comprises the cationic lipid, cholesterol, DOPE, DPPE-PEG2000, and the compound represented by Formula II in a ratio of 30:30:30:5:5 mol%.

9. The lipid nanoparticle of any one of claims 1-8, wherein a nucleic acid is encapsulated in the lipid nanoparticle.

10. A method of delivering a nucleic acid to a cell, comprising contacting the cell with the lipid nanoparticle of claim 9.

11. A method of delivering a nucleic acid to a subject in need thereof, comprising administering to the subject the lipid nanoparticle of claim 9.

12. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 1-9, and a pharmaceutically acceptable carrier.

13. A cationic lipid selected from the group consisting of Compound Nos. 31 to 38 having the structures shown in the following Table:NDJP.023WO2 PATENT TableNDJP.023WO2 PATENT.

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