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.023WO 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.NDJP.023WO PATENT

[0005] There is a continuing need for lipid molecules for efficient delivery of nucleic acids and other agents to target cells and organs. 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: -NDJP.023WO PATENT wherein the arrow indicates a bond to A1, wherein n4 is 1, 2, 3, 4, or 5, wherein n5 is 0, 1, 2, 3, 4, or 5, wherein B2is a 5- or 6-membered nitrogen-containing saturated aliphatic heterocyclic 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 optionally substituted by a hydroxy group, 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.023WO 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 44 shown in the following Table 1: Table 1 Compound No. Structure4NDJP.023WO PATENTNDJP.023WO PATENTNDJP.023WO PATENTNDJP.023WO PATENTNDJP.023WO PATENTNDJP.023WO PATENTNDJP.023WO PATENTNDJP.023WO PATENT.

[0014] Embodiment [7]. A lipid nanoparticle comprising the compound of any one of Embodiments [1] to [6] or pharmaceutically acceptable salt thereof.NDJP.023WO PATENT

[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), and wherein the lipid nanoparticle further comprises a compound of Formula IINDJP.023WO PATENT .

[0011] , wher cally ; OPE,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 O O OO O OO H HON O O N N N O OO NN H H O O O O O O ONH HN ONOON O OO OO O O O Formula II.

[0020] Embodiment

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

[0012] , wherein a nucleic acid is encapsulated in the lipid nanoparticle.NDJP.023WO PATENT ll, c ed th C a c lt thFormula 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: - X R11R111 n4 N n4N R 21 B2R2 13, R andn5,NDJP.023WO PATENT hatic at is and lipid and s to a lipid rtain cond zable moie lipid may ipid” refer ange.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, nonadecadienyl, icosadienyl, decatrienyl, undecatrienyl, dodecatrienyl, tridecatrienyl,NDJP.023WO PATENT te l, no l, C l, C l, C 9 al l, C he gr d w[ ] n one em o ment, s t e same as . n anot er em o ment, is 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.

[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 aNDJP.023WO PATENT C1 to C4 alkyl. As used h “ trogen-containing saturated aliphatic heterocyclyl” refe atic heterocyclyl having at least one nitrogen atom. taining saturated aliphatic heterocyclyl includes, but olidinyl, 3-pyrrolidinyl, 1- piperidinyl, 2-piperidinyl, 3 nyl, 4-thiomorpholinyl, and 1-piperazinyl. In some em trogen-containing saturated aliphatic heterocyclyl selecR17R14R14N N NN R16 N N R16 N O- R15R15, , , , , X , R17R17NN O- - X , and X 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. [003 oup consisting of: N, , , 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, isoxazolyl, triazolyl and isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. In some embodiments, B2isNDJP.023WO PATENT N N , 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 these production methods and appropriately selecting raw material compounds, reagents, andNDJP.023WO PATENT reaction conditions, one skilled in the art can easily produce any lipids included in the range of general formula I.[ ] n t e const tuent p s o t e p nanopart c es accor ng to t e 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 (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-NDJP.023WO PATENTGlycolipids 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, d i ti id d nanoparticles according to the present dsc osure, n a t on to t e cat on c p s o t e 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 r 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.

[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,NDJP.023WO PATENT 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, 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,NDJP.023WO PATENT 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.023WO PATENT O O OO H O OO HON N O O O N N OO NN H H O O O O O O ONH HN ON O O N O OO OO O O O Formula II

[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.023WO 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.023WO 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.023WO PATENT toco arged subs sitive char h as stear can inclu rine, phos mple, mem these subs o the purp esent discl less. As u erage parti light scatt ment, etc. o thepresent 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.023WO PATENT m nt is he pr A, or or ph es ac e- st id na in th in th ed in ic D g of ay be he ge

[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.023WO 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,NDJP.023WO 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.023WO 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. EXAMPLES AbbreviationsEDC·HCl: N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hydrochlorideNDJP.023WO PATENT DMAP: 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 Example 1: Synthesis of cationic lipids Synthesis of Compounds 1, 2, 3, 6, 7, 8, 24, 25, 26, 27 of Table 1

[0080] Compound 1 was prepared according to Scheme 1. HO O O O NBoc + Et3NCl CHCl3 N BocO HO Intermediate 1 O O O OO O OOOMSA . OH DCMNHMSA N DCM, DIEAO O O O Intermediate 2 O O NH O N2O H N N N EDC, DMAP & DCM O O O 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 chloride (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 pressureNDJP.023WO PATENT 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 N2gas. 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 wasexchanged 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 inNDJP.023WO PATENT 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. OHOSiSi SiOHN OOBr Si SiHNClOBocClN O N O N OHO SiHSiNH MSASNH oOiC N Boc DCMO 2Pyr, O to RT DCM, Et3Intermediate 3Scheme 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). For 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.NDJP.023WO PATENT

[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 N2 gas. 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 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 andNDJP.023WO PATENT 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)+.

[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)+.NDJP.023WO PATENT OH as O as the 1- asoil. ESI MS (m / z): 825.66 (M+H)+. Synthesis of Compounds 5, 13, 14 of Table 1

[0099] Compound 13 was prepared according to Scheme 4. O O O O O 1. MSA, DCM OH BocN N O2. Et3N, succinic anhydrideO O O O Intermediate 1 Intermediate 2 H N 2NO O O H N HATU, DIEA N N DCM O O O Scheme 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. WONDJP.023WO PATENT 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 using 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

[0104] Compound 9 was prepared according to Scheme 5. O O HO NHBoc O O MSA N NHBoc N Boc O DCMEtN, EO O O3 DC.HCl, DMAPO O Intermediate 2 Intermediate 1 O O 1. MSA, DCM H N N N 2. EDC.HCl, DIEA, DMAP O OO OHO N O Scheme 5NDJP.023WO PATENT

[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 ph , and the crude product was p mn was eluted with (0-60)% ncentrated via rotary evapor (m / z): 697.70 (M+H)+

[0107] ) was added to a solution of I5 ml) under N2gas. 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, 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 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 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.)NDJP.023WO PATENT 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

[0109] Compound 11 was prepared according to Scheme 6. O O HO ONHBoc OMSA O N Boc N NHBoc DCM O Et3N, EDC.HCl, DMAP O O O Intermediate 2 O Intermediate 1 O 1. MSA, DCM O O 2. EDC.HCl, DIEA, DMAP N N N O O O H HO N O 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) 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 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 (0-60)% 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 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 amino propionic acid (102 mg, 0.87, 1.25NDJP.023WO PATENT 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 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 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)+. Synthesis of Compounds 15, 16, 17, and 18 of Table 1

[0114] Compound 15 was prepared according to Scheme 7. O O O HO Oleic Acid O EDC.HCl, DMAP MSA, DCM HN BocN BocN O HO O O O Intermediate 1 Intermediate 2O O O O O O N OH2NOON N N OH N Et3N O O EDC, O O H HCl, DMAP DCM O O 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 driedNDJP.023WO PATENT 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 t. Fractions were concentrated and .0 yield).

[0116] old solution of intermediate 1 in methane sulfonic acid (2.48 g, 21 r 1 hour. LC / MS showed that the aturated Na2CO3(100 mL). The c ). The combined organic phase w d under reduced pressure using an 100% yield).

[0117] of Intermediate 2 in Scheme 7 (4.4 2 mL, 8.7 mmol)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 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 (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 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.023WO PATENT methanol grad cuum to yield Compound 15

[0119] 7 (0.48 g, 0.65 mmol) was dis ol) and DMAP (0.04 g, 0.033 g, 0.68 mmol) was added to m temperature overnight. Th . Solvent was evaporated un g residue was suspended in er (30 mL) and brine solution d. Solvent was concentrated u was purified by flash chromato0-5) % DCM – 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 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 – 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 dilutedNDJP.023WO PATENT 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 18 (120 mg, 56 % yield). ESI MS (m / z) 860.98 (M+ H)+. Synthesis of Compound 19 of Table 1

[0122] Compound 19 was prepared according to Scheme 8. (Z) O OH (Z)Int [0 heme 8 is the same mmol) in anhydrou d, and the reaction complete deprotectly 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,NDJP.023WO PATENT 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 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

[0125] Compound 20 was prepared according to Scheme 9. O O OH O OH O O 1. MSA, DCM NO N OEDC.HCl, DMAP (cat) 2. Et3N, O DCM OH O O Intermediate 1 O O O O O O O OH DIEA, HATU, DCMO H N N O N N N H2N O O O O O Intermediate 2Scheme 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.NDJP.023WO PATENT Fraction diate 1 as solid (6g [ mediate 1 in Schem mol, 2eq) was adde onfirmed complete succinic anhydrid ture. The reaction ction was complete as added to the m M (2x25 mL). Th nic phase was drie provide Intermed

[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 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 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 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.023WO PATENT Synthesis of Compound 22 of Table 1

[0130] Compound 22 was prepared according to Scheme 10. O O HO Oleic Acid O O EDC.HCl, D MSA, DCM BocN MAP BocN HN HO O O O O Intermediate 1 Intermediate 2O O OO N O H N O2O H N OH N N Et3N N DCM O O O O EDC,HCl, DMAP O O O DCM O Intermediate 3 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 ,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 ed with water (50 mL) and O4 and filtered. Solvent was ude product was purified by luted with a (0-5) % DCM –NDJP.023WO 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

[0133] Compound 23 was prepared according to Scheme 11. (Z) (Z) O (Z) (Z) O O O O 1. MSA, DCM BocN NHBoc N O2.NEBt3oNc,GHlAycTiU,O (Z) (Z) neO(Z) (Z)O Intermediate 1 Intermediate 2 (Z) (Z) O 1. MSA, DCM O O H N N N 2. DIEA, EDC.HCl O O HO N(Z) (Z)O O 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, 2 eq), and diisopropylethylamine (117 , 0.66 mmol, 2eq) were sequentially added. After 15NDJP.023WO PATENT 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

[0136] Compound 28 was prepared according to Scheme 12. O HO O 1. MSA, DCM Et3N, DCM NBoc NBoc O Myristoyl chloride2. Et3N,O Succinic anhydride HO Intermediate 1 O O O OH2NO O H OHNN N N N O O HATU, DIEA, DCM O O O O 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 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- 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)+.

[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) wasNDJP.023WO PATENT 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 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 and filtered. Solvent wa tem using a silica colum nd (0-20)% DCM-MeO aporator to yield CompNDJP.023WO PATENT Synthesis of Compound 29 of Table 1

[0140] Compound 29 was prepared according to Scheme 13. HO O EDC.HCl, DMAP O NBoc 1. MSA, DCM NBoc DCM O 2. DCM, DIEA, HATU HO 2-hexyloctanoic acid O O NHBoc Intermediate 1 HO O O O O 1. MSA, DCM OO HNHBoc N N N N O 2. DCM, DIEA, HATU O O O HO O N Intermediate 2 O 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 Na2SO4 and 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 aqueous NaHCO3 (20 mL), water (20 mL), and brine (20 mL). The organic phase was dried withNDJP.023WO PATENT 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 acetatedried 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 29 as solid (63 mg, 44% yield). ESI MS (m / z): 752.7 (M+H)+.

[0144] Synthesis of Compound 30 of Table 1: Compound 30 was prepared according to Scheme 14. O O OO HOONHNN Methyl iodide NN N O O I O O 1 O O30Scheme 14

[0145] To a suspension of Compound 1 (170 mg, 0.24 mmol) in acetonitrile (2 mL), methyl iodide (0.5 mL, excess) was added. The reaction mixture was stirred overnight. Next day, LC / MS showed the formation of desired product. The solvent was concentrated and the reaction mixture was purified by ISCO / MPLC system using a silica column (25 g). ColumnNDJP.023WO PATENT 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 30 as solid (105 mg, 52% Yield). ESI MS (m / z): 710.11 (M-I)+

[0146] Synthesis of Compounds 43 and 31 of Table 1: Compound 31 was prepared according to Scheme 15. O O O O O N O N HO N N OH O O O HATU, DIEA O O DCM O 43 O Intermediate 2 O O 1. MeI, acetonitrile, RT O Cl N N O Ionexchange O O resin [HCl] 31 O Scheme 15 Synthesis of Compound 43 in Scheme 15: To a mixture of Intermediate 2 in Scheme 1 (400 mg, 0.63 mmol, 1eq), N,N’-dimethyl aminoethanol (85 mg, 0.95 mmol, 1.5 eq) in anhydrous DCM (20 mL), HATU (359 mg, 0.94 mmol, 1.5eq) and DIEA (0.25 mL, 1.26, 2 eq mmol) were added at room temperature. The resulting solution was stirred at ambient temperatures under N2gas. Next day, LC / MS showed that the reaction was complete conversion. The reaction mixture was diluted with DCM (20 mL) and then washed with water (20 ml) and brine (20 mL). The solvent was concentrated, and the residue 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 43 as solid (208 mg, 46% yield). ESI MS (m / z): 697.7 (M+H)+

[0147] To a suspension of Compound 43 (300 mg, 0.43 mmol, 1eq) in acetonitrile (0.5 mL), MeI (0.5 mL, 4.3 mmol) was added. The resulting mixture was stirred at room temperature 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 purified on ISCO MPLC using 24 g silica column. Column was eluted with (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated to yield Intermediate as iodide counterNDJP.023WO PATENT ion. This intermediate was dissolved to a minimum volume of DCM and passed through a column of ion exchange anion resin (activated with HCl). Column was eluted with 100% DCM uum ESI ared OH Br N le (1mL), bromoethanol (0.7 mL, 10.0 mmol) was added at room temperature. The reaction flask was heated at 65-70oC and stirred overnight under N2 gas. Next day, LC / MS showed that most of the starting material was completely converted to bromide salt. The solvent was evaporated, and the residue was purified on ISCO MPLC using 40 g silica column. Column was eluted with (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated to yield Compound 32 as bromide counter ion. (560 mg, 68% yield). MS ESI (m / z): 741.42 (M-Br)+

[0150] Synthesis of Compound 33 of Table 1: Compound 33 was prepared according to Scheme 17. O O O O 1. MSA, DCM H N NHBoc N N N 2. DIEA, EDC.HCl O O O O O DMAP, N,N'-dimethylglycine O 44 O Intermediate 2 O in Scheme 5 O 1. MeI H 40, acetonitrile, oCN N N 2.Anion resin (HCl)O O OCl33 O Scheme 17NDJP.023WO PATENT Synthesis of Compound 44 in Scheme 17: MSA (93 1.43 mmol, 2.0 eq) was added to a solution of Intermediate 2 in scheme 5 (500 mg, 0.71 mmol, 1eq) in DCM (5 ml) 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 (124 mg, 0.89 mmol, 1.25 eq.), EDC.HCl (205 mg, 1.07 mmol, 1.5 eq), and DIEA (0.63 mL, 3.5 mmol, 5eq) were sequentially added. After 15 min, the ice-bath was removed, and the reaction was stirred overnight. LC / MS confirmed the 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 Na2SO4 and filtered. Solvent was concentrated, and the crude product was purified by ISCO / M ith (0-100)% Hexane-ethy ombined and concentrated % yield). ESI MS (m / z): 6 [015 n acetonitrile (0.5 mL), M 40oC stirred overnight. N converted to methyl iodid ied on ISCO MPLC usin OH gradient. Fractions w ter ion. This intermediate a column of anion ion ex DCM and all fractions we high vacuumto give Compound 33 as chloride counter ion (90 mg, 42% yield). MS ESI (m / z): 696.7 (M- Cl)+

[0152] Synthesis of Compounds 44 and 34 of Table 1: Compound 34 was prepared according to Scheme 18.NDJP.023WO PATENT O O O OH H O Bromoethanol N N H NoN N N O O O acetonitrile, 65 C O O O 44 O Br O 34 Scheme 18

[0153] To a suspension of Compound 44 (300 mg, 0.43 mmol) in acetonitrile (3 mL), bromoethanol (0.3 mL, 4.3 mmol) was added at room temperature. The reaction flask was heated at 65-70oC and stirred overnight under N2gas. Next day, LC / MS showed that most of the starting material was completely converted to bromide salt. The solvent was evaporated, and the residue was purified on ISCO MPLC using 40 g silica column. Column was eluted with (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated to yield Compound 34 as bromide counter ion (220 mg, 68% yield). MS ESI (m / z): 726.68 (M-Br)+

[0154] Synthesis of Compound 35 of Table 1: Compound 35 was prepared according to Scheme 19. O O O O1.MeI, acetonitrileO N N 2.An O N ion resin (HCl) H N N O O N O H O O Cl 15 O 35 Scheme 19

[0155] To a suspension of Compound 15 (320 mg, 0.39 mmol, 1eq) in acetonitrile (2 mL), MeI (0.5 mL, excess) was added. The resulting mixture was stirred at room temperature 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 purified by ISCO MPLC using 24 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 anion ion exchange resin (activated with HCl). Column was eluted with 100% DCM and all fractions were combined and concentrated. The obtained product was dried under highNDJP.023WO PATENT v d). M ed ac OH N Br (3 m skwas heated at 65-70oC and stirred overnight under N2gas. Next day, LC / MS showed that most of the starting material was completely converted to bromide salt. The solvent was evaporated, and the residue was purified by ISCO MPLC using 40 g silica column. Column was eluted with (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated to yield Compound 36 as bromide counter ion. (310 mg, 68% yield). MS ESI (m / z): 848.79 (M-Br)+

[0158] Synthesis of Compounds 37 and 38 of Table 1: Compounds 37 and 38 were prepared according to Scheme 21. HO O O + TEA O Cl N Boc DCM, rt Intermediate 1 N Boc Palmitoyl chloride Myristoyl chloride HO N-Boc diethanol amine HOEt3N, DCMO O O O O 1. MSA, DCM N N Boc OH 2S.uDcIcEinAi,c anhydrideO O O O O Intermediate 2 Intermediate 3 N H2N EDC.HCl, DMAP DCMN HOEDC.HCl, DMAPDCM O O O O O O N H N N O O O N OO N37 O 38 ONDJP.023WO PATENT Scheme 21

[0159] Synthesis of Intermediate 1 in Scheme 21: To a solution of N-Boc diethanol amine (2.4 g, 121 mmol) in anhyd DCM (100 mL), triethyl amine (11.3 mL, 81 mmol) was added. The round bottom flask was cooled with an ice bath, and then myristoyl chloride (11 mL, 40.5 mmol) was slowly added. The mixture was stirred at room temperature for 4h by which time LC / MS confirmed that the reaction was completed. The reaction was quenched with water (40 mL) and the product was extracted with DCM (1x50 mL). The organic layer was washed with water (50 mL) and brine (50 mL) solution. The organic phase was separated and dried with Na2SO4and filtered. The crude residue was purified by ISCO / MPLC system using a silica column (120 g). Column was eluted with (0-70)% Hexane- Ethyl acetate gradient. Fractions were combined and concentrated via rotary evaporator to yield Intermediate 1 as solid (10.3 g, 61% Yield). ESI MS (m / z): 416.56 (M+H)+

[0160] Synthesis of Intermediate 2 in Scheme 21: To a solution of Intermediate 1 (3.4 g, 8.1 mmol) in anhyd DCM (50 mL), triethyl amine (2.3 mL, 16.3 mmol) was added. The round bottom flask was cooled with an ice bath, and then pulmonoyl chloride (2.6 mL, 9.8 mmol) was slowly added. The mixture was stirred at room temperature for 2h by which time LC / MS confirmed that the reaction was completed. The reaction was quenched with water (20NDJP.023WO PATENT temperature. Next day, LC / MS 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 water (25 mL) and saturated brine solution (25 ml). The extracted aqueous solution was washed with DCM (100 ml), dried with Na2SO4, filtered and concentrated via rotary evaporator to yield crude oil. The precipitationof product was obtained by addition of n-hexanes and placement of material in 5 C cold room.The product was filtered via Buchner funnel and dried fully under high vacuum to yield Intermediate 3 as white crystalline solid (2.5 g, quantitative yield). ESI MS (m / z): 654.71 (M+H)+

[0162] Synthesis of Compound 37 in Scheme 21: To a mixture of Intermediate 3 (1.2 g, 1.8 mmol), N,N-dimethylenediamine (240 mg, 2.7 mmol) in anhydrous DCM (20 mL), EDC.HCl (500 mg, 2.7 mmol) and DMAP (22 mg, 0.18 mmol) were added at room temperature. The resulting solution was stirred at ambient temperatures under N2gas. Next day, LC / MS showed that the reaction was complete conversion. The reaction mixture was diluted with DCM (20 mL) and then washed with water (20 ml) and brine (20 mL). The solvent was concentrated and the residue 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 37 as solid (750 mg, 57% Yield). ESI MS (m / z): 724.76 (M+H)+

[0163] Synthesis of Compound 38 in Scheme 21: To a mixture of Intermediate 3 (1.2 g, 1.8 mmol), N,N-dimethylethanol (240 mg, 2.7 mmol) in anhydrous DCM (20 mL), EDC.HCl (500 mg, 2.7 mmol) and DMAP (22 mg, 0.18 mmol) were added at room temperature. The resulting solution was stirred at ambient temperatures under N2gas. Next day, LC / MS showed that the reaction was complete conversion. The reaction mixture was diluted with DCM (20 mL) and then washed with water (20 ml) and brine (20 mL). The solvent was concentrated and the residue 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 38 as solid (860 mg, 66% Yield). ESI MS (m / z): 725.76 (M+H)+NDJP.023WO PATENT

[0164] Synthesis of Compound 39 of Table 1: Compound 39 was prepared according to Scheme 22. O O O O O 1.MeI, ac O N H etonitrile N 2.Anion resin (HCl) N H O NO N OO N37 O 39 OClScheme 22. , converted to methyl iodide salt. The excess MeI was evaporated and the residue was purified by ISCO MPLC using 24 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 anion ion exchange resin (activated with HCl). Column was eluted with 100% DCM and all fractions were combined and concentrated. The obtained product was dried under high vacuum to give a desired product Compound 39 as chloride counter ion (161 mg, 75% yield). MS ESI (m / z): 738.78 (M-Cl)+

[0166] Synthesis of Compound 40 of Table 1: Compound 40 was prepared according to Scheme 23. OH N

[0067] o a suspens on o Compoun 37 (350 mg, 0. 8 mmo ) n aceton tr e (2 mL), bromoethanol (0.34 mL, 4.8 mmol) was added at room temperature. The reaction flask was heated at 65-70oC and stirred overnight under N2 gas. Next day, LC / MS showed that mostNDJP.023WO PATENT of the starting material was completely converted to bromide salt. The solvent was evaporated and the residue was purified by ISCO MPLC using 40 g silica column. Column was eluted with (0-20)% DCM-MeOH gradient. Fractions were combined and concentrated to yield Compound 40 as bromide counter ion (220 mg, 54% yield). MS ESI (m / z): 768.82 (M-Br)+

[0168] Synthesis of Compound 41 of Table 1: Compound 41 was prepared according to Scheme 24. O O O O O N 1.MeI, acetonitrile O O 2.Anion resin (HCl) N O O N O O OO N38 Cl 41O Scheme 24

[0169] To a suspension of Compound 38 (250 mg, 0.34 mmol, 1eq) in acetonitrile (2 mL), MeI (0.2 mL, mmol) was added. The resulting mixture was stirred at room temperature 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 purified on ISCO MPLC using 24 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 anion ion exchange resin (activated with HCl). Column was eluted with 100% DCM and all fractions were combined and concentrated. The obtained product was dried under high vacuum to give a desired product Compound 41 as chloride counter ion (118 mg, 45% yield). MS ESI (m / z): 739.74 (M-Cl)+

[0170] Synthesis of Compound 42 of Table 1: Compound 42 was prepared according to Scheme 25. O O O O O N bromoethanol O OH O N O O N acetonitroO ile, 65-70 C O 38 OO NBr 42 ONDJP.023WO PATENT [01 etonitrile (2 mL), brom action flask was heated d that most of the start evaporated and the res was eluted with (0-20 ed to yield Compound (M-Br)+Example 2: In vitro expression of Fluc mRNA

[0172] LNP formulations with Fluc mRNA were prepared with the following compositions: (Test compound / cholesterol / DSPC / DMG-PEG2000) = 50 / 38 / 10 / 2 (mol%)

[0173] 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.

[0174] 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 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 2 LNP Formulation cationic lipid PS (nm) PDI %EE yield (%) LNP 1a Compound 1 98 0.171 96 45 LNP 2a Compound 2 98 0.163 93 47NDJP.023WO PATENT LNP F d (%) LNP 3 LNP 4 LNP 5 LNP 6 LNP 7 LNP 8 LNP 9 LNP 1 LNP 1 LNP 1 LNP 1 LNP 1 LNP 1 LNP 1 LNP 1 LNP 18a Compound 18 129 0.195 93 86 LNP 19a Compound 19 163 0.095 79 82 LNP 20a Compound 20 143 0.142 99 70 LNP 21a Compound 21 143 0.154 99 74 LNP 22a Compound 22 181 0.111 93 72 LNP 23a Compound 23 144 0.141 78 80 LNP 24a Compound 24 214 0.176 99 73 LNP 25a Compound 25 225 0.143 100 73 LNP 26a Compound 26 181 0.161 97 83 LNP 27a Compound 27 163 0.116 96 77 LNP 28a Compound 28 152 0.149 97 79 LNP 29a Compound 29 183 0.135 97 70 LNP R1 HEDC 110 0.174 95 60 LNP R2 HEDC-M1 245 0.170 98 41

[0175] In vitro expression of Fluc mRNA were measured according to the following protocol 1 or 2: Protocol 1:

[0176] 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 usedNDJP.023WO PATENT 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% 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) 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 cence values were obtaine 4-parameter logistic mode uding one of Compounds Protocol 2: [0177 upplemented with 10% HI 004), EMEM media (ATC ) were used respectively.n ay , ce s were p a e n w e opaque -we - rea e plates (USA 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% CO2for 24 hours. On day 2, the ONE-Glo EX Luciferase Assay System (Promega Ref # E8130) was equilibrated to room temperature and combined. Plates were prepared according to Promega kit guidelines by adding 3 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-responseNDJP.023WO PATENT curve e LNP formul ording to Prot Table LNP Form LNP LNP LNP LNPLNP 5a 20 4 60 LNP 6a 30 300 104200 LNP 7a 170 510 70 LNP 8a 80 640 70 LNP 9a 70 20 40 LNP 10a 460 900 60 LNP 11a 250 140 100 LNP 12a 60 10 80 LNP 13a 380 20 900 LNP 14a 510 510 130 LNP 15a 210 130 90 LNP 16a 60 100 140 LNP 17a 1660 140 130 LNP 18a 390 260 140 LNP 19a 110 1330 200 LNP 20a 3 50 40 LNP 21a 140 760 10 LNP 22a 4310 330 120 LNP 23a 70 3350 3090 LNP 24a 170 40 40 LNP 25a 300 400 170 LNP 26a 110 110 50 LNP 27a 340 110 190 LNP 28a 290 100 140 LNP 29a 100 80 50 LNP R1a 90 30 240 LNP R2a 250 10 ND ND: No DataNDJP.023WO PATENT o A549 cells, H a, LNP 6a, LN he EC50 value w . When LNP 3a 50 value was 1. LNP 1a, LNP 2a NP 14a, LNP 1 NP 24a, LNP 25 he EC50value was about 1.2 to 24 -fold lower than when LNP R1a was applied to the same cells.

[0179] 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 EC50 value 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 EC50value was about 1.4 to 2.5-fold lower than when LNP R2a was applied to the same cells. Example 3: In vitro expression of Fluc mRNA

[0180] 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%)

[0181] 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.NDJP.023WO PATENTILMTMUser Guide by Precision NanoSystems. Table 4 LNP Formulation cationic lipid PS (nm) PDI %EE yield (%) LNP 1b Compound 1 87 0.161 96 87 LNP 2b Compound 2 98 0.195 95 77 LNP 3b Compound 3 83 0.171 76 85 LNP 4b Compound 4 95 0.179 72 59 LNP 5b Compound 5 84 0.179 82 80 LNP 6b Compound 6 75 0.168 95 70 LNP 7b Compound 7 74 0.171 97 69 LNP 8b Compound 8 81 0.140 91 85 LNP 10b Compound 10 81 0.154 97 81 LNP 11b Compound 11 82 0.136 97 77 LNP 12b Compound 12 85 0.163 86 73 LNP 13b Compound 13 89 0.152 86 60 LNP 14b Compound 14 92 66 92 66 LNP 15b Compound 15 177 0.121 75 75 LNP 16b Compound 16 174 0.149 80 81 LNP 17b Compound 17 155 0.137 87 73NDJP.023WO PATENT LNP Formulation cationic lipid PS (nm) PDI %EE yield (%) LNP 18b Compound 18 143 0.221 78 110 LNP 19b Compound 19 169 0.166 51 94 LNP 21b Compound 21 143 0.208 89 68 LNP 22b Compound 22 138 0.248 76 72 LNP 23b Compound 23 159 0.134 60 80 LNP 24b Compound 24 147 0.154 93 94 LNP 26b Compound 26 139 0.116 86 89 LNP 27b Compound 27 161 0.137 86 85 LNP 28b Compound 28 157 0.159 77 84 LNP 29b Compound 29 199 0.175 78 69 LNP R2b HEDC-M1 157 0.159 77 84

[0183] 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 5 LNPEC50 (nM) EC50 (nM) EC50 (nM)Formulation in A549 in HEP3B in Panc-1 LNP 1b 4.40 0.23 ND LNP 2b ND 0.47 ND LNP 3b 0.60 0.33 0.20 LNP 4b 1.44 0.98 0.80 LNP 5b 0.24 0.14 0.20 LNP 6b 1.04 0.30 0.70 LNP 7a 0.34 0.91 0.42 LNP 8b 1.73 0.62 ND LNP 10b ND 0.99 0.71 LNP 11b 0.82 0.26 1.07 LNP 12b 0.30 0.27 0.21 LNP 13b 0.15 0.16 0.52 LNP 14b 0.05 0.08 0.17 LNP 15b 0.08 0.47 0.20 LNP 16b 0.06 0.08 0.18 LNP 17b 0.14 0.23 0.23NDJP.023WO PATENT LNPEC50 (nM) EC50 (nM) EC50 (nM)Formulation in A549 in HEP3B in Panc-1 LNP 18b 0.41 0.31 0.17 LNP 19b 2.28 0.32 0.14 LNP 21b 1.04 1.24 0.09 LNP 22b 0.20 0.72 0.14 LNP 23b 0.60 63.00 25.00 LNP 24b 1.28 1.12 0.77 LNP 26b 0.85 0.75 1.17 LNP 27b 0.55 1.65 0.63 LNP 28b 5.07 2.64 0.29 LNP 29b 0.08 0.03 0.33 LNP R2b 0.87 0.87 1.09 ND: No Data

[0184] 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 EC50 value was about 1.1 to 17-fold lower than when LNP R2b was applied to the same cells When LNP 1b, LNP2b, LNP 3b, LNP P15b, LNP16b, LNP17b, LNP EP3B cells, the EC50value wa the same cells. When LNP3b 13b, LNP14b, LNP15b, LN 24b LNP27b, LNP28b or L 1.4 to 12 -fold lower than wh

[0185] T e term compr s ng as used ere n s synonymous w th “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0186] 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 practiceNDJP.023WO PATENT 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.

[0187] 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 precedencNDJP.023WO PATENT WHAT IS 1. of: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: - X R11R114N 12n N n4R2R12 13, R andn5B , wherein the arrow indicates a bond to A1, wherein n4 is 1, 2, 3, 4, or 5, 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 optionally substituted by a hydroxy group,NDJP.023WO PATENT lkyl, and 2. of, wherein n3 is 1, 2, 3. of, wherein R1and R24. of, wherein R1and R2, , , , and , wherein the arrow indicates a bond to C(O). 5. The compound of claim 1 or pharmaceutically acceptable salt thereof, wherein B2is selected from the group consisting of: R14N16R15NN R,, and , 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. 6. The compound of claim 1 or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of Compound Nos. 1 to 44 shown in the following Table 1:NDJP.023WO PATENT Table 1 Compound No.StructureO O O H N 1 N NNDJP.023WO PATENT Compound No.StructureO O O 6 N N N OOHO O O O 7 N N N OOHO O O O 8 N N N OOHO O O H 9 N N N OO OO O O H 10 N N N O O O ONDJP.023WO PATENT Compound No.StructureO O O N 11 N N O O H O O O O N N 12 N O O H ONDJP.023WO PATENTN 17 O O H O O O O N N N 18 O O H O O OO ON N N H 19 O O O O O H 20NN N O O ONDJP.023WO PATENT Compound No.StructureO O O H 21NN NH N N N 23 O O O O O O N N N 24 H O ONDJP.023WO PATENT C28 O O O O OO HN N NNDJP.023WO PATENTO OO ClO 34 O O OH H N N N O O O Br O 35 O O O N N N O H O Cl ONDJP.023WO PATENT Compound No.Structure36 O OH O O N N N H O O Br O 37 O O O N H N OO NO 38 O O O N O OO NO 39 O O O N H N OO NOCl40 O O O OH N H N OO NBr O

Claims

NDJP.023WO PATENT Compound No.Structure41 O O O O ONCl 42 OH OO NBr O 43 O O O O N N O O O 44 O O H N N O.

7. A lipid nanoparticle comprising the compound of claim 1 or pharmaceutically acceptable salt thereof.

8. The lipid nanoparticle of claim 7, further comprising a sterol, a phospholipid, and a polyalkylene glycol-modified lipid.

9. The lipid nanoparticle of claim 8, wherein the phospholipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-NDJP.023WO PATENT 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).

10. The lipid nanoparticle of claim 8, wherein the lipid nanoparticle comprises: 40-60 mol% of the compound of claim 1 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; DOP total 11.e p nanopart c e o c a m , w ere n t e p osp o p s se ecte rom t e 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), is selected from the group cons ,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), and wherein the lipid nanoparticle further comprises a compound of Formula II O O OO H O OO HON O O N NO NN O O N H H O O O O O O ONH HN ON O O N O OO OO O O O Formula II.NDJP.023WO PATENT 12. The lipid nanoparticle of claim 8, wherein the lipid nanoparticle comprises: alt E, up he he . he ell ngNDJP.023WO PATENT ng7. A lipid nanoparticle comprising the compound of claim 1 or pharmaceutically acceptable salt thereof.

8. The lipid nanoparticle of claim 7, further comprising a sterol, a phospholipid, and a polyalkylene glycol-modified lipid.

9. The lipid nanoparticle of claim 8, wherein the phospholipid is selected from the group consisting of l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1 ,2-dioleoyl-sn-g1ycero-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 -methoxypoly ethylene glycol (DMG-PEG).

10. The lipid nanoparticle of claim 8, wherein the lipid nanoparticle comprises:40-60 mol% of the compound of claim 1 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; and1-5 mol% of the polyalkylene glycol-modified lipid that is DMG-PEG to the total lipid amount of the lipid nanoparticle.

11. The lipid nanoparticle of claim 8, wherein the phospholipid is selected from the group consisting of l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-distearoyl- sn-glycero-3-phosphorylethanolamine (DSPE), and l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamme (DPPE), wherein the polyalkylene glycol-modified lipid is selected from the group consisting of N-[carbonyl-methoxypolyethylene glycol]-! ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE-PEG), N-[carbonyl-methoxypoly ethylene glycol]- 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG), and N-[carbonyl- m ethoxy pol yethy lene glycol] - 1 ,2-distearoy l-sn-glycero-3 -phosph oethanolamine(DSPE-PEG), and wherein the lipid nanoparticle further comprises a compound of Formula IIFormula II.

12. The lipid nanoparticle of claim 8, wherein the lipid nanoparticle comprises:20-60 mol% of the compound of claim 1 or pharmaceutically acceptable salt thereof to the total lipid amount of the lipid nanoparticle;5-50 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-mod ified lipid selected from the group consisting of DPPE -PEG, DMPE-PEG, and DSPE-PEGto the total lipid amount of the lipid nanoparticle, and0.5-10 mol% of the compound of Formula II to the total lipid amount of the lipid nanoparticleFormula II.

13. The lipid nanoparticle of claim 7, wherein a nucleic acid is encapsulated in the lipid nanoparticle.

14. A method of delivering a nucleic acid to a cell, comprising contacting the cell with the lipid nanoparticle of claim 7.

15. A method of delivering a nucleic acid to a subject in need thereof, comprising administering to the subject the lipid nanoparticle of claim 7.

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