Lipid compounds, compositions, and uses thereof

Abstract

The present disclosure provides lipid compounds and compositions (e.g., lipid nanoparticle (LNP) compositions) comprising lipid compounds of the present disclosure. The present disclosure provides methods of delivering an active agent (e.g., polynucleotide) to a cell or tissue in a subject, preferably an extrahepatic cell or tissue, comprising administering to the subject an effective amount of a lipid nanoparticle of the present disclosure, wherein the lipid nanoparticle comprises lipid compounds of the present disclosure and the active agent (e.g., polynucleotide).

Description

LIPID COMPOUNDS, COMPOSITIONS, AND USES THEREOFRELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 653,050, filed May 29, 2024, the entire contents of which are hereby incorporated by reference in their entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure provides lipid compounds and compositions (e.g., lipid nanoparticle (LNP) compositions) comprising lipid compounds of the present disclosure. The present disclosure also provides methods of delivering an active agent (e.g., polynucleotide) to a cell or tissue in a subject, such as an extrahepatic cell or tissue, comprising administering to the subject an effective amount of a lipid nanoparticle of the present disclosure, wherein the lipid nanoparticle comprises lipid compounds of the present disclosure and the active agent (e.g., polynucleotide).BACKGROUND

[0003] Many biologically active agents can be difficult to deliver into cells. These active agents include polynucleotide-based therapies (such as DNA -based therapies or RNA -based therapies like mRNA or siRNA), as well as CRISPR / Cas9-based gene editing therapies. Recently, lipid nanoparticles (LNPs) have been developed as encapsulating vehicles for delivering these types of biologically active agents to cells and tissues of interest. For example, LNPs comprising ionizable lipids can serve as vehicles for delivering biologically active agents across cell membranes and directly into target cells and for directing the active agents to preferred tissues of interest.

[0004] However, LNPs may benefit from improvement, such as extended stability and cellular or tissue specificity. For example, many LNPs strongly favor hepatic cells and tissues, and have limited effectiveness in targeting extrahepatic (i.e., non-hepatic) cells and tissues. A need therefore exists for stable LNPs compositions that effectively target extrahepatic cells and tissues in a subject.SUMMARY

[0005] The present disclosure provides lipid compounds and compositions (e.g., lipid nanoparticle (LNP) compositions) comprising lipid compounds of the present disclosure that may provide, inter alia, improved stability and / or extrahepatic targeting.

[0096] In certain embodiments, the present disclosure provides a lipid compound of the following formula (Formula (I)):or a pharmaceutically acceptable salt thereof; wherein each of R1and Rris independently - (C1-C9 alkylene)-R?; each R5is independently C2-C12 alkyl, C2-C12 alkenyl, -CH(R6)R', - C(O)O-(CH2)P-CH-(R8)R9, -OC(O)-(CH2)q-CH-(R8)R9, or -OC(O)O-(CH2)P-CH(RS)R9; each R° and R'7is independently hydrogen, C7-C12 alkoxy, or C7-C12 alkenoxy, provided that R6and Rzare not both hydrogen; each R8and R9is independently hydrogen, Ci-C 12 alkyl, C2- C12 alkenyl, C2-C12 alkynyl, or C1-C12 alkylsulfide, provided that R8and R9are not both hydrogen and not both C1-C12 alkylsulfide; n is 0-5: p is 0-3: q is 0-3: X1is O, CH2, NH, bl(C 1-C.3 aikyi)d wherein each L is independently -(CH2)m-C(R2)(R2'’)-(CH2)t-, or -(CHzWCs-Cs cycloaikyiene-(CH2)t- , or -(CH2)m-(3 -6-membered heterocyclylene)-(CH?)t-; R2is hydrogen, C1-C12 alkyl, or Ci- C12 alkoxy; R2’ is optionally substituted C1-C12 alkyl, Ci-C’12 alkoxy, optionally substitutedC3-C12 cycloalkyl, (CAC4 alkylene)-(optionally substituted C3-C12 cycloalkyl), optionally substituted C5-C6 aryl, or (C1-C4 alkylene)-(optionally substituted C5-C6 aryl); and m and t are each independently selected from 0-4; and wherein-membered to 8- membered heterocyclyl containing one or more N atoms substituted with CJ-CI2aikyi; R3and R4are each independently Ci-CY alkyl; or R3and R4, together with the nitrogen atom to which they are attached, form a 5-6 membered heterocycle comprising one or more N atoms.

[0007] In certain embodiments, the present disclosure provides a lipid compound of the following formula (Formula (I)):or a pharmaceutically acceptable salt thereof; wherein each of R1and R1is independently - (Cj-C? alkylene)-R5; each R' is independently C2-C12 alkyl, C2-C12 alkenyl, -CH(R6)RZ, ■■each R6and R' is independently hydrogen, C7-C12 alkoxy, or C7-C12 alkenoxy, provided that R° and R' are not both hydrogen; each R° and R9is independently hydrogen, C1-C12 alkyl, C2- C12 alkenyl, C2-C12 alkynyl, or C1-C12 alkylsulfide, provided that R8and R9are not both hydrogen and not both Cj-Ci2 alkylsulfide; n is 0-5; p is 0-3; q is 0-3; X1is O, CH?, NH, N- (C1-C3 alkyl), or X2; X2isor fft, and wherein each L is independently -(CH?)m-C(R2)(R2")- (CH2)t-, or --(CH?)m-C3“C8 cycloalkylene-(CH2)r, or -(CH2)m-(3-6-membered heterocyclylene)-(CH2)t-; R2is hydrogen, C1-C12 alkyl, or C1-C12 alkoxy; R2” is optionally substituted C1-C12 alkyl, C1-C12 alkoxy, optionally substituted C3-C12 cycloalkyl, (C1-C4 alkylene)-(optionally substituted C3-C12 cycloalkyl), optionally substituted C5-C6 aryl, or (Ci- C4 alkylene)-(optionally substituted C5-C6 aryl); and m and t are each independently selected from 0-4; and wherein ¥ isor a 3-membered to 8-membered heterocyclyl containing one or more N atoms substituted with C1-C12 alkyl; R3and R4are each independently Ci-Ce alkyl; or R3and R4, together with the nitrogen atom to which they are attached, form a 5-6 membered heterocycle comprising one or more N atoms.

[0008] In certain embodiments, the present disclosure provides a lipid compound of the following formula (Formula (I)):

[0099] or a pharmaceutically acceptable salt thereof; wherein each of R1and Rris independently -(C1-C9 alkylene)-R'8; each R5is independently C2-C12 alkyl, C2-C12 alkenyl, ■■CH(R8)R9; each R6and R7is independently hydrogen, C7-C12 alkoxy, or C7-C12 alkenoxy, provided that R6and R ' are not both hydrogen; each R8and R9is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 alkylsulfide, provided that R8and R9are not both hydrogen and not both C1-C12 alkylsulfide; n is 0-5; p is 0-3; q is 0-3; X1is O,CH2, NH, N-(CI-C3 alkyl),wherein each L is independently -(CH2)m-C(R2)(R2”)-(CH2)t-, or ~(CH2)m-C3-C8 cycloaikyiene-((3H2)t-, or -(CH2)m-(3 -6-membered heterocyclylene)-(CH2)t-; R2is hydrogen, C1-C12 alkyl, or Ci-C’12 alkoxy; R2" is optionally substituted Ci-Ci2 alkyl, Cf-Ci2 alkoxy, optionally substituted C3-C12 cycloalkyl, (C1-C4 alkylene)-(optionally substituted C3-C12 cycloalkyl), optionally substituted C5-C6 aryl, or (C1-C4 alkylene)-(optionally substituted Cs-Ce aryl); and m and t are each independently selected from 0-4; and whereinor a 3 -membered to 8-membered heterocyclyl containing one or more N atoms substituted with C1-C12 alkyl; R1and R4are each independently C1-C6 alkyl; or R1and R4, together with the nitrogen atom to which they are attached, form a 5-6 membered heterocycle comprising one or more N atoms.

[0010] In certain embodiments, the present disclosure provides a lipid compound of the following formula (Formula (I)):

[0011] or a pharmaceutically acceptable salt thereof; wherein each of R? and Rris independently -(C1-C9 alkylene)-R5; each R5is independently C2-C12 alkyl, C2-C12 alkenyl, -CH(R6)R7, -C(O)O-(CH2)P-CH-(R8)R9, -OC(O)-(CH2)q-CH-(R8)R9, or -OC(O)O-(CH2)p- CH(R°)R9; each R° and R7is independently hydrogen, C?-Ci2 alkoxy, or C7-C12 alkenoxy, provided that R° and Rzare not both hydrogen; each R8and R9is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 alkylsulfide, provided that R8and R9are not both hydrogen and not both C1-C12 alkylsulfide; n is 0-5; p is 0-3; q is 0-3; X1is O, CH2, NH, N-(CI-C3 alkyl),wherein each L is independently -(CH2)<n-C(R2)(R2')-(CH2)t-, or ~-(CH2)ir.-C.3-C8 cycloalkylene-(CH2)t~, or -(CH2)m-(3-6-membered heterocyclylene)-(CH2)t-; R2is hydrogen, Cf-Ci2 alkyl, or Ci-Ci2 alkoxy; R‘ is optionally substituted Ci-Cf2 alkyl, C1-C12. alkoxy, optionally substituted C3-C12 cycloalkyl, (C1-C4 alkylene)-(optionally substituted C3-C12 cycloalkyl), optionally substituted C5-C6 aryl, or (Ci-CU alkylene)-(optionally substituted Cs- C6 aryl); and m and t are each independently selected from 0-4; and wherein ¥or a 3- membered to 8 -membered heterocyclyl containing one or more N atoms substituted with Ci- C12 alkyl; R3and R4are each independently Ci-Ce alkyl; or R3and R4, together with the nitrogen atom to which they are attached, form a 5-6 membered heterocycle comprising one or more N atoms.

[0012] In certain embodiments,2)ra-C(R2)(Rz')-(Cl-h)t- or -(CH2)m-C3-Cs cycloalkylene-(CH2)t-; R2is hydrogen or C1-C12 alkyl; R2’ is Ci-C12 alkyl;certain embodiments, L is -(CH2)m-C(R2)(R2")"(CH2)t-. In certain embodiments, R‘ is hydrogen. In certain embodiments, t is 0. In certain embodiments, RJand R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl. In certain embodiments, Rfis -(C1-C9 alkylene)-R5; R?is ■■ O(3(O)-(CH2)q-CH-(R8)R9; and each R8and R9is independently Ci-Ci2 alkyl. In certain embodiments, R8is n -hexyl; and R9is n-butyl. In certain embodiments, R8is C1-C12 alkylsulfide; and R9is C1-C4 alkyl. In certain embodiments, R8is C4-C8 alkylsulfide; and R9is C1-C4 alkyl. In certain embodiments, Rsis Cs, Ck, or Cs alkylsulfide; and R9is methyl.

[0013] In certain embodiments, q is 0. In certain embodiments, R' and R? are the same. In certain embodiments, Rris -(Ci -Co alkylene)-R5; R5is -CH(R6)RZ; and R° and R7is independently C7-C12 alkenoxy.

[0014] In certain embodiments,are each independently Ci-Cs alkyl, or R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl. In certain embodiments, n is 2-3. In certain embodiments, X1is O, n is 2-3, and R3and R4are each independently C1-C6 alkyl; optionally C1-C2 alkyl. In certain embodiments, X‘ is CH2, n is 1-3. and R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl. In certain embodiments, R1is -(C1-C9 alkylene)- R5; R3is -C(O)O-(CH2)p-CH-(R8)R9; and each R8and R9is independently Ci-Ci2 alkyl or C2- C12 alkenyl. In certain embodiments, Rsis C1-C12 alkylsulfide; and R9is C1-C4 alkyl. In certain embodiments, R8is CU-Cs alkylsulfide; and R9is C1-C4 alkyl. In certain embodiments, R8and R9are the same. In certain embodiments, R8and R9are n-pentyl. In certain embodiments, p is 2. In certain embodiments, R8and R9are u-heptyl. In certain embodiments, R8and R9are l-hepten-7-yl. In certain embodiments, p is 0. In certain embodiments, R1and R1are the same. In certain embodiments, Rris -(C1-C9 alkylene)-R3; R5is -CH(R6)R'; and each R° and R7is independently C7-C12 alkenoxy. In certain embodiments, R1is -(C1-C9 alkylene)-R5; and R3is C4-C12 dienyl. In certain embodiments, R1’ is -(C1-C9 alkylene)-R5; R5is -CH(R6)R'; and each R° and R7is independently C7-C12 alkoxy. In certain embodiments, R1and R1are the same.

[0015] In certain embodiments, R1is -(C1-C9 alkylene)-R5; R5is -OC(O)-(CH2)q-CH- (R8)R9; each Rsand R9is independently C1-C12 alkyl or C1-C12 alkylsulfide (but not both Ci-Ci 2 alkylsulfide);(CHalm-Cs-Cx cycloalkylene- (CHfit; m yrt, is 0; and t is 0. In certain embodiments, n is 2; ¥ is ; and R’ and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl. In certain embodiments, q is 0. In certain embodiments, R8is n-hexyl; and R9is ?r-butyl. In certain embodiments, Rsis C1-C12 alkylsulfide; and R9is C1-C4 alkyl. In certain embodiments, R8is C4-C8 alkylsulfide;and R9is C1-C4 alkyl. In certain embodiments, Rrand R' are the same. In certain embodiments, L is cyclopentylene or cyclo hexylene.

[0016] In certain embodiments,{( 'i I;):-: R2is hydrogen; R2" is Cf-Ci2 alkyl; m is 2: t is 0; n is 0; and Y is a 3-membered to 8- membered heterocyclyl containing one or more N atoms substituted with Ci-Ci2 alkyl. In certain embodiments, R1is -(C1-C9 alkylene)-R5; R5is -OC(O)-(CH2)q-CH-(R8)R9; and each R8and R9is independently C1-C12 alkyl. In certain embodiments, R8is n-hexyl; and R9is n- butyl. In certain embodiments, q is 0. In certain embodiments, R1and R1are the same.

[0017] In certain embodiments, the compound is selected from any one of Compounds 1- 31. In certain embodiments, the compound is selected from any one of Compounds 1-18. In certain embodiments, the compound is Compound 1, 2, 3, 4, 5, 6, 7, 10, 12, 13, 14, 15, 16, 17, or 18.

[0018] In certain embodiments, R1is -(Ci-Cs alkylene)-!!5; R5is -CH(R6)RZ, -C(O)O-certain embodiments, R5is -CH(R°)R / ; and each R6and R7is independently C7-C12 alkoxy or C7-C12 alkenoxy. In certain embodiments, Rris -(Cr-Cg alkylene)-R5; R5is -C(O)O-(CH2)p-CH- (R8)R9or -OC(O)-(CH2)q-CH-(R8)R9; and each R8and R9is independently C1-C12 alkyl. In certain embodiments, R1’ is -(C1-C9 alkylene)-R’; and R5is C2-C12 alkenyl. In certain embodiments, R5is -C(O)O-(CH2)p-CH-(R8)R9; and each R° and R9is independently C1-C12 alkyl or C2-C12 alkenyl. In certain embodiments, R3is -OC(O)-(CH2)q-CH-(R8)R9; and each R8and R9is independently C1-C12 alkyl. In certain embodiments, R!and R1are the same. In certain embodiments, n is 2. In certain embodiments, R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl.

[0019] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising a compound of the present disclosure. In certain embodiments, the lipid nanoparticle comprises a compound of the present disclosure; a phospholipid; a cholesterol; and a polyethylene glycol lipid. In certain embodiments, the lipid nanoparticle comprises about 2.0-80 mol% of the compound of the present disclosure, about 7.5-40 mol% of phospholipid, about 6-50 mol% of cholesterol, and about 1-4 mol% of PEG lipid. In certain embodiments, the lipid nanoparticle comprises about 40 mol% of the compound of thepresent disclosure, about 30 mol% of phospholipid, about 28.5 mol% of cholesterol, and about 1.5 mol% of PEG lipid. In certain embodiments, the lipid nanoparticle comprises about 45-50 mol% of the compound of the present disclosure, about 10 mol% of phospholipid, about 38-42 mol% of cholesterol, and about 2 -3 mol% of PEG lipid. In certain embodiments, the lipid nanoparticle comprises about 47.5 mol% of the compound of the present disclosure, about 10 mol% of phospholipid, about 40 mol% of cholesterol, and about 2.5 mol% of PEG lipid. In certain embodiments, the lipid nanoparticle comprises about 47.5-52.5 mol% of the compound of the present disclosure, about 10 mol% of phospholipid, about 37-40 mol% of cholesterol, and about 1-2 mol% of PEG lipid. In certain embodiments, the lipid nanoparticle comprises about 50 mol% of the compound of the present disclosure, about 10 mol% of phospholipid, about 38.5 mol% of cholesterol, and about 1.5 mol% of PEG lipid. In certain embodiments, the lipid nanoparticle comprises about 57.5-62.5 mol% of the compound of the present disclosure, about 10 mol% of phospholipid, about 26-29 moi% of cholesterol, and about 2-3 mol% of PEG lipid. In certain embodiments, the lipid nanoparticle comprises about 60 mol% of the compound of the present disclosure, about 10 mol% of phospholipid, about 27.5 mol% of cholesterol, and about 2.5 mol% of PEG lipid. In certain embodiments, the lipid nanoparticle comprises about 45-50 mol% of the compound of the present disclosure, about 10 mol% of phospholipid, about 37.5-40.5 mol% of cholesterol, and about 3-4 mol% of PEG lipid. In certain embodiments, the lipid nanoparticle comprises about 47.5 mol% of the compound of the present disclosure, about 10 mol% of phospholipid, about 39 mol% of cholesterol, and about 3.5 mol% of PEG lipid.

[0020] In certain embodiments, the lipid nanoparticle further comprises a targeting component. In certain embodiments, the targeting component is a targeting lipid. In certain embodiments, the targeting component is an active targeting component. In certain embodiments, the active targeting component is an antibody, an antigen-binding fragment of an antibody, a protein, a peptide, or a small molecule.

[0021] In certain embodiments, the lipid nanoparticle further comprises one or more polynucleotides encapsulated within the lipid nanoparticle. In certain embodiments, the one or more polynucleotides comprises RNA. In certain embodiments, the one or more polynucleotides comprises DNA. In certain embodiments, the one or more polynucleotides comprises DNA and RNA.

[0022] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising the lipid nanoparticle of the present disclosure, and a pharmaceutically acceptable excipient.

[0023] In certain embodiments, the present disclosure provides a method of delivering a polynucleotide to an extrahepatic cell or tissue in a subject, wherein the method comprises administering to the subject an effective amount of a lipid nanoparticle of the present disclosure or a pharmaceutical composition of the present disclosure. In certain embodiments, the extrahepatic cell or tissue comprises a brain cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a lung cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a bone marrow cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a spleen cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a kidney cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a heart cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a pancreatic cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a muscle cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises an immune cell or tissue.

[0024] In certain embodiments, the present disclosure provides a method of treating a disease in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0025] In certain embodiments, the present disclosure provides a method of producing a therapeutic composition, wherein the method comprises encapsulating an active agent within a lipid nanoparticle, wherein the lipid nanoparticle comprises a compound of the present disclosure. In certain embodiments, the active agent comprises DNA. In certain embodiments, the active agent comprises RNA. In certain embodiments, the active agent comprises DNA and RNA.

[0026] In certain embodiments, the present disclosure provides a method of producing a vaccine or prophylactic composition, wherein the method comprises encapsulating an active agent within a lipid nanoparticle, wherein the lipid nanoparticle comprises a compound of the present disclosure. In certain embodiments, the active agent comprises DNA. In certain embodiments, the active agent comprises RNA. In certain embodiments, the active agent comprises DNA and RNA.

[0027] In certain embodiments, the present disclosure provides a lipid nanoparticle or pharmaceutical composition of the present disclosure for use in delivering an active agent (e.g., one or more polynucleotides) to an extrahepatic cell or tissue in a subject.

[0028] In certain embodiments, the present disclosure provides a use of a lipid nanoparticle or pharmaceutical composition of the present disclosure in the manufacture of amedicament for delivering an active agent (e.g., one or more polynucleotides) to an extrahepatic cell or tissue in a subject.

[0029] In certain embodiments, the present disclosure provides a lipid nanoparticle or pharmaceutical composition of the present disclosure for use in treating a disease in a subject.

[0030] In certain embodiments, the present disclosure provides a use of a lipid nanoparticle or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease in a subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 shows mean total flux in several organs from mice, six hours following intravenous administration of firefly luciferase mRNA encapsulated LNP formulations comprising MC3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, or Compound 10.

[0032] FIG. 2 shows mean total flux in several organs from mice, six hours following intravenous administration of firefly luciferase mRNA encapsulated LNP formulations comprising MC3, Compound 5, or Compound 7.

[0033] FIG. 3 shows mean total flux in several organs from mice, six hours following intravenous administration of firefly luciferase mRNA encapsulated LNP formulations comprising MC3, Compound 3, or Compound 10.

[0034] FIG. 4A, FIG. 4B, and FIG. 4C show unique molecular identifier counts in the femur (FIG. 4A), spleen (FIG. 4B), or muscle (FIG. 4C) of cynomolgus monkeys following intravenous administration of LNP formulations comprising MC3, Compound 1 , Compound 2, Compound 3, Compound 4, Compound 6, or Compound 10.

[0035] FIG. 5A, FIG. SB, and FIG. 5C show unique molecular identifier counts in the femur (FIG. 5A), spleen (FIG. 5B), or muscle (FIG. 5C) of cynomolgus monkeys following intravenous administration of LNP formulations comprising MC3, Compound 5, or Compound 7.

[0036] FIG. 6 shows a time course of green fluorescent protein (GFP) expression inCD 14+ monocytes of a non- human primate (NHP) following intravenous (IV) administration of an LNP formulation comprising Compound 10.

[0037] FIG. 7 shows single-cell sequencing of GFP protein-positive (GFP+) cell types from the bone marrow aspirate in NHPs by LNP formulations comprising Compound 10.DETAILED DESCRIPTIONI. LIPID COMPOUNDS

[0038] Without being bound by theory, the lipid compounds disclosed herein facilitate delivery of an active agent to a desired target in a subject, e.g., to extrahepatic cells or tissues in the subject, when the active agent is encapsulated in an LNP comprising said lipid compound(s).

[0039] In certain embodiments, the present disclosure provides lipid compounds of Formula (I):wherein each of R1and R1is independently -(C1-C9 alkylene)-R°; each R5is independently C2-C12 alkyl, C2-C12 alkenyl, -CH(R6)R7, -C(O)O-(CH2)P-CH-(R8)R9, -OC(O)-(CH2)q-CH- (RS)R9, or -OC(O)O-(CH2)p-CH(R8)R9; each R6and Rzis independently hydrogen, C?-Ci2 alkoxy, or C7-C12 alkenoxy, provided that R6and R' are not both hydrogen; each Rsand R9is independently hydrogen, Cj-Ci2 alkyl, C2-C12 alkenyl, Ca-Cr? alkynyl, or C1-C12 alkylsulfide, provided that R8and R9are not both hydrogen, and not both C1-C12 alkylsulfide; n is 0-5; p ismembered heterocyclene)-(CH2)t-; Rzis hydrogen, C1-C12 alkyl, or C1-C12 alkoxy; R2’ is optionally substituted Cj-Ci2 alkyl, C1-C12 alkoxy, optionally substituted Cs-Cr? cycloalkyl, (C1-C4 alkylene)-(optionally substituted C3-C12 cycloalkyl), optionally substituted C5-C6 aryl, or (C1-C4 alkylene)-(optionally substituted C5-C6 aryl); and m and t are each independentlyselected from 0-4; and wherein-membered to 8-membered heterocyclyl containing one or more N atoms substituted with C1-C12 alkyl; R3and R4are each independently Ci-Ce alkyl; or R3and R4, together with the nitrogen atom to which they are attached, form a 5-6 membered heterocycle comprising one or more N atoms.

[0040] In certain embodiments, the present disclosure provides lipid compounds of Formula (I):wherein each of R* and R1' is independently -(C1-C9 alkylene)-R5; each R’ is independently C2-C12 alkyl, C2-C12 alkenyl, -CH(R6)R7, -C(O)O-(CH2)P-CH-(R8)R9, -OC(O)-(CH2)q-CH- (R8)R9, or -OC(O)O-(CH2)p-CH(R8)R9; each R6and R' is independently hydrogen, C7-C12 alkoxy, or C7-C12 alkenoxy, provided that R° and R7are not both hydrogen; each R8and R9is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, or C2-C12 alkynyl, provided that Rsand R9are not both hydrogen; n is 0-5; p is 0-3; q is 0-3; X’ is O, CH2, NH, N-(Ci-C.3 alkyl), or X’-; X2ismembered heterocyclene)-(CH2)t-; R2is hydrogen, Ci-Ci2 alkyl, or C1-C12 alkoxy; R2' is optionally substituted C1-C12 alkyl, C1-C12 alkoxy, optionally substituted C3-C12 cycloalkyl, (Ci-Q alkylene)-(optionally substituted C3-C12 cycloalkyl), optionally substituted C5-C6 aryl, or (C1-C4 alkylene)-(optionally substituted C5-C6 aryl); and m and t are each independently selected from 0-4; and wherein-membered to 8-membered heterocyclylcontaining one or more N atoms substituted with C1-C12 alkyl; R3and R4are each independently C1-C6 alkyl; or R;and R4, together with the nitrogen atom to which they are attached, form a 5-6 membered heterocycle comprising one or more N atoms.

[0041] In certain embodiments, X!of the lipid compound of Formula (I) iscycloalkylene - (CH2)t-; R2is hydrogen or C1-C12 alkyl; R2” is C1-C12 alkyl; m is 2; t is 0 or 2; n is 2; and Y is

[0042] In certain embodiments, L is ~-(CH2)m-C(R2)(R2' )-(CH2)t-.

[0043] In certain embodiments, L is -(CHclm-Cb-Cs cycloalkylene-(CH2)t~. In certain embodiments, m and t are 2. In certain embodiments, L is -(CH2)m-cyclohexylene-(CH2)t-. In certain embodiments, L is -{CH2)2-cyclohexylene-(CH2)2~.

[0044] In certain embodiments, R2is hydrogen.

[0045] In certain embodiments, R2is C1-C12 alkyl. In certain embodiments, R2is C1-C3 alkyl, n certain embodiments,is methyl.

[0046] In certain embodiments, t is 0. In certain embodiments, t is 2.

[0047] In certain embodiments, R ’ and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl.

[0048] In certain embodiments, R2" is C2-C6 alkyl. In certain embodiments, R2” is methyl, ethyl, n-propyl, n-butyl, or n-hexyl.

[0049] In certain embodiments, R' is -(Ci-Ch alkylene)-R5; R3is -OC(O)-(CH2)q-CH- (R8)R9; and each R8and R9is independently C1-C12 alkyl. In certain embodiments, each R8and R9is independently C3-C7 alkyl. In certain embodiments, R8is n-hexyl and R9is n-butyl. In certain embodiments, q is 0. In certain embodiments, R1and R1are the same. In certain embodiments, R? is -(C1-C9 alkylene)-R5; R is -CI I( R6)R and each R6and R7is independently C7-C12 alkenoxy. In certain embodiments, each R6and Rzis octenoxy. In certain embodiments, R” and Rzare cis-5-octen-l-oxy. In certain embodiments, R1is -(C5-C7 alkylene)-R5. In certain embodiments, R1is -«-pentylene-R3, -?r-hexylene-R5, or -n-hexylene- R5.

[0050] In certain embodiments, R1is -(Ci-Ch alkylene)-R5; R3is -C(O)O-(CH2)p-CH- (R8)R9; and each R8and R9is independently C1-C12 alkyl. In certain embodiments, each R8and R9is independently C3-C7 alkyl. In certain embodiments, R8and R9are n-pentyl. In certain embodiments, R8and R9are H-heptyl. In certain embodiments, q is 0. In certain embodiments, q is 2. In certain embodiments, R!’ and R1are the same. In certain embodiments, R1is -(C3-C7 alkylene )-R5. In certain embodiments, R1is -7?-butylene-R5.

[0051] In certain embodiments, R1or R1’ is -(C1-C9 alkylene)-R5; R5is -C(O)O-(CIl2)P- CH-(R8)R9; and each R8and R9is independently C1-C12 alkyl or C1-C12 alkylsulfide (but not both C1-C12 alkylsulfide). In certain embodiments, one of R8and R9is C1-C12 alkyl and the other is Ci-C’12 alkylsulfide. In certain embodiments, R8is C1-C12 alkylsulfide; and R9is Ci- C4 alkyl. In certain embodiments, R8is C4-C8 alkylsulfide; and R9is C1-C4 alkyl. In certain embodiments, R8is Cs alkylsulfide; and R9is methyl. In certain embodiments, R8is Cs alkylsulfide; and R9is methyl. In certain embodiments, R8is Cs alkylsulfide; and R9is methyl. In certain embodiments, R / ’ and R1are the same.

[0052] In certain embodiments,are each independently Ci-C-6 alkyl. In certain embodiments, R-’ and R4are ethyl. In certain embodiments, n is 2-3. In certain embodiments,R4are each independently Ci-C-6 alkyl. In certain embodiments, X' is O, n is 2-3, Y isare each independently Ci-Ch alkyl. In certain embodiments, R3and R4are ethyl. In some embodiments, R1is -(C1-C9 alkylene)-R5; R5is -C(O)O-(CH?)p- CH-(RS)R9; and each R8and R9is independently Ci-Ci2 alkyl or C2-C12 alkenyl. In certain embodiments, R8and R9are the same. In certain embodiments, R8and R9are n-pentyl. In certain embodiments, p is 2. In certain embodiments, R1’ is -(C1-C9 alkylene)-R5; R5is - CH(R6)R'J and each R6and R' is independently C7-C12. alkenoxy. In certain embodiments, each R° and R7is octenoxy. In certain embodiments, R6and R7are crv-5-octen-l -oxy. In certain embodiments, R1is -(C5-C7 alkylene)-^. In certain embodiments, R1is -n-pentylene- R5, -n-hexylene-R5, or -zj-hexylene-R5.

[0053] In certain embodiments, each R8and R9is independently Cs-C? alkyl. In certain embodiments, R8and R9are n-heptyl. In certain embodiments, each R8and R9isindependently C3-C7 alkenyl. In certain embodiments, R8and R9are l-hepten-7-yl. In certain embodiments, p is 0. In certain embodiments, R‘ and R!are the same.

[0054] In certain embodiments,together with the nitrogen atom to which they are attached, form a pyrrolidinyl. In certain embodiments, n is 2-3. In certain embodiments,and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl. In certain embodiments, R1is -(C1-C9 alkylene)-R0; R5is -C(O)O-(CH2)p-CH-(R8)R9; and each Rsand R9is independently C1-C12 alkyl or C2-C12 alkenyl. In certain embodiments, R8and R9are the same. In certain embodiments, each R8and R9is independently C3-C7 alkyl. In certain embodiments, R8and R9are «-pentyl. In certain embodiments, p is 2. In certain embodiments, R!is -(C1-C9 alkylene)-R°; R5is -CH(R°)R7; and each R6and R7is independently C7-C12 alkenoxy. In certain embodiments, each R6and R / is octenoxy. In certain embodiments, R6and R7are cks-5-octen-l-oxy. In certain embodiments, R8and R9are H-heptyl. In certain embodiments, each R8and R9is independently C3-C7 alkenyl. In certain embodiments, Rsand R9are l-hepten-7-yl. In certain embodiments, p is 0. In certain embodiments, R1and R1are the same. In certain embodiments, R‘ is -(C5-C7 alkylene)-R5. In certain embodiments, R1is -«-pentylene-R5, -n-hexylene-R5, or -n-heptylene-R5.

[0055] In certain embodiments,are each independently Ci-C-6 alkyl or R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl; R!is -(C1-C9 alkylene)-R5; and R5is C4-Ci? dienyl. In certain embodiments, R5is (2Z, 5Z)-undeca-2, 5-dienyl. In certain embodiments, R’ is -(C1-C9 alkylene)-R5; R5is -CH(R6)R'; and each R6and R7is independently C7-C12 alkoxy. In certain embodiments, R.° and R7are octoxy. In certain embodiments, Rrand R1are the same. In certain embodiments, R1is -(C5-C7 alkylene)^5. In certain embodiments, R1is -n-pentylene- R5, -n-hexylene-R5, or -n-heptylene-R5.

[0056] In certain embodiments, R1is -(C1-C9 alkylene)-R’’: R5is -OC(O)-(CH2)q-CH- (R8)R9; each R8and R9is independently C1-C12 alkyl; X!iscycloaIkyIene-(CH?.)t; m is 0; and t is 0. In certain embodiments,together with the nitrogen atom to which they are attached, form a pyrrolidinyl. In certain embodiments, q is 0. In certain embodiments, each R8and R9is independently C3-C7 alkyl. In certain embodiments, R8is n- hexyl and R9is n-butyl. In certain embodiments, R1and R1are the same. In certain embodiments, L is cyclopentylene or cyclohexylene. In certain embodiments, R!is -(C5-C7 alkylene)-!!5. In certain embodiments, R1is -n-hexylene-R5.

[0057] In certain embodiments, m and t are 2.

[0058] In certain embodiments, R6or Rzis hydrogen. In certain embodiments, only one ofR6and R7is hydrogen. In certain embodiments, I!8or R9is hydrogen. In certain embodiments, only one of R8and R9is hydrogen.

[0059] In certain embodiments,(CH2)t-; R2is hydrogen: R2is C1-C12 alkyl; rn is 2; t is 0; n is 0; and Y is a 3-membered to 8- membered heterocyclyl containing one or more N atoms substituted with Ci-Ci2 alkyl. In certain embodiments, R2’ is C2-C6 alkyl. In certain embodiments, Rz' is n-hexyl. In certain embodiments, Y is A’-Ci-CY alkyl-piperidinyl or 2V-C1-C12 alkyl-pyrrolidinyl. In certain embodiments, Y is A-Ci-Cs alkyl-piperidinyl or TV-Cf-C.? alkyl-pyrrolidinyl. In certain embodiments, Y is A-methylpiperidinyl or A-methylpyrrolidinyl.

[0060] In certain embodiments, R1is -(Ci-C? alkylene)-!!5; R5is -OC(O)-(CH2)q-CH-( R8)R9; and each Rsand R9is independently C1-C12 alkyl. In certain embodiments, each Rsand R9is independently C3-C7 alkyl. In certain embodiments, R8is ? / -hexyl and R9is ra-butyl. In certain embodiments, q is 0. In certain embodiments, R1and R1are the same. In certain embodiments, R1is -(C5-C7 alkylene)-!!5. In certain embodiments, R1is -n-hexylene-R5.

[0061] In certain embodiments, R1is -(C1-C9 alkylene)- R5; R5is -CH(R6)R7, -C(O)O-

[0062] In certain embodiments, R’ is -(C1-C9 alkylene)- R?; R5is -CHIR^R1'; and each R6and R' is independently C?-Cf2 alkoxy or C7-C12 alkenoxy. In certain embodiments, R6and Rzare octenoxy. In certain embodiments, R6and R7are octoxy. In certain embodiments, R1is --(C1-C.3 alkylene)-R5. In certain embodiments, R? is -ethylene-R5.

[0063] In certain embodiments, Rris -(C1-C9 alkyl ene)-Ri; R5is -C(O)O-(CH2)p-CH- (R8)R9or -OC(O)-(CH2)q-CH-(Rs)R9; and each R8and R9is independently C1-C12 alkyl. In certain embodiments, Rris -(C5-C7 alkylene)-R5. In certain embodiments, R!is -n- pentylene-R5. In certain embodiments, R1is -n-hexylene-R5. In certain embodiments, R5is - C(O)O-(CH?)p-CH-(R8)R9. In certain embodiments, p is 2. In certain embodiments, each R8and R9is independently C1-C12 alkyl. In certain embodiments, each R8and R9is independently C3-C7 alkyl. In certain embodiments, R® and R9are n-pentyl. In certain embodiments, R5is -OC(O)-(CH2)q-CH-(R8)R9. In certain embodiments, q is 0. In certain embodiments, each R8and R9is independently C3-C7 alkyl. In certain embodiments, R8is n- hexyl and R9is n-butyl.

[0064] In certain embodiments, Rris -(C1-C9 alkyl ene)-R5; and R® is C2-C12. alkenyl. In certain embodiments, Rris -(C5-C7 alkylene)-R5. In certain embodiments, R1is -w-hexylene- R®. In certain embodiments, R5is C4-C12 dienyl. In certain embodiments, R® is (2Z, 52)- undeca-2, 5 -dienyl.

[0065] In certain embodiments,certain embodiments,is 0. In certain embodiments, R2is hydrogen. In certain embodiments, R2” is C1-C12 alkyl. In certain embodiments, R2’ is C3-C7 alkyl. In certain embodiments, R2' is n-hexyl. In certain embodiments, R3and R4are each independently Ci-Ce alkyl. In certain embodiments, R3and R4are ethyl. In certain, embodiments, R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl.

[0066] In certain embodiments, R' is -(C1-C9 alkylene)-R5; R5is -C(O)O-(CH2)P-CH- (R8)R9; and each R8and R9is independently C1-C12 alkyl or C2-C12. alkenyl. In certain embodiments, R1is -(C3-C7 alkylene)-R5. In certain embodiments, R1is -n-pentylene-R3. In certain embodiments, each R8and R9is independently Cs-C? alkyl. In certain embodiments,R8and R9are u-heptyL In certain embodiments, R8and R9are heptenyl. In certain embodiments, R1and R1are the same. In certain embodiments, n is 2. In certain embodiments, X!is CH2. In certain embodiments, R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl.

[0067] In certain embodiments, R1is -(C1-C9 alkylene)- R5; R5is -OC(O)-(CH2)q-CH- (R8)R9; and each Rsand R9is independently C1-C12 alkyl. In certain embodiments, R1is - (C3-C7 alkylene)-Rrt In certain embodiments, R1is -«-hexylene-R5. In certain embodiments, each Rsand R9is independently C3-C7 alkyl. In certain embodiments, R8is «-hexyl and R9is n-butyl. In certain embodiments, R!and R1are the same. In certain embodiments, n is 2.

[0068] In certain embodiments, R1is --{C1-C9 alkylene j-R3; R5is -OC(O)-(CH2)q-CH- (R8)R9: and each R8and R9is independently Cf-Ci2 alkyl or Cf-Ci2 alkylsulfide (but not both C1-C12 alkylsulfide). In certain embodiments, one of R8and R9is C1-C12 alkyl and the other is Ci-Ci2 alkylsulfide. In certain embodiments, R8is C1-C12 alkylsulfide: and R9is C1-C4 alkyl. In certain embodiments, R8is C4-C8 alkylsulfide: and R9is C1-C4 alkyl. In certain embodiments, Rsis C5 alkylsulfide; and R9is methyl. In certain embodiments, R8is Ce alkylsulfide; and R9is methyl. In certain embodiments, R8is Cs alkylsulfide; and R9is methyl. In certain embodiments, R1’ and R’ are the same.

[0069] In certain embodiments,(CH2)t-; m is 2; and t is 0. In certain embodiments, R2is hydrogen. In certain embodiments, R2' is Cs -Ci 2 alkyl. In certain embodiments, R2' is Cs-C? alkyl. In certain embodiments, R2’ is zj-hexyl. In certain embodiments, R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl.

[0070] In certain embodiments, the lipid compound of Formula (I), or pharmaceutically acceptable salt thereof, is an ionizable lipid compound.

[0071] Examples of lipid compounds of Formula (I) are presented in Table 1. Pharmaceutically acceptable salts of the compounds presented in Table 1 are also encompassed.Table 1. Exemplary Compounds

[0072] In certain embodiments, the lipid compound is selected from any one of Compounds 1 -26 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is selected from any one of Compounds 1-27 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is selected from any one of Compounds 1-31 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is selected from any one of Compounds 28-31 from Table 1, or a pharmaceutically acceptable salt thereof. In certainembodiments, the lipid compound is selected from any one of Compounds 1 -1 1 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is selected from any one of Compounds 12-18 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 1 , 2, 3, 4, 5, 6, 7, 10, 12, 13, 14, 15, 16, 17, or 18 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 1, 2, 3, 4, 5, 6, 7, or 10 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 12, 13, 14, 15, 16, 17, or 18 from Table 1, or a pharmaceutically acceptable salt thereof.

[0073] In certain embodiments, the lipid compound is Compound 3, 10, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, or 26 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 10, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, or 26 from Table 1, or a pharmaceutically acceptable salt thereof

[0074] In certain embodiments, the lipid compound is Compound 1, 2, 4, 5, 6, 7, 8, 9, or 11 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 1, 2, 6, 7, 8, or 9 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 1 , 2, 8, or 9 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 1 or 2 from Table 1 , or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 4, 5, or 11 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 4 or 5 from Table 1, or a pharmaceutically acceptable salt thereof.

[0075] In certain embodiments, the lipid compound is Compound 16 or 17 from Table 1, or a pharmaceutically acceptable salt thereof.

[0076] In certain embodiments, the lipid compound is Compound 24 or 25 from Table 1 , or a pharmaceutically acceptable salt thereof.

[0077] In certain embodiments, the lipid compound is Compound 1 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 2 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 3 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 4 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 5 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 6 from Table 1 or a pharmaceuticallyacceptable salt thereof. In certain embodiments, the lipid compound is Compound 7 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 10 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 12 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 13 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 14 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 15 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 16 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 17 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 18 from Table 1 or a pharmaceutically acceptable salt thereof.

[0078] In certain embodiments, the pharmaceutically acceptable salt is a pharmaceutically acceptable acid addition salt. Examples of acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate), lactate, malate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitoate, pectinate, persulfate, 3 -phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. In certain embodiments, the basic nitrogen-containing groups may be quatemized with such agents as lower alkyl halides such as, but not limited to, methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides such as, but not limited to, decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid and such organic acids as acetic acid, fumaric acid, maleic acid, 4-methylbenzenesulfonic acid, succinic acid, and citric acid.

[0079] In certain embodiments, the pharmaceutically acceptable salt is a pharmaceutically acceptable base addition salt. Basic addition salts may be prepared in situ during the final isolation and purification of the compounds by reacting a carboxylic acid-containing moietywith a suitable base such as, but not limited to, the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary, or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as, but not limited to, lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetra-ethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, and the like. Other examples of organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.II. LIPID COMPOSITIONSLipid Nanopartides (LNP)

[0080] In certain embodiments, the present disclosure provides lipid compositions (e.g., a lipid nanoparticle (LNP)) comprising at least one lipid compound of Formula (I), or a pharmaceutically acceptable salt thereof. In certain embodiments, the present disclosure provides lipid nanoparticles (LNPs) comprising at least one lipid compound of Formula (I), or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid nanoparticles are microspherical vesicles (i.e., liposomes) that comprise liposomal lamellar phase lipid bilayers (unilamellar or multilamellar) which encompass an interlamellar space. In certain embodiments, the lipid nanoparticles are nanospherical vesicles that comprise liposomal lamellar phase lipid bilayers (unilamellar or multilamellar).

[0081] In certain embodiments, the lipid nanoparticle (LNP) comprises: (i) at least one lipid compound of Formula (I), or a pharmaceutically acceptable salt thereof: (ii) at least one phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) at least one helper lipid (e.g., cholesterol); and (iv) at least one PEG lipid. In certain embodiments, the LNP comprises: (i) at least one lipid compound of Formula (I), or a pharmaceutically acceptable salt thereof; (ii) at least one phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) at least one cholesterol; and (iv) at least one PEG lipid. In certain embodiments, the lipid nanoparticle (LNP) comprises one or more additional lipid components.

[0082] In certain embodiments, the lipid composition (e.g., LNP) comprises any one of Compounds 1-11 from Table 1 , or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises any one of Compounds 12-18 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipidcomposition (e.g., LNP) comprises any one of' Compounds 1-26 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises any one of Compounds 1-31 from Table 1, or a pharmaceutically acceptable salt thereof.

[0083] In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 1 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 2 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 3 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 4 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 5 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 6 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 7 from Table I, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 10 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 12 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 13 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 14 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 15 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 16 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 17 from Table I, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 18 from Table 1 , or a pharmaceutically acceptable salt thereof.

[0084] Phospholipids for use in a lipid composition (e.g., LNP) of the present disclosure can be neutral, uncharged, or zwitterionic phospholipids. Examples of phospholipids for use in a lipid composition include, but are not limited to: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidyicholine (DMPC), phosphatidylcholine (PLPC), i,2-distearoyl-sn- glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine(EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1- myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-palmitoyl-2 -myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2- diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1 ■■ stearoyl- 2 -palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanol amine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In certain embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC).

[0085] Helper lipids for use in a lipid composition (e.g., LNP) of the present disclosure include steroids, sterols, and alkyl resorcinols. Examples of helper lipids for use in a lipid composition include, but are not limited to, cholesterol, cholesterol hemisuccinate, and 5- heptadecylresorcinol. In certain embodiments, the helper lipid is cholesterol.

[0086] PEG lipids for use in a lipid composition (e.g., LNP) of the present disclosure include compounds which comprise a lipid moiety bound to a PEG-based polymer moiety (i.e., PEG moiety). In certain embodiments, the lipid moiety of the PEG lipid is derived from diacylglycerol or diacylglycamide. In certain embodiments, the lipid moiety of the PEG lipid is derived from a dialkylglycerol or dialkylglycamide group. In certain embodiments, the di alkylglycerol or dialkylglycamide group has alkyl chain length from about C4 to about C40 saturated or unsaturated carbon atoms. In certain embodiments, the alkyl chain length is from about CIO to about C20. In certain embodiments, the dialkylglycerol or dialkylglycamide group comprises one or more functional groups (e.g., amide or ester). In certain embodiments, the dialkylglycerol or dialkylglycamide group comprises one or more substituted alkyl groups.

[0087] PEG moieties of the PEG lipid can include any polyethylene glycol (PEG) or other polyalkylene ether polymers, including optionally substituted linear or branched polymers of ethylene glycol or ethylene oxide. PEG moieties can have a molecular weight of: about 130 to about 50,000 Da; about 150 to about 25,000 Da; about 150 to about 15,000 Da; about 150 to about 10,000 Da; about 150 to about 5,000 Da; about 150 to about 4,000 Da; about 150 to about 3,000 Da; about 150 to about 2,500 Da; about 150 to about 2,000 Da; about 500 to about 3,000 Da; about 500 to about 2,000 Da; about 1,000 to about 3,000 Da; or about 1,000to about 2,000 Da. In certain embodiments, the PEG moiety comprises PEG2000 (having 2,000 Da).

[0088] In certain embodiments, the lipid composition comprises about 15-80 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 20-80 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises about 40-70 mol% of at least one lipid compound of Formula (I), or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 45-65 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 45-50 mol% (e.g., about 47.5 mol%) of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 47.5-52.5 mol% (e.g., about 50 mol%) of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 50-55 mol% (e.g., about 52.5 mol%) of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 52.5-57.5 mol% (e.g., about 55 mol%) of a lipid compound of Formula ( I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 55-60 mol% (e.g., about 57.5 mol%) of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 57.5-62.5 mol% (e.g., about 60 mol%) of a lipid compound of Formula ( I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 60-65 mol% (e.g., about 62.5 mol%) of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 62.5-67.5 rnol% (e.g., about 65 mol%) of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0089] In certain embodiments, the lipid composition comprises about 7.5-45 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)). In certain embodiments, the lipid composition comprises about 7.5-40 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)). In certain embodiments, the lipid composition (e.g., LNP) comprises about 7.5-12.5 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)). In certain embodiments, the lipid composition comprises about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)).

[0090] In certain embodiments, the lipid composition (e.g., LNP) comprises about 6-50 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition (e.g., LNP) comprises about 6-48.5 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition (e.g., LNP) comprises about 6-45 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition (e.g., LNP) comprises about 20-50 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition comprises about 25-45 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition comprises about 26-29 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition comprises about 27.5 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition comprises about37-40 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition comprises about 38.5 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition comprises about 37.5-40.5 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition comprises about 39 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition comprises about38-42 mol% of a helper lipid (e.g., cholesterol). In certain embodiments, the lipid composition comprises about 40 mol% of a helper lipid (e.g., cholesterol).

[0091] In certain embodiments, the lipid composition (e.g., LNP) comprises about 1-4 mol% of a PEG lipid. In certain embodiments, the lipid composition comprises about 1-2 mol% (e.g., 1.5 mol%) of a PEG lipid. In certain embodiments, the lipid composition comprises about 1 .5-2.5 mol% (e.g., 2 mol%) of a PEG lipid. In certain embodiments, the lipid composition comprises about 2-3 mol% (e.g., 2.5 mol%) of a PEG lipid. In certain embodiments, the lipid composition comprises about 2.5-3.5 mol% (e.g., 3 mol%) of a PEG lipid. In certain embodiments, the lipid composition comprises about 3-4 mol% (e.g., 3.5 mol%) of a PEG lipid. In certain embodiments, the PEG lipid comprises 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene giycol-2000 (DMG-PEG 2000, also referred to herein as PEG-DMG).

[0092] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 15- 80 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 7.5-45 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC )); (iii) about 6-50 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1-4 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 20-80 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 7.5-40 mol% of a phospholipid (e.g., distearoylphosphatidylcholine(DSPC)); (iii) about 6-50 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1-4 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 20-80 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 7.5-40 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 6-45 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1-4 mol% of a PEG lipid (e.g., PEG-DMG).

[0093] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 40- 70 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 7.5-12.5 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 20-50 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1-4 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 45-65 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC));(iii) about 25-45 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1 -4 mol% of a PEG lipid (e.g., PEG-DMG).

[0094] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 45- 50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 38-42 mol% of a helper lipid (e.g., cholesterol); and (iv) about 2-3 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 47.5 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 40 mol% of a helper lipid (e.g., cholesterol); and (iv) about 2.5 mol% of a PEG lipid (e.g., PEG-DMG).

[0095] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 40- 50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 10-30 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 18-49 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1-2 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 40-50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 20-30 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 18-28 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1-2 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable saltthereof; (ii) about 30 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 18.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1.5 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 40 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidy Icholine (DSPC)); (iii) about 48.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1.5 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 20 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 28 mol% of a helper lipid (e.g., cholesterol ); and (iv) about 2 mol% of a PEG lipid (e.g., PEG- DMG). In certain embodiments, the lipid composition comprises: (i) about 50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 30 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 18 mol% of a helper lipid (e.g., cholesterol); and (iv) about 2 rnol% of a PEG lipid (e.g., PEG-DMG).

[0096] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 47.5-52.5 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC));(iii) about 37-40 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1-3 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 37.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 2.5 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 38 mol% of a helper lipid (e.g., cholesterol); and (iv) about 2 mol% of a PEG lipid (e.g., PEG- DMG).

[0097] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 47.5-52.5 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC));(iii) about 37-40 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1-2 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii)about 38.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1.5 mol% of a PEG lipid (e.g., PEG-DMG).

[0098] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about57.5 -62.5 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 26-29 mol% of a helper lipid (e.g., cholesterol); and (iv) about 2-3 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 60 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 27.5 mol% of a helper lipid (e.g., cholesterol ); and (iv) about 2.5 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 60 mol% of a lipid compound of Formula (1) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 28 mol% of a helper lipid (e.g., cholesterol); and (iv) about 2 mol% of a PEG lipid (e.g., PEG- DMG).

[0099] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 45- 50 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC); (iii) about37.5-40.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 3-4 mol% of a PEG lipid. In certain embodiments, the lipid composition comprises: (i) about 47.5 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC); (iii) about 39 moi% of a helper lipid (e.g., cholesterol); and (iv) about 3.5 mol% of a PEG lipid.

[0100] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 35- 40 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 25-30 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 28-39 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1.5 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 35 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 25 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC));(iii) about 38.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1.5 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 40 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 30 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC));(iii) about 28.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1.5 mol% of a PEG lipid (e.g., PEG-DMG).

[0101] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 20- 35 moi% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 40 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 25-38 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1-3 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 20 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof;(ii) about 40 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 37.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 2.5 mol% of a PEG lipid (e.g., PEG-DMG). In certain embodiments, the lipid composition comprises: (i) about 33 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 40 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 25.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 1 .5 mol% of a PEG lipid (e.g., PEG-DMG).

[0102] In certain embodiments, the lipid compound of the lipid composition is any one of Compounds 1-31 of Table 1 , or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound of the lipid composition is any one of Compounds 1-26 of Table 1 , or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound of the lipid composition is any one of Compounds 1-7, 10, and 12-18 of Table 1, or a pharmaceutically acceptable salt thereof.

[0103] In certain embodiments, lipid compositions of the present disclosure (e.g., LNP) further comprise a targeting component. In certain embodiments, the targeting component is a passive targeting component. In certain embodiments, the passive targeting component comprises a targeting lipid. In certain embodiments, the targeting lipid comprises at least one cationic targeting lipid. Examples of cationic targeting lipids for use in a lipid composition (e.g., LNP) of the present disclosure include, but are not limited to, l,2-dioleoyl-3- trimethylammonium propane (DOTAP) and / V"[l-(2,3-dioleyloxy)propyl]-A'GV,Af- trimethylammonium chloride (DOTMA). In certain embodiments, the targeting lipid comprises at least one anionic targeting lipid. Examples of anionic targeting lipids for use in a lipid composition (e.g., LNP) of the present disclosure include, but are not limited to, phosphatidic acid (PA), Bis(monoacylglycero)phosphate (BMP), hemi- bis(monoacylglycero)phosphates (hemi-BMP), and bis(diacylglycero)phosphates (BDP).

[0104] In certain embodiments, the targeting component of a lipid composition (e.g., a lipid nanoparticie (LNP)) disclosed herein is an active targeting component. In certain embodiments, the active targeting component comprises an antibody. In certain embodiments, the active targeting component comprises an antigen-binding fragment of an antibody. In certain embodiments, the active targeting component comprises a protein. In certain embodiments, the active targeting component comprises a peptide. In certain embodiments, the active targeting component comprises a small molecule.Active Agents

[0105] In certain embodiments, the lipid compositions (e.g., a lipid nanoparticle (LNP)) disclosed herein comprise at least one active agent (e.g., RNA, DNA). In certain embodiments, the present disclosure provides lipid compositions (e.g., a lipid nanoparticie (LNP)) comprising at least one lipid compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one active agent (e.g., RNA, DNA) within the lipid composition, e.g., within the LNP.

[0106] In certain embodiments, the active agent comprises one or more polynucleotides. In certain embodiments, the active agent comprises one or more RNA. In certain embodiments, the active agent comprises one or more DNA. In certain embodiments, the active agent comprises one or more RNA and one or more DNA.

[0107] In certain embodiments, the active agent comprises mRNA. In certain embodiments, the active agent comprises an mRNA encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease, such as Cas9).

[0108] In certain embodiments, the active agent comprises gRN A. In certain embodiments, the active agent comprises dgRNA or sgRNA.

[0109] In certain embodiments, the active agent comprises an inhibitory polynucleotide, e.g., siRNA (i.e., a non-coding, short interfering RNA molecule).Compositions and Methods[OHO] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid composition (e.g., a lipid nanoparticie (LNP)) of the present disclosure. In certain embodiments, the pharmaceutical composition comprises a lipid composition (e.g., a lipid nanoparticie (LNP)) of the present disclosure, and at least one pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition is an aqueous solution or suspension. In certain embodiments, the pharmaceutical composition is an oil-based solution or suspension.

[0111] In certain embodiments, the present disclosure provides a method of delivering an active agent (e.g., polynucleotide) to a target cell or tissue (e.g., extrahepatic cell or tissue). In certain embodiments, the method comprises administering to a subject an effective amount of a lipid composition (e.g., LNP) of the present disclosure. In certain embodiments, the method comprises administering to a subject an effective amount of a pharmaceutical composition comprising a lipid composition (e.g., LNP) of the present disclosure.

[0112] In certain embodiments, the target cell or tissue is an extrahepatic cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises one or more of (e.g., all of): brain cell or tissue; lung cell or tissue; bone marrow cell or tissue; spleen cell or tissue; lymph node cell or tissue; ovarian / testicular cell or tissue; muscle cell or tissue; kidney cell or tissue; pancreatic cell or tissue; heart cell or tissue; and / or immune cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises one or more of (e.g., all of): brain cell or tissue; lung cell or tissue; and / or bone marrow cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a brain cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a lung cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a bone marrow cell or tissue. In certain embodiments, the bone marrow cell or tissue is in a femur. In certain embodiments, the extrahepatic cell or tissue comprises a spleen cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a lymph node cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises an ovarian or testicular cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a muscle cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a kidney cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a pancreatic cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises a heart cell or tissue. In certain embodiments, the extrahepatic cell or tissue comprises an immune cell or tissue. In some embodiments, the extrahepatic cell comprises a neutrophil, a monocyte (e.g., a CD 14+ monocyte), a macrophage, a B-cell, an erythroid progenitor, a hematopoietic stem cell, a CD3+ cell, an NK cell, or a combination thereof.

[0113] In certain embodiments, the present disclosure provides a method of treating a disease in a subject or a lipid composition (e.g., LNP) for use in a method of treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a lipid composition (e.g., LNP) of the present disclosure. In certain embodiments, the lipid composition (e.g., LNP) comprises a lipid compound of Formula (I), or a pharmaceutically acceptable salt thereof, and an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP)comprises any one of Compounds 1-31 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises any one of Compounds 1-26 from Table 1 , or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises any one of Compounds 1-11 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises any one of Compounds 12-18 from Table 1 , or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 1 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 2 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 3 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 4 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 5 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 6 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 7 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 10 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprisesCompound 12 from Table 1 , or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 13 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 14 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 15 from Table 1, or apharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA).In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 16 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 17 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 18 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA).

[0114] In certain embodiments, the present disclosure provides a method of producing a therapeutic composition, the method comprising encapsulating an active agent (e.g., DNA, RNA) within a lipid composition (e.g., LNP) of the present disclosure. In certain embodiments, the present disclosure provides a method of producing a prophylactic composition, the method comprising encapsulating an active agent (e.g., DNA, RNA) within a lipid composition (e.g., LNP) of the present disclosure. In certain embodiments, the present disclosure provides a method of producing a vaccine, the method comprising encapsulating an active agent (e.g., DNA, RNA) within a lipid composition (e.g., LNP) of the present disclosure.

[0115] In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises a lipid compound of Formula (I), or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises any one of Compounds 1 -31 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises any one of Compounds 1-26 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises any one of Compounds 1-11 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises any one of Compounds 12-18 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 1 from Table 1, or apharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA).In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 2 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 3 from Table 1 , or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 4 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 5 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 6 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 7 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 10 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 12 from Table 1 , or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 13 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 14 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 15 from Table 1 , or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA).In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 16 from Table 1 , or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 17 from Table 1 , or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 18 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the active agent (e.g., DNA, RNA) is encapsulated into the lipid nanoparticle (LNP) by mixing a solution comprising the active agent (e.g., DNA, RNA) with a solution / suspension comprising the lipid nanoparticles, or comprising precursor elements of the lipid nanoparticles. Examples of solutions or solvents that can be used in forming LNPs or in encapsulating active agents into LNPs include, but are not limited to: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, and isopropanol.

[0116] In certain embodiments, the active agent comprises DNA. In certain embodiments, the active agent comprises RNA. In certain embodiments, the active agent comprises DNA and RNA. In certain embodiments, the RNA comprises mRNA. In certain embodiments, the RNA comprises inhibitory RNA, e.g., siRNA.III. DEFINITIONS

[0117] Unless stated otherwise, the following terms and phrases have the meanings described below. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present disclosure.

[0118] Administering: As used herein, the term "administering" refers to providing a composition to a subject.

[0119] Alkenoxy: As used herein, the term "alkenoxy" refers to an alkenyl moiety attached through a divalent oxygen bridge (e.g., -O-C2-20 alkenyl). Examples of such groups include, but are not limited to, ethenoxy, propenoxy, and the like.

[0120] Alkenyl: As used herein, the term "alkenyl" refers to an unsaturated hydrocarbon chain (branched or unbranched) having one or more carbon-carbon double bonds within the chain. In certain embodiments, an alkenyl group may be optionally substituted with one or more substituents. Examples of alkenyl groups include, but are not limited to, ethylenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0121] Alkenylene: As used herein, the term "alkenylene" refers to a divalent alkenyl group. Examples of alkenylene groups include, but are not limited to, ethenyiene, propenylene, butenylene, pentenylene, hexenylene, and the like.

[0122] Alkoxy: As used herein, the term "alkoxy" refers to an alkyl moiety attached through a divalent oxygen bridge (e.g., R-O-C1-20). Examples of such groups include, but are not limited to, methoxy, ethoxy, propoxy, and the like.

[0123] Alkyl: As used herein, the term "alkyl" refers to a saturated hydrocarbon chain (branched or unbranched). In certain embodiments, an alkyl group may be optionally substituted with one or more substituents (i.e., the alkyl group may be unsubstituted or may be substituted with one or more substituents). In certain embodiments, an alkyl group may be optionally substituted with one or more halo groups (e.g., F). Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (n-propyl, iso-propyl), butyl (n-butyl, sec-butyl, iso-butyl, tert-butyl), pentyl (n-pentyl, isopentyd, neopentyl), and the like.

[0124] Alkylene'. As used herein, the term "alkylene" refers to divalent alkyl group. Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene (n-propylene, iso-propylene), butylene (n-butylene, sec-butylene, iso-butylene, tert-butylene), pentylene (n-pentylene, isopentylene, neopentylene), and the like.

[0125] Alkynyl: As used herein, the term "alkynyl" refers to an unsaturated hydrocarbon chain (branched or unbranched) having one or more carbon-carbon triple bonds within the chain. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like.

[0126] Alkynylene." As used herein, the term "alkynylene" refers to a divalent alkynyl group. Examples of alkynylene groups include, but are not limited to, ethynylene, propynylene, butynylene, pentynylene, hexynylene and the like.

[0127] Approximately / About: As used herein, the terms "approximately" and "about" are used interchangeably and refer to a value that is w ithin + / ■■ 10% of the recited value as applied to one or more values of interest. In certain embodiments, the term refers to a range of values that fall within + / - 10%, + / - 9%, + / - 8%, + / - 1%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1%, or less of the stated reference value, unless otherwise expressly s tated or otherwise clearly evident from the context.

[0128] Aryl: As used herein, the term "aryl" refers to a monocyclic aromatic hydrocarbon ring or a multicyclic group that contains at least one aromatic hydrocarbon ring. In certain embodiments, an aryl group may be optionally substituted with one or more substituents. Examples of aryl groups include, but are not limited to, phenyl and naphthyl.

[0129] Arylene: The term "arylene" is a multivalent (e.g., divalent or trivalent) aryl group.

[0130] Cycloalkyl: As used herein, the term "cycloalkyl" refers to a saturated monocyclic or multicyclic (e.g., bicyclic or tricyclic) hydrocarbon ring. In certain embodiments, a cycloalkyl group may be optionally substituted with one or more substituents. In certain embodiments, a cycloalkyl group may be optionally substituted with one or more C1-C12 alkyl groups (e.g., methyl, ethyl, / ?-pentyl, / ?-butyl, n-pentyl, etc.). The number of ring atoms in the cycloalkyl ring can be specified using "Cx-Cycycloalkyl" nomenclature where x and y are integers specifying the number of ring atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl and the like. In certain embodiments, a cycloalkyl group comprises a fused cycloalkyl group (e.g., fused bicyclic or fused tricyclic group). In certain embodiments, a cycloalkyl group comprises a bridged cycloalkyl group (e.g., bridged bicyclic group). In certain embodiments, a cycloalkyl group comprises a spiro cycloalkyl group (e.g., spiro bicyclic group).

[0131] Cycloalkylene: As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group. Examples of such groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, adamantylene, and the like.

[0132] Dienyl: As used herein, the term "dienyl" refers to an unsaturated hydrocarbon chain (branched or unbranched) having one or more carbon-carbon double bonds within the chain. Examples of dienyl groups include, but are not limited to, 1,3-pentadienyl, 1,4- hexadienyl, (2Z,5Z)-undeca-2,5-dienyl, and the like.

[0133] Effective amount: As used herein, the term "effective amount" or "therapeutically effective amount" of an agent is an amount sufficient to effect a beneficial or desired result (e.g., biological, medical, or clinical result). As such, an effective amount depends upon the context in which it is being applied (e.g., route of administration, seriousness of the condition, biochemistry, and medical history of subject, etc.), and can be determined bystandard clinical techniques by those with skill in the art (e.g., extrapolated from doseresponse curves derived from testing).

[0134] Halo: As used herein, the term "halo" refers to fluoro, chloro, bromo, and / or iodo.

[0135] Heterocyclic / Heterocycle / Heterocyclyl: As used herein, the term "heterocyclic," "heterocycle," or "heterocyclyl" refers to a saturated or unsaturated non-aromatic ring (monocyclic or bicyclic) containing one or more (e.g., from 1 to 4) heteroatoms (e.g., N, O, or S). The number of ring atoms in the heterocyclic ring can be specified using "x-y membered" nomenclature where x and y are integers specifying the number of ring atoms. For example, a 3-6 membered heterocycle group refers to a saturated or unsaturated 3- to 6-membered ring structure containing one or more heteroatoms, such as nitrogen, oxygen, and sulfur. Examples of heterocyclic groups include, but are not limited to, pyrrolinyl, pyrrolidinyl, pyrazolidinyl, oxazolyl, thiazolyl, piperidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, and the like.

[0136] Heterocyclylene: As used herein, the term "heterocyclylene" refers to a divalent heterocycle group. Examples of such groups include, but are not limited to, pyrrolinylene, pyrrolidinylene, pyrazolidinylene, oxazolylene, thiazolylene, piperidinylene, piperazinylene, morpholinylene, and the like.

[0137] Hydroxyalkyl: As used herein, the term "hydroxyalkyl" refers to an R-Ci-20-OH group. Examples of such groups include, but are not limited to, hydroxymethyl, 2- hydroxyethyl, 3-hydroxypropyl, and the like.

[0138] Lipid nanoparticle'. As used herein, the terms "lipid nanoparticle" or "LNP" refer to particles comprising a plurality of lipid molecules physically associated with each other by intermolecular forces. In certain embodiments, lipid nanoparticles are microspherical vesicles that comprise liposomal lamellar phase lipid bilayers (unilamellar or multilamellar). In other embodiments, lipid nanoparticles are nanospherical vesicles that comprise liposomal lamellar phase lipid bilayers (unilamellar or multilamellar).

[0139] mol %>'. As used herein, the terms "mole percentage" or "mol %" refer to the mole fraction of a specific element within a mixture, stated as a percentage of the total number of moles in the mixture. The mole fraction is the number of moles of one ingredient in the given mixture relative to the total number of moles in the mixture.

[0140] Pharmaceutically acceptable'. As used herein, the terms "pharmaceutically acceptable" or "therapeutically acceptable" are used to describe compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0141] Pharmaceutically acceptable excipients: As used herein, the term "pharmaceutically acceptable excipient" refers to an ingredient in a composition capable of suspending, carrying, diluting, stabilizing, controlling, encapsulating, or otherwise supplementing a compound or composition of the present disclosure (e.g., LNP) in a pharmaceutical composition. Pharmaceutically'' acceptable excipients are substantially nontoxic, noninflammatory, and otherwise pharmaceutically acceptable (as defined above) in a subject.

[0142] Pharmaceutically acceptable salt: As used herein, the phrase "pharmaceutically acceptable salt" refers to salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like and are commensurate with a reasonable benefit / risk ratio. Compounds as described herein may contain either a basic or an acidic functionality, or both, and can be converted to a pharmaceutically acceptable salt, when desired, by using a suitable acid or base. The salts may be prepared in situ during the final isolation and purification of the compounds.

[0143] Phospholipid'. As used herein, the term "phospholipid" refers to a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more double or triple bonds in the carbon chains (e.g., one or more unsaturations).

[0144] Preventing or prophylaxis'. As used herein, the term "preventing," "prevention," or "prophylaxis" refers to partially or completely delaying onset of a disease or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a disease or condition; partially or completely delaying progression of a disease or condition; and / or decreasing the risk of developing pathology associated with a disease or condition. In certain embodiments, "preventing," "prevention," or "prophylaxis" of a disease or condition may be considered a subset within the meaning of the term "treatment" or "treating" of the disease or condition.

[0145] Subject: As used herein, the term "subject" refers to any organism to winch a composition in accordance with the present disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects comprise animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. The subject may seek or need treatment, require treatment, is receiving treatment, will receive treatment, or is under care by a trained professional for a particular disease or condition.

[0146] The Same Substituents: As used herein, when two substituents are "the same," the substituents have the same molecular formula and sequence of bonded atoms, but may or may not differ in the three-dimensional orientations of their atoms in space. In certain embodiments, when each of the same substituents contains one or more double bonds, the configuration of each of the one or more double bonds in one substituent is the same as the configuration of the corresponding one or more double bonds in the other substituent.

[0147] Treating'. As used herein, the term "treating" or "treatment" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, reducing incidence of, and / or preventing one or more symptoms or features of a particular disease or condition. Treatment may be administered to a subject who does not exhibit signs of a disease or condition and / or to a subject who exhibits only early signs of a disease or condition for the purpose of decreasing the risk of developing pathology (or further pathology) associated with the disease or condition.General Considerations

[0148] At various places in the present disclosure, substituents, or properties of compounds of the present disclosure are disclosed in groups or in ranges. It is intended that the present disclosure comprises each and every individual or sub -combination of the members of such groups and ranges, and that such groups or ranges include the endpoints. By way of nonlimiting example, if a group or range is from about 1 to about 10, then the group or range includes both the value of about 1 and the value of about 10.

[0149] Articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that comprise "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure can include embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure can include embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

[0150] The term "comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the terms "consisting of" and "consisting essentially of are also encompassed and disclosed.

[0151] The abbreviation, "e.g.," is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g.," is synonymous with the term "for example." The abbreviation, "i.e.," is derived from the Latin id est, and is used herein to indicate a non-limiting rewording or clarification. Thus, the abbreviation "i.e.," is synonymous with the term "that is."

[0152] Where a variable is provided in the context of an organic chemical structure as having a range of numbers, that variable is understood to be an integer value in that range, inclusive of the end points. For example, “n is 0-3” means that n is 0, 1, 2, or 3,

[0153] Any embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Any embodiment of the agents, methods, and / or compositions of the present disclosure can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0154] The present specification will control in instances where publications, patent applications, patents, and other references mentioned herein are incorporated by reference and are in conflict with the present specification.

[0155] Section headings, materials, methods, and examples are illustrative only and not intended to be limiting.EXAMPLESExample 1 - General Materials and Methods for Syntheses

[0156] All temperatures are in degrees Celsius and are uncorrected. Reagent grade chemicals and anhydrous solvents were purchased from commercial sources and unless otherwise mentioned, were used without further purification. The names of the products were determined using the naming software included in the Biovia electronic lab notebook. Silica gel chromatography was performed on Teledyne Isco instruments using pre-packaged disposable SiCh stationary phase columns with eluent flow-rate ranges of 15 to 200 mL / min, UV detection (254 and 280 nm). Reverse phase purification was carried out using C 18 columns, UV detection (214 and 254 nm). The chemical shifts are reported in parts-per- million and are referenced to solvent peaks, which in1H NMR appear at 7.26 ppm for CDCh, 2.50 for DMSO-rA, and 3.31 ppm for CD3OD.

[0157] Terms and abbreviations:4-PP Y 4-pyrrolidin- 1 -ylpyridine;Ac acetyl; aq aqueous;Bn benzyl;DBU 1 ,8-diazabicyclo[5.4.0]undec-7-ene;DCM dichloromethane;DIP E A N,N- diisopropyl ethylamine ;D M AP 4- di m ethylamin opy ri dine ;EDCI l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;Et ethyl;EtOAc ethyl acetate;h hour(s); HC1 hydrochloride; HPLC high performance liquid chromatography; LCMS liquid chromatography mass spectrometry; mCPBA 3 -chloroperbenzoic acid; Me methyl; min minute(s); NaHCCh sodium bicarbonate; \a2SO . sodium sulfate; PPTS pyridinium p-toluenesulfonate; Py pyridine; TBS teH-buty I dimethylsily l ; TEA triethylamine; TEMPO l-oxidanyl-2,2,6,6-tetramethyl-piperidine; Tol toluene; NH4Q ammonium chloride; NM:R nuclear magnetic resonance; P-TSOH-H2O para-toluenesulfonic acid monohydrate; sat. saturated:THF tetrahydrofuran.Example 2 - Synthesis of Compound 1: 07-[3-[4,4”bis[(Z)~oct~5-enoxy]butanoyioxy]"2-[3"P(diethylamino)propoxycarbonyloxy]azetidin"l~yI]propyI] Ol”(3-pentytectyl) heptanedioate

[0158] Step 1 :

[0159] To a solution of undecan-6-one (10 g, 58.72 mmol, 1 eq) in THF (100 mL) was added NaH (3.05 g, 76.34 mmol, 60% purity, 1.3 eq) slowly at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 1 h under N2 atmosphere.Then ethyl 2-diethoxyphosphorylacetate (17. 11 g, 76.34 mmol, 15.15 mL, 1 .3 eq) in THF(100 mL.) was added to the above reaction mixture dropwise at 25 °C and the reaction mixture was stirred for another 11 h at 25 °C under N? atmosphere. The reaction mixture wasadded sat. NHaCl (200 mL) slowly under N2 and stirred for another 10 min after addition. The reaction mixture was poured into H2O (200 mL) and extracted with EtOAc 600 mL (3*200 mL). The combined organic layers were dried over Na2S(>4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate™ 100 / 1 to 10 / 1). The organic phase was evaporated under reduced pressure to get a residue. Compound ethyl 3-pentyloct- 2-enoate (10 g, 41.60 mmol, 70.84% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCh) 8 ppm 5.62 (s, 1 H), 4.14 (q, .1 7.2 Hz, 2 H), 2.65 - 2.51 (m, 2 H), 2.20 - 2.09 (m, 2 H), 1.48 - 1.26 (m, 15 H), 0.90 (t, J=6.4 Hz, 6 H).SNT3

[0160] Step 2:

[0161] To a solution of ethyl 3-pentyloct-2-enoate (10 g, 41.60 mmol, 1 eq) in THF (100 mL) was added Pd / C (44.27 g, 41.60 mmol, 10% purity, 1 eq) under Ar atmosphere. The suspension was stirred under H2 (15 Psi) at 25 °C for 12 hr. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOc, Petroleum ether / Ethyl acetate:=:100 / T to 10 / 1). Compound ethyl 3-pentyloctanoate (8.5 g, 35.07 mmol, 84.29% yield) was obtained as a colorless oil.1H X.V1 R (400 MHz, CDCb) 8 ppm 4.16-4.10 (m, 2H), 2.23-2.22(m, 2H), 1.86- 1.84(m, 1H), 1.31-1.24(m, 19H), 0.91-0.87(t, J ==3.4 Hz, 6H).

[0162] Step 3:

[0163] To a solution of LiAlH4 (2.5 M, 28.05 mL, 2 eq) in THF (85 mL) was added ethyl 3 -pentyl octanoate (8.5 g, 35.07 mmol, 1 eq) slowly at 0 °C under N2 atmosphere. The reaction mixture was stirred for 12 h at 25 °C under N? after addition. The reaction mixture was added sat. NHUCl (100 mL) slowly at 0 °C under N2 and stirred for another 10 min after addition. The reaction mixture was poured into H2O (100 mL) and extracted with EtOAc 600 mL (3*200 mL). The organic layer was concentrated under reduced pressure to get a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound 3-pentyloctan-l-ol (5 g, 24.96 mmol, 71.17% yield) wasobtained as a colorless oil.!H NMR (400 MHz, CDCh) S ppm 3.68-3.64(m, 2H), 1.55-1.42(m, 4H), 1.31 -1.26(m, 16H), 0.90-0.87(t, J =3.4Hz, 6H).

[0164] Step

[0165] To a solution of 3-pentyloctan-l-ol (5 g, 24.96 mmol, 1 eq) in DCM (50 mL) was added EDCI (5.74 g, 29.95 mmol, 1.2 eq), heptanedioic acid (7.99 g, 49.91 mmol, 2 eq), DIPEA (8.06 g, 62.39 mmol, 10.87 mL, 2.5 eq) and DMAP (304.87 mg, 2.50 mmol, 0.1 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O (200 mL) and extracted with DCM 600 mL (200 mL * 3). The combined organic layers were washed with DCM 100 mL (50 mL * 2), dried over NacSOq filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:::100 / 1 to 0 / 1). Compound 7-oxo-7-(3-pentyloctoxy) heptanoic acid (5 g, 14.60 mmol, 58.50% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCI3) δ ppm 4.09 (t, J = 7.0 Hz, 2H), 2.37 (t, J = 7.4 Hz, 2H), 2.31 (s, 2H), 1.66 (s, 4H), 1.57 (d, J = 6.6 Hz, 2H), 1.44 - 1.36 (m, 3H), 1.35 - 1.10 (m, 17H), 0.89 ( t, J = 6.8 Hz, 6H).

[0166] Step 5:

[0167] To a solution of 4,4- dimethoxybutanenitrile (20 g, 154.85 mmol, 1 eq) and (Z)-oct- 5-en-l-ol (49.63 g, 387.13 mmol, 2.5 eq) in Tol. (200 mL) was added PPTS (9.73 g, 38.71 mmol, 0.25 eq) under N2 atmosphere. The reaction mixture was stirred at 120 °C for 34 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O ( 1000 mL) and extracted with DCM 1500 mL (500 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=50 / l to 1 / 1). Compound 4,4- bis[(Z)-oct-5-enoxy]butanenitrile (10 g, 31.10 mmol, 20.09% yield) was obtained as acolorless oil. 'H NMR (400 MHz, CDCI3) δ ppm 5.54 - 5.20 (m, 4H), 4.56 (t, J - 5.2 Hz, 1H), 3.74 ■■ 3.56 (m, 2H), 3.51 ■■ 3.34 (m, 2H), 2.43 (t, J = 7.4 Hz, 2H), 2.1 1 ■■ 2.01 (m, 8H), 1.99 - 1.90 (m, 211). 1.65 - 1.56 (m, 4H), 1.49 - 1.35 (m, 4H), 0.97 (t, J - 7.4 Hz, 6H).

[0168] Step 6:1807 !NT8

[0169] To a solution of 4,4-bis[(Z)-oct-5-enoxy]butanenitrile (10 g, 31.10 mmol, 1 eq) in EtOH (50 mL) and H2O (50 mL) was added KOH (5.24 g, 93.31 mmol, 3 eq) under N2 atmosphere. The reaction mixture was stirred at 110 °C for 12 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with water (500 mL), adjusted to PH::::7 by using 1 M HC1 at 0 °C under N2 and extracted with EtOAc (500 mL * 3). The combined organic layers were washed with aq. NaCl (300 mL * 2), dried over Na2SC)4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=50 / T to 1 / 1). Compound 4,4-bis[(Z)-oct-5- enoxy]butanoic acid (5 g, 14.68 mmol, 47.21% yield, 100% purity) was obtained as a colorless oil.!H NMR (400 MHz, CDCh) S ppm 5.48 - 5.25 (m, 4H), 4.53 (t, J = 5.4 Hz, 1H), 3.65 - 3.55 (m, 2H), 3.49 - 3.36 (m, 2H), 2.46 (t, J = 7.4 Hz, 2H), 2.10 - 2.00 (m, 8H), 1.99 ■■ 1.92 (m, 2H), 1.64 ■■ 1.54 (m, 4H), 1.47 ■■ 1.37 (m, 4H), 0.96 (t, J = 7.4 Hz, 6H).[01701 Step 7: ^T9

[0171] To a solution of l,3-dihydroxypropan-2-one (20 g, 222.03 mmol, 1 eq) in DMF(200 mL) was added imidazole (6.05 g, 88.81 mmol, 0.4 eq) under N2 atmosphere, then a solution of TBSCI (10.04 g, 66.61 mmol, 8.20 mL, 0.3 eq) in DMF (200 mL) was added to the above reaction mixture under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (500 mL) and extracted with DCM 1000 mL (500 mL * 2). The combined organic layers were dried over NazSCM, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOc, Petroleum ether / Ethyl acetate=50 / l to 5 / 1). Compound l-[tert-butyl(dimethyl)silyl]oxy-3-hydroxy-propan-2-one (6g, 29.36 mmol, 44.08% yield) was obtained as a colorless oiL 'H NMR (400 MHz, CDCb) 8 ppm 4.51 (s, 2H), 4.32 (s, 211). 0.93 (s, 9H), 0.10 (m, 6H).

[0172] Step 8:!?mo !fm1

[0173] lb a solution of l-[tert-butyl(dimethyl)silyl]oxy-3-hydroxy-propan-2-one (5 g, 24.47 mmol, 1 eq) in DCM (50 mL) was added EDCI (5.63 g, 29.36 mmol, 1.2 eq), DIPEA (7.91 g, 61.17 mmol, 10.66 mL, 2.5 eq) and DMAP (298.93 mg, 2.45 mmol, 0.1 eq) under N2 atmosphere, then 7-oxO”7-(3-penty-loctoxy)heptanoic acid (10.06 g, 29.36 mmol, 1.2 eq) was added to the above reaction mixture under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H 2O (100 mL) and extracted with DCM 200 ml, (100 ml, * 2), The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=50 / l to 5 / 1 ). Compound O7-[3-[tert-butyl(dimethyl)silyl]oxy-2-oxo-propyl] Ol-(3- pentyloctyl) heptane-dioate (9.5 g, 17.96 mmol, 73.41% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCL3) 8 ppm 4.96 (s, 2H), 4.27 (s, 2H), 4.08 (t, J - 7.2 Hz, 2H), 2.45 (t, J = 7.4 Hz, 2H), 2.31 (t, J = 7.6 Hz, 2H), 1.76 - 1.64 (m, 4H), 1.58 (s, 2H), 1.47 - 1.39 (m, 3H), 1.31 - 1.23 (m, 1611). 0.93 (s, 9H), 0.89 (t, J - 6.8 Hz, 6H), 0.11 (s, 6H).

[0174] Step 9:ibmi SNT12

[0175] To a solution of O7-[3-[tert-butyl(dimethyl)silyl]oxy-2-oxo-propyl] 01 -(3- pentyloctyl) heptanedioate (8 g, 15.13 mmol, 1 eq) in THF (80 mL) was added triethylamine trihydrofluoride (3.16 g, 19.63 mmol, 3.20 mL, 1 .30 eq) dropwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 0 °C for 2 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (250 mL) and extracted with DCM 500 mL (250 mL * 2). The combined organic layers were dried over NazSCh, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography(SiOz, Petroleum ether / Ethyl acetate::::50 / l to 5 / 1). Compound O7-(3-hydroxy-2-oxo-propyl) 01 -(3 -pentyloctyl) heptanedioate (4 g, 9.65 mmol, 63.78% yield) was obtained as a colorless oil.

[0176] Step 10:

[0177] To a solution of O7-(3-hydroxy-2-oxo-propyl) 01 -(3 -pentyloctyl) heptanedioate (4 g, 9.65 mmol, 1 eq) in DCM (40 mL) was added EDCI (2.22 g, 11 .58 mmol, 1.2 eq), DIPEA (3.12 g, 24.12 mmol, 4.20 mL, 2.5 eq) and DMAP (117.87 mg, 964.85 pmol, 0.1 eq) under N2 atmosphere, then 4,4-bis[(Z)-oct-5-enoxy]butanoic acid (3.29 g, 9.65 mmol, 1 eq) was added to the above reaction mixture under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM (100 mL * 2). The combined organic layers were dried over Na2SC)4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleumether / Ethylacet- ate=50 / l to 5 / 1). Compound O7-[3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2-oxo-propyl] 01- (3-pentyloctyl) heptanedioate (3.7 g, 5.02. mmol, 52,03% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDCL3) 5 ppm 5.49 - 5.24 (m, 4H), 4.76 (d, J === 4.0 Hz, 4H), 4.52 (t, J = 5.4 Hz, 1 H), 4.19 - 4.02 (m, 4H), 3.63 - 3.55 (m, 2H), 3.46 - 3.37 (m, 2H), 2.52 (t, J = 7.4 Hz, 2H), 2.44 (t, J === 7.4 Hz, 2H), 2.31 (t, J === 7.4 Hz, 2H), 2.08 - 2.03 (m, 8H), 2.00 - 1 .94 (m, 2H), 1.72 - 1.64 (m, 4H), 1.63 - 1.55 (m, 10H), 1.46 - 1.37 (m, 8H), 1.35 - 1.22 (m, 22H), 0.96 (t, J - 7.4 Hz, 6H), 0.89 (t, J - 7.0 Hz, 6H).

[0178] Step 11 :

[0179] To a solution of O7-[3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2-oxo-propyl] 01- (3 -pentyloctyl) heptanedioate (3.7 g, 5.02 mmol, 1 eq), azetidin-3-ol (659.95 mg, 6.02 mmol,1.2 eq, HC1 salt), NaBIhCN (630.93 mg, 10.04 mmol, 2 eq) in DC VI (35 mL.) and MeOH (10 ml) was added TEA (609.56 mg, 6.02 mmol, 838.46 pL, 1.2 eq) slowly at 0 °C under Na atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (50 mL.) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were dried over NaiSCh, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane / Methanol=50 / 1 to 5 / 1). Compound O7-[3-[4,4-bis[(Z)-oct-5- enoxy]butanoyloxy]-2-(3-hydroxyazetidin-l-yl)propyl] Ol-(3-pentylo-ctyl)heptanedioate(2.9 g, 3.65 mmol, 72.74% yield) was obtained as a colorless oil.!H NMR (400 MHz,CDCh) 8 ppm 5.44 5.27 (m, 4H), 4.52 ■■ 4.47 (m, 1 H ), 4.13 4.01 (m, 6H), 3.81 ■■ 3.72 (m, 2H), 3.62 - 3.53 (m, 2H), 3.46 - 3.36 (m, 2H), 3.17 - 3.04 (m, 2H), 2.77 - 2.67 (m, 1H), 2.42 (t, J = 7.6 Hz, 2H), 2.37 ■■ 2.28 (m, 4H), 2.09 ■■ 2.01 (m, 811 ). 1.96 ■■ 1.90 (m, 2H), 1 .68 ■■ 1.55 (m, 12H), 1.44 - 1.24 (m, 241 h. 0.96 (t, J - 7.4 Hz, 6H), 0.89 (t, J - 7.0 Hz, 6H).

[0180] Step 12:

[0181] To a solution of O7-[3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxyj-2-(3- hydroxyazetidin-1 ~yl)propyl] Ol-(3-pentyloctyl) heptanedioate (2.9 g, 3.65 mmol,1 eq) in DCM (29 mL) was added TEA (1.11 g, 10.96 mmol, 1 .52 mL, 3 eq) and CDI (888.18 mg, 5.48 mmol, 1.5 eq) under N2 atmosphere. The mixture was stirred at 20 °C for 2 hr under N2 atmosphere. Then 3-(diethylamino)propan-l-ol (5.75 g, 43.82 mmol, 6.53 mL, 12 eq) was added to the above reaction mixture under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 h under N? atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (20 mL) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO?, Petroleum ether / Ethyl acetate=50 / l to 0 / 1). Compound O7-[3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2-[3- [3(diethylamino)propoxycarbonyloxy]azetidin- 1 -yljpropyl] 01 -(3 -pentyloctyl) heptanedioate (0.1 g, 105.11 pmol, 2.88% yield)was obtained as a colorless oil.!H NMR (400 MHz,CD3OD) 8 ppm 5.44 - 5.27 (m, 4H), 5.03 - 4.94 (m, 1H), 4.52 (t, J === 5.4 Hz, 1H), 4.22 - 4.01 (m, 9H), 3.82 ■■ 3.73 (m, 2H), 3.65 3.56 (m, 2H), 3.50 ■■ 3.41 (m, 2H), 2.84 ■■ 2.74 (m, 1H), 2.65 - 2.53 (m, 6H), 2.44 - 2.31 (m, 6H), 2.13 - 2.00 (m, 8H), 1.94 - 1.81 (m, 4H), 1.67 - 1.55 (m, 10H), 1.48 ■■ 1.22 (m, 26H), 1.06 (t, J = 7.2 Hz, 6H), 0.96 (t, J = 7.6 Hz, 6H), 0.91 (t, J = 7.0 Hz, 6H).Example 3 - Synthesis of Compound 2: 07"[3"[4,4~bis[(Z)~oet~5~enoxy]butanoyloxy]~2~[3-(4-pyrrolidin-l-ylbutanoyioxy)azetidine-l-yl]propyl] 01%3-pentyIoetyI) heptanedioate

[0182] Synthetic scheme:Compeursd 2

[0183] To a solution of O7-[3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2-(3- hydroxyazetidin-l-yl)propyl] Ol -(3-pentyloctyl) heptanedioate (0.6 g, 755.53 pmol, 0.3 eq) in DCM (6 mL) was added EDCI (579.34 mg, 3.02 mmol, 1.2 eq), DIPEA (1.14 g, 8.81 mmol, 1 .54 mL, 3.5 eq) and DMAP (30.77 mg, 251.84 pmol, 0.1 eq) under N2 atmosphere, then 4-pyrrolidin-l-ylbutanoic acid (487.74 mg, 2.52 mmol, 1 .00 eq, HC1) was added to the above reaction mixture under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (200 mL) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were dried over NaaSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane / Methanol=;:50 / 1 to 5 / 1). Compound O7-[3- [4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2-[3-(4-pyrrolidin- 1 -ylbutanoyloxy)azetidine- 1 - yl]propyl] 01 -(3 -pentyloctyl) heptanedioate (0.1 g, 107.14 pmol, 4.25% yield, 100% purity) was obtained as a colorless oil.!H NMR (400 MHz, CDCh) 8 ppm 5.45 - 5.26 (m, 4H), 5.11 - 4.99 (m, 1H), 4.49 (t, J 5.4 Hz, 1H), 4.12 - 4.02 (m, 6H), 3.82 - 3.73 (m, 2H), 3.62 - 3.52 (m, 2H), 3.47 - 3.37 (m, 2H), 3.23 ■■ 3.16 (m, 2H), 2.70 (t, J = 5.0 Hz, 1H), 2.62 ■■ 2.53 (m, 4H), 2.44 - 2.37 (m, 4H), 2.36 - 2.28 (m, 4H), 2.09 - 2.00 (m, 8H), 1.96 - 1.87 (m, 4H), 1.82(s, 4H), 1.70 - 1.54 (m, 16H), 1.46 - 1.35 (m, 8H), 1.30 - 1.23 (m, 15H), 0.96 (l.. / 7.6 1 k 6H), 0.89 (t, 7= 7.0 Hz, 6H).Example 4 - Synthesis of Compound 3: Compound [7»[3“[4,4»bis[(Z)“Oct“5~ enoxy] bo tanoyloxyj -2“[3" [4~(2~pyrroBdme~l" ylethylcarbamoyloxy)decaaoyioxy]axetidm”l“yl]propoxy]“7”oxo~heptyl] 2- botyloetauoate

[0184] Step

[0185] To a solution of 5-hexyltetrahydrofuran~2-one (100 g, 587.38 mmol, 1 eq) in H2O (500 mL) was added NaOH (24.67 g, 616.75 mmol, 1 .05 eq) slowly under N2 atmosphere.The reaction mixture was stirred at 100 °C for 12 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove solvent. Compound 4- hydroxydecanoyloxysodium(90 g, crude) was obtained as a white solid and it was used in next step directly.

[0186] Step

[0187] To a solution of 4-hydroxydecanoyloxy sodium (10 g, 47.56 mmol, 1 eq) in DMSO (100 mL) was added bromomethylbenzene (8.13 g, 47.56 mmol, 5.65 mL, 1 eq) dropwise under N2 atmosphere. The reaction mixture was stirred at 25 °C for 5 min under N2 atmosphere. The reaction mixture was diluted with sat. NaCl (100 mL) and extracted with EtOAc (200 mL) (100 mL * 2). The combined organic layers were dried over NazSCM, filtered and the filtrate was concentrated under reduced pressure to give a residue. Compound benzyl 4-hydroxydecanoate (12 g, crude) was obtained as a pale yellow oil and it was used in next step quickly.

[0189] To a solution of benzyl 4- hydroxydecanoate (12 g, 43.1 1 mmol, 1 eq) in DCM (120 niL) was added (4-nitrophenyl) carbonochloridate (17.38 g, 86.21 mmol, 2 eq) and Py. (6.82 g, 86.21 mmol, 6.96 mL, 2 eq) slowly at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 1 hr under N2 atmosphere. The reaction mixture was diluted with petroleum ether (100 mL), filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCb., Petroleum ether / Ethyl acetate:::T()() / T to 10 / 1). Compound benzyl 4-(4- nitrophenoxy)carbonyloxydecanoate (10 g, 22.55 mmol, 52.31% yield) was obtained as a colorless oil.]H NMR (400 MHz, CDCh) 8 ppm 8.30-8.25 (m, 2H), 7.46-7.33 (m, 7H), 5.14 (s, 2H), 4.92-4.85 (m, 1H), 2.51 (t, J = 8.0 Hz, 2H), 2.16-2.08 (m, 1H), 2.03-1.96 (m, 1H), 1.79-1.62 (m, 2H), 1.47-1.28 (m, 8H), 0.90 (t, J = 6.8 Hz, 3H).

[0190] Step 4:imr19 !NT29

[0191] To a solution of benzyl 4-(4-nitrophenoxy)carbonyloxydecanoate (10 g, 22.55 mmol, 1 eq) in DCM (100 mL) was added 2-pyrrolidin-l-ylethanamine (7.72 g, 67.65 mmol, 3 eq), DMAP (550.94 mg, 4.51 mmol, 0.2 eq) and DIPEA (8.74 g, 67.65 mmol, 11 .78 mL, 3 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL *2). The combined organic layers were concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate=50 / 1 to 0 / 1). Compound benzyl 4-(2-pyrro lidin- 1- ylethylcarbamoyloxy) decanoate (7 g, 16.72 mmol, 74.17% yield) was obtained as a colorless oil.

[0193] To a suspension of Pd / C (2 g, 1.88 mmol, 10% purity, 0.112 eq) in THF (140 niL) was added benzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)decanoate (7 g, 16.72 mmol, 1 eq) under Nz atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under H2 (15 Psi). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM / MeOH:::50 / l to 3 / 1). Compound 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)decanoic acid (3 g, 9.13 mmol, 54.62% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCh) 3 ppm 9.18 (b. 1 H), 6.58 (d, J - 3.6 Hz, 1H), 4.72 (d. J - 4.8 Hz, 1H), 3.68-3.61 (m, 1H), 3.21- 3.15 (m, 1H), 3.09-3.00 (m, 5H), 2.89-2.85 (m, 1H), 2.37-2.20 (m, 2H), 2.03-1.97 (m, 5H), 1.83-1.72 (m, 1H), 1.83-1.73(m, 1H), 1.62-1.55(m, 1H), 1.51-1.44(m, 1H), 1.30-1.25 (m, 8H), 0.86(t, .1 == 6.0 Hz, 3H).

[0195] To a solution of heptane- 1,7-diol (98.99 g, 748.81 mmol, 3 eq) in DCM (980 ml.) was added EDCI (71.77 g, 374.41 mmol, 1.5 eq), DMAP (15.25 g, 124.80 mmol, 0.5 eq), DIPEA (96.78 g, 748.81 mmol, 130.43 ml, 3 eq) and 2-butyloctanoic acid (50 g, 249.60 mmol, 1 eq) under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (1000 mL) and extracted with DCM 1000 mL (500 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOz, Petroleum ether / Ethyl acetate=l / O to 10 / 1 ).Compound 7-hydroxyheptyl 2 -butyloctanoate (30 g, 95.39 mmol, 38.22% yield) was obtained as a colorless oil.fH NMR (400 MHz, CDCh) 8 ppm 4.08 (t, J:;= 6.6 Hz, 2H ), 3.72 - 3.57 (m, 2H), 2.37 - 2.16 (m, 1H), 1.71 - 1.50 (m, 6H), 1.49 - 1.34 (m, 8H), 1.32 - 1.16 (m, 12H), 0.95 - 0.81 (m, 6H).

[0196] Step 7:

[0197] To a solution of 7-hydroxyheptyl 2-buty I octanoate (30 g, 95.39 mmol, 1 eq) in zXCN (150 mL) and H2O (150 mL) was added [acetoxy(phenyl)-iodanyl] acetate (76.81 g, 238.47 mmol, 2.5 eq) and TEMPO (9.00 g, 57.23 mmol, 0.6 eq) under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (500 mL) and extracted with DCM 500 mL (250 mL * 2). The combined organic layers were dried over NacSCh, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=;:l / O to 10 / 1). Compound 7-(2- butyloctanoyloxy)heptanoic acid (16 g, 48.56 mmol, 50.91% yield, 99.7% purity) was obtained as a colorless oil.fH NMR (400 MHz, CDCh) 8 ppm 4.13 - 4.04 (m, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.36 - 2.28 (m, 111)., 1.76 ■■ 1.55 (m, 6H), 1.51 - 1.36 (m, 6H), 1.34 - 1.19 (m, 12H), 0.98 - 0.83 (m, 6H).

[0198] Step 8:

[0199] A mixture of 7-(2-butyloctanoyloxy)heptanoic acid (3 g, 9.13 mmol, 1.2 eq), 1- [tert-butyl(dimethyl)silyl]oxy-3-hydroxy-propan-2-one (1.56 g, 7.61 mmol, 1 eq), EDCI (1.75 g, 9.13 mmol, 1.2 eq), DIPEA (2.46 g, 19.03 mmol, 3.31 mL, 2.5 eq) and DMAP (92.98 mg, 761.07 pmol, 0.1 eq) in DCM (40 mL) was stirred at 20 °C for 12 hr under Na atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 150 mL (50 mL * 3). The combined organic layers were dried over NacSCM, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOc, Petroleum ether / Ethyl acetate= 100 / 1 to 5 / 1). Compound [7-[3-[tert-butyl(dimethyl)silyl]ox-y-2-oxo-propoxy]-7- oxo-heptyl]2-butyloctanoate (2 g, 3.88 mmol, 51.05% yield) was obtained as a colorless oil.1H NMR (400 MHz, CDCh) 8 ppm 4.96 (s, 2H), 4.27 (s, 2H), 4.07 (t, J - 6.6 Hz, 2H), 2.44(t J = 7.4 Hz, 2H), 2.36 ■■ 2.26 (m, 1H), 1.71 - 1.57 (m, 6H), 1.49 ■■ 1.35 (m, 6H), 1.34 1.18(m, 1211), 0.93 (s, 9H), 0.91 - 0.85 (m, 6H), 0.11 (s, 6H).

[0200] Step 9:

[0201] A mixture of [7-[3-[tert-butyl(dimethyl)silyl]oxy-2-oxo-propoxy]-7-oxo-heptyl] 2- butyloctanoate (2 g, 3.88 mmol, 1 eq) and N,N-diethylethanamine;trihydrofluoride (1.25 g, 7.77 mmol, 1.27 mL, 2 eq) in THE (20 mL) was stirred at 20 °C for 12 hr underN2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over NasSCfi, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 20 / 1). Compound [7-(3-hydroxy-2-oxo-propoxy)-7-oxo-heptyl] 2 -butyloctanoate (1 g, 2.50 mmol, 64.26% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCh) 8 ppm 4.77 (s, 2H), 4.38 (s, 2H), 4.08 (t, J = 6.6 Hz, 2H), 2.45 (t, J = 7.4 Hz, 2H), 2.34 - 2.27 (m, 1H), 1.71 - 1.57 (m, 6H), 1.47 - 1.37 (m, 6H), 1.30 - 1.21 (m, 12H), 0.91 - 0.86 (m, 6H)

[0202] Step 10:

[0203] A mixture of [7-(3-hydroxy-2-oxo-propoxy)-7-oxo-heptyl]2-butyloctanoate (1 g, 2.50 mmol, 1 eq), 4,4”bis[(Z)-oct-5-enoxy]butanoic acid (1.02 g, 3.00 mmol, 1.2 eq), EDCI (574.32 mg, 3.00 mmol, 1.2 eq), DIPEA (806.66 mg, 6.24 mmol, 1.09 mL,2.5 eq) and DMAP(37.00 mg, 249.66 pmol, 0.1 eq) in DCM (20 mL) was stirred at 20°C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 150 mL (50 mL * 3). The combined organic layers were dried over NazSCM, filtered and the filtrate was concentrated under reduced pressure to give a residue.The residue was purified by column chromatography (SiOc, Petroleum ether / Ethylacetate:::10 / l to 0 / 1). Compound [7-[.3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2-oxo- propoxy]-7-oxo-heptyl] 2 -butyloctanoate (0.7 g, 968.15 pmol, 38.78% yield) was obtained as a colorless oil.1H NMR (400 MHz, CDCb) 8 ppm 5.47 - 5.24 (m, 4H), 4.77 - 4.75 (m, 4H),4.52 (t J = 5.4 Hz, 1H), 4.07 (t, J = 6.6 Hz, 2H), 3.63 ■■ 3.55 (m, 2H), 3.46 ■■ 3.39 (m, 2H),2.52 (t, J - 7.4 Hz, 2H), 2.46 - 2.42 (m, 2H), 2.34 - 2.29 (m, 1H), 2.05 (d, J - 7.4 Hz, 6H), 2.01 - 1.95 (m, 2H), 1.70 - 1.56 (m, 16H), 1.45 - 1.38 (m, 10H), 1.30 - 1.23 (m, 14H), 0.96 (t, J = 7.6 Hz, 6H), 0.90 - 0.87 (m, 6H)

[0204] Step 11 :

[0205] A mixture of azetidin-3-ol (212.13 mg, 1.94 mmol, 2 eq, HC1), (7-[3-[4,4-bis[(Z)- oct-5-enoxy]butanoyloxy]-2-oxo-propoxy]-7-oxo-heptyl]2-butyloctanoate (0.7 g, 968.15 pmol, 1 eq), TEA (117.56 mg, 1.16 mmol, 161.71 pL, 1.2 eq) and NaBH(OAc)s (246.23 mg, 1.16 mmol, 1.2 eq) in DCM (14 mL) and MeOH (4 mL) was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were dried over NacSCfi, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOa, Petroleum ether / Ethyl acetate=10 / l to 0 / 1). Compound 7- (3-((4,4-bis((Z)-oct-5-en-l-yloxy)butanoyl)oxy)-2-(3-hydroxyazetidin-l-yl)propoxy)-7- oxohepty!2-butyloctanoate (0.7 g, 897.29 pmol, 92.68% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCb) δ ppm 5.47 - 5.22 (m, 4H), 4.52 - 4.44 (m, 2H), 4.09 - 4.05 (m, 6H), 3.77 ■■ 3.71 (m, 2H), 3.61 - 3.54 (m, 2H), 3.45 ■■ 3.38 (m, 2H), 3.08 (t, J = 6.2 Hz, 2H), 2.73 - 2.66 (m, 1H), 2.41 (t, J - 7.6 Hz, 2H), 2.36 - 2.29 (m, 4H), 2.09 - 2.01 (m, 8H), 1.97 - 1.90 (m, 2H), 1.68 - 1.53 (m, 14H), 1.45 - 1.34 (m, 12H), 1.32 - 1.22 (m, 16H), 0.96 (t, J = 7.6 Hz, 6H), 0.90 - 0.87 (m, 6H).

[0206] Step 12: Compound 3

[0207] A mixture of [7-[3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2-(3-hydroxyazetidin- 1 yl)propoxy]-7-oxo-heptyl] 2 -butyloctanoate (0.7 g, 897.29 pmol, 1 eq) , 4-(2-pyrrolidin-l- ylethylcarbamoyloxy)decanoic acid (442.07 mg, 1.35 mmol, 1.5 eq), EDCI (206.41 mg, 1.08 mmol, 1.2 eq), DIPEA (289.92 mg, 2.24 mmol, 390.73 pL, 2,5 eq) and 4-pyrrolidin-l- ylpyridine (13.30 mg, 89.73 pmol, 0.1 eq) in DCM (20 ml) was stirred at 20 °C for 12 hr under Nz atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 150 mL (50 mL * 3). The combined organic layers were dried over NazSO, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOc, Petroleum ether / Ethyl acetate::::10 / l to 0 / 1). Compound [7-[3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2-[3-[4-(2 -pyrrolidine- 1- ylethylcarbamoyloxy)decanoyloxy]azetidin-l-yl]propoxy]-7-oxo-heptyl] 2-butyloctanoate (0.11 g, 100.87 pmol, 11.24% yield) was obtained as a colorless oil.NMR (400 MHz, CDCh) 8 ppm 5.34 - 5.20 (m, 4H), 5.04 - 4.93 (m, 1 H), 4.68 (s, 1 H), 4.42 (t, J - 5.4 Hz, 1H), 4.01 - 3.96 (m, 6H), 3.70 (t, J:;= 7.2 Hz, 2H), 3.56 - 3.45 (m, 2H), 3.38 - 3.30 (m, 2H), 3.25 (d, J = 4.8 Hz, 2H), 3.16 - 3.08 (m, 2H), 2.65 - 2.61 (m, 1H), 2.59 - 2.51 (m, 4H), 2.36 - 2.19 (m, 8H), 2.04 - 1.92 (m, 8H), 1.90 - 1.78 (m, 4H), 1.74 (s, 4H), 1.61 - 1.48 (m, 14H), 1.37 - 1.28 (m, 10H), 1.19 (s, 20H), 0.88 (t, J = 7.4 Hz, 6H), 0.83 - 0.79 (m, 9H).Example 5 - Synthesis of Compound 4: [2~[3-[3~(diethytamiHo)propoxycarbonyIoxy]azetid"m~l~yI]~3~(4,4~dio£toxybutanoyIoxy)propyl](9Z,12Z)»octadeea”9,12”dienoate

[0208] Step 1 :

[0209] To a solution of 4,4-dimethoxybutanenitrile (20 g, 154.85 mmol, 1 eq) and octan- l-ol (50.41 g, 387.13 mmol, 61.18 mL, 2.5 eq) in Tol. (200 niL) was added PPTS (9.73 g, 38.71 mmol, 0.25 eq) under N2 atmosphere. The reaction mixture was stirred at 110 °C for 36 hr under N? atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (1000 mL) and extracted with DCM 1500 mL (750 mL * 2). The combined organic layers were dried over NaaSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=50 / l to 1 / T). Compound 4,4-dioctoxybutanenitrile (30 g, 92.16 mmol, 59.51% yield) was obtained as a colorless oil.

[0210] Step 2:^T30 i^T31

[0211] To a solution of 4,4-dioctoxybutanenitrile (30 g, 92.16 mmol, 1 eq) in EtOH (300 mL) and H2O (300 mL) was added KOH (20.68 g, 368.63 mmol, 4 eq) under N2 atmosphere. The reaction mixture was stirred at 110 °C for 12 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with water (500 mL), adjusted to PH=7 by using 1 M HC1 at 0 °C under N2 and extracted with EtOAc (500 mL * 3). The combined organic layers were washed with sat. NaCl (300 mL * 2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO?, Petroleum ether / Ethyl acetate=50 / l to 1 / 1). Compound 4,4-dioctoxybutanoic acid (19 g, 55.15 mmol, 59.84% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCb) 8 ppm 5.51 - 5.38 (m, 1 H), 3.78 - 3.69 (m, 1H), 3.57 (t, J - 6.6 Hz, 2H), 3.49 - 3.41 (m, 1 H), 2.69 - 2.53 (m,1H), 2.43 - 2.21 (rn, 2H), 2.14 - 1.99 (m, 1H), 1.60 - 1.43 (m, 4H), 1.29 - 1.12 (m, 20H), 0.88 ■■ 0.69 (m, 6H).

[0212] Step 3:

[0213] To a solution of l-[tert-butyl(dimethyl)silyl]oxy-3-hydroxy-propan-2-one (4.5 g, 22.02 mmol, 1 eq) in DCM (45 mL) was added EDCI (5.07 g, 26.43 mmol, 1.2 eq), DIPEA (7.12 g, 55.06 mmol, 9.59 mL, 2.5 eq) and DMAP (269.04 mg, 2.20 mmol, 0.1 eq) under N2 atmosphere, then (9Z,12Z)~octadeca~9,12~dienoic acid (7.41 g, 26.43 mmol, 7.41 mL, 1.2 eq) was added to the above reaction mixture. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL. * 2). The combined organic layers were dried over NasSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate =50 / 1 to 5 / 1 ). Compound [3-[tert-butyl(dimethyl)silyl]oxy-2-oxo-propyl] (9Z,12Z)- octadeca-9,12-dienoate (5.5 g, 11.78 mmol, 53.51% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCh) 8 ppm 5.45 - 5.28 (m, 4H), 4.96 (s, 2H), 4.28 (s, 2H), 2.78 (t, J = 6.4 Hz, 2H), 2.44 (t, J = 7.6 Hz, 2H), 2.11 - 1.98 (m, 4H), 1.73 - 1.63 (m, 2H), 1.38 - 1.29 (m, 14H), 0.94 (s, 9H), 0.90 (t, J = 6.8 Hz, 3H), 0.11 (s, 6H).

[0214] Step

[0215] To a solution of [3-[tert-butyl(dimethyl)silyl]oxy-2-oxo-propyl] (9Z, 12Z)- octadeca-9,12-dienoate (5.5 g, 1 1.78 mmol, 1 eq) in THF (55 mL) was added N,N- diethylethanamineitrihydrofluoride (2.85 g, 17.67 mmol, 2.88 mL, 1.5 eq) dropwise at 0 °C under N2 atmosphere. The mixture was stirred at 0 °C for 2 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL *2). The combined organic layers were dried over NasSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOz, Petroleum ether / Ethyl acetate =50 / 1 to 5 / 1). Compound (3-hydroxy- 2-oxo-propyl) (9Z,12Z)-octadeca-9,12-dienoate (2.5 g, 7.09 mmol, 60.19% yield) was obtained as a colorless oil.

[0216] Step 5:

[0217] To a solution of (3-hydroxy-2-oxo-propyl) (9Z,12Z)-octadeca-9,12-dienoate (2.5 g, 7.09 mmol, 1 eq) in DCM (25 mL) was added EDCI (1.63 g, 8.51 mmol, 1.2. eq), DIPEA (2.29 g, 17.73 mmol, 3.09 mL, 2.5 eq) and DMAS’ (86.64 mg, 709.21 pmol, 0.1 eq) under Nc atmosphere, then 4,4-dioctoxybutanoic acid (2.44 g, 7.09 mmol, 1 eq) was added to the above reaction mixture. The reaction mixture was stirred at 20 °C for 12 hr under Ns atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over NacSCM, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=50 / T to 0 / 1). Compound [3-(4,4- dioctoxybutanoyloxy)-2-oxo-propyl] (9Z,12Z)-octadeca-9,12-dienoate (1.8 g, 2.65 mmol, 37.38% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) δ ppm 5.42 - 5.32 (m, 4H), 4.76 (d, J == 1 .8 Hz, 4H), 4.52 (t, J == 5.4 Hz, 1 H), 3.63 - 3.53 (m, 2H), 3.48 - 3.37 (m, 2H), 2.78 (t, J = 6.4 Hz, 2H), 2.53 (t, J = 7.4 Hz, 2H), 2.43 (t, J = 7.4 Hz, 2H), 2.09 - 2.05 (m, 4H), 2.01 - 1.94 (m, 2H), 1.69 - 1.63 (m, 2H), 1.57 (s, 8H), 1.35 - 1.28 (m, 30H), 0.91 - 0.87 (m, 9H).

[0218] Step 6:sm'ss

[0219] A mixture of [3-(4,4-dioctoxybutanoyloxy)-2-oxo-propyl] (9Z, 12Z)-octadeca-9, 12- dienoate (1.8 g, 2.65 mmol, 1 eq), azetidin-3-ol (348.50 mg, 3.18 mmol, 1.2 eq, HC1), TEA (321 .89 mg, 3.18 mmol, 442.76 pL, 1 .2 eq) in DCM (14 mL) and MeOH (4 ml.) was stirred at 20 °C for 1 h under N2 atmosphere. Then NaBHjCN (249.88 mg, 3.98 mmol, 1.5 eq) was added to the above reaction mixture slowly at 0 °C under N2 atmosphere. The reaction mixture was stirred at 200C for 11 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography(SiOz, Dichloromethane / Methanoi:::50 / 1 to 5 / 1). Compound [3-(4,4-dioctoxybutanoyloxy)-2- (3-hydroxyazetidin- 1 -yi)propyl] (9Z,12Z)-octadeca-9,12-dienoate (0.7 g, 950.94 pmol, 35.87% yield) was obtained as a colorless oil.1H NMR (400 MHz, CDCb) 8 ppm 5.47 - 5.28 (m, 4H), 4.54 ■ 4.45 (m, 2H), 4.24 ■■ 3.94 (m, 4H), 3.80 3.73 (m, 2H), 3.61 ■■ 3.52 (m, 2H), 3.46 - 3.36 (m, 2H), 3.14 - 3.10 (m, 1H), 2.78 (t, J = 6.4 Hz, 2H), 2.72 (t, J = 5.0 Hz, 1H), 2.42 (t, J === 7.4 Hz, 2H), 2.32 (t, J === 7.6 Hz, 2H), 2.09 - 2.03 (m, 4H), 1 .96 - 1.91 (m, 2H), 1.65 - 1.54 (m, 6H), 1.41 - 1.21 (m, 36H), 0.93 - 0.85 (m, 9H).

[0220] Step 7:Compound 4

[0221] To a solution of [3-(4,4~dioctoxybutanoyloxy)-2-(3-hydroxyazetidin-l-yl)propyl] (9Z,12Z)-octadeca-9,12-dienoate (0,7 g, 950.94 pmol, 1 eq) in DCM (7 ml) was added TEA (288.67 mg, 2.85 mmol, 397.08 uL, 3 eq) and GDI (231.29 mg, 1.43 mmol, 1.5 eq) under Ns atmosphere. The reaction mixture was stirred at 20 °C for 2 hr under Ns atmosphere. Then 3- (diethylamino)propan-l-ol (1.50 g, 11.41 mmol, 1.70 mL, 12 eq) was added to the above reaction mixture. The reaction mixture was stirred at 20 °C for 12 h under Ns atmosphere.The reaction mixture was diluted with HsO (50 mL) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were dried over NasSCU, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOs, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound [2-[3-[3- (diethylamino)propoxycarbonyloxy]azetid-in-l-yl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate (0.1 g, 111.94 pmol, 11.77% yield) was obtained as a colorless oil.JH NMR (400 MHz, CDCh) 6 ppm 5.44 - 5.27 (m, 4H), 5.06 - 4.99 (m, 1H), 4.49 (t, J = 5.4 Hz, 1H), 4.19 (t, J = 6.4 Hz, 2H), 4. 12 ■ 3.99 (m, 4H), 3.83 ■■ 3.75 (m, 2H), 3.60 - 3.53 (m, 2H), 3.46 - 3.35 (m, 2H), 3.28 - 3.21 (m, 2H), 2.78 (t, J - 6.4 Hz, 2H), 2.74 - 2.67 (m, 1H), 2.58 ■■ 2.53 (m, 4H), 2.41 (t, J = 7.6 Hz, 2H), 2.31 (t, J = 7.6 Hz, 211). 2.11 ■■ 2.02 (m, 4H), 1.96 - 1.90 (m, 2H), 1.88 - 1.80 (m, 2H), 1.65 - 1.59 (m, 4H), 1.59 - 1.53 (m, 4H), 1 .37 - 1.20 (m, 34H), 1 .04 (t, J - 7.2 Hz, 6H), 0.94 - 0.83 (m, 9H).Example 6 ~ Synthesis of Compound 5: [3"(4,4~dioctoxybutanoytoxy)~2~[3~(4~pyrroMm~ l~ylbutanoyioxy)azetidm"l"yl]propyl] (9Z,12Z)"Octadeea~9,12"dienoate

[0222] Synthetic scheme:Compound 5

[0223] A mixture of [3 -(4,4 -dioctoxyb utanoyloxy)-2-(3 -hydroxyazetidin- 1 -yl)propyl](9Z,12Z)-octadeca-9,12-dienoate (800.00 mg, 1.09 mmol, 0.3 eq), 4-pyrrolidin-l-ylbutanoic acid (841.92 mg, 4.35 mmol, 1.2 eq, HC1), EDCI (833.35 mg, 4.35 mmol, 1.2 eq), DIPEA (1.64 g, 12.68 mmol, 2.21 ml, 3.5 eq) and 4-pyrrolidin- 1 -ylpyridine (107.38 mg, 724.53 pmol, 0.2 eq) in DCM (20 ml) was stirred at 20 °C for 12 hr under Na atmosphere. The reaction mixture was diluted with H2O (100 ml.) and extracted with DCM 200 ml (100 ml.. * 2). The combined organic layers were dried over Na2SC.fi, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOa, Petroleum ether / Ethyl acetate=10 / l to 0 / T). Compound [3-(4,4- dioctoxybutanoyloxy)-2-[3-(4-pyrrolidin-l~ylbutanoyloxy)azetidin~l-yl]propyl] (9Z.12Z)- octadeca-9,12-dienoate (0.12 g, 137.09 pmol, 3.78% yield) was obtained as a colorless oil.fH NMR (400 MHz, CDClr) 8 ppm 5.55 ■■ 5.17 (m, 4H), 5.06 (m, 1H), 4.49 (t, J= 5.2 Hz, 1 H), 4.23 - 3.97 (m, 4H), 3.78 (t, . / 7.0 Hz, 2H), 3.64 - 3.49 (m, 2H), 3.46 - 3.34 (m, 2H), 3.20 (t, j 6.8 Hz, 2H), 2.78 (t, J = 6.2 Hz, 2H), 2.70 (t, J= 4.8 Hz, 1H), 2.65 - 2.44 (m, 6H), 2.43 ■■ 2.25 (m, 6H), 2.05 (m, 4H), 1.96 - 1.89 (m, 2H), 1.87 - 1.82 (m, 2H), 1.81 - 1.71 (m, 8H), 1.64 ■■ 1.52 (m, 6H), 1.29 (d, J= 11.4 Hz, 34H), 0.94 ■■ 0.84 (m, 9H).Example 7 ~ Synthesis of Compound 6: 01~[3"[7"(l"hepM®etoxy)~7"OxO"heptmioyl[oxy~2~[3”(4~pyrrolidm~l"ylbatasioyloxy)azetidin~l~yl]propyl] O7~(l-heptyloctyl) heptanedioate

[0224] Step 1 : INT36

[0225] To a solution of pentadecan-8-one (25 g, 110.43 mmol, 1 eq) in MeOH (250 mL) was added NaBEk (8.36 g, 220.85 mmol, 2 eq) slowly at 0 °C under N?. atmosphere. The reaction mixture was stirred at 20 °C for 2 hr under N2 atmosphere. The reaction mixture was added sat. NH4CI slowly at 0 °C under N2 atmosphere, diluted with H2O (300 mL) and extracted with EtOAc 600 mL (200 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound pentadecan-8-ol (20 g, 87.56 mmol, 79.29% yield) was obtained as a colorless oil.

[0226] Step 2:

[0227] To a solution of pentadecan- 8- ol (5 g, 21.89 mmol, 1 eq) in DCM (50 mL) was added EDCI (5.46 g, 28.46 mmol, 1.3 eq), heptanedioic acid (17.53 g, 109.45 mmol, 5 eq), DIPEA (7.07 g, 54.73 mmol, 9.53 mL, 2.5 eq) and DMAP (267.43 mg, 2.19 mmol, 0.1 eq) under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H?O (100 mL) and extracted with DCM 300 mL (100 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1).Compound 7-(l-heptyloctoxy)-7-oxo4ieptanoic acid (2 g, 5.40 mmol, 24.66% yield) was obtained as a colorless oil.

[0228] Step .3:

[0229] To a solution of l,3-dihydroxypropan-2-one (546.93 mg, 6.07 mmol, 0.45 eq) in DCM (50 ml) was added DMAP (164.84 mg, 1 .35 mmol, 0.1 eq), DIPEA (4.36 g, 33.73 mmol, 5.88 mL, 2.5 eq) and EDCI (3.10 g, 16.19 mmol, 1.2 eq), then 7-(l- heptyloctoxy)-7-oxo-heptanoic acid (5 g, 13.49 mmol, 1 eq) was added to the above reaction mixture under Ni atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H?O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over NacSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, dichloromethane / Methanol==50 / l to 5 / 1). Compound 01- [3- [7 -( 1 -heptyloctoxy)-7-oxo-heptanoyl]oxy-2 -oxo-propyl] O7-( 1 -heptyloctyl) heptanedioate (4 g, 5.03 mmol, 82.85% yield) was obtained as a colorless oil. 'H NMR (400 MHz, CDCh)8 ppm 4.91 4.82 (m, 2H), 4.75 (s, 4H), 2.44 (t, J = 7.6 Hz, 4H), 2.30 (t, J = 7.6 Hz, 4H), 1.75 - 1.60 (m, 16H), 1.51 (d, J - 5.4 Hz, 6H), 1.43 - 1.38 (m, 4H), 1.27 (s, 34H), 0.88 (t, J - 6.8 Hz, 12H)

[0230] Step 4:

[0231] To a solution of Ol-[3-[7-(l-heptyloctoxy)-7-oxo-heptanoyl]oxy-2-oxo-propyl] O7-(l -heptyloctyl) heptanedioate (4 g, 5.03 mmol, 1 eq), azetidin-3-ol (661.31 mg, 6.04 mmol, 1.2 eq, HC1), TEA (610.82 mg, 6.04 mmol, 840.19 uL, 1.2 eq) in DCM (35 mL) and MeOH (10 mL) was added NaBH(OAc)3 (2.13 g, 10.06 mmol, 2 eq) slowly at 0 °C under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (200 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by columnchromatography (SiO2, Dichloromethane / Methanol=50 / 1 to 5 / 1). Compound Ol-[3-[7-(l- heptyloctoxy)- 7 -oxo-heptanoyl] oxy-2- (3 -hydro xyazetidin- 1 -yl)propy I] O7~( 1 ■ heptyloctyi) heptanedioate (2.2 g, 2.58 mmol, 51 .32% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCis) 8 ppm 4.86 (t, J = 6.2 Hz, 2H), 4.46 (t, J = 5.8 Hz, 1H), 4. 18 - 4.00 (m, 4H), 3.76 (l. J - 6.8 Hz, 2H), 3.39 (t, J - 7.2 Hz, 2H), 3.14 (d, J - 5.4 Hz, 2H), 2.45 - 2.24 (rm 10H), 2.02 (d, J = 7.8 Hz, 2H), 1.73 - 1.58 (m, 8H), 1.51 (d, J = 5.6 Hz, 6H), 1.40 - 1.24 (rm 42H), 0.88 (t, J = 6.8 Hz, 12H).

[0232] Step 5 :

[0233] To a solution of Ol-[3-[7-(l-heptyloctoxy)-7-oxo-heptanoyl]oxy-2-(3- hydroxyazetidin-l-yl)propyl] O7-(l -heptyloctyl) heptanedioate (2.2 g, 2.58 mmol,1 eq) in DCM (22 mL) was added EDCI (593.81 mg, 3.10 mmol, 1.2 eq), DIPEA (1.17 g, 9.03 mmol, 1.57 mL, 3.5 eq) and DMAP (31.54 mg, 258.13 pmol, 0.1 eq) under N2 atmosphere, then 4-pyrrohdin- 1-yibutanoic acid (499.93 mg, 2.58 mmoi, 1 eq, HC1) was added to the above reaction mixture under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane / Methano 1=50 / 1 to 5 / 1). Compound Ol-[3-[7-(l-heptyloctoxy)-7-oxo-heptanoyl]oxy-2-[3-(4-pyrrolidin-l- ylbutanoyloxy)azetidin- l-yi]propyl] O7-(l -heptyloctyl) heptanedioate (0.1 g, 100.86 pmol, 3.91% yield, 100% purity) was obtained as a colorless oil.rH XX1R (400 MHz, CDCb) 8 ppm 5.12 - 5.00 (m, IH), 4.92 ■■ 4.80 (m, 2H), 4. 16 - 3.98 (m, 4H), 3.77 (t, J = 7.2 Hz, 2H),3.22 (t, J - 6.4 Hz, 2H), 3.16 - 3.09 (m, 2H), 2.75 - 2.68 (m, H i). 2.51 (t, J - 6.6 Hz, 2H), 2.36 ■■ 2.26 (m, 8H), 2.23 ■■ 2.07 (m, 6H), 1.75 ■■ 1.58 (m, 10H), 1.50 (d, J = 5.4 Hz, 10H), 1.40 - 1.23 (m, 44H), 0.88 (t, J === 6.8 Hz, 12H).Example 8 - Synthesis of Compound 7: Ol~[3"[7"(l"hept"6"enyioct~7"en®xy)~7"0x®" heptaffioyl]oxy~2”[3“(4”pyrrolidm”l“ylbutauoyloxy)azetidin”l”yi]propyl] O7-(l~hept~6- enyioct-7-esyl) heptanedioate

[0234] Step

[0235] To a suspension of Mg (1.44 g, 59.29 mmol, 1.05 eq) in THF (60 mL) was added b (143.33 mg, 564.71 pmol, 113.75 pL, 0.01 eq) under Nc atmosphere and then the reaction mixture was stirred at 45 °C for 0.5 h under N? atmosphere. Then 7- bromohept - bene ( 10 g, 56.47 mmol, 1 eq) was added to the above reaction mixture dropwise at 60 °C for 0.5 h under N2 atmosphere. The reaction mixture was stirred at 60 °C for 2 h. Then the reaction mixture was cooled to 0 °C and ethyl formate (3.97 g, 53.65 mmol, 4.32 mL, 0.95 eq) was added to the above reaction mixture dropwise under N2 atmosphere. The reaction mixture was stirred at 20 °C for 2 hr under N2 atmosphere. Then the reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (3*150 ml). The combined organic layers were dried over NazSCM, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was dissolved in EtOH (150 mL) and a solution of KOH (15.84 g, 282.36 mmol, 5 eq) in H2O (150 mL) was added to the above reaction mixture. The reaction mixture was stirred for 2 hr at 20 °C under N2 atmosphere. Then the reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (3*150 ml). The combined organic layers were dried over NasSOa, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:::1 / 0 to 20 / 1). Compound pentadeca- l,14-dien-8-ol (7.5 g, 33.43 mmol, 19.73% yield) was obtained as a colorless oil.5H NMR (400 MHz, CDCls) 8 ppm 5.91 ■ 5.74 (m, 2H), 5.13- 4.77 (m, 4H), 3.68 - 3.54 (m, 1H), 2.12 - 1.98 (m, 4H), 1.47 - 1.31 (m, 16H).

[0236] Step 2:

[0237] To a solution of pentadeca- 1,14-dien-8-ol (5 g, 22.28 mmol, 1 eq) in DCM (230 mL) was added EDCI (5.55 g, 28.97 mmol, 1.3 eq), heptanedioic acid (17.85 g, 11 1.42 mmol, 5 eq), DMAP (272.23 mg, 2.23 mmol, 0.1 eq) and DIPEA (7.20 g, 55.71 mmol, 9.70 ml, 2.5 eq) under N?_ atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (200 mL.) and extracted with DCM 500 mL (250 mL * 2). The combined organic layers were dried over NasSCE, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOc, Petroleum ether / Ethyl acetate=l / O to 0 / 1).Compound 7-(l-hept-6-enyloct-7-enoxy)-7-oxo-heptanoic acid (2.5 g, 6.82 mmol, 30.61% yield) was obtained as a colorless oil. 'H NMR (400 MHz, CDCh) 8 ppm 5.92 - 5.72 (m, 2H), 5.04 - 4.91 (m, 4H), 4.90 - 4.84 (m, 1H), 2.37 (t, J= 7.4 Hz, 2H), 2.30 (t, J= 7.4 Hz, 2H), 2.08 - 2.01 (m, 4H), 1.69 - 1.63 (m, 4H), 1.52 (d, J ------ 5.4 Hz, 4H), 1 .44 - 1 .28 (m, 14H).

[0238] Step 3 :

[0239] To a solution of 7-(l -hept-6-enyloct-7-enoxy)-7-oxo-heptanoic acid (2 g, 5.46 mmol, 1 eq) in DCM (22 mL) was added EDCI (1.26 g, 6.55 mmol, 1.2 eq), DMAP (66.66 mg, 545.65 pmol, 0.1 eq), l,3-dihydroxypropan-2-one (221.18 mg, 2.46 mmol, 0.45 eq) and DIPEA (1.76 g, 13.64 mmol, 2.38 mL, 2.5 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (l OOmL) and extracted with DCM 200 mL (50 mL * 4). The combined organic layerswere dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:::l / 0 to 0 / 1). Compound Ol-[3-[7-(l-hept-6-enyloct-7-enoxy)-7-oxo- heptanoyl]oxy-2- oxo-propyl] O7-(l-hept-6-enyloct-7-enyl) heptanedioate (1.1 g, 1.40 mmol, 25.61% yield) was obtained as a colorless oil.]H NMR (400 MHz, CDCh) 8 ppm 5.91 - 5.68 (m, 4H), 5.06 - 4.83 (m, 10H), 4.75 (s, 4H), 2.44 (t, J --- 7.6 Hz, 4H), 2.30 (t, J --- 7.6 Hz, 4H), 2.07 - 2.00 (m, 8H), 1.72 - 1.63 (m, 8H), 1.54 - 1.48 (m, 8H), 1.43 - 1.35 (m, 12H), 1.33 - 1.23 (m, 16H).

[0240] Step 4:SNT4S

[0241] To a solution of Ol-[3-[7-(l-hept-6-enyloct-7-enoxy)-7-oxo-heptanoyl]oxy-2-oxo- propyl] O7-(1-hept-6-enyloct-7-enyl) heptanedioate (0.5 g, 635.23 umol, 1 eq) in DCM (3.88 mL) and MeOH (1.11 ml) was added azetidin-3-ol (83.51 mg, 762.28 pmol, 1.2 eq, HC1), TEA (77.13 mg, 762.28 pmol, 106.10 pL, 1.2 eq) and NaBH(OAc)3(538.53 mg, 2.54 mmol, 4 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (50 ml.) and extracted with DCM 50 mL (25 mL * 2). The combined organic layers were dried over Na?.SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound Ol-[3-[7-(l-hept-6-enyloct-7- enoxy)- 7-oxo- heptanoyl ]oxy-2- (3-hydroxyazetidin-l-yl) propyl] O7-(1 -hept-6-enyloct-7-enyl) heptanedioate (0.2 g, 236.91 pmol, 37.29% yield) was obtained as a colorless oil.fH NMR (400 MHz, CDCh) 8 ppm 5.90 - 5.72 (m, 4H), 5.04 - 4.91 (m, 8H), 4.90 - 4.83 (m, 2H), 4.52 - 4.41 (m, 1H), 4.13 - 4.03 (m, 4H), 3.76 (t, . / 6.8 Hz, 2H), 3.11 (s, 2H), 2.75 ■■ 2.64 (m, 1H), 2.35 ■■ 2.27 (m, 8H), 2.07 ■■ 2.01 (m, 8H), 1.69 ■■ 1.62 (m, 10H), 1.54 - 1.48 (m, 8H), 1.41 - 1.35 (m, 12H), 1.32 - 1.26 (rn, 14H).

[0242] Step 5 :Compound 7

[0243] To a solution of Ol-[3-[7-(l-hept-6-enyloct-7-enoxy)-7-oxo-heptanoyl]oxy-2-(3- hydroxyazetidin-1-yl)propyl] O7-(l-hept-6-enyloct-7-enyl) heptanedioate (0.4 g, 473.82 pmol, 1 eq) in DCM (4 mL.) was added 4-pyrrolidin-l-ylbutanoic acid (110.12 nig, 568.58 pmol, 1.2 eq, HQ), EDCI (109.00 mg, 568.58 pmol, 1.2 eq), 4-pyrrolidin-l-ylpyridine (7.02 mg, 47.38 pmol, 0.1 eq) and DIPEA (214.33 mg, 1.66 mmol, 288.86 pL, 3.5 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (50mL) and extracted with DCM 100 mL (25mL * 4). The combined organic layers were dried over NasSO-i, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM MeOH =100 / T to 1 / 1). Compound Ol-[3-[7-(l-hept-6-enyloct- 7-enoxy)-7-oxo-heptanoyl]oxy-2-[3-(4-pyrrolidin- 1 -ylbutanoyioxylazetidin- 1 -yl]propyl] 07- (l-hept-6-enyloct-7-enyl) heptanedioate (0,15 g, 152.53 pmol, 32.19% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCh) 8 ppm 5.91 - 5.69 (m, 4H), 5.35 - 5.24 (m, 1H), 5.04 - 4.91 (m, 8H), 4.89 - 4.82 (m, 2H), 4.66 - 4.59 (m, 2H), 4.51 - 4.42 (m, 2H), 4.34 - 4.20 (m, 4H), 3.88 - 3.78 (m, 2H), 3.68 (d, J--- 4.0 Hz, 1H), 3.25 - 3.16 (m, 2H), 2.90 - 2.80 (m, 2H), 2.59 (t, .7= 6.4 Hz, 2H), 2.42 - 2.36 (m, 4H), 2.30 ft, J= 7.6 Hz, 4H), 2.12 ■ 2.02 (m, 20H), 1.68 - 1.62 (m, 811). 1.52 (d, J ------ 6.2 Hz, 6H), 1 .40 - 1.27 (m, 281 i).Example 9 - Synthesis of Compound 8: O7~[2"[3~[3"(diethytammo)propoxyc~ arbonyloxy] azetidin-l-yl] ~3~ [7~oxo~7~(3~penty'Ioctoxy)heptanoyI] oxy-propyl] Ol-(3- pentyloctyl) heptanedioate

[0244] Step 1 :m

[0245] To a solution of l,3-dihydroxypropan-2-one (25 g, 277.54 mmol, 1 eq) in DCM (500 ml) was added tert-butyl-chloro-dimethyl-silane (92.03 g, 610.58 mmol, 75.12 ml, 2.2 eq) and TEA (61.80 g, 610.69 mmol, 85.00 ml, 2.20 eq) under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (1000 mL) and extracted with DCM 1000 mL (500 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=50 / l to 1 / 1). Compound l,3-bis[[tert- butyl(dimethyl)silyl]oxy]propan~2~one (50 g, 156.94 mmol, 56.55% yield) was obtained as a colorless oil.]H NMR (400 MHz, CDCh) 8 ppm 4.42 (s, 4H), 0.93 (s, 18H), 0.10 (s, 12H).

[0246] Step 2: ™ ™

[0247] To a solution of l,3-bis[[tert-butyl(dimethyl)silyl]oxy]propan-2-one (50 g, 156.94 mmol, 1 eq), azetidin-3-ol (20.63 g, 188.32 mmol, 1.2 eq, HC1), TEA (19.06 g, 188.38 mmol, 26.22 mL, 1.20 eq) in DCM (105 mL) and MeOH (30 mL) was added NaBH(OAc).? (66.64 g, 314.43 mmol, 2.00 eq) slowly at 0 °C under N2 atmosphere. The reaction mixture was stirred at 20C'C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (1000 mL) and extracted with DCM 1000 mL (500 mL * 2). The combined organic layers were dried over Na2SOs, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Si()2, Dichloromethane / Methanol=50 / lto 5 / 1). Compound l-[2~[tert”butyd(dimethyl)silyl]oxy~l" [[tert-butyl(dimethyl)silyl]oxymethyl]ethyl]azetidin-3-ol (20 g, 53.24 mmol, 33.92% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCh) 8 ppm 4.49 ■■ 4.35 (m, 1H), 4.02- 3.86 (m, 2H), 3.70 - 3.56 (m, 4H), 3.55 - 3.43 (m, 2H), 2.74 - 2.62 (m, 1H), 0.90 (s, 18H), 0.06 (s, 12H).

[0248] Step 3:

[0249] To a solution of 1 - [2-[tert-butyl(dimethyl)silyl]oxy- 1 -[[tert- butyl(dimethyl)silyl]oxymethyl]ethyl]azetidin-3-ol (10 g, 26.62 mmol, 1 eq) in DCM (100 mL) was added TEA (8.08 g, 79.85 mmol, 11.11 ml, 3 eq) and CDI (4.32 g, 26.62 mmol, 1 eq) under N2 atmosphere. The reaction mixture was stirred at 20 °C for 2 hr under N2 atmosphere. Then 3-(diethylamino)propan-l-ol (10.48 g, 79.85 mmol, 11.91 mL, 3 eq) was added to the above reaction mixture under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (200 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over NaaSOw filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyi acetate=50 / 1 to 0 / 1 ) . Compoun d [ 1 - [2- [tert-butyl(dimeth yl) si 1 y 1] oxy- 1 - [ [tert- butyl(dimethyl)silyl]oxymethyl]ethyl]azetidin-3-yl] 3-(diethyiamino)propyi carbonate (2.5 g, 4.69 mmol, 17.62% yield) was obtained as a colorless oil.

[0250] Step 4:^T49 ^T50

[0251] To a solution of [l-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymethyl]ethyl]azetidin-3-yl] 3-(diethylamino)propyl carbonate (2.5 g, 4.69 mmol, 1 eq) in THF (25 mL) was added N,N-diethylethanamine;trihydrofluoride (1.51 g, 9.38 mmol, 1.53 mL, 2 eq) dropwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 20 °C for 5 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by columnchromatography (SiO2, Dichloromethane / Methanol::::50 / 1 to 5 / 1). Compound3- (diethylamino)propyl [l-[2-hydroxy~l"(hydroxymethyl)ethyl]azetidin-3"yl] carbonate (1.2 g,3.94 mmol, 84.04% yield) was obtained as a colorless oil. ’H NMR (400 MHz, D2O) 8 ppm 5.36 ■■ 5.26 (m, 1H), 4.72 ■■ 4.63 (m, 2H), 4.44 (s, 2H), 4.28 (t J = 5.8 Hz, 2H), 3.86 ■■ 3.77 (m, 4H), 3.66 (t, J - 6.0 Hz, 2H), 3.58 - 3.47 (m, 1H), 3.16 - 3.13 (m, 4H), 2.14 - 2.07 (m, 2H),1.95 - 1.79 (m, 2H), 1.28 (s, 6H)

[0252] Step 5:Compound §

[0253] To a solution of 7-oxo-7-(3-pentyloctoxy)heptanoic acid (675.16 mg, 1.97 mmol, 2 eq) in DCM (3 ml) was added 2,4,6-trichlorobenzoyl chloride (480.78 mg, 1.97 mmol, 307.99 uL, 2 eq) and TEA (299.20 mg, 2.96 mmol, 411 .55 pL, 3 eq) at 0 °C under N? atmosphere. The reaction mixture was stirred at 20 °C for 2 hr under N2 atmosphere. Then DM AP (12,04 mg, 98.56 pmol, 0.1 eq) and 3-(diethylamino)propyl [l-[2-hydroxy-l- (hydroxymethyl)ethyl]azetidin-3-yl] carbonate (0.3 g, 985.61 pmol, 1 eq) were added to the above reaction mixture at 0 °C under N2 atmosphere. The reaction mixture was stirred at 20 °C for 10 hr under N2 atmosphere. The reaction mixture was diluted with H2O (50 ml) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were dried over NazSO, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography(S1O2, Dichloromethane / Methanol:::50 / 1 to 5 / 1). Compound O7-[2-[3-[3- (diethylamino)propoxyc- arbonyloxy] azetidin- 1 --y 1] -3-[7~oxo-7~(3 ■ pentyloctoxy)heptanoyl]oxy-propyl] Ol-(3~pentyloctyl) heptanedioate (0.1 g, 104,89 nmol, 10.64% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCh) 8 ppm 5.08 - 4.98(m, 1H), 4.22 (t, J - 6.0 Hz, 2H), 4.11 - 4.03 (m, 8H), 3.84 - 3.75 (m, 2H), 3.31 - 3.22 (m, 2H), 2.80 (s, 4H), 2.72 ■■ 2.68 (m, 1H), 2.35 - 2.28 (m, 8H), 2.02 (s, 2H), 1.70 ■■ 1.54 (m, 14H), 1.48 - 1.11 (m, 50H), 0.89 (t, J - 6.8 Hz, 12H).Example 10 - Synthesis of Compound 9: O7"[3"[7"0x0"7"(3"pentyloct®xy)heptauoyi]oxy"2“[3~(4-pyrroIidm-l~yIbataHoyloxy)azetidiB"l"yI]propyl] Ol-(3~pentyloctyl) heptanedioate

[0254] Step 1 :!OT5UNITS!

[0255] A mixture of 7-oxo- / -(3-pentyloctoxy)heptanoic acid (10 g, 29.20 mmol, 1 eq), l,3-dihydroxypropan-2-one (1.18 g, 13.14 mmol, 0.45 eq), DIPEA (9.43 g, 72.99 mmol, 12.71 ml, 2.5 eq), EDC1 (6.72 g, 35.04 mmol, 1.2 eq) and DMAP (356.68 mg, 2.92 mmol, 0.1 eq) in DCM (100 mL) was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (200 ml.) and extracted with DCM 600 mL (300 mL * 2). The combined organic layers were dried over NasSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound O7-[2-oxo~ 3-[7-oxo-7-(3-pentyloctoxy)heptanoyl]oxy-propyl] 01 -(3-pentylocty I) heptanedioate (7.2 g,9.74 mmol, 33.37% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCh) 8 ppm4.75 (s, 4H), 4.09 (t, J - 7.2 Hz, 4H), 2.44 (t, J - 7.4 Hz, 4H), 2.31 (t, J === 7.4 Hz, 4H), 1 .731.55 (in, 14H), 1.44 1.37 (m, 6H), 1.31 1.21 (in, 30H), 0.89 (t, J =;:6.8 Hz, 12H).

[0256] Step 2:

[0257] To a solution of O7-[2-oxo-3-[7-oxo-7-(3-pentyloctoxy)heptanoyl]oxy-propyl] 01- (3 -pentyloctyl) heptanedioate (3.5 g, 4.74 mmol, 1 eq), TEA (575.04 mg, 5.68 mmol, 1.2 eq), azetidin-3-ol (622.58 mg, 5.68 mmol, 1.2 eq, HC1) in DCM (12.25 mL) and MeOH (3.5 mL)was added NaBH(0Ac)3 (2.01 g, 9.47 mmol, 2 eq) at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 hours under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over NaaSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound O7-[2-(3-hydroxyazetidin-l- yl)-3-[7-oxo-7-(3-pentyloctoxy)heptanoyl]oxy-propyl] 01 -(3-pentyloctyl) heptanedioate (3.2 g, 4.02 mmol, 41.26% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCb) 8 ppm 4.47 (t, J = 5.8 Hz, 1H), 4.13 - 4.03 (m, 8H), 3.75 (m, 2H), 3.10 (t, J = 6.8 Hz, 2H), 2.69 (t, J - 4.8 Hz, 1 H), 2.32 (m, 8H), 1.67 - 1.54 (m, 12H), 1 .41 - 1 .22 (m, 40H), 0.89 (l. J == 6.8 Hz, 12H).

[0258] Step 3:SNT52 Compound 9

[0259] A mixture of 07- [2-(3-hydroxyazetidin- 1 -yl)-3-[7-oxo-7-(3- pentyloctoxy)heptanoyl]oxy-propyl] 01 -(3 -pentyloctyl) heptanedioate (2.7 g, 3.39 mmol,1 eq), 4-pyrrolidin-l-yl butanoic acid (639.77 mg, 4.07 mmol, 1.2 eq), DIPEA (1.53 g, 11.87 mmol, 2.07 mL, 3.5 eq), EDCI (780.13 mg, 4.07 mmol, 1.2 eq) and 4-pyirolidin-l-ylpyridine (50.26 mg, 339.13 pmol, 0.1 eq) in DCM (27 mL) was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2S(>4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOz, Petroleum ether / Ethyl acetate::::100 / T to 0 / 1).Compound O7-[3-[7-oxo-7-(3-pentyloctoxy)heptanoyl]oxy-2-[3-(4-pyrrolidin-l- ylbutanoyloxy)azetidin-l-yl]propyl] 01 -(3-pentyloctyl) heptanedioate (0.1 g, 105.84 pmol, 3.12% yield, 99% purity) was obtained as a colorless oil.!H NMR (400 MHz, CDCh) 8 ppm 5.06 (t, J - 5.8 Hz, H i). 4.16 - 3.99 (m, 8H), 3.78 (m, 2H), 3.24 - 3.13 (m, 2H), 2.70 (s, 1H), 2.66 - 2.47 (m, 5H), 2.40 (t, J:;= 7.4 Hz, 2H), 2.32 (m, 8H), 1.94 - 1.78 (m, 6H), 1 .68 - 1 .55 (m, 16H), 1.26 (s, 40H), 0.89 (t, J = 6.9 Hz, 12H).Example 11 - Synthesis of Compound 10: [7“[3"[7~(2~butytoctanoyloxy)heptanoytoxy]~2“ [3~[4~(2~pyrrolidm"l~ylethykarbamoyloxy)deeanoyioxy]azetidm~l~yl]propoxy]"7"oxo~ heptyl] 2-bwtyIoctaHoate

[0260] Step l:

[0261] A mixture of 7-(2-butyloctanoyloxy)heptanoic acid (2 g, 6.09 mmol, 1 eq), 1 ,3- dihydroxypropan-2-one (274.22 mg, 3.04 mmol, 0.5 eq), EDCI (1.40 g, 7.31 mmol,1.2 eq), DIPEA (1.97 g, 15.22 mmol, 2.65 mL, 2.5 eq) and DMAP (74.38 mg, 608.86 pmol, 0.1 eq) in DMF (50 mL) was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 20 / 1). Compound [7-[3-[7-(2- buty loctanoyloxy)he-ptanoyloxy] -2-oxo-propoxy] -7-oxo-hepty 1] 2-butyl

[0262] octanoate(1 .1 g, 1.55 mmol, 25.41% yield) was obtained as a colorless oil. 'H NMR (400 MHz, CDCI3) δ ppm 4.76 (s, 4H), 4.07 (t, J = 6.6 Hz, 4H), 2.44 (t, J = 7.4 Hz, 4H), 2.31 (m, 2H), 1.73 - 1.54 (m, 14H), 1.49 - 1.37 (m, 12H), 1.36 - 1.21 (m, 26H), 0.88 (m, 12H).

[0263] Step 2:

[0264] To a mixture of [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2-oxo-propoxy]-7- oxo-heptyl] 2-butyloctanoate (1.1 g, 1.55 mmol, 1 eq), azetidin-3-ol (203.39 mg, 1.86 mmol, 1.20 eq, HC1) and TEA (187.86 mg, 1.86 mmol, 258.40 pL, 1.2 eq) in DCM (14 ml.) and MeOH (4 mL) was added NaBH(OAc)s (655.78 mg, 3.09 mmol, 2 eq) at 0 °C under N2 atmosphere, the reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL *2). The combined organic layers were dried over NacSCE, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 1 / 1). Compound [7-[3-[7-(2- butyloctanoyloxy)heptanoyloxy]-2-(3-hydroxyazetidin-l-yl)propoxy]-7-oxo-heptyl] 2- butyloctanoate (0.5 g, 650,95 umol, 42.08% yield) was obtained as a colorless oil. 'H NMR (400 MHz, CDCh) 8 ppm 4.61 - 4.33 (m, 1H), 4.23 - 3.95 (m, 8H), 3.78 (s, 2H), 3.12 (s, 2H), 2.79 - 2.64 (m, 1H), 2.38 - 2.27 (m, 6H), 1.77 - 1.53 (m, 15H), 1.52 - 1.16 (m, 38H), 0.88 (m, 12H)

[0265] Step 3 :Compound 10A mixture of [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2-(3-hydroxyazetidin-l- yl)propoxy]-7-oxo-heptyl] 2 -butyloctanoate (0.5 g, 650.95 pmol, 1 eq), 4-(2-pyrrolidin-l- ylethylcarbamoyloxy)decanoic acid (256.56 mg, 781.14 pmol, 1.2 eq), EDCI (149.74 mg, 781.14 pmol, 1.2 eq), DIPEA (210.33 mg, 1.63 mmol, 283.46 pL, 2.5 eq) and DMAP (9.65 mg, 65,09 umol, 0.1 eq) in DCM (10 mL) was stirred at 20 °C for 12 hr under Nz atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 50 mL (25 mL * 2). The combined organic layers were dried over NazSCM, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOa, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound [7-[3-[7-(2- butyloctanoy I oxy)heptanoy I oxy] -2-[3 - [4-(2-pyrrol idin- 1 - ylethylcarbamoyloxy)decanoyloxy]azetidin- l-yl]propoxy]-7-oxo-heptyl] 2 -butyloctanoate (0.13 g, 120.53 pmol, 18.52% yield) was obtained as a colourless oil. ‘H NMR (400 MHz, CDCh) 8 ppm 5.48 ■■ 5.19 (m, 1H), 5.06 (m, 1H), 4.75 (s, 1H), 4.10 (s, 8H), 3.85 ■■ 3.66 (m.2H), 3.33 (d, J - 4.8 Hz, 2H), 3.24 - 3.13 (m, 2H), 2.75 - 2.42 (m, 7H), 2.41 - 2.27 (m, 8H), 1.99 ■■ 1.70 (m, 8H), 1.67 ■■ 1.53 (m, 14H), 1.49 - 1.34 (m, 14H), 1.33 ■■ 1.17 (m, 34H), 0.92 ■ 0.85 (m, 15H).Example 12 - Synthesis of Compound 11: [2-[3-[3~(diethytammo)propoxy€arbonytoxy]a^etidm”l“yI]“3~[(9Z512Z)-o£tadeca-9,12- dienoyl] oxy-propyl] (9Z,12Z)-oetadeca-9,l 2-dieooate

[0267] Synthetic scheme:Compound 11

[0268] To a solution of (9Z,12Z)~octadeca-9,12-dienoic acid (184.27 mg, 657.07 nmol, 184.27 uL, 1 eq) in DCM (5 ml.) was added 2,4,6-trichlorobenzoyl chloride (16.0.3 mg, 65.71 pmol, 10.27 pL, 0.1 eq) and DIPEA (212.30 mg, 1.64 mmol, 286.12 uL, 2.5 eq) slowly at 0 °C under N2 atmosphere. Then 3-(diethylamino)propyl [ 1 -[2-hydroxy- 1 - (hydroxymethyl)ethyl]azetidin-3-yl] carbonate (0.2 g, 657.07 pmol, 1 eq) was added to the above reaction mixture under N2 atmosphere and the reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (50 ml) and extracted with DCM 60 mL (30 mL * 2). The combined organic layers were dried over NazSO, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography(SiOz, Dichloromethane / Methanoi:::50 / 1 to 5 / 1). Compound [2-[3-[3- (diethylamino)propoxycarbonyloxy]azetidin-l-yl]-3-[(9Z,12Z)-octadeca-9,12-dienoyl]oxy- propyl] (9Z,l2Z)-octadeca-9,12-dienoate (0.08 g, 96.47 pmol, 14.68% yield) was obtained as a colorless oil. 'H NMR (400 MHz, CDCI3) 8 ppm 5.46 - 5.26 (m, 8H), 5.03 (t, J = 5.8 Hz, 1H), 4.20 (t, J= 6.4 Hz, 2H), 4.06 (t, J= 5.6 Hz, 4H), 3.84 - 3.74 (m, 2H), 3.30 - 3.22 (m,2H), 2.78 (t, . / 6.4 Hz, 4H), 2.71 (t, J ------ 5.0 Hz, 1H), 2.59 (s, 4H), 2.32 (t, . / 7.6 Hz, 4H),2.10 ■■ 2.02 (m, 8H), 1.92 ■■ 1.83 (m, 2H), 1.67 ■■ 1.57 (m, 6H), 1.38 ■■ 1.28 (m, 28H), 1.11 ■1.01 (m, 6H), 0.90 (l. J -- 6.8 Hz, 6H).Example 13 - Synthesis of Compound 12: [7“[3"[7~(2"botyi"octanoyloxy)heptanoyloxy]" 2~[3"[4”(2-pyrroIidin"l”ytethyIcarbamoyioxy)octunoyloxy]uzetidm"l-yI]prop"Oxy]"7”Oxo~ heptyl] 2-butyIoctanoate

[0269] Step l :

[0270] To a solution of 5-butyltetrahydrofiiran-2-one (50 g, 352.10 mmol, 1 eq) in H2O (250 mL) was added NaOH (14.8 g, 369.7 mmol, 1.05 eq) under N2 atmosphere. The reaction mixture was stirred at 100 °C for 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. Compound 4- hydroxyoctanoyloxysodium (25 g, crude) was obtained as a white solid and it was used in next step directly without further purification.

[0271] Step 2:IMT56 [NT57

[0272] To a solution of 4-hydroxyoctanoyloxysodium (25 g, 147.38 mmol, 1 eq) in DMSO (250 mL) was added BnBr (25.20 g, 147.38 mmol, 18.91 mL, 1 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 10 min under N2 atmosphere. The reaction mixture was diluted with H2O (200 mL) and extracted with ethyl acetate 800 mL (400 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. Compound benzyl 4- hydroxyoctanoate (35 g, crude) was obtained as a colorless oil.

[0273] Step 3:

[0274] To a solution of benzyl 4-hydroxyoctanoate (35 g, 91.88 mmol, 1 eq) and (4- nitrophenyl) carbonochloridate (37.04 g, 183.76 mmol, 2 eq) in DCM (350 mL) was added Py. (14.54 g, 183.76 mmol, 14.83 mL, 2 eq) dropwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 1 h under N2 atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 10 / 1). Compound benzyl 4-(4-nitrophenoxy)carbonyloxyoctanoate (12.5 g, 31.81 mmol, 21.94% yield) was obtained as a yellow oil.

[0275] Step 4:

[0276] A mixture of benzyl 4-(4-nitrophenoxy)carbonyloxyoctanoate (12.5 g, 31.81 mmol, 1 eq), 2-pyrrolidin-l-ylethanamine (7.26 g, 63.62 mmol, 2 eq), DMAP (389 mg, 3.18 mmol, 0.1 eq) and DIEA (12.33 g, 95.42 mmol, 46.54 mL, 3 eq) in DCM (350 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:::100 / 1 to 1 / 1). Compound benzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)octanoate (3.75 g, 9.60 mmol, 31.90% yield) was obtained as a yellow oil.

[0277] Step 5 :INT59 INTSO

[0278] To a suspension of benzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)octanoate (3.75 g, 9.60 mmol, 1 eq) in THF (400 mL) was added Pd / C (1.02 g, 960.03 pmol, 10% purity', 0.1 eq) under Ar atmosphere. The reaction mixture was stirred at 25 °C for 12 h under H2 (15 Psi). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane / Methanol^lOO / l to 10 / 1). Compound 4-(2-pyrrolidin-l- ylethylcarbamoyloxy)octanoicacid (1.25 g, 3.74mmol, 43.43% yield) was obtained as a brown oil. 1H NMR (400 MHz, CDC13) 8 ppm 7.51 - 7.33 (m, 1H), 6.41 (d. J - 3.6 Hz, 1H), 4.74 (d, J = 2.4 Hz, 1H), 3.71 ■■ 3.58 (m, 1H), 3.23 ■■ 3.13 (m, 1H), 3.07 ■■ 2.94 (m, 4H), 2.89 ■■ 2.81 (m, 1H), 2.71 - 2.63 (m, 1H), 2.38 - 2.18 (m, 2H), 2.01 - 1.93 (m, 4H), 1.87 - 1.72 (m, 2H), 1.67 ■ 1.56 (m, 1H), 1.54 ■■ 1.44 (m, 1H), 1.38 ■ 1.23 (m, 4H), 0.93 ■■ 0.81 (m, 3H).

[0279] Step 6:

[0280] A mixture of [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2-(3-hydroxyazetidin- 1 - yl)propoxy]-7-oxo-heptyl]2-butyloctanoate (0.85 g, 1.11 mmol, 1 eq), 4-(2-pyrrolidin-l- ylethylcarbamoyloxy)octanoic acid (332.42 mg, 1.11 mmol, 1 eq}, EDO (254.57 mg, 1.33mmol, 1.2 eq), DMAP (13.52 mg, 110.66 pmol, 0.1 eq) and DIPEA (357.55 mg, 2.77 mmol, 481.88 pL, 2.5 eq) in DCM (8.5 mL) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under Ns atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over NacSOu filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1) to give the compound. Then the compound was dissolved in EtOAc(5 ml) and EtOH(5 ml) respectively and filtered. The filtrate was concentrated under reduced pressure to give the final product. Compound [7-[3-[7-(2-butyl-octanoyloxy)heptanoyloxy]- 2-[3-[4-(2-pyrrolidin-l-ylethylcarbamoyloxy)octanoyloxy]azetidin-l-yl]prop-oxy]-7-oxo- heptyl] 2-butyloctanoate (0.15 g, 142.79 nmol, 12.90% yield) was obtained as a colorless oil. 'H NMR (400 MHz, CDCh) 8 ppm 5.19 (s, 1H), 5.06 (t, . / 5.8 Hz, 1 H), 4.75 (s, H i). 4.09 - 4.05 (m, 8H), 3.80 ■■ 3.75 (m, 2H), 3.30 (d, J= 5.4 Hz, 2H), 3.22 - 3.18 (m, 2H), 2.70 (t, J= 5.0 Hz, 1H), 2.61 (s, 2H), 2.54 (s, 4H), 2.41 - 2.36 (m, 2H), 2.35 - 2.30 (m, 6H), 1.87 (s, 8H), 1.65 ■■ 1.56 (m, 14H), 1.46 ■■ 1.41 (m, 4H), 1.38 ■■ 1.35 (m, 611). 1.32 ■■ 1.24 (m, 28H), 0.90 - 0.86 (m, 15H).Example 14 - Synthesis of Compound 13: ((2~(3"((4~(((2~(pyrrolidm"l" yI)ethyi)carbamoyl)oxy)hexanoyi)oxy)azetidin"l“yI)propaBe~l,3"diyI)bis(oxy))bis(7” oxoheptane~7,l~diyi) bis(2~butytoctanoate)

[0282] A mixture of 5-ethyltetrahydrofuran-2-one (20 g, 175.24 mmol, 20.00 mL, 1 eq) and NaOH (736 g, 184.00 mmol, 1 .05 eq) in H2O (100 mL) was stirred at 100 °C for 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. Compound 4-hydroxyhexanoyloxysodium (27 g, 1 / 5.17 mmol, 99.97% yield) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) 5 ppm 3.55 - 3.39 (m, 1H), 2.39 - 2.16 (m, 2H), 1.83 - 1.34 (m, 4H), 0.94 (t, J - 7.2 Hz, 3H).

[0283] Step 2:

[0284] To a solution of 4-hydroxyhexanoyloxysodium (27 g, 175.17 mmol, 1 eq) in DMSO (270 mL) was added BnBr (29.97 g, 175.17 mmol, 20.82 mL, 1 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 10 min under N2 atmosphere. The reaction mixture was diluted with H2O (1000 mL) and extracted with ethyl acetate 2,000 mL (500 mL * 4). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate™ 100 / 1 to 20 / 1). Compound benzyl 4-hydroxyhexanoate (35 g, 125.97 mmol, 71.91% yield, 80% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 7.39 - 7.30 (m, 5H), 5.13 (s, 2H), 3.67 - 3.42 (m, 1H), 2.59 - 2.49 (m, 2H), 1.90 - 1.82 (m, 2H), 1.77 - 1.65 (m, 1H), 1.55 - 1.40 (m, 2H), 0.94 (t, J = 7.2 Hz, 3H).

[0285] Step 3:

[0286] A mixture of benzyl 4-hydroxyhexanoate (35 g, 157.47 mmol, 1 eq) and (4- nitrophenyl) carbonochloridate (63.47 g, 314.91 mmol, 2 eq) in DCM (350 mL) was added Py. (24.90 g, 314.91 mmol, 14.52 mL, 2 eq) dropwise at 0C'C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 2 h under N2 atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 30 / 1). Compound benzyl 4-hydroxyhexanoate benzyl 4-(4- nitrophenoxy)carbonyloxyhexanoate (12.5 g, 18.71 mmol, 20% yield, 90% purity) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 8 ppm 8.35 ■ 8.22 (m, 2H), 7.54 - 7.29 (m, 7H), 5.30 - 5.01 (m, 2H), 4.54 - 4.37 (m, 1H), 2.58-2.51 (m, 2H), 2.38 - 2.25 (m, 1H), 2.04 - 1.85 (m, 1 H), 1.75 - 1 .50 (m, 2H), 1.03 - 0.99 (m, 3H).

[0287] Step 4:

[0288] To a solution of benzyl 4-(4-nitrophenoxy)carbonyloxyhexanoate (12.5 g, 32.27 mmol, 1 eq) and 2-pyrrolidin-l-ylethanamine (7.36 g, 64.52 mmol, 2 eq) in DCM (125 ml) was added DIEA (12.5 g, 96.80 mmol, 16.86 mL, 3 eq) and DMAP (394.19 mg, 3.22 mmol, 0.1 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (300 mL) and extracted with DCM 500 mL (250 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate::::100 / 1 to 1 / 1). Compound benzyl 4-(2-pyrrolidin-l ~ylethylcarbamoyloxy)hexanoate (6.5 g, 14.31 mmol, 53.9% yield, 78% purity) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 6 ppm 7,55-7.15 (m, 5H), 5.12 (s, 2H), 4.77-4.62 (m, 1H), 3.34 - 3.18 (m, 2H), 2.64 - 2.39 (m, 8H), 2.00 - 1.82 (m, 2H), 1.80 - 1.72 (m, 4H), 1.56 (dd, J = 6.4, 11.2 Hz, 2H), 0.90 (t, J = 7.2 Hz, 3H).

[0289] Step 5:INT65 INT66

[0290] To a solution of benzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)hexanoate (6.5 g, 17.93 mmol, 1 eq) in THF (100 mL) was added Pd / C (2.00 g, 1.88 mmol, 10% purity, 0.1 eq) under Ar atmosphere. The reaction mixture was stirred at 30 °C for 12 h under H2 (30 Psi).The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane / MethanofolOO / l to 10 / 1 ). Compound 4-(2-pyrrolidin-l- ylethylcarbamoyloxy)hexanoic acid (5 g, 17.71 mmol, 94.37% yield, 96.436% purity) was obtained as a brown oil. 1 H NMR (400 MHz, CDC13) 8 ppm 10.66 (s, 1H), 6.65-6.49 (m, 1H), 4.87 ■■ 4.39 (m, 1H), 3.72 ■ 3.53 (m, 1H), 3.26 ■■ 3.02 (m, 3H), 3.00 2.74 (m, 3H), 2.40 ■■ 2.14 (m, 2H), 2.03 - 1.72 (m, 6H), 1.67 - 1.45 (m, 2H), 0.89 (t, J - 7.6 Hz, 3H).

[0291] Step 6:

[0292] A mixture of [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2-(3-hydroxyazetidin-l- yl)propoxy]-7-oxo-heptyl] 2- butyloctanoate (1 g, 1.04 mmol, 1 eq), 4-(2-pyrro lidin- 1- ylethylcarbamoyloxy)hexanoic acid (340.37 mg, 1 .25 mmol, 1 .2 eq), EDCI (299.49 mg, 1.56 mmol, 1.2 eq), DMAP (12.72 mg, 104.15 pmol, 0.1 eq) and DIPEA (336.52 mg, 2.60 mmol, 453.53 pL, 2.5 eq) in DCM (10 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate::::100 / 1 to 0 / 1). Then the compound was dissolved in EtOAc(5 ml) and EtOH(5 ml) respectively and filtered. The filtrate was concentrated under reduced pressure to give the final product. Compound [7-[3-[7-(2- buty I octanoyloxy)heptanoyloxy] -2-[3- [4-(2-pyrrolidin- 1 - ylethy lcarbamoyloxy)hexanoyloxy] azetidin- 1 -y l]propoxy] -7-oxo-heptyl] 2-buty loctanoate (0.15 g, 146.71 pmol, 100.00% yield, 100% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.18 - 5.04 (m, 1H), 5.01 - 4.96 (m, 1H), 4.68 - 4.57 (m, 1H), 4.02 - 3.94 (m, 811). 3.73 - 3.67 (m, 2H), 3.22 (d, J == 5.6 Hz, 211). 3.15 - 3.10 (m, 2H), 2.63 (t, J === 5.0 Hz, 1H), 2.56 ■■ 2.39 (m, 611). 2.34 ■■ 2.21 (m, 8H), 1.87 ■■ 1.69 (m, 611). 1.60 ■■ 1.47 (m, 16H), 1.39 - 1.33 (m, 4H), 1.31 - 1.26 (m, 8H), 1.23 - 1.16 (m, 22H), 0.86 - 0.78 (m, 15H).Example 15 - Synthesis of Compound 14: [6“[3"[6~(2~butytoctasi0ytoxy)hexanoytoxy]~2~ [3~[4~(2~pyTroIidm"l~ylethykarbamoyIoxy)deeanoyioxy]azetidm~l~yI]propoxy]"6"Oxo~ hexyl] 2-batyloctaHoate

[0293] Step l:

[0294] To a solution of 2-butyloctanoic acid (15.0 g, 74.88 mmol, 1.0 eq) and hexane- 1 ,6- diol (26.6 g, 224.64 mmol, 3.0 eq) in DCM (200 mL) was added EDO (21.5 g, 1 12.32 mmol, 1.5 eq), DMAP (4.6 g, 37.44 mmol, 0.5 eq) and DIPEA (29.0 g, 224.64 mmol, 3.0 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (500 ml.) and extracted with DCM 800 mL (400 mL.* 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 5 / 1 ). Compound 6- hydroxyhexyl 2-butyloctanoate (15.2 g, 50.58 mmol, 67% yield) was obtained as a colorless oil. 1 H NMR (400 MHz, CDCL3) 5 ppm 4.08 (t, J - 6.4 Hz, 2H), 3.69 - 3.61 (m, 2H), 2.37 - 2.27 (m, 1H), 1.66 ■ 1.55 (m, 6H), 1.46 ■■ 1.36 (m, 6H), 1.31 1.21 (m, 12H), 0.93 ■■ 0.83 (m, 6H).

[0295] Step 2:

[0296] To a solution of 6-hydroxyhexyl 2-butyloctanoate (15.2 g, 50.58 mmol, 1.0 eq) in MeCX (75 mL) and H2O (75 mL) was added TEMPO (791 mg, 5.06 mmol, 0.1 eq) and [acetoxy(phenyi)-iodanyi] acetate (35.8 g, 111.28 mmol, 2.2 eq) at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (500 mL) and extracted with DCM 2000 mL (1000 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 8 / 1). Compound 6-(2- butyloctanoyioxy)hexanoic acid (16.0 g, 50.88 mmol, 72% yield) was obtained as a colorlessoil. 1 H NMR (400 MHz, CIXT3) 5 ppm 4.08 (t, J - 6.8 Hz, 2H), 2.38 (t, J - 7.2 Hz, 2H), 2.35 ■■ 2.28 (m, 1H), 1.71 ■■ 1.63 (m, 4H), 1.62 ■■ 1.54 (m, 2H), 1.48 ■■ 1.40 (m, 4H), 1.33 ■■ 1.22 (m, 1211), 0.93 - 0.84 (m, 6H).

[0297] Step 3 :

[0298] To a solution of 6-(2-butjdoctanoyloxy)hexanoic acid (5.0 g, 15.90 mmol, 1.0 eq) and l,3-dihydroxypropan-2-one (716 mg, 7.95 mmol, 0.5 eq) in DCM (50 ml.) was added EDCI (3.6 g, 19.08 mmol, 1 .2 eq), DMAP (194 mg, 1.59 mmol, 0.1 eq) and DIPEA (5.1 g, 39.75 mmol, 2.5 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 ml) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 5 / 1). Compound [6-[3-[6-(2-butyloctanoyloxy)hexanoyloxy]-2-oxo-propoxy]-6-oxo-hexyl] 2 -butyloctanoate (3.3 g, 4.74 mmol, 73% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 4.76 (s, 4H), 4.08 (t, J - 6.4 Hz, 4H), 2.45 (t, J - 7.2 Hz, 4H), 2.37 ■■ 2.26 (m, 2H), 1.77 1.63 (m, 8H), 1.62 ■■ 1.57 (m, 4H), 1.49 ■■ 1.39 (m, 8H), 1.34 ■■ 1.21 (m, 24H), 0.94 - 0.82 (m, 12H).

[0299] Step 4:

[0300] To a solution of [6-[3-[6-(2-butyloctanoyloxy)hexanoyloxy]-2-oxo-propoxy]-6- oxo-hexyl] 2-butyloctanoate (3.3 g, 4.74 mmol, 1.0 eq) in DCM (48 mL) and MeOH (12 mL) was added azetidin-3-ol (623 mg, 5.69 mmol, 1.2 eq, HO) and TEA (576 mg, 5.69 mmol, 1.2 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 1 h under N2 atmosphere. Then NaBH(OAc)3 (2.0 g, 9.48 mmol, 2.0 eq) was added into the above reaction mixture and stirred at 25 °C for 11 h under N2 atmosphere. The reaction mixture was quenched by addition of H2O (100 mL) slowly at 0 °C under N2 atmosphere and it was extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 1 / 1). Compound [6-[3-[6-(2-butyloctanoyloxy)hexanoyloxy]-2-(3- hydroxyazetidin-l-yl)-propoxy]-6-oxo-hexyl] 2-butyloctanoate (900 mg, 1.22 mmol, 29% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 4.52 ■■ 4.42 (m, 1 H), 4.12 - 4.04 (m, 8H), 3.74 (td, J - 6.4, 2.0 Hz, 211). 3.08 (t, J - 6.4 Hz, 211). 2.72 - 2.65 (m, 1H), 2.37 ■ 2.28 (m, 6H), 1.71 ■■ 1.54 (m, 12H), 1.48 ■■ 1.38 (m, 8H), 1.31 ■■ 1.23 (m, 24H), 0.93 - 0.85 (m, 12H).

[0391] Step 5:

[0392] To a solution of [6-[3-[6-(2-butyloctanoyioxy)hexanoyloxy]-2-(3-hydroxyazetidin- l-yl)propoxy]-6-oxo-hexyl] 2-butyrloctanoate (900 mg, 1.22 mmol, 1.0 eq) and 4-(2- pyn,olidin-l-yletbylcarbamoyloxy)decanoic acid (401 mg, 1.22 mmol, 1.0 eq) in DCM (9 ml) was added EDCI (281 mg, 1.46 mmol, 1.2 eq), DMAP (15 mg, 122 pmol, 0.1 eq) and DIPEA (393 mg, 3.05 mmol, 2.5 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (50 ml) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:::100 / 1 to 0 / 1). Then the compound was dissolved in EtOAc(5 ml) and EtOH(5 ml) respectively and filtered. The filtrate was concentrated under reduced pressure to give the final product. Compound [6-[3-[6-(2-butyloctanoyloxy)hexanoyloxy]-2-[3-[4-(2-pyrrolidin- 1 ■■ylethylcarbamoyloxy)decanoyloxy]azetidin- 1 -yl]propoxy]- 6 -oxo-hexyl] 2-butyloctan-oate (160 mg, 625.04 pmol, 51.40% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.15 (s, 1H), 5.06 (t, J - 5.8 Hz, 1H), 4.75 (s, 1H), 4.09 - 4.04 (m, 8H), 3.80 - 3.75 (m, 2H), 3.28 - 3.17 (m, 4H), 2.70 (t, J = 5.2 Hz, 1H), 2.59 - 2.52 (s, 6H), 2.40 - 2.30 (m, 8H), 1.90 -1.78 (m, 6H), 1.70 - 1.55 (m, 14H), 1.46 - 1.38 (m, 10H), 1.31 - 1.22 (m, 34H), 0.90 - 0.86 (m, 15H).Example 16 - Synthesis of Compound 15: [8“[3"[S~(2“bwtyioctanoyIoxy)octanoyIoxyJ"2“[3~[4~(2~pyrrolidm"l~ylethykarbamoyloxy)decanoyioxy]azetidm~l~yl]propoxy]"8”oxo~ octyl] 2-butyloctawate

[0303] Step ! :

[0304] To a solution of 2-butyloctanoic acid (6.85 g, 34.19 mmol, 1 eq) and octane- 1,8- diol (15 g, 102.58 mmol, 3 eq) in DCM (150 mL) was added EDO (7.18 g, 37.44 mmol, 1 .5 eq), DMAP (1.52 g, 12.48 mmol, 0.5 eq) and DIEA (9.68 g, 74.88 mmol, 13.04 mL, 3 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 ml. * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 10 / 1). Compound 8-hydroxyoctyl 2-butyloctanoate (15 g, 44.72 mmol, 59.72% yield, 97.95% purity) was obtained as a colorless oil.

[0305] Step 21NT73

[0306] To a solution of 8-hydroxyoctyl 2- butyloctanoate (15 g, 44.72 mmol, 1 eq) in MeCN (75 ml) and H2O (75 rnL) was added TEMPO (0.72 g, 4.56 mmol, 0.1 eq) and [acetoxy(phenyl)"iodanyl] acetate (32.37 g, 100.45 mmol, 2.2 eq) at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (500 ml) and extracted with DCM 2000 mL (1000 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate wasconcentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 1 / 1). Compound 8-(2- butyl octanoyloxy)octanoic acid (7.5 g, 19.91 mmol, 43.61% yield, 90.94% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 4.07 (t, J = 6.4 Hz, 2H), 2.40 - 2.27 (m, 3H), 1.68 - 1.55 (m, 6H), 1.48 - 1.25 (m, 20H), 0.92 - 0.85 (m, 6H).

[0307] Step 3:

[0308] To a solution of 8-(2-butyloctanoyloxy)octanoic acid (5.5 g, 16.06 mmol, 1 eq) and l,3-dihydroxypropan-2-one (630 mg, 7.22 mmol, 0.5 eq) in DCM (55 mL) was added EDCI (4.14 g, 21.64 mmol, 1 .2 eq), DMAP (88 mg, 720.32 pmol, 0.1 eq) and DIEA (3.74 g, 28.86 mmol, 5.02 ml., 2.5 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 5 / 1). Compound [8-[3-[8-(2-butyloctanoyloxy)octanoyloxy]-2-oxo- propoxy] -8 -oxo-octyl] 2- butyloctanoate (1.4 g, 1.89 mmol, 26.25% yield) was obtained as a colorless oil. 1 H NMR (400 MHz, CDC13) 8 ppm 4.76 (s, 4H), 4.07 (t, J - 6.4 Hz, 4H), 2.43 (t, J = 7.6 Hz, 4H), 2.35 ■■ 2.27 (m, 2H), 1.73 ■■ 1.59 (m, 12H), 1.49 - 1.25 (m, 40H), 0.88 (dt, J - 2.0, 6.8 Hz, 12H).

[0309] Step 4:

[0310] A mixture of [8-[3-[8-(2- butyloctanoyloxy)octanoyloxy]-2-oxo-propoxy]-8-oxo- octyl] 2 -butyloctanoate (1.4 g, 1.89 mmol, 1 eq), azetidin-3-ol (250 mg, 2.28 mmol, 1.2 eq, HC1) and TEA (230 mg, 2.28 mmol, 316.92 pL, 1.2 eq) in DCM (14 mL) and MeOH (3.5 mL) was stirred at 25 °C for 2 h under N2 atmosphere. Then NaBH(OAc)3 (804 mg, 3.80 mmol, 2 eq) was added into the above reaction mixture and stirred at 25 °C for 12 h under N2atmosphere. The reaction mixture was quenched by addition of H2O (100 mL) at 0 °C under N2 atmosphere. The mixture was extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / "Ethyl acetate 100 1 to 0 / 1). Compound [8-[3-[8-(2- butyloctanoyloxy)octanoyloxy]-2-(3-hydroxyazetidin-l-yl)propoxy]-8-oxo-octyl] 2- butyloctanoate (830 mg, 957.85 pmol, 50.57% yield, 91.88% purity)was obtained as a colorless oil. 1H NM R (400 MHz, CDC13) 8 ppm 4.47 (t, J = 6.0 Hz, 1H), 4.12 - 4.03 (m, 8H), 3.81 - 3.68 (m, 2H), 3.12 - 2.99 (m, 2H), 2.68 (q, J = 4.8 Hz, 1H), 2.40 - 2.27 (m, 6H), 2.24 - 2.09 (m, 1H), 1.77 - 1 .51 (m, 16H), 1.50 - 1.26 (m, 36H), 0.88 (dt, J =;:2.0, 6.8 Hz, 12H).

[0312] A mixture of [8-[3 - [8-(2- butyloctanoyloxy)octanoyloxy]-2-(3-hydroxyazetidin- 1 - yl)propoxy]-8-oxo-octyl] 2 ■■butyloctanoate (630 mg, 791.29 pmol, 1 eq), 4-(2-pyrrolidin-l- ylethylcarbamoyloxy)decanoic acid (260 mg, 0.79 mmol, 1 eq), EDCI (182 mg, 0.95 mmol, 1.2 eq), DMAP (10 mg, 81.86 pmol, 0.1 eq) and DIEA (256 mg, 1.98 mmol, 345 pL, 2.5 eq) in DCM (6 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Then the compound was dissolved inEtOAc(5 ml) and EtOH(5 ml) respectively and filtered. The filtrate was concentrated under reduced pressure to give the final product. Compound [8--[3 - [8-(2- buty I octanoyloxy)octanoyloxy] -2-[3-[4-(2-pyrrolidin- 1 -ylethylcarbamoy I oxy)deca- noyloxy]azetidin-l-yl]propoxy]-8-oxo-octyl] 2.-butyloctanoate (150 mg, 124.71 pmol, 12% yield, 93.71% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.17 (s, 1H), 5.06 (t, J - 5.8 Hz, 1H), 4.75 (s, 1H), 4.10 - 3.99 (m, 8H), 3.84 - 3.73 (m, 2H), 3.33 - 3.11 (m, 4H), 2.70 (t, J = 5.2 Hz, 1H), 2.60 (t, J = 5.8 Hz, 2H), 2.52 (s, 4H), 2.42 - 2.35 (m, 2H), 2.34 - 2.26 (m, 6H), 1.90 (d, J == 7.6 Hz, 1 H), 1.78 (s, 6H), 1.68 - 1.55 (m, 12H), 1.48 - 1.40 (m, 4H), 1.39 - 1.17 (m, 44H), 0.97 - 0.81 (m, 15H).Example 17 - Synthesis of Compound 16: [l-[2-[7"(2“butyIortanoyIoxy)heptanoyIoxy]-l- [7”(2-bHtyIoctaHoyloxy)heptanoyIoxymethyl]ethyI]azetidiH”3-yI] 3~(2-pyrrolidm-l~ ylethylcarbamoyloxyXydupentanecarboxylate

[0313] Step l :

[0314] To a solution of benzyl 3-oxocyclopentanecarboxylate (10 g, 45.82 mmol, 1 eq) in THF (100 mL), toluene (25 mL) and H2O (50 mL) was added NaBH4 (8.67 g, 229.10 mmol, 5 eq) portion wise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 0 °C for 1 h under N2 atmosphere. The reaction mixture was quenched by addition HC1 (IM lOOmL) slowly at 0 °C under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue.The residue was purified by column chromatography (SiO2, Petroleum etherZEthyl acetate=100 / l to 0 / 1). Compound benzyl 3-hydroxycyclopentanecarboxylate (9.6 g, 42.71 mmol, 93.22% yield, 98% purity) was obtained as a colorless oil.

[0315] Step

[0316] To a solution of benzyl 3-hydroxycyclopentanecarboxylate (9.6 g, 14.53 mmol, 1 eq) and (4-nitrophenyl) carbonochloridate (5.86 g, 29.06 mmol, 2 eq) in DCM (30 mL) was added Py. (2.30 g, 29.06 mmol, 2.35 mL, 2 eq) dropwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 1 h under N2 atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 30 / 1). Compound benzyl 3-(4-nitrophenoxy)carbonyloxycyclopentanecarboxylate (10 g, 25.69 mmol, 58.94% yield, 99% purity) was obtained as a yellow' oil. 1H NMR (400 MHz, CDC13) 8 ppm 8.33 - 8.08 (m, 2H), 7.37 - 7.10 (m, 7H), 5.15-5.09 (m, 1H), 5.08 (s, 2H), 3.03 - 2.67 (m, 1H), 2.35 - 2.17 (m, 2H), 2.07 - 1 .84 (m, 4H).

[0317] Step 3:

[0318] A mixture of benzyl 3-(4-nitrophenoxy)carbonyloxycyclopentanecarboxylate (10 g, 25.94 mmol, 1 eq), 2-pyrrolidin-1-ylethanamine (5.92 g, 51.90 mmol, 2 eq), DIEA (10.06 g, 77.84 mmol, 13.56 mL, 3 eq) and DMAP (158.51 mg, 1.30 mmol, 0.1 eq) in DCM (50 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:::100 / 1 to 1 / 1). Compound benzyl 3-(2-pyrrolidin-l- ylethyicarbamoyloxylcyclopentanecarboxylate (8.1 g, 22.24 mmol, 85.73% yield, 99% purity) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 8 ppm 7.45-7.27 (m, 5H), 5.13 (s, 2H), 5.10 - 5.02 (m, 1 H), 3.32-3.19 (m, 2H), 2.89 - 2.78 (m, 1H), 2.61 - 2.48 (m, 6H), 2.38 - 2.22 (m, 1H), 2.11 - 1.71 (m, 10H).

[0319] Step 4:

[0320] To a solution of benzyl 3-(2-pyrrolidin-l- ylethylcarbamoyloxy)cyclopentanecarboxylate (9 g, 24.97 mmol, 1 eq) in THF (150 mL) was added Pd / C (2.66 g, 2.50 mmol, 10% purity, 0.1 eq) under Ar atmosphere. The mixture was stirred at 30 °C for 12 h under H2 (30 Psi). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane / Methanol=20 / 1 to 1 / 1). Compound 3-(2- pyrrolidin-l-ylethylcarbamoyloxy)cyclo-pentanecarboxylic acid (5.5 g, 18.92 mmol, 75.78% yield, 93% purity-) was obtained as a pale brown solid. 1H NMR (400 MHz, CDC13) 5 ppm 7.11-6.82 (m, 1H), 5.13 - 4.88 (m, 1H), 3.60 - 3.32 (m, 2H), 3.29 - 2.82 (m, 6H), 2.78 - 2.54 (m, 1H), 2.41 - 1.51 (m, 10H).

[0321] Step 5:

[0322] To a solution of [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2-(3- hydroxyazetidin-l-yl)propoxy]-7-oxo-heptyl] 2-buty I octanoate (1.0 g, 1.30 mmol, 1.0 eq) and 3-(2-pyrrolidin-l-ylethylcarbamoyloxy) cyclopentanecarboxylic acid (351 mg, 1.30 mmol, 1 .0 eq) in DCM (10 mL) was added EDCI (298 mg, 1.56 mmol, 1.2 eq), DIPEA (420 mg, 3.25 mmol, 2.5 eq) and DMAP (16 mg, 130 pmol, 0.1 eq) under N?. atmosphere. Thereaction mixture was stirred at 25 °C for 12 h under Ns atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over NasSO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOz, Petroleum ether / Ethyl aeetate:::T00 / T to 0 / 1). Then the compound was dissolved in EtOAc(5 ml) and EtOH(5 ml) respectively and filtered. The filtrate was concentrated under reduced pressure to give the final product. Compound [l-[2-[7-(2- butyl octanoyloxy)heptanoyloxy] - 1 - [7-(2- bulyloctanoyioxy)heptanoyloxymethyl]ethyl]azetidin-3-yl] 3-(2-pyrrolidin-l- ylethylcarbamoyloxy)cyclopentanecarboxylate (160 mg, 758.71 pmol, 52.98% yield) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCh) 8 ppm 5.80 (s, 1H), 5.07 (d, J = 5.8 Hz, 1H), 4.06 (t, . / 6.6 Hz, 8H), 3.82 - 3.74 (m, 2H), 3.48 (d, . / 5.4 Hz, 2H), 3.21 - 3.14 (m, 2H), 3.02 ■■ 2.91 (m, 4H), 2.80 ■■ 2.68 (m, 2H), 2.37 ■■ 2.29 (m, 6H), 2.28 ■■ 2.21 (m, 1H), 2.11 - 1.75 (m, 10H), 1.65 - 1.53 (m, 12H), 1.49 - 1.22 (m, 38H), 0.90 - 0.85 (m, 12H).Example 18 - Synthesis of Compound 17: [l~[2"[7"(2~bKtyioetan©ytoxy)heptanoyIoxy]~l~ [7”(2“bHtytoctaHoyIoxy)»heptanoyloxy methyl] ethyl] azetidin-3-yI] 3“(2»pyTrolidm»l“ ylethykarbamoyloxy)eyctohexanecarboxylate

[0323] Step

[0324] To a solution of 3 -oxocyclohexanecarboxylic acid (10 g, 35.17 mmol, 1 eq) inMeCN (100 ml) was added DBU (22.48 g, 147.72 mmol, 22.28 mL, 2 eq) and BnBr (13.24 g, 77.38 mmol, 9.2mL, 1.1 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with ethyl acetate 200 mL (100 mL * 2). The combined organic layers were dried over Na2S(M filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetateM 00 / 1

[0325] to 5 / 1). Compound benzyl 3-oxocyclohexanecarboxyiate (16 g, 68.88 mmol, 97.92% yield) was obtained as a yellow oil. ‘H NMR (400 MHz, CDCh) 8 ppm 7.30 - 7.42 (m, 5 H), 5.15 (s, 2 H), 2.81 ■■ 2.91 (m, 1 H), 2.58 (d, J= 8.0 Hz, 2 H), 2.37 (d, J= 5.2 Hz, 2 H), 2.00 - 2.19 (m, 2 H), 1.80 - 1.92 (m, 1 H), 1.67 - 1.80 (m, 1 H).INT83

[0326]

[0327] To a solution of benzyl 3-oxocyclohexanecarboxylate (16 g, 68.88 mmol, 1 eq) inTHF (160 mL), toluene (40 mL) and H2O (80 mL) was added NaBH4 (13.03 g, 344.42 mmol, 5 eq) portionwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 0°C for 1 h under N2 atmosphere. The reaction mixture was quenched by addition HQ (IM: lOOmL) slowly at 0 °C under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with ethyl acetate 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 3 / 1). Compound benzyl 3- hydroxycyclohexanecarboxylate (12.6 g, 53.78 mmol, 63.00% yield) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 8 ppm 7.32 7.40 (m, 5 H), 5.13 (s, 2 H), 3.55 ■■ 3.69 (m, 1 H), 2.43 (s, 1 H), 2.24 (d, J - 12.4 Hz, 1 H), 1.90 - 2.00 (m, 2 H), 1.86 (dd, J - 9.6, 3.4 Hz, 1 H), 1.21 - 1.50 (m, 4 H).

[0329] To a solution of benzyl 3-hydroxycyclohexanecarboxylate (12.6 g, 53.78 mmol, 1 eq), (4-nitrophenyl) carbonochloridate (10.84 g, 53.78 mmol, 1 eq) in DCM (160 mL) was added Py. (8.5 g, 107.56 mmol, 8.68 mL, 2 eq) dropwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 1 h under N2 atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 3 / 1). Compound benzyl 3-(4-nitrophenoxy)carbonyloxycyclohexanecarboxylate (20 g, 50.08 mmol, 80.00% yield) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 8 ppm8.25 - 8.31 (m, 2 H), 7.31 - 7.42 (m, 7 H), 5.14 (d, J - 1.6 Hz, 2 H), 4.65 - 4.79 (m, 1 H), 2.39 ■■ 2.59 (m, 2 H), 2.12 ■■ 2.20 (m, 1 H), 1 .92 ■■ 2.04 (m, 2 H), 1.37 ■■ 1.61 (m, 4 H).

[0331] A mixture of benzyl 3~(4~nitrophenoxy)carbonyloxycyclohexanecarboxylate (20 g, 50.08 mmol, 1 eq), 2-pyrrolidin-l-ylethanamine (5.72 g, 50.08 mmol, 1 eq), DIPEA (19.42 g, 150.22 mmol, 16.16 mL, 3 eq) , DMAP (611.76 mg, 5.00 mmol, 0.1 eq) in DCM (200 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (300 mL) and extracted with DCM 600 mL (300 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 1 / 1). Compound benzyl 3-(2-pyrrolidin-l- ylethylcarbamoyloxy)cyclohexanecarboxylate (17.2 g, 45.40 mmol, 90.66% yield) was obtained as a yellow oil.

[0332] Step 5:

[0333] To a solution of benzyl 3-(2-pyrrolidin- 1 - ylethylcarbamoyloxy)cyclohexanecarboxylate (17.2 g, 45.40 mmol, 1 eq) in THF (340 mL) was added Pd / C (4.83 g, 4.54 mmol, 10% purity, 0.1 eq) under Ar atmosphere. The mixture was stirred at 25 °C for 12 h under H2 (30 Psi). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane / Methanol= 100 / 1 to 1 / 1). Compound 3- (2-pyrrolidin-l-ylethylcarbamoyloxy)cyclohexanecarboxylic acid (1 1 g, 37.83 mmol, 83.34% yield, 97.8% purity) ’was obtained as a brown oil. 1H NMR (400 MHz, CDC13) 5 ppm 10.04 (s, 1 H), 6.95 (s, 1 H), 4.45 - 4.61 (m, 1 H), 3.57 (id. J - 7.6, 2.6 Hz, 1 H), 3.29 (ddd, J 10.8,7.8, 3.4 Hz, 1 H), 2.83 - 3.17 (m, 6 H), 2.12 - 2.32 (m, 2 H), 1.90 - 2.10 (m, 5 H), 1.74 - 1.86 (m, 2 H), 1.61 (d, J=11.6 Hz, 1 H), 1.16 ■■ 1.47 (m, 3 H).

[0334] Step 6:

[0335] A mixture of [7~[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2~(3-hydroxyazetidin- 1 yl)propoxy]-7-oxo-hept-yl] 2-butyloctanoate (1 g, 1.30 mmol, 1 eq), 3-(2-pyrrolidin-l- ylethylcarbamoyloxy) cyciohexanecarboxylic acid (740.39 mg, 2.60 mmol, 2 eq), EDCI (299.49 mg, 1.56 mmol, 1.2 eq), DMAP (15.90 mg, 130.19 pmol, 0.1 eq) and DIPEA (420.65 mg, 3.25 mmol, 566.91 pL, 2.5 eq) in DCM (10 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:;= 100 / 1 to 0 / 1). Then the compound was dissolved in EtOAc(5 ml) and EtOH(5 ml) respectively and filtered. The filtrate was concentrated under reduced pressure to give the final product. Compound [l-[2- [7"(2~butyrloctanoyloxy)heptanoyloxy]~l-[7-(2"butyloctanoyloxy)-heptanoyloxy methyl]ethyl]azetidin-3-yl] 3-(2-pyrrolidm-l-ylethylcarbamoyloxy)cyc[ohexanecarboxylate (0.16 g, 154.67 pmol, 33.33% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.19 - 5.06 (m, 1H), 4.97 (t, J = 5.8 Hz, 1H), 4.60 - 4.48 (m, 1H), 4.04 - 3.95 (m, 8H), 3.74 - 3.68 (m, 2H), 3.22 (d, J = 4.8 Hz, 1H), 3.11 - 3.05 (m, 2H), 2.62 (t, J:;= 5.0 Hz, 1H), 2.56 - 2.38 (m, 6H), 2.37 - 2.31 (m, 1H), 2.27 - 2.21 (m, 6H), 2.19 (d, J = 5.6 Hz, 1H), 1.96 * 1.90 (m, 1H), 1.80 (d, J == 14.2 Hz, 2H), 1.71 (s, 4H), 1.59 - 1.49 (m, 12H), 1.40 - 1.34 (m, 4H), 1.32 - 1.25 (m, 10H), 1.23 - 1.14 (m, 28H), 0.83 - 0.78 (m, 12H).Example 19 - Synthesis of Compound 18: [7“[3"[7~(2-butyioctan«)yI«)xy) heptanoyioxy]- 2~[3-[4~[2~(2"pyrroIidin~l~yIethykarbamoyloxy) ethyl] octanoyloxy] azetidin-l-yi] propoxy] -7-oxo-heptyI] 2-butylGctanoate Dess-Martin

[0336] Step 1 : ™ ™

[0337] A solution of 4-butylcyclohexanol (25 g, 160.00 mmol, 1 eq) in DCM (250 ml) was degassed and purged with N2 for 3 times. Then DMP (74.65 g, 176 mmol, 54.5 ml.., 1.1 eq) was added to the solution. The mixture was stirred at 25 °C for 2 h under N2 atmosphere.The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 1 / 0 to 15 / 1). Compound 4-butylcyclohexanone (25 g, 158.83 mmol, 99.28% yield, 98% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 2.42 - 2.25 (m, 4H), 2.08 - 1.98 (m, 2H), 1.75 - 1.62 (m, 1H), 1.44 - 1.27 (m, 8H), 0.99 - 0.81 (m, 3H).

[0338]

[0339] To a solution of 4-butylcyclohexanone (25 g, 162.10 mmol, 1 eq) in DCM (250 ml) was added m-CPBA (46.05 g, 226.9 mmol, 85% purity, 1.4 eq) slowly at 0 °C. The mixture was stirred at 25 °C for 2 h under N2 atmosphere. The reaction mixture was quenched by addition aq. Na2SO3 (100 ml). It was extracted with DCM 450 ml. (150 rnL * 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 330 g Sepa Flash® Silica Flash Column, Eluent of 8-13% Ethyl acetate / Petroleum ether gradient at 150 mL / min). Compound 5-butyloxepan-2- one (27 g, 158.59 mmol, 97.85% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 4.34 - 4.23 (m, 1H), 4.20 - 4.12 (m, 1H), 2.77 - 2.47 (m, 2H), 2.14 - 1.79 (m, 2H), 1 -64 - 1.41 (m, 2H), 1.38 * 1.20 (m, 7H), 1.07 - 0.61 (m, 3H).

[0341] To a solution of 5-butyloxepan-2-one (27 g, 158.58 mmol, 1 eq) in H2O (270 mL) was added NaOH (6.66 g, 166.53 mmol, 1.05 eq), then the mixture was stirred at 100 °C for 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced to give a crude product. Compound 4-(2 -hydroxyethyl) octanoyloxysodium (30 g, crude) was obtained as a white solid.

[0342] Step 4: INT91

[0343] To a solution of 4-(2-hydroxyethyl) octanoyloxysodium (30 g, 142.68 mmol, 1 eq) in DMSO (150 niL) was added BnBr (8.13 g, 47.56 mmol, 5.65 mL, 1 eq). The mixture was stirred at 25 °C for 0.5 h under N2 atmosphere. The reaction mixture was diluted with water (1500 mL) and extracted with ethyl acetate 1500 mL (500 mL * 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 220 g Sepa Flash® Silica Flash Column, Eluent of 15-25% Ethyl acetate / Petroleum ether gradient at 100 mL / min). Compound benzyl 4-(2-hydroxyethyl) octanoate (32 g, 114.82 mmol, 80.47% yield, 99.89% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 5 ppm 7.53 ■■ 7.27 (m, 5H), 5.12 (s, 2H), 3.75 ■■ 3.60 (m, 2H), 2.38 (t, J = 8.0 Hz, 2H), 1.69 - 1.46 (m, 6H), 1.31-1.23 (m, 6H), 0.97 - 0.78 (m, 3H).

[0345] A mixture of benzyl 4-(2-hydroxyethyl) octanoate (22 g, 79.02 mmol, 1 eq) and (4- nitrophenyl) carbonochloridate (31.86 g, 158.06 mmol, 2 eq) in DCM (200 mL) was degassed and purged with N2 for 3 time. Then Py. (12.5 g, 158.06 mmol, 12.76 mL, 2 eq) was added dropwise to the mixture at 0 °C. The mixture was stirred at 25 °C for 1 h under N2 atmosphere. The reaction mixture was diluted with petroleum ether (500 mL) and filtered.The filtrate was concentrated under reduced pressure to give a residue. The residue waspurified by flash silica gel chromatography (ISCO®; 220 g Sepa Flash® Silica Flash Column, Eluent of 5-10% Ethyl acetate / Petroleum ether gradient at 100 mL / min). Compound benzyl 4-[2-(4-nitrophenoxy) carbonyioxyethyl] octanoate (28 g, 61 .87 mmol, 78.29% yield, 98% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 5 ppm 8.54 - 7.76 (m, 2H), 7.47 - 7.22 (m, 7H), 5.13 (s, 2H), 4.44 - 4.06 (m, 2H), 2.44 - 2.33(in, 2H), 1.79 - 1.66 (m, 4H), 1.58 - 1.46 (in, 1H), 1.40 - 1.23 (m, 6H), 0.95 - 0.85 (in, 3H).

[0346] Step 6:

[0347] To a mixture of benzyl 4-[2-(4-nitrophenoxy) carbonyioxyethyl] octanoate (28 g, 63.12 mmol, 1 eq) and 2-pyrrolidin-l-yle thanamine (14.4 g, 126.28 mmol, 2 eq) in DCM (300 mL) was added DIEA (24.48 g, 189.40 mmol, 33 mL, 3 eq) and DMAP (771.32 mg, 6.32 mmol, 0.1 eq). The mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction was concentrated under reduced pressure to give a residue. The residue was diluted with water (1000 mL) and extracted with ethyl acetate 1500 mL (500 mL * 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 330 g Sepa Flash® Silica Flash Column, Eluent of 70-80% Ethyl acetate / Petroleum ether gradient at 120 mL / min). Compound benzyl 4-[2"(2~pyrrolidin-l- ylethy I carbamoyloxy) ethyl] octanoate (26 g, 61.74 mmol, 97.80% yield, 99.40% purity) was obtained as a pale yellow oil.

[0348] Step 7:

[0349] To a solution of benzyl 4-[2-(2-pyrroHdin-l-ylethylcarbamoyloxy) ethyl] octanoate (26 g, 62.12 mmol, 1 eq) in THF (400 mL) was added Pd / C (6.61 g, 6.21 mmol, 10% purity, 0.1 eq) under Ar atmosphere. The suspension was degassed and purged with H2 for 3 times. The mixture was stirred at 30 °C for 12 h under H2 (30 Psi) atmosphere. The suspension was filtered through a pad of celite and the filtrate was concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO2, DCM / MeOH =20 / 1 to 5 / T). Compound 4-[2-(2-pyrrolidin-l-ylethylcarbamoyloxy) ethyl] octanoic acid (14 g, 39.22 mmol, 63.13% yield, 92% purity) was obtained as a pale yellow oil. 1H NMR (400 MHz, CDC13) 8 ppm 8.36 ■■ 8.13 (m, 1H), 6.39 (s, 1H), 4.30 3.76 (m, 2H), 3.45 ■■ 3.27 (m, 2H), 3.05 - 2.77 (m, 6H), 2.49 - 2.16 (m, 2H), 1.97-1.92 (m, 4H), 1.67 - 1.45 (m, 5H), 1 .38- 1.17 (m, 6H), 1.08 - 0.50 (m, 3H).

[0350] Step 8:Compound 1g

[0351] To a mixture of [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-(3- hydroxyazetidin-l-yl) propoxy]-7-oxo-heptyl] 2-butyloctanoate (1.0 g, 1.30 mmol, 1.0 eq) and 4-[2-(2-pyrrolidin-l-ylethylcarbamoyloxy) ethyl] octanoic acid (512 mg, 1.56 mmol, 1.2 eq) in DCM (10 ml) was added EDCI (299 mg, 1.56 mmol, 1 .2 eq), DIPEA (420 mg, 3.25 mmol, 2.5 eq) and DMAP 16 mg, 130 pmol, 0.1 eq). The reaction mixture was diluted with H2O (100 ml) and extracted with ethyl acetate 300 mL (100 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PE / EtOAc=10 / l to 1 / 3). Compound [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-[3-[4-[2- (2-pyrrolidin-l-ylethylcarbamoyloxy) ethyl] octanoyloxy] azetidin-l-yl] propoxy]-7-oxo- heptyl] 2-butyloctanoate (150 mg, 96.3% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.20 (s, 1H), 5.08 - 5.02 (m, 1 H), 4.12 - 4.01 (m, 10H), 3.80 - 3.76 (m, 2H), 3.29 (s, 2H), 3.22 - 3.16 (m, 2H), 2.73 - 2.68 (m, 1H), 2.58 (t, >6.0 Hz, 2H), 2.51(s, 4H), 2.37 - 2.26 (m, 8H), 1.87 - 1.70 (m, 6H), 1.67 - 1.54 (m, 16H), 1.52 - 1.16 (m, 46H), 0.95 ■■ 0.83 (m, 1511).Example 20 - Synthesis of Compound 19: [7”[3”[7“(2»butylo£tanoyl0xy) heptanoyloxy]» 2"[3"[4,4"dimethyL6"(2~pyrTOlidin“l"yiethyIcarbam©ytoxy) hexanoyi] oxyazetidm-l-yl] propoxy] -7-oxo-heptyI] 2-butyIoetanoate

[0352] Step$353^ To a solution of 4,4-dimethylcyclohexanone (40 g, 316.9 / mmol, 1 eq) in DCM (400 ml) was added m-CPBA (102.96 g, 507.15 mmol, 85% purity, 1.6 eq) at 0 °C. The mixture was stirred at 25 °C for 2 hr under N2 atmosphere. The reaction mixture was quenched by addition aq. Na2SO3 (800 ml) slowly. It was extracted with DCM 500 mL (100 mL *5). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate::::100 / 1 to 0 / 1). Compound 5,5- dimethyloxepan-2-one (18 g, 126.59 mmol, 39.94% yield) was obtained as a colorless oil.

[0354] Step

[0355] A mixture of 5,5-dimethyloxepan-2-one (18 g, 126.59 mmol, 1 eq) and NaOH (5.32 g, 132.92 mmol, 1.05 eq) in H2O (90 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 100 °C for 12 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a crude product. Compound (6- hydroxy-4,4-dimethyl-hexanoyl) oxysodium (14 g, crude) was obtained as a white solid.

[0356] INT97 Step 3: INTOT

[0357] To a solution of (6-hydroxy-4,4-dimethyl-hexanoyl) oxysodium (14 g, 76.84 mmol, 1 eq) in DMSO (140 mL) was added BnBr (13.14 g, 76.84 mmol, 9.13 mL, 1 eq). Themixture was stirred at 25 °C for 5 min. The reaction mixture was diluted with H2O (200 mL) and extracted with ethyl acetate 600 mL (200 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a crude product. Compound benzyl 6-hydroxy-4,4-dimethyl-hexanoate (12 g, crude) was obtained as a colorless oil.

[0358] Step 4:

[0359] To a solution of benzyl 6-hydroxy-4,4-dimethyl-hexanoate (12 g, 47.94 mmol, 1 eq), (4-nitrophenyl) carbonochloridate (19.32 g, 95.87 mmol, 2 eq) in DCM (120 mL) was added Py. (7.58 g, 95.87 mmol, 7.74 mL, 2 eq) at 0 °C. The mixture was stirred at 0 °C for 0.5 hr. The reaction mixture was diluted writh petroleum ether (200 mL) and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 5 / 1). Compound benzyl 4,4-dimethyl-6-(4-nitrophenoxy) carbonyloxy-hexanoate (10 g, 24.07 mmol, 50.21% yield) was obtained as a colorless oil.

[0360] Step 5:

[0361] A mixture of benzyl 4, 4-dimethyl-6-(4-nitrophenoxy) carbonyloxy-hexanoate (10 g, 24.07 mmol, 1 eq), 2-pyrrolidin-l-ylethanamine (5.50 g, 48.14 mmol, 2 eq), DMAP (294.07 mg, 2.41 mmol, 0.1 eq) and DIPEA (9.33 g, 72.21 mmol, 12.58 mL, 3 eq) in DCM (100 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (200 mL) and extracted with ethyl acetate 600 mL (200 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 3 / 1). Compound benzyl 4,4-dimethyl-6-(2-pyrrolidin-l-ylethylcarbamoyloxy) hexanoate (6 g, 15.36 mmol, 63.83% yield) was obtained as a colorless oil.

[0362] Step 6:

[0363] To a suspension of Pd / C (4.91 g, 4.61 mmol, 10% purity, 0.3 eq) in THF (120 mL) was added benzyl 4,4-dimethyl-6-(2-pyrrolidin-l-ylethylcarbamoyloxy) hexanoate (6 g, 15.36 mmol, 1 eq). The mixture was stirred at 30 °C for 12 hr under H2 (15 Psi) atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:::100 / 1 to 0 / 1). Compound 4,4-dimethyl-6-(2-pyrrolidm-l- ylethylcarbamoyloxy) hexanoic acid (3 g, 9.99 mmol, 65.00% yield) was obtained as a colorless oil.

[0364] Step 7 :Compound 19

[0365] To a solution of [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-(3- hydroxyazetidin-1-yl) propoxy]-7-oxo-heptyl] 2-butyloctanoate (0.8 g, 1.04 mmol, 1 eq) in DCM (12 mL) was added EDCI (239.59 mg, 1.25 mmol, 1.2 eq), 4-pyrrolidin-l-ylpyridine (15.44 mg, 104.15 umol, 0.1 eq), 4,4-dimethyl-6-(2-pyrrolidin-l -ylethylcarbamoyloxy) hexanoic acid (438.01 mg, 1.46 mmol, 1.4 eq) and DIPEA (336.52 mg, 2.60 mmol, 453.53 p.L, 2.5 eq). The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 150 mL (50 mL *3). Thecombined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate- 100 / 1 to 0 / 1). Compound [7-[3-[7-(2- butyloctanoyloxy) heptanoyloxy]-2-[3-[4,4-dimethyl-6-(2-pyrrolidin-l-ylethylcarbamoyloxy) hexanoyl] oxyazetidin-l-yl] propoxy]-7-oxo-heptyl] 2-butyloctanoate (0.15 g, 98% purity) was obtained as a colorless oil. HI NMR (400 MHz, CDC13) 8 ppm 5.38 (s, 1 H), 5.08-5.02 (m, 1H), 4.19 - 3.96 (m, 10H), 3.80 - 3.76 (m, 2H), 3.33 (d, J=5.38 Hz, 2H), 3.23 - 3.16 (m, 2H), 2.71 (t, J 5. 1 Hz, 1H), 2.68 - 2.57 (m, 4H), 2.38 - 2.25 (m, 8H), 1.81 (s, 6H), 1.67 - 1.53 (m, 16H), 1.49 - 1.22 (m, 38H), 0.92 (s, 6H), 0.90 -0.86 (m, 12H).Example 21 - Synthesis of Compound 20: [7"[3"[7~(2"butylo£tanoyloxy) heptanoy Ioxy]~ 2“[3”[3»[l“[2~(2~pyrrolidiH“l~ylethyIcarbamoyIoxy) ethyl] cyclohexyl] propanoyloxy] azetidin~l~yl] propoxy] -T-oxo-heptyl] 2~butylocta

[0366] Step 1 :m 03

[0367] To a solution of spiro [5.5] undecan-3-one (3.5 g, 21.05 mmol, 1 eq) in DCM (70 ml) was added m-CPBA (6.84 g, 33.68 mmol, 85% purity, 1.6 eq). The mixture was stirred at 25 °C for 48 hr under N2 atmosphere. The reaction mixture was quenched by addition aq. Na2SO3 (20 mL). It was diluted with H2O (40 mL) and extracted with ethyl acetate 120 mL (40 mL x 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 5 / 1). Compound 9- oxaspiro [5.6] dodecan- 10-one (7 g, 38.41 mmol, 93% yield) was obtained as a yellow oil.1H NMR (400 MHz, CDC13) 5 ppm 4.27 - 4.15 (m, 2H), 2.64 - 2.54 (m, 2H), 1.75 - 1.68 (m, 2H), 1.66 - 1.58 (m, 2H), 1.50 - 1.36 (m, 10H).

[0368] Step 2:

[0369] To a solution of 9-oxaspiro [5.6] dodecan- 10-one (7 g, 38.40 mmol, 1 eq) in H2O (70 mL) was added NaOH (1.62 g, 40.32 mmol, 1.05 eq). The mixture was stirred at 100 °C for 12 hr under N2 atmosphere. The reaction mixture was concentrated to give a crudeproduct. Compound 3-[l-(2-hydroxyethyl) cyclohexyl] propanoyloxysodium (8 g crude) was obtained as a white solid.

[0370]

[0371] To a solution of 3-[l-(2-hydroxyetbyl) cyclohexyl] propanoyloxysodium (8 g,35.99 mmol, 1 eq) in DMSO (90 mL) was added BnBr (6.16 g, 35.99 mmol, 4.28 mL, 1 eq).The mixture was stirred at 25 °C for 5 min under N2 atmosphere. The reaction mixture was diluted with H2O (80 ml) and extracted with ethyl acetate 240 mL (80 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 5 / 1). Compound benzyl 3-[l- (2-hydroxyethyl) cyclohexyl] propanoate (5.5 g, 18.94 mmol, 52.62% yield) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 5 ppm 7.40 - 7.33 (m, 5H), 5.12 (s, 2H), 3.67 (t, J - 7.6 Hz, 2H), 2.38 - 2.29 (m, 2H), 1 .69 - 1 .64 (m, 2H), 1.59 - 1.53 (m, 2H), 1 .48 - 1.40 (m, 6H), 1.32 ■ 1.25 (m, 4H).

[0372] Step 4:

[0373] To a solution of benzyl 3-[l-(2-hydroxyethyl) cyclohexyl] propanoate (5.5 g, 18.94 mmol, 1 eq) and (4-nitrophenyl) carbonochloridate (5.73 g, 28.41 mmol, 1.5 eq) in DCM (55 mL) was added Py. (3.00 g, 37.88 mmol, 3.06 mL, 2 eq) at 0 °C. The mixture was stirred at 25 °C for 1 hr under N2 atmosphere. The reaction mixture was diluted with petroleum ether (200 mL) and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 5 / 1). Compound benzyl 3-[l-[2-(4-nitrophenoxy) carbonyloxyethyl] cyclohexyl] propanoate (6.8 g, 14.93 mmol, 78.82% yield) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 5 ppm 8.30 - 8.25 (m, 2H), 7.41 - 7.32 (m, 7H), 5.12 (s, 2H), 4.32 (t, J = 7.6 Hz, 2H), 2.41 ■■ 2.28 (m, 2H), 1.79 ■■ 1.68 (m, 4H), 1.51 ■■ 1.41 (m, 6H), 1.37 ■■ 1.30 (m, 4H).

[0374] Step 5 :INT106 8HT1O7

[0375] A mixture of benzyl 3-[l-[2-(4-nitrophenoxy) carbonyloxyethyl] cyclohexyl] propanoate (6.8 g, 14.92 mmol, 1 eq), 2-pyrrolidin-l-ylethanamine (3.40 g, 29.86 mmol, 2 eq), DIPEA (5.78 g, 44.78 mmol, 7.8 mL, 3 eq), DMAP (182 mg, 1.49 mmol, 0.1 eq) in DCM (68 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (40 mL) and extracted with ethyl acetate 120 mL (40 mL x 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 1 / 4). Compound benzyl 3-[l-[2-(2-pyrrolidin-l-ylethylcarbamoyloxy) ethyl] cyclohexyl] propanoate (5.3 g, 12.31 mmol, 82.45% yield) was obtained as a yellow oil.

[0377] To a solution of benzyl 3~[l-[2~(2-pyrrolidin- 1-ylethylcarbamoyloxy) ethyl] cyclohexyl] propanoate (5.3 g, 12.31 mmol, 1 eq) in THF (53 mL) was added Pd / C (1.31 g, 1.23 mmol, 10% purity, 0.1 eq) under N2 atmosphere. The suspension was degassed and purged with H2 for 3 times. The suspension was stirred at 25 °C for 12 hr under H2 (15 Psi) atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM / MeOH =100 / 1 to 5 / 1). Compound 3-[ l-[2-(2-pyrro lidin- 1- ylethylcarbamoyloxy) ethyl] cyclohexyl] propanoic acid (3 g, 8.22 mmol, 66.81% yield) was obtained as a yellow7oil. 1 H NMR (400 MHz, CDC13) 8 ppm 6.70 (s, 1H), 6.00 (s, 1H), 4.11 (t, J = 5.6 Hz, 2H), 3.48 ■■ 3.36 (m, 2H), 3.05 (s, 4H), 2.97 (t, J = 5.2 Hz, 2H), 2.23 ■■ 2.14 (m, 2H), 1.98 (s, 4H), 1.72 - 1.65 (m, 2H), 1.61 (t, J = 5.6 Hz, 2H), 1.49 - 1.22 (m, 10H).

[0378] Step 7:Compound 2®

[0379] A mixture of [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2-(3-hydroxyazetidin-l- yl)propoxy]-7-oxo-heptyl] 2 -butyloctanoate (1 g, 1.30 mmol, 1 eq), 3-[l-[2-(2-pyrrolidin-l- ylethylcarbamoyloxy)ethyl]cyclohexyl]propanoic acid (664.86 mg, 1.95 mmol, 1.5 eq), DIPEA (420.65 mg, 3.25 mmol, 566.91 uL, 2.5 eq), DMAP (15.90 mg, 130.19 pmol, 0.1 eq) and EDCI (299.49 mg, 1.56 mmol, 1 .2 eq) in DCM (10 ml) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (10 ml) and extracted with ethyl acetate 30 mL (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 100 / 1 to 3 / 1). Compound [7-[3-[7-(2-butyloctanoyloxy) heptanoy loxy] -2-[3 - [3 - [ 1 - [2-(2-pyrroli din- 1 -ylethylcarbamoy loxy) ethyl] cyclohexyl] propanoyloxy] azetidin-l-yl] propoxy] -7-oxo-heptyl] 2 -butyloctanoate (150 mg, 92.6% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.28 (s, 1H), 5.07 ■■ 5.04 (m, 1H), 5.05 (t, >5.8 Hz, 1 H), 4.11 ■■ 3.99 (m, 10H), 3.80 - 3.76 (m, 2 H), 3.30 (d, >5.6 Hz, 211). 3.23 - 3.18 (m, 21 h. 2.71 (t, >5.00 Hz, 1 H), 2.59 (t, >6.0 Hz, 2H), 2.52 (s, 4H), 2.37 ■ 2.25 (m, 8H), 1.78 (s, 4H), 1.69 ■■ 1.54 (m, 18H), 1.46 1.22 (m, 46H), 0.90 - 0.86 (m, 12H).Example 22 - Synthesis of Composed 21: [7“[3"[7~(2~bwtyioctan«)yl«)xy) heptanoytoxy]- xyazetidio-l-yl]

[0380] Step 1 :

[0381] A mixture of 5, 5-dimethyltetrahydrofiiran-2-one (10 g, 87.61 mmol, 1 eq) and NaOH (3.68 g, 91.99 mmol, 1.05 eq) in H2O (50 ml) was degassed and purged with N2 for 3 times. The mixture was stirred at 100 °C for 12 hr under N2 atmosphere. The mixture was concentrated under reduced pressure to give a crude product. Compound (4-hydroxy-4~ methyl -pentanoyl) oxysodium (12 g, crude) was obtained as a white solid.

[0383] A mixture of (4-hydroxy-4-methyl -pentanoyl) oxysodium (12 g, 77.85 mmol, 1 eq) and BnBr (13.32 g, 77.85 mmol, 9.25 ml, 1 eq) in DMSO (60 ml) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 5 min under N2 atmosphere. The mixture was quenched by addition H2O (500 mL). It was extracted with ethyl acetate (100 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a crude product. Compound benzyl 4-hydroxy-4-methyl-pentanoate (14.8 g, crude) obtained as a colorless oil.

[0384] Step 3 :

[0385] To a mixture of benzyl 4-hydroxy-4-methyl-pentanoate (14.8 g, 66.58 mmol, 1 eq) and (4- nitrophenyl) carbonochloridate (26.84 g, 133.17 mmol, 2 eq) in DCM (150 mL) was added Py. (10.53 g, 133.17 mmol, 10.75 mL, 2 eq) at 0 oC. The mixture was wanned to 25 °C and stirred for 1 hr under N2 atmosphere. The mixture was diluted with petroleum ether (500 mL) and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=0 / 1 to 1 / 10). Compound benzyl 4-methyl-4-(4-nitrophenoxy) carbonyloxy-pentanoate (19 g, 49.05 mmol, 73.66% yield) obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 = 8.33 - 8.19 (m, 211), 7.41 - 7.30 (m, 7H), 5.15 (s, 2H), 2.58 - 2.48 (m, 2H), 2.26 - 2.15 (m, 2H), 1.58 (s, 6H).

[0386] Step 4:

[0387] A mixture of benzyl 4-methyl-4-(4-nitrophenoxy) carbonyl oxy -pentanoate (19 g, 49.05 mmol, 1 eq), 2-pyrrolidin-l-ylethanamine (11.20 g, 98.09 mmol, 2 eq), DIEA (19.02 g, 147.14 mmol, 25.63 ml, 3 eq), and DMAP (599.21 mg, 4.90 mmol, 0.1 eq) in DCM (170 ml) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The mixture was quenched by addition H2O (500 mL). It was extracted with DCM 300 mL (100 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PE / EtOAc:::100 / 1 to 3 / 1). Compound benzyl 4~methyl-4"(2~pyrrolidin"l-ylethylcarbamoyloxy) pentanoate (15 g, 44.14 mmol, 71% yield) obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 8 - 7.42 ■■ 7.27 (m, 5H), 5.12 (s, 2H), 5.02 (s, 1H), 3.29 ■ 3.16 (m, 2H), 2.60 ■■ 2.48 (m, 6H), 2.48 - 2.41 (m, 2H), 2.11 - 2.05 (m, 2H), 1.77 (t, J = 3.2 Hz, 4H), 1.45 (s, 6H).

[0388] Step 5 :

[0389] To a solution of benzyl 4-methyl-4-(2-pyn-olidin-l-ylethylcarbamoyloxy) pentanoate (15 g, 44.14 mmol, 1 eq) in THE (300 mL) was added Pd / C (4.70 g, 4.41 mmol, 10% purity, 0.1 eq) under N2 atmosphere. The suspension was degassed and purged with H2 for 3 times. The mixture was stirred at 25 °C for 12 hr under H2 (15 Psi) atmosphere. The reaction mixture was filtered and the filtrate was concentrated to give a residue. The residue was purified by silica gel column chromatography (DCM / MeOH, from 0 / 1 to 1 / 8).Compound 4-methyl-4-(2-pyrrolidin-l -ylethylcarbamoyloxy) pentanoic acid (6 g, 96.2% purity) was obtained as a brown oil.

[0390] Step 6:

[0391] A mixture of [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-(3-hydroxyazetidin- 1-yi) propoxy]-7- oxo-heptyl] 2-butyloctanoate (800 mg, 1.04 mmol, 1 eq), EDCI (239.60 mg, 1.25 mmol, 1.2 eq), 4-methyl-4-(2-pyrrolidin-l -ylethylcarbamoyloxy) pentanoic acid (340.38 mg, 1.24 mmol, 1.2 eq), DIEA (336.52 mg, 2.60 mmol, 453.54 pL, 2.5 eq) and DMAP (12.72 mg, 104.16 mmol, 0.1 eq) in DCM (10 mL). The mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with water (50 mL) and extracted with ethyl acetate 60 mL (20 mL * 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 25 g Sepa Flash® Silica Flash Column, Eluent of 60~70% Ethyl acetate / Petroleum ether gradient at 60 mL / min). Compound [7-[3- [7-(2-butyloctanoyloxy) heptanoyloxy] -2- [3 - [4-methyl-4-(2-pyrroli din- 1 - ylethylcarbamoyloxy) pentanoyl] oxyazetidin-1 -yl] propoxy]-7-oxo-heptyl] 2-butyloctanoate (150 mg, 90.8% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.09 - 5.03 (m, 2H), 4.09 - 4.04 (m, 8H), 3.80 - 3.76 (m, 2H), 3.26 - 3.18 (m, 4H), 2.73 - 2.68 (mJ H ). 2.61 - 2.50 (m, 6H), 2.43 - 2.38 (m, 2 H), 2.36 - 2.28 (m, 6H), 2.09 - 2.02 (m, 2H), 1.79 (s, 4 H), 1.66 - 1.55 (m, 16H), 1 .50 - 1 .43 (m, 10H), 1.40 - 1.20 (m, 36H), 0.90 - 0.86 (m, 12H).Example 23 - Synthesis of Composed 22: O6"[3“[6"0x0"6“(3"pentytoetoxy) hexanoyl] oxy"2"[3“[4“(2"pyrrolidiH“l“yIethylcarbamoyIoxy) decanoyloxy] azetidio-l-yl] propyl] Ol~(3~pentytoctyl) hexanedioate

[0392] Step ] -INT115 INT116

[0393] To a solution of undecan-6-one (70 g, 411.06 mmol, 1 eq) in THF (1400 mL) was added NaH (21.37 g, 534.38 mmol, 60% purity, 1.3 eq) and ethyl 2- diethoxyphosphorylacetate (138.23 g, 616.59 mmol, 122.33 mL, 1.5 eq) at 0 °C. The mixture was stirred at 80 °C for 24 hr under N2 atmosphere. The reaetion mixture was quenched by addition H2O (2000 mL) at 0 °C. It was extracted with ethyl acetate 4500 mL (1500 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound ethyl 3- pentyloct-2-enoate (40 g, 166.40 mmol, 40.48% yield) was obtained as a white solid.

[0394] Step 2:

[0395] To a suspension of Pd / C (116.64 mmol, 10% purity, 0.1 eq) in THF (800 mL) was added ethyl 3-pentyloct-2~enoate (40 g, 166.40 mmol, 1 eq). The mixture was stirred at 25 °C for 12 hr under H2 atmosphere. The reaction mixture filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound ethyl 3-pentyloctanoate (20 g, 82.51 mmol, 49.58% yield) was obtained as a colorless oil.

[0396] Step 3:

[0397] To a solution of ethyl 3-pentyloctanoate (20 g, 82.51 mmol, 1 eq) in THF (200 mL) was added LAH (2.5 M, 39.60 mL, 1.2 eq) by dropwise. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was quenched by addition H2O (20 mL) at 0 °C. It was diluted with water (300 mL) and extracted with ethyl acetate 1500 mL(500 mL. * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Etbyl acetate:::100 / 1 to 1 / 1). Compound 3- pentyloctan-1 -ol (8 g, 39.93 mmol, 48.39% yield) was obtained as a white solid.

[0398] Step 4:

[0399] A mixture of 3-pentyloctan-l-oi (8 g, 39.93 mmol, 1 eq), adipic acid (29.18 g, 199.64 mmol, 33.15 mL, 5 eq), EDCI (9.19 g, 47.91 mmol, 1.2 eq), DMAP (487.79 mg, 3.99 mmol, 0.1 eq) and DIPEA (12.90 g, 99.82 mmol, 17.39 mL, 2.5 eq) in DCM (40 mL) and THE (40 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (120 mL) and extracted with ethyl acetate 120 mL (40 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / T). Compound 6-oxo-6-(3-pentyloctoxy) hexanoic acid (4 g, 12.18 mmol, 30.50% yield) was obtained as a colorless oil.

[0490] Step 5 :

[0401] A mixture of 6-oxo-6-(3-pentyloctoxy) hexanoic acid (2 g, 6.09 mmol, 1 eq), 1,3- dihydroxypropan-2-one (219.38 mg, 2.44 mmol, 0.4 eq), EDCI (1.40 g, 7.31 mmol, 1.2 eq), DMAP (74.38 mg, 608.86 pmol, 0.1 eq) and DI PEA (1 .97 g, 15.22 mmol, 2.65 mL, 2.5 eq) in DCM (20 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (20 mL) and extracted with ethyl acetate 60 mL (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue.The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound O6-[2-oxo-3-[6-oxo-6-(3-pentyloctoxy) hexanoyl] oxypropyl] 01 -(3 -pentyloctyl) hexanedioate (1 .4 g, 1.97 mmol, 32.34% yield) was obtained as a colorless oil.

[0402] Step 6:

[0403] A mixture of 06- [2-oxo-3-[6-oxo-6-(3 -pentyloctoxy) hexanoyl] oxy -propyl] 01- (3 -pentyloctyl) hexanedioate (1.4 g, 1.97 mmol, 1 eq), azetidin-3-ol (161.79 mg, 1.48 mmol, 0.75 eq, HC1), NaBH(OAc)3 (417.31 mg, 1.97 mmol, 1 eq) and TEA (119.55 mg, 1.18 mmol, 164.44 pL, 0.6 eq) in DCM (7 mL) and MeOH (28 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The mixture was diluted with H2O (20 mL) and extracted with ethyl acetate 60 mL (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound O6-[2-(3- hydroxyazetidin-l-yl)-3-[6-oxo-6-(3-pentyloctoxy) hexanoyl] oxy-propyl] Ol-(3-pentyloctyl) hexanedioate (0.6 g, 781.14 pmol, 39.67% yield) was obtained as a colorless oil.

[0494] Step 7 :Compound 21

[0405] A mixture of 06- [2-(3-hydroxyazetidin- 1 -yl)-3-[6-oxo-6-(3-pentyloctoxy) hexanoylj oxy-propyl] Ol-(3-pentyloctyl) hexanedioate (0.6 g, 781.14 gmol, 1 eq), 4-(2- pyrrolidin-l-ylethylcarbamoyloxy) decanoic acid (307.87 mg, 937.36 pmol, 1.2 eq), EDCI (179.69 mg, 937,36 nmol, 1 .2 eq), DMAP (9.54 mg, 78.11 pmol, 0.1 eq) and DIPEA (252.39 mg, 1.95 mmol, 340.15 pL, 2.5 eq) in DCM (6 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (20 mL) and extracted with ethyl acetate 60 mL (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound O6-[3-[6- oxo-6-(3 -pentyloctoxy) hexanoyl] oxy-2-[3-[4-(2-pyrrolidin-l-ylethylcarbamoyloxy) decanoyloxy] azetidin- 1-yl] propyl] Ol-(3-pentyloctyl) hexanedioate (0.15 g, 137.69 pmol, 17.63% yield, 99% purity) was obtained as a colorless oil. 1 H NMR (400 MHz, CDC13) 8 ppm 5.46 ■■ 5.13 (m, 1H), 5.09 5.03 (m, 1H), 4.76 (s, 1H), 4.14 ■■ 4.01 (m, 8H), 3.83 3.72 (m, 2H), 3.32 (s, 2H), 3.23 - 3.16 (m, 2H), 2.74 - 2.50 (m, 6H), 2.42 - 2.28 (m, 10H), 1.97 - 1.76 (m, 6H), 1.68 - 1.64 (m, 8H), 1.62 - 1.55 (m, 10H), 1.44 - 1.38 (m, 211). 1.32 - 1.24 (m, 36H), 0.97 - 0.83 (m, 15H).Example 24 - Synthesis of Compound 23: 01~[3”[6”(l”heptytoctoxy)~6”Oxo-hexaHGyl] oxy-2-[3-H"(2“PyrroIidin-l"ytethyIcarbamoyioxy) decanoyloxy] azetidin-l-yl] propyl] O6~(l"heptyloctyl) hexanedioate

[0406] Step l :1NT122 INT123

[0497] A mixture of adipic acid (19.19 g, 131.34 mmol, 21.81 mL, 5 eq), pentadecan-8-ol (6 g, 26.27 mmol, 1 eq), EDCI (6.04 g, 31.52 mmol, 1.2 eq), DMAP (320.91 mg, 2.63 mmol, 0.1 eq) and DI PEA (8.49 g, 65.67 mmol, 11.44 mL, 2.5 eq) in DCM (251 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (200 mL) and extracted with ethyl acetate 600 ml (200 mL * 3). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1).Compound 6-(l-heptyloctoxy)-6-oxo-hexanoic acid (8 g, 22.44 mmol, 85.42% yield) was obtained as a colorless oil.

[0408] Step 2:

[0409] To a solution of 6-(l-heptyloctoxy)-6-oxo-hexanoic acid (4 g, 11.22 mmol, 1 eq) in DCM (45 mL) was added EDCI (2.58 g, 13.46 mmol, 1.2 eq), DMAP (137.06 mg, 1.12 mmol, 0.1 eq), l,3-dihydroxypropan-2-one (505.29 mg, 5.61 mmol, 0.5 eq) and DIPEA (3.62 g, 28.05 mmol, 4.89 mL, 2.5 eq). The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate::::100 / 1 to 0 / 1). Compound 01 - [3 -[6-( 1 -heptyloctoxy)-6-oxo-hexanoyl] oxy-2-oxo-propyl] O6-(l -heptyloctyl) hexanedioate (2 g, 2.61 mmol, 23.24% yield) was obtained as a colorless oil.

[0410] Step 3:

[0411] To a mixture of Ol-[3-[6-(l-heptyloctoxy)-6-oxo-hexanoyl] oxy-2-oxo-propyl]O6-(1 -heptyloctyl) hexanedioate (1.5 g, 1.96 mmol, 1 eq) and azetidin-3-ol (171 .51 mg, 2.35mmol, 1.2 eq) in DCM (21 mL) and MeOH (5.25 mL.) was added TEA (237.43 mg, 2.35 mmol, 326.59 pL, 1.2 eq) and NaBH(0Ac)3 (828.84 mg, 3.91 mmol, 2 eq). The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The mixture was diluted with 1120 (100 mL) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=10 / l to 0 / 1). Compound 01 -[3-[6-(l-heptyloctoxy)~6-oxo-hexanoyl] oxy-2-(3- hydroxyazetidin-l-yl) propyl] O6-(l -heptyloctyl) hexanedioate (0.8 g, 970.62 pmol, 49.64% yield) was obtained as a colorless oil.

[0412] Step 4:Compound 23

[0413] To a solution of Ol-[3-[6-(l-heptyloctoxy)-6-oxo-hexanoyl] oxy-2-(3- hydroxyazetidin-l-yl) propyl] O6-(l -heptyloctyl) hexanedioate (0.7 g, 849.29 pmol, 1 eq) in DCM (10 mL) was added EDCI (195.37 mg, 1.02 mmol, 1.2 eq), DMAP (10.38 mg, 84.93 pmol, 0.1 eq), 4-(2-pyrrolidin-l-ylethylcarbamoyloxy) decanoic acid (334.73 mg, 1 .02 mmol, 1.2 eq) and DIPEA (274.41 mg, 2.12 mmol, 369.83 uL, 2.5 eq). The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The mixture was diluted with H2O (50 mL) and extracted with DCM 100 mL (50 mL *2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound 01 -[3-[6-(l -heptyloctoxy)-6-oxo-hexanoyl] oxy-2-[3-[4-(2-pyrrolidin-l-ylethylcarbamoyloxy) decanoyloxy] azetidin-l-yl] propyl] O6-(l -heptyloctyl) hexanedioate (0.15 g, 97.02% purity) wus obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.19 (s, 1H), 5.10 - 5.03 (m, 1H), 4.90 - 4.84 (m, 211). 4.81 - 4.69 (m, 1H), 4.13 - 3.97 (m, 4H), 3.83 - 3.71 (m, 2H), 3.34 ■■ 3.24 (m, 2H), 3.23 ■■ 3.14 (m, 2H) 2.72 ■■ 2.67 (m, 1H), 2.60 (t, >5.4 Hz, 211). 2.54 (s, 4H), 2.44 - 2.27 (m, 10H), 1.95 - 1.87 (m, 1H), 1.84 - 1.74 (m, 5H), 1.69 - 1.64 (m, I OH ). 1.51 (d, >5.6 Hz, 8 H), 1.31 - 1.21 (m, 46H), 0.95 - 0.81 (m, 15H).Example 25 - Synthesis of Compound 24: l-[3-[l-[2"[7"(2-butyloctanoyIoxy) heptanoyloxy]-l~[7"(2~butyloctanoyloxy) heptanoytoxymethyl] ethyl] azetidm-3- yl] oxy-3-oxo-propyI] hep

[0414] Step 1 : ™m26

[0415] To a mixture of benzyl 4-hydroxydecanoate (2.5 g, 8.98 mmol, 1 eq) and 1- methylpiperidine-4-carboxylic acid (2.57 g, 17.95 mmol, 2 eq) in DCM (25 ml) was added EDCI (2.07 g, 10.78 mmol, 1 .2 eq), DMAP (1 10.00 mg, 900.41 pmol, 0.1 eq) and DIEA (2.9 g, 22.44 mmol, 3.91 mL, 2.5 eq) at 25 oC. The mixture was stirred at 25 oC for 12 h under N2 atmosphere. The mixture was quenched by addition H2O (200 mL). It was extracted with ethyl acetate (50 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (ethyl acetate in petroleum ether, from 30% to 55%). Product l-(3-benzyloxy-3-oxo-propyl) heptyl 1 -methylpiperidine ■■ 4-carboxylate (3 g, 7.27 mmol, 80.92% yield, 97.75% purity) was obtained as a yellow oil.

[0416] Step 2:

[0417] To a solution of 1 -(3 -benzyloxy-3 -oxo-propyl) heptyl l-methylpiperidine-4- carboxylate (3 g, 7.43 mmol, 1 eq) in THF (30 mL) was added Pd / C (1.03 g, 967.86 pmol, 10% purity, 0.13 eq) at 25 oC. The suspension was stirred at 25 oC for 12 h under H2 (15 Psi) atmosphere. The suspension was filtered and the filtrate was concentrated to give a residue. The residue was purified by silica gel column chromatography(dichloromethane / methanol, from 10 / 1 to 5 / 1). Product 4-(l-methylpiperidine-4-carbony I) oxydecanoic acid (2 g, 6.10 mmol, 82.09% yield, 95.64% purity) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 8 - 8.14 (s, 1H), 5.00 - 4.86 (m, 1 H), 3.06 (d, J === 11 .2 Hz, 2H), 2.41 (s, 3H), 2.37 - 2.16 (m, 5H), 2.03 - 1.76 (m, 6H), 1.53 (s, 2H), 1.37 - 1.15 (m, 8H), 0.95 - 0.77 (m, 3H).

[0418] Step 3:

[0419] To a mixture of [7-[3-[7-(2- butyloctanoyloxy)heptanoyloxy]-2-(3- hydroxyazetidin-1-yl)propoxy]-7-oxo-heptyl] 2-butyloctanoate (800 mg, 1.04 mmol, 1 eq) and 4-(l -methylpiperidine -4-carbonyl)oxydecanoic acid (490 mg, 1.56 mmol, 1.5 eq) in DCM (8 mL) was added EDCI (240 mg, 1.25 mmol, 1.2 eq), DMAP (13 mg, 106.41 umol, 0.1 eq) and DIEA (337 mg, 2.61 mmol, 454.18 pL, 2.5 eq) at 25 oC. The mixture was stirred at 25 oC for 12 h under N2 atmosphere. The mixture was quenched by addition of H2O (50 mL). It was extracted with ethyl acetate (20 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give a residue. The residue was purified by silica gel column chromatography (ethyl acetate in petroleum ether, from 30% to 80%). Product l-[3-[l-[2-[7-(2-butyloctanoyloxy) heptanoyloxy]-l-[7-(2- butyloctanoyloxy) heptanoyloxymethyl] ethyl] azetidin-3- yl] oxy -3 -oxo-propyl] heptyl 1- methylpiperidine-4-carboxylate (150 mg, 95.65% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.11 - 5.01 (m, 1 H), 4.94 - 4.86 (m, 1H), 4.11 - 4.02 (m, 8 H), 3.78 (t, >7.2 Hz, 2H), 3.24 - 3.15 (m, 2H), 2.83 ( d, >11.0 Hz, 2H), 2.71 (t, >5.0 Hz, 1H), 2.35 - 2.27 (m, 10 H), 2.04 -1.88 (m, 5H), 1.86 - 1.75 (m, 3H), 1.67 - 1.54 (m, 16H), 1.46 - 1.34 (m, 12H), 1.33 - 1.23 (m, 34 H), 0.90 - 0.86 (m, 15 H).Example 26 - Synthesis of Compound 25: l~[3"[l~]2"[7“(2"butyloetenoy!oxy) heptaooyloxy]~l~[7"(2~butyioetanoyioxy) heptanoyi oxymethyl] ethyl] azetidio~3~ yl] oxy-3-oxo-propyl] heptyl l-methyIpyrroUdme-3-carboxylate

[0420] Step 1 : ™

[0421] To a mixture of benzyl 4-hydroxydecanoate (2.5 g, 8.98 mmol, 1 eq) and 1 - methylpyrrolidine-3 -carboxy lie acid (2.32 g, 17.96 mmol, 2 eq) in DCM (25 mL) was added EDCI (2.07 g, 10.78 mmol, 1.2 eq), D1EA (2.90 g, 22.45 mmol, 3.91 mL, 2.5 eq) and DMAP (110.00 mg, 900.41 pmol, 0.1 eq) at 25 oC. The mixture was stirred at 25 oC for 12 h. The mixture was quenched by addition of H2O (50 mL). It was extracted with ethyl acetate (50 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give a residue. The residue was purified by silica gel column chromatography (ethyl acetate in petroleum ether, from 30% to 55%). Product l-(3- benzyloxy-3 -oxo-propyl) heptyl l-methylpyrrolidme-3-carboxylate (3 g, 7.38 mmol, 82.23% yield, 95.88% purity) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 8 = 7.43 ■■ 7.29 (m, 5H), 5.12 (s, 2H), 4.99 - 4.84 (m, 1H), 3.09 - 2.95 (m, 1H), 2.84 (q, J === 8.4 Hz, 1H), 2.71 - 2.53 (m, 2H), 2.48 (q, J - 8.0 Hz, 1H), 2.43 - 2.30 (m, 5H), 2.14 - 2.02 (m, 2H), 2.02 - 1.84 (m, 2H), 1.65 - 1.45 (m, 2H), 1.27 (s, 8H), 0.88 (t, J = 6.8 Hz, 3H).

[0423] To a suspension of Pd / C (1.07 g, 1.00 mmol, 10% purity, 0.13 eq) in THF (30 mL) was added 1 -(3 -benzyloxy-3 -oxo-propyl) heptyl 1 -methylpyrrolidine-3- carboxylate (3 g, 7.38 mmol, 1 eq). The mixture was stirred for 12 h at 25 oC under H2(50 Psi) atmosphere. The suspension was filtered and the filtrate was concentrated to give a residue. The residue was purified by silica gel column chromatography (dichloromethane / methanol= 10 / Ito 5 / T).Product 4-(l-methylpyrroiidine-3-carbonyl) oxydecanoic acid (2 g, 6.45 mmol, 83.71% yield, 96.51% purity) was obtained as a yellow oil.

[0424] Step 3:ompoun

[0425] To a mixture of [7-[3-[7-(2- butyloctanoyloxy)heptanoyloxy]-2-(3- hydroxyazetidin-l -yl)propoxy]-7-oxo-heptyl] 2-butyloctanoate (800 mg, 1.04 mmol, 1 eq) and 4-(l-methylpyrrolidine-3-carbonyl)oxydecanoic acid (468 mg, 1.56 mmol, 1.5 eq) in DCM (8 mL) was added EDC1 (240 mg, 1.25 mmol, 1.2 eq), DMAP (13 mg, 106.41 pmol, 0.1 eq) and DIEA (336 mg, 2.60 mmol, 452.83 pL, 2.5 eq) at 25 oC. The mixture was stirred at 25 oC for 12 h under N2 atmosphere. The mixture was quenched by addition H2O (50 mL). It was extracted with ethyl acetate (20 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (ethyl acetate in petroleum ether, from 30% to 60%). Product 1 -[3-[ l-[2-[7-(2- butyloctanoyloxy) heptanoyloxy]~l"[7-(2-butyioctanoyloxy) heptanoy 1 oxymethyl] ethyl] azetidin-3- yl] oxy-3 -oxo-propyl] heptyl l-methylpyrrolidine-3 -carboxylate (150 nig, 100% purity) was obtained as a colorless oil. IH NMR (400 MHz, CDC13) 8 ppm 5.09 - 5.03 (m, 1H), 4.94 - 4.85 (m, 1H), 4.11 - 4.02 (m, 8H), 3.82 - 3.74 (m, 2H), 3.22 - 3.18 (m, 2H), 3.07 - 2.99 (m, IH), 2.89 - 2.84 (m, 1H), 2.74 - 2.59 (m, 3H), 2.51 (q, J=8.0 Hz, IH), 2.37 (d, J==2.8 Hz, 3 H), 2.36 - 2.30 (m, 7H), 2.14 - 2.05 (m, 2H), 1.97 - 1.89 (m, IH), 1.89 - 1.81 (m, IH), 1.70 (s, IH), 1.68 - 1.54 (m, 14H), 1.47 - 1.22 (m, 46H), 0.92 - 0.85 (m, 15H).

[0426] Example 27 - Synthesis of Compound 26: [7"[3~[7"(2~butyloetan©y§©xy) heptanoyIoxy]-2-[3-[4-[2-(2-pyrrolidiK”l“yIethytearbamoyloxy) ethyl] octanoyloxy] azetidin-l-yl] propoxy] -7-oxo-heptyl] Z-bntyloetanoate

[0427] Step !: SNT138 WT131

[0428] A mixture of 5-propyltetrahydrofuran-2-one (20 g, 156.04 mmol, 1 eq) and NaOH(6.56 g, 163.84 mmol, 1 .05 eq) in H2O (100 mL) was degassed and purged with N2 for 3times. The mixture was stirred at 100 °C for 12 hr under N2 atmosphere. The mixture was concentrated under reduced pressure to give a crude product. Compound 4- hydroxyheptanoyloxysodium (22 g, crude) was obtained as a white solid.

[0429] Step 2:

[0430] A mixture of 4-hydroxyheptanoyloxysodium (22 g, 130.95 mmol, 1 eq) and BnBr (22.40 g, 130.95 mmol, 1.0 eq) in DMSO (220 ml) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 5 min under N2 atmosphere. The mixture was quenched by addition H2O (500 niL). It was extracted with ethyl acetate (200 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (ethyl acetate in petroleum ether, from 0 / 1 to 1 / 10). Compound benzyl 4-hydroxyheptanoate (27 g, 114 mmol, 88% yield) obtained as a colorless oil. 1H NMR (400 MHz, CDCI3) 5 - 7.42 - 7.30 (m, 5H), 5.13 (s, 2H), 3.70 - 3.57 (m, 1H), 2.56 - 2.48 (m, 2H), 1.92 ■ 1.81 (m, 1H), 1.78 ■■ 1.67 (m, 1H), 1.50 1.40 (m, 3H), 1.39 ■■ 1.29 (m, 1H), 0.98 - 0.89 (m, 3H).

[0431] Step

[0432] To a mixture of benzyl 4-hydroxyheptanoate (27 g, 114 mmol, 1 eq) and (4- nitrophenyl) carbonochloridate (45.83 g, 228 mmol, 2 eq) in DCM (270 mL.) was added Py. (18.01 g, 228 mmol, 2 eq) by dropwise at 0 oC. The mixture was allowed to warm to 25 oC and stirred for 1 h under N2 atmosphere. The mixture was diluted with petroleum ether 500 mL and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (ethyl acetate in petroleum ether, from 0 / 1 to 1 / 10). Compound benzyl 4-(4-nitrophenoxy) carbonyloxyheptanoate (16.6 g, 43 mmol, 81.39% yield, 99.95% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 6 - 8.37 - 8.12 (m, 2H), 7.44 - 7.27 (m, 6H), 5.14 (s, 2H), 4.96 - 4.85 (m, 1H), 2.52 (t, J = 7.6 Hz, 2H), 2.17 ■■ 2.07 (m, 1 H), 2.06 ■■ 1.95 (m, 111 ). 1.80 ■■ 1.68 (m, 1 H), 1.67 ■■ 1.58 (m, 1H), 1 .52 - 1.36 (m, 2H), 0.97 (t, J - 7.2 Hz, 3H).

[0433] Step 4:

[0434] A mixture of benzyl 4-(4-nitrophenoxy) carbonyloxyheptanoate (16.6 g, 41.35 mmol, 1 eq), 2-pyrrolidin-l- ylethanamine (9.44 g, 82.71 mmol, 2 eq), DMAP (505.23 mg, 4.14 mmol, 0.1 eq) and DIEA (16.03 g, 124.06 mmol, 21.61 mL, 3 eq) in DCM (150 ml) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The mixture was concentrated under reduced pressure to give a residue. The residue was diluted with water (200 ml.) and extracted with ethyl acetate (500 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (ethyl acetate in petroleum ether, from 1 / 5 to 2 / 1). Compound benzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy) heptanoate (12 g, 31.87 mmol, 77.07% yield) was obtained as a yellow oil. 1H NMR (400 MHz, CDC13) 8 = 7.40 7.31 (m, 5H), 5.12 (s, 2H), 4.79 (s, 1H), 3.35 - 3.20 (m, 2H), 2.58 (t, J = 6.0 Hz, 2H), 2.51 (s, 4H), 2.46 * 2.40 (m, 2H), 2.25 (s, 1H), 2.00 - 1.90 (m, 1 H), 1.89 - 1.81 (m, 1H), 1 .80 - 1.73 (m, 4H), 1.64 - 1.41 (m, 2H), 1.40 - 1.29 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H).

[0435] Step 5:

[0436] To a solution of benzyl 4-(2-pyrrolidm-l -ylethylcarbamoyloxy) heptanoate (12 g, 31.87 mmol, 1 eq) in THF (240 mL) was added Pd / C (3.39 g, 3.19 mmol, 10% purity, 0.1 eq) under N2 atmosphere. The suspension was stirred at 25 °C for 12 hr under H2 (15 Psi) atmosphere. The suspension was filtered and the filtrate was concentrated reduced pressure to give a residue. The residue was purified by silica gel column chromatography (MeOH in DCM, from 0 / T to 1 / 8). Compound 4-(2-pyrrolidin-l-ylethylcarbamoyloxy) heptanoic acid (6 g, 20.95 mmol, 65.74% yield) obtained as a brown oil. 1H NMR (400 MHz, CDC13) 8 = 6.84 (s, 1H), 6.60 (d, J = 4.8 Hz, 1H), 4.82 - 4.67 (m, 1 H), 3.74 - 3.59 (m, 1H), 3.23 - 2.96 (m,6H), 2.94 - 2.86 (m, 1H), 2.37 - 2.19 (m, 2H), 2.07 - 1.93 (m, 5H), 1.85 - 1.72 (m, 1H), 1.66 - 1.54 (m, 1H), 1.52 ■■ 1.41 (m, 1H), 1.40 ■■ 1.29 (m, 2H), 0.90 (t J = 7.2 Hz, 3H).

[0437] Step 6:

[0438] To a mixture of (7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2~(3- hydroxyazetidin-l-yl) propoxy]-7-oxo-heptyl] 2-butyloctanoate (900 mg, 1.17 mmol, 1.0 eq) and 4-(2-pyrrolidin-l -ylethylcarbamoyloxy) heptanoic acid (436 mg, 1.52 mmol, 1.3 eq) in DCM (9 rnL) was added EDCI (269 mg, 1 .40 mmol, 1.2 eq), DMAP (15 mg, 117 pmol, 0.1 eq) and DIPEA (378 mg, 2.93 mmol, 2.5 eq) at 25 °C under N2 atmosphere. The mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with water (50 mL) and extracted with ethyl acetate 100 mL (50 mL * 2). The combined organic layers were dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PE / EtOAc=10 / l to 1 / 4). Compound [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-[3-[4-[2-(2-pyrrolidin- 1 - ylethylcarbamoyloxy) ethyl] octanoyloxy] azetidin-l-yl] propoxy] -7-oxo-heptyl] 2- butyloctanoate (150 mg, 95.99% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 5 ppm 5.16 (s, 1H), 5.06 (m, 1H), 4.78 (s, 1H), 4.11 ■■ 4.02 (m, 8H), 3.83 ■ 3.73 (m, 2H), 3.29 (d, >5.8 Hz, 2H), 3.23 - 3.17 (m, 2H), 2.70 (m, 1H), 2.59 (t, >6.0 Hz, 2H), 2.52 (s, 4H), 2.42 ■■ 2.27 (m, 8H), 1.95 ■■ 1.86 (m, 1H), 1.78 (s, 4H), 1.58 (d, >8.6 Hz, 16H), 1.50 ■■ 1.23 (m, 40H), 0.94 - 0.83 (m, 15H).Example 28 - Synthesis of Composed 27: [7“[3"[7~(2“bntytoctan«)yl«)xy)heptanoyloxy]~2“

[0440] A mixture of benzyl 4-hydroxydecanoate (5 g, 14.37 mmol, 1 eq), 4-pyrro lidin- 1- ylbutanoic acid (3.06 g, 15.81 mmol, 1.1 eq, HC1), EDCI (3.31 g, 17.24 mmol, 1.2 eq), DMAP (175.54 mg, 1.44 mmol, 0.1 eq) and DIPEA (7.43 g, 57.47 mmol, 10.01 mL, 4 eq) in DCM (40 mL) was degassed and purged with N2 for 3 times, and then the reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was added H20 (50 mL) and extracted with ethyl acetate (50 mL "< 3). The combined organic layers were washed with brine (100 mL x 2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:=:100 / 1 to 3 / 1). Compound benzyl 4-(4-pyrrolidin-l- ylbutanoyloxy)decanoate (2.3 g, 5.51 mmol, 38.33% yield) was obtained as yellow oil.lHNMR (400 MHz, CDC13) 8 ppm 7.30 - 7.40 (m, 5 H), 5.12 (s, 2 H), 4.85 - 4.96 (m, 1 H), 2.31 - 2.52 (m, 10 H), 1.73 - 1.96 (m, 8 H), 1.45 - 1.59 (m, 2 H), 1.27 (m, 8 H), 0.88 (m, 3 H).

[0441] Step 2:

[0442] To a suspension of benzyl 4-(4-pyrrolidin-l-ylbutanoyloxy)decanoate (4.2 g, 10.06 mmol, 1 eq) in THF (42 mL) was added Pd / C (1.07 g, 1.01 mmol, 10% purity, 0.1 eq) under Ar atmosphere. The suspension was degassed and purged with H2 for 3 times. The reaction mixture was stirred under H2 (15 Psi) at 25 °C for 12 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was used directly in the next step without further purification. Compound 4-(4-pyrrolidin-l- ylbutanoyloxy)decanoic acid (2.6 g, 7.94 mmol, 78.94% yield) was obtained as yellow oil.1H NMR (400 MHz, CDC13) 8 ppm 4.90 - 5.02 (m, 1 H), 3.01 (s, 6 H), 2.66 - 2.82 (m, 1 H),2.41 ■■ 2.57 (m, 1 H), 2.29 (m, 3 H), 1.97 (m, 8 H), 1.46 ■■ 1.66 (m, 2 H), 1.27 (m, 8 H), 0.87 (t, J - 6. Hz, 3 H).

[0443] Step 3 :

[0444] A mixture of 4-(4-pyrrolidin-1-ylbutanoyloxy)decanoic acid (554.21 mg, 1.69 mmol, 1.3 eq) , [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2-(3-hydroxyazetidin-l- yl)propoxy]-7-oxo-heptyl] 2 -butyloctanoate (1.00 g, 1.30 mmol, 1 eq), EDCI (299.49 mg, 1.56 mmol, 1.2 eq), DMAP (15.90 mg, 130.19 nmol, 0.1 eq) and DIPEA (420.65 mg, 3.25 mmol, 566.91 pL., 2.5 eq) in DCM (10 mL) was degassed and purged with N2 for 3 times, and then the reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was added H2O (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine (10 mL x 2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 1 / 4). Compound [7-[3-[7-(2- buty I octanoyloxy)heptanoyloxy] -2-[3 - [4-(4-pyrrolidin- 1 - ylbutanoyloxy)decanoyloxy]azetidin-l-yl]-propoxy]-7-oxo-heptyl] 2-butyloctanoate (150 mg, 96,32% purity) was obtained as colorless oil. 1 H NMR (400 MHz, CDC13) 8 ppm 5.06 (m, 1 H), 4.86 4.95 (m, 1 H), 4.02 - 4.10 (m, 8 H), 3.78 (m, 2 H), 3.15 ■■ 3.24 (m, 2 H), 2.70 (m, 1 H), 2.45 - 2.58 (m, 6 H), 2.27 - 2.38 (m, 10 H), 1.82 - 1.96 (m, 4 H), 1.77 - 1.82 (m, 4 H), 1.54 - 1.67 (m, 13 H),1.21 - 1.49 (m, 49 H), 0.86 - 0.90 (m, 15 H).Example 29 - Synthesis of Compound 28: [l-[2-[7~(3-butyIsulfauyI-2"methyl~ propauoyl)oxyheptanoyIoxy]~l~[7~(3"bMtj4suIfamd~2~methyI" propauoyl)oxyheptanoyloxymethyI]ethyl]aze-tidiK”3“yI] 4-(2-pyrroIidm-l" ylethyicarbamoyloxy)decauoate

[0445] Step

[0446] A mixture of 7-hydroxyheptanoic acid (20 g, 136.82 mmol, 1 eq), BnBr (23.39 g, 136.81 mmol, 16.24 mL, 1 eq) and K2CO3 (28.36 g, 205.22 mmol, 1.5 eq) in DMF (200 mL) was stirred at 25 °C for 4 h under N2 atmosphere. The reaction mixture was diluted with H2O (1000 mL) and extracted with Ethyl acetate 2000 mL (1000 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 2 / 1). Compound benzyl 7-hydroxyheptanoate (15 g, 63.48 mmol, 46.40% yield, 100% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 7.42 - 7.30 (m, 5H), 5.12 (s, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.37 (t, J - 7.2 Hz, 2H), 1 .73 - 1 .63 (m, 2H), 1.59 - 1.52 (m, 2H), 1 .42 - 1 .32 (m, 4H).

[0447] Step 2:

[0448] To a solution of benzyl 7-hydroxyheptanoate (15 g, 63.48mmol, 1 eq) and TEA (16.05 g, 158.69 mmol, 22.09 ml, 2.5 eq) in DCM (150 mL) was added 2-methylprop-2- enoyl chloride (7.96 g, 76.17 mmol, 7.44 mL, 1.2 eq) dropwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 2 h under N2 atmosphere. The reaction mixture was diluted with H2O (500 mL) and extracted writh DCM 1000 mL (500 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate::::100 / 1 to 10 / 1). Compound benzyl 7- (2-methylprop-2-enoyloxy)hept-anoate ( 15 g, 49.13 mmol, 79% yield, 99.7% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 7.42 - 7.29 (m, 5H), 6.10 (s, 1H), 5.55 (s, 1H), 5.12 (s, 2H), 4.13 (t, J = 6.4 Hz, 2H), 2.37 (t, J = 7.6 Hz, 2H), 1.95 (s, 3H), 1.72 - 1.63 (m, 4H), 1.45 - 1.33 (m, 4H).

[0449] Step 3 :

[0450] To a solution of benzyl 7-(2-methylprop-2-enoyloxy)heptanoate (5 g, 16.43 mmol, 1 eq) and NaOH (65.71 mg, 1.65 mmol, 0.1 eq) in DMSO (50 mL) was added butane- 1 -thiol(1.47 g, 16.39 mmol, 1.75 mL, 1 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 ml. * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound benzyl 7-(3-butydsulfanyl-2-methyl- propanoyl)oxyheptanoate (5.7 g, 14.30 mmol, 87.06% yield, 99% purity) was obtained as a colorless oil. 1H XVI R (400 MHz, CDC13) 8 ppm 7.45 - 7.29 (m, 5H), 5.12 (s, 2H), 4.08 (t, J = 6.4 Hz, 2H), 2.89 - 2.80 (m, 1H), 2.70 - 2.54 (m, 2H), 2.54 - 2.33 (m, 4H), 1.70-1.62 (m, 4H), 1 .59 - 1.51 (m, 2H), 1.46 - 1.34 (m, 6H), 1.25 (d, J == 6.8 Hz, 3H), 0.91 (t, J - 7.2 Hz, 3H).

[0451] Step 4:

[0452] To a solution of benzyl 7-(3-butylsulfanyl-2-methyl-propanoyl)oxyheptanoate (5.7 g, 14.45 mmol, 1 eq) in THE (100 mL) was added Pd / C (3.07 g, 2.89 mmol, 10% purity, 0.2 eq) under Ar atmosphere. The reaction mixture was stirred at 25 °C for 12 h under H2 (30 Psi). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound 7-(3 -butylsulfanyl-2-methyl- propanoyl)-oxyheptanoic acid (4 g, 13.01 mmol, 90.04% yield, 99% purity) was obtained as a colorless oil. 1 H NMR (400 MHz, CDC13) 8 ppm 4.10 (t, J - 6.4 Hz, 2H), 2.92 - 2.45 (rn, 5H), 2.36 (t, J = 7.6 Hz, 2H), 1.70 ■■ 1.61 (m, 4H), 1.60 ■■ 1.52 (m, 2H), 1.45 ■■ 1.34 (m, 6H), 1.25 - 1.25 (m, 111). 1.25 (d, J - 7.2 Hz, 211). 0.91 (t, J - 7.2 Hz, 311).

[0453] Step 5 :INT142 SNTU3

[0454] To a solution of 7-(3-butylsulfanyl-2-methyl-propanoyl)oxyheptanoic acid (4 g, 13.14 mmol, 2.22 eq), l,3-dihydroxypropan-2-one (532.58 mg, 5.91 mmol, 1 eq), DMAP(144.46 mg, 1.18 mmol, 0.4 eq) and EDCI (3.73 g, 19.50 mmol, .3.3 eq) in DCM (80 mL) was added DIEA (5.05 g, 39.02 mmol, 6.80 mL, 6.6 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound [3-[7-(3-butylsulfanyl-2-methyl- propanoyl)oxyheptanoyloxy]-2-oxo-propyl] 7-(3-butylsulfanyl-2-methyl- propanoyl)oxyheptanoate (2.2 g, 2.72 mmol, 46.03% yield, 82% purity) was obtained as a colorless oil. 1 H NMR (400 MHz, CDC13) 8 ppm 4.76 (s, 4H), 4.10 (t, J === 6.8 Hz, 4H), 2.88 - 2.78 (m, 2H), 2.71 ■ 2.61 (m, 2H), 2.61 ■■ 2.38 (m, 10H), 1.71-1.62 (m, 8H), 1.59 - 1.51 (m, 4H), 1.47 - 1.35 (m, 12H), 1.25 (d. J - 7.2Hz. 6H), 0.91 (t, J - 7.2 Hz, 6H).

[0455] Step 6:SNT143 -NTU4

[0456] To a solution of [3-[7-(3-butylsulfanyl-2-methyl-propanoyl)oxyheptanoyloxy]-2- oxo-propyl] 7-(3-butylsulfanyl-2-methyl-propanoyl)oxyheptanoate (2.2 g, 3.32 mmol, 1 eq) in DCM (14 mL) / MeOH (3.5 mL) was added azetidin-3-ol (485.13 mg, 6.64 mmol, 2 eq) and TEA (738.77 mg, 7.30 mmol, 1.02 mL, 2.2 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 0.5 h under N2 atmosphere. Then NaBH(OAc)3 (1.41 g, 6.64 mmol, 2 eq) was added to the above reaction mixture under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was quenched by addition of H2O (100 mL) slowly at 0 °C under N2 atmosphere and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound [3-[7-(3 -butylsulfany 1-2-methyl-propanoy l)oxy heptanoy loxy] -2-(3 -hy droxyazetidin- 1 - ylipropyl] 7-(3-butylsu-lfanyl-2-methyl-propanoyl)oxyheptanoate (1.2 g, 1.50 mmol, 45.20% yield, 90% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 4.53- 4.40 (m, 1 H), 4.18 - 4.03 (m, 8H), 3.83 - 3.64 (m, 2H), 3.18-3.03 (m, 2H), 2.88 - 2.75 (m,2H), 2.71 - 2.47 (m, 9H), 2.33 (t, J - 7.2 Hz, 4H), 1.71 - 1.51 (m, 12H), 1.45 - 1.30 (m, 12H), 1.24 (d, J = 6.8 Hz, 6H), 0.91 (t J = 7.2 Hz, 6H).

[0457] Step 7:Compound 28

[0458] A mixture of [3-[7-(3-butylsulfanyl-2-methyl-propanoyl)oxyheptanoyloxy]-2-(3- hydroxyazetidin- 1 -yl)p-ropyl] 7 -(3 -butylsulfany 1-2-methyl-propanoy l)oxyheptanoate (1.2 g, 1.67 mmol, 1 eq), 4-(2-pyrrolidin-l-ylethyicarbamoyloxy)decanoic acid (1.09 g, 3.33 mmol, 2 eq), EDCI (479.23 mg, 2.50 mmol, 1.5 eq), DIEA (646.19 mg, 5.00 mmol, 870.87 pL, 3 eq) and DMAP (40.72 mg, 333.32 pmol, 0.2 eq) in DCM ( 15 niL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (50 ml) and extracted with DCM 80 ml. (40 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1) to give the compound. Then the compound was filtered with EtOAc(5 ml) and EtOH(5 ml) respectively and the filtra te was concentrated under reduced pressure to give the final product. Compound [ 1 -[2-[7-(3-butylsulfanyl-2-methyl-propanoyl)oxyheptanoyloxy]-l - [7"(3~butyrlsulfanyl~2-methyi-propanoyl)oxyheptanoyloxymethyl]ethyl]aze-tidin”3-yi] 4-(2- pyrrolidin-l-yletbylcarbamoyloxy)decanoate (150 mg, 145.56 pmol, 8.73% yield, 90% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 5 ppm 5.21 (s, 1H), 5.12 - 5.01 (m, i l l). 4.75 (d. J - 1.8 Hz, H i). 4.14 - 4.01 (m, 811 ). 3.80 - 3.7.3 (m, 2H), 3.30 (d. J - 5.4 Hz, 2H), 3.23 ■■ 3.16 (m, 2H), 2.87 ■■ 2.78 (m, 2H), 2.74 ■■ 2.61 (m, 4H), 2.57 ■■ 2.50 (m,8H), 2.42 - 2.28 (m, 6H), 1.90 (d, J === 8.0 Hz, 2H), 1.79 (s, 6H), 1 .69 - 1.49 (m, 14H), 1.46 - 1.34 (m, 12H), 1.31 ■■ 1.23 (m, 16H), 0.94 - 0.85 (m, 9H).Example 30 - Synthesis of Compound 29: [l”[2"[7“(2»methyI”3“pentylsnIfaHyI“ propanoyl)oxyheptanoyioxy] -1 - [7"(2~methyb3"pentyfeulfanyb propanoyl)oxyheptanoyloxy-methyl] ethyl] azetidin-3-y I] 4~(2“pyrrolidm~l~ ylethylcarbamoyloxy)decanoate

[0459] Step !:

[0460] A mixture of benzyl 7-(2-methylprop-2-enoyloxy)heptanoate (5 g, 16.43 mmol, 1 eq), pentane- 1 -thiol (8.56 g, 87.15 mmol, 5 eq) and TEA (9.09 g, 89.81 mmol, 12.50 ml, 5.47 eq) was stirred at 60 °C for 10 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 ml) and extracted with DCM 200 mL (100 ml * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound benzyl 7-(2-methyl-3- pentylsulfanyl-propanoyl)oxyheptano-ate (3.5 g, 7.59 mmol, 46.20% yield, 88.6% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CD3OD) 8 ppm 7.39 ■■ 7.28 (m, 5H), 5.11 (s, 2H), 4.16 - 4.01 (m, 2H), 2.80 - 2.71 (m, 1H), 2.71 - 2.64 (m, 1H), 2.64 - 2.56 (m, 1H), 2.51 (t, J == 7.2 Hz, 2H), 2.37 (t, J - 7.2 Hz, 2H), 1.69 - 1.51 (m, 6H), 1 .44 - 1 .28 (m, 8H), 1.20 (d, J = 6.8 Hz, 3H), 0.98 - 0.84 (m, 3H).

[0461] Step 2:5NT145 INTU6

[0462] To a solution of benzyl 7-(2-methyl-3-pentylsulfanyl-propanoyl)oxyheptanoate (3 g, 7.34 mmol, 1 eq) in THF (60 ml) was added Pd / C (3.91 g, 3.67 mmol, 10% purity, 0.5 eq) under Ar atmosphere. The reaction mixture was stirred at 25 °C for 4 h under H2 (15 Psi). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound 7-(2-methyl-3-pentylsulfanyl-propanoyl)- oxyheptanoic acid (2.0 g, 5.34 mmol, 72.70% yield) was obtained as a colorless oil. 1H NMR(400 MHz, CDC13) 8 ppm 4.10 (t, J - 6.8 Hz, 2H ), 2.87 - 2.78 (m, H I). 2.71 - 2.62 (m, 1H),2.60 ■■ 2.55 (m, 1H), 2.52 (t, J = 7.6 Hz, 2H), 2.37 (t, J = 7.6 Hz, 2H), 1.66 (t, J = 6.4 Hz, 4H),1.61 - 1.54 (m, 2H), 1.42 - 1.31 (m, 8H), 1.25 (d, J - 6.8 Hz, 3H), 0.94 - 0.86 (m, 3H).

[0463] Step 3 :

[0464] A mixture of l,3-dihydroxypropan-2-one (220 mg, 2.44 mmol, 1 eq), 7-(2-methyl- 3-pentylsulfa-nylpropanoyl)oxyheptanoic acid (2.0 g, 5.34 mmol, 2.19 eq), EDO (1.17 g,6.11 mmol, 2.5 eq), DMAP (29.84 mg, 244.23 nmol, 0.1 eq) and DIPEA (1.26 g, 9.77 mmol, 1.70 mL, 4 eq) in DCM (20 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound [3--[7-(2- methy 1-3 -pentyl sulfany I -propanoyl)oxyheptanoyloxy] -2-oxo-propy I ] 7-(2-methyl-3 - pentylsulfanyl-propanoyl)oxyhe-ptanoate (1 g, 1.45 mmol, 59.25% yield, 100% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 4.76 (s, 4H), 4.10 (t, J = 6.8 Hz, 4H), 2.88 ■■ 2.78 (m, 2H), 2.71 - 2.62 (m, 2H), 2.61 - 2.54 (m, 2H), 2.54 ■ 2.49 (m, 4H), 2.44 (t, J = 7.6 Hz, 4H), 1.73 - 1.63 (m, 8H), 1.62 - 1.59 (m, 2H), 1.57 - 1.54 (m, 2H), 1.45 - 1.31 (m, 16H), 1.25 (d, J =;:6.8 Hz, 6H), 0.95 - 0.86 (m, 6H).

[0465] Step 4:

[0466] A mixture of [3-[7-(2-methyl-3-pentylsulfanyl-propanoyl)oxyheptanoyloxy]-2- oxopropyl] 7-(2-methyl-3-pentylsulfanyl-propanoyl)oxyheptanoate (I g, 1 .45 mmol, I eq), azetidin-3-ol (317.09 mg, 2.89 mmol, 2 eq, HO), TEA (322.17 mg, 3.18 mmol, 443.15 pL, 2.2 eq) in DCM (14 mL) and MeOH (3.5 mL) was stirred at 25 °C for 0.5 h under N2 atmosphere. Then NaBH(OAc)3 (613.44 mg, 2.89 mmol, 2 eq) was added to the above reaction mixture and the reaction mixture was stirred at 25 °C for 1 1.5 h under N2atmosphere. The reaction mixture was quenched by slowly addition of H2O (100 mL) at 0 °C under N2 atmosphere and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate::::100 / 1 to 0 / 1). Compound [2-(3-hydroxyazetidin-l-yl)-3-[7-(2- methyl -3 -pentyl sulfanyl -propanoyl)-oxyheptanoyloxy]propyl] 7 -(2-methy 1-3 -pentyl sulfany I- propanoyl)oxyheptanoate (700 mg, 921.69 nmol, 63.69% yield, 98.50% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 4.47 (t, J - 6.0 Hz, 1H), 4.17 - 4.02 (m, 8H), 3.80 - 3.68 (m, 2H), 3.13 - 3.01 (m, 2H), 2.88 - 2.76 (m, 2H), 2.72 - 2.61 (m, 3H), 2.60 - 2.48 (m, 6H), 2.33 (t, J - 7.6 Hz, 4H ), 1 .69 - 1 .63 (m, 8H), 1.60 - 1.54 (m, 4H), 1 .43 - 1.29 (m, 16H), 1.25 (d, J = 6.8 Hz, 6H), 0.97 - 0.86 (m, 6H).

[0467] Step 5:Compound 29

[0468] A mixture of [2-(3-hydroxyazetidin-l-yl)-3-[7-(2-methyl-3-pentylsulfanyl- propanoyl)oxyheptanoyloxy]-propyl] 7-(2-methyl-3-pentylsulfanyl-propanoyl)oxyheptanoate (700 mg, 935.73 pmol, 1 eq), 4-(2~pyrroiidin- 1 - ylethylcarbamoyloxy)decanoic acid (368.80 mg, 1.12 mmol, 1.2 eq), EDCI (269.07 mg, 1.40 mmol, 1.5 eq), DIPEA (302.34 mg, 2.34 mmol, 407.46 pL, 2.5 eq) and DMAP (11.43 mg, 93.57 pmol, 0.1 eq) in DCM (7 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Then it was purified by prep-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H2O (0.1%TFA)-ACN: THF-1 :!]; gradient: 30%-55% B over 14.0 min). Compound [l-[2-[7"(2-methyl-3-pentylsulfanyl- propanoyl)oxyheptanoyloxy]-l-[7-(2-rnethyl-3-pentylsulfanyl-propanoyl)oxyheptanoyloxy- methyl]ethyl]azetidin-3-yl] 4-(2-pyrrolidin-l -ylethylcarbamoyloxy)decanoate (0.15 g, 127.93 pmol, 15.05% yield, 100% purity, TFA salt) was obtained as a colorless oil. IH NMR (400 MHz, CDC13) 8 ppm 12.12 (s, IH), 6.82 - 6.59 (m, IH), 5.43 - 5.18 (m, IH), 4.80 - 4.71 (m, IH), 4.67 (d, J = 9.6 Hz, 2H), 4.46 - 4.39 (m, 2H), 4.35 - 4.15 (m, 4H), 4.12 - 4.07 (m, 4H), 3.91 - 3.82 (m, 2H), 3.60 - 3.50 (m, 2H), 3.27 (s, 2H), 3.18 - 3.01 (m, IH), 2.97 - 2.76 (m, 4H), 2.71 - 2.64 (m, 2H), 2.59 - 2.49 (m, 4H), 2.45 - 2.31 (m, 6H), 2.13 (d, J = 5.0 Hz, 4H), 2.02 - 1.90 (m, I H), 1.84 - 1 .71 (m, 2H), 1.69 - 1.54 (m, 12H), 1 .53 - 1.16 (m, 32H), 1.04 - 0.78 (m, 9H).Example 31 - Synthesis of Compound 30: [l"[2-[7-(3"heptyisulfenyl-2"methyI- propa® oyl)oxyheptanoyloxy] -1 - [7"(3~heptyIsHlfa-i?yl~2~ methyIpropanoyI)oxyheptaHoyioxymethyI] ethyl] azetidin~3~yl] 4~(2~pyrrolidin~l~ yIethylcarbamoyIoxy)decaHoate

[0469] Step !:

[0470] To a solution of benzyl 7-(2-methylprop-2- enoyloxy)heptanoate (5 g, 16.42 mmol, 1 eq), NaOH (131.42 mg, 3.28 mmol, 0.2 eq) in DMSO (50 ml) was added heptane- 1 -thiol (3.04 g, 23.00 mmol, 3.60 mL, 1.4 eq) dropwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with aq. NaHCO3 (100 mL) and extracted with Ethyl acetate 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate- 100 / 1 to 10 / 1). Compound benzyl 7- (3-heptylsulfanyl-2-methyl-propanoyl)oxyheptanoate (6 g, 13.47 mmol, 81.98% yield, 98% purity) was obtained as a colorless oil. I H NMR (400 MHz, CDC13) 8 ppm 7.41 - 7.30 (m, 5H), 5.12 (s, 2H), 4.09 (t, J = 6.8 Hz, 2H), 2.83 (dd, J = 6.8, 12.8 Hz, IH), 2.67 - 2.65 (m, J = 6.8 Hz, H i). 2.60 - 2.48 (m, 3H), 2.37 (t, J - 7.6 Hz, 2H), 1 .71 - 1 .58 (m, 6H), 1.39 - 1.24 (m, 15H), 0.89 (t, J == 6.8 Hz, 3H).

[0471] Step 2:

[0472] To a suspension of Pd / C (7.32 g, 6.87 mmol, 10% purity, 0.5 eq) in THF (120 mL) was added benzyl 7-(3-heptylsulfanyl-2-methyl-propanoyl)oxyheptanoate (6 g, 13.47 mmol, 1 eq) under Ar atmosphere. The reaction mixture was stirred at 25 °C for 10 h under H2 (15 Psi). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 10 / 1). Compound 7-(3-heptylsulfanyl-2-methyl- propanoyl)-oxyheptanoic acid (4 g, 11.42 mmol, 83.08% yield, 98.9% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 4.10 (t, J = 6.8 Hz, 2H), 2.83 (dd, J = 7.2, 12.8 Hz, 1H), 2.66 (dd, J - 6.8 Hz, 1H), 2.60 - 2.47 (m, 3H), 2.37 (t, J - 7.2 Hz, 2H), 1.70 ■■ 1.52 (m, 6H), 1.44 ■■ 1.34 (m, 6H), 1.32 ■■ 1.22 (m, 9H), 0.94 ■■ 0.84 (m, 3H).

[0473] Step 3:

[0474] To a solution of 7-(3-heptylsulfanyl-2-methylpropanoyl)oxyheptanoic acid (4 g, 11 .42 mmol, 1 eq) and 1 ,3-dihydroxypropan-2-one (500 mg, 5.56 mmol, 0.4 eq) in DCM (45 mL) was added EDCI (3.22 g, 17.30 mmol, 1.5 eq), DMAP (144 mg, 1.06 mmol, 0.2 eq) and DIEA (4.47 g, 34.61 mmol, 6.05 mL, 3 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate^ 100 / 1 to 0 / 1). Compound [3-[7-(3-heptylsulfanyl-2-methyl- propanoyl)oxyheptanoyloxy] -2-oxo-propyl] 7 -(3-hepty Isulfa-ny 1-2- methylpropanoyl)oxyheptanoate (3 g, 3.69 mmol, 32.00% yield, 92% purity) was obtained as a colorless oil. 1 H NMR (400 MHz, CDCI3) 5 ppm 4.75 (s, 4H), 4.09 (t, J - 6.8 Hz, 4H), 2.82 (dd, J = 7.2, 12.8 Hz, 2H), 2.70 ■■ 2.60 (m, 2H), 2.59 ■■ 2.47 (m, 6H), 2.43 (t, J = 7.2 Hz,4H), 1.73 - 1.61 (m, 10H), 1.56 (quin, J - 7.6 Hz, 4H), 1.43 - 1.33 (m, 12H), 1.30 - 1.23 (m, 16H), 0.88 (t, J = 6.8 Hz, 6H).

[0475] Step 4:isms'! ssm 52

[0476] A mixture of [3-[7-(3-hepty4sulfanyl-2-methylpropanoyl)oxyheptanoyloxy]-2-oxo- propyl] 7-(3-heptylsulf-anyl-2-methyl-propanoyl)oxyheptanoate (3 g, 4.02 mmol, 1 eq), TEA (892 mg, 8.82 mmol, 1.23 ml, 2.2 eq) and azetidin-3-ol (880 mg, 8.04 mmol, 2 eq, HC1 salt) in DCM (28 mL) and MeOH (7 mL) was stirred at 25 °C for 0.5 h under N2 atmosphere.Then NaBH(OAc)3 (1.70 g, 8.04 mmol, 2 eq) was added to the above reaction mixture and the reaction mixture was stirred at 25 °C for 11.5 h under N2 atmosphere. The reaction mixture was quenched by slowly addition of H2O (100 ml.) at 0 °C under N2 atmosphere and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound [3-[7-(3-heptylsulfanyl-2-methyl- propanoy I ioxyheptanoyloxy] -2-(3 - hyd-roxyazetidin- 1 -y l)propyl]7-(3 -heptylsul fanyl-2- methyl-propanoyl)oxyheptanoate (1.4 mg, 1.50 mmol, 43.28% yield) was obtained as a yellow oil. 1 H NMR (400 MHz, CDCI3) 5 ppm 4.76 (s, 1 H), 4.46 (q, J - 5.6 Hz, 1H), 4.12 - 4.05 (m, 8H), 3.74 (dd, J = 6.4, 8.4 Hz, 2H), 3.11 (dd, J = 6.0, 8.4 Hz, 2H), 2.87 ■■ 2.77 (m, 2H), 2.73 - 2.47 (m, I OH ). 2.33 (t, J 7.2 Hz, 4H), 1.72 - 1.50 (rn, 14H), 1.41 - 1.32 (m, 14H), 1.28 ■■ 1.22 (m, 12H), 0.92 ■■ 0.84 (m, 6H).

[0477] Step 5:

[0478] To a solution of [3-[7-(3-hepty4sulfanyl-2-methylpropanoyl)oxyheptanoyloxy]-2- (3 -hydroxy azetidin- 1 -yl)propyl] 7 -(3 -heptylsulfany l-2-methylpropanoyl)oxyheptanoate (1.2 g, 1 .49 mmol, 1 eq) and 4-(2-pyrrolidin-l-ylethylcarbamoyloxy) decanoic acid (490 mg, 1 .49 mmoi, 1 eq) in DCM (12 mL) was added EDCI (429 mg, 2.25 mmol, 1.5 eq), DMAP (36.46 mg, 298.44 pmol, 0.2 eq) and DIEA (579 mg, 4.47 mmol, 779.73 p.L, 3 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 80 mL. (40 ml. * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1) to give the compound. Then the compound was filtered with EtOAc(5 ml) and EtOH(5 ml) respectively and the filtrate was concentrated under reduced pressure to give the final product. Compound [l-[2- [7- (3 -heptylsulfanyl -2 -methyl-propanoyl)oxyheptanoyloxy]~ 1 ■■ [7 ■ (3 -heptylsuifa-nyi-2- methylpropanoyl)oxyheptanoyloxymethyl]ethyl] azetidin-3-yl] 4-(2-pyrrolidin-l- ylethylcarbamoyloxy)decanoate (160 nig, 131.63 pmol, 15.12% yield, 90.61 % purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.42 - 5.27 (rn, 1H), 5.06 (t, J = 5.8 Hz, 1H), 4.75 (s, 1H), 4.15 ■■ 4.01 (m, 8H), 3.86 - 3.67 (m, 2H), 3.32 (d, J = 5.4 Hz, 2H), 3.24 - 3.15 (m, 2H), 2.88 - 2.80 (m, 2H), 2.73 - 2.47 (m, 14H), 2.38 - 2.32 (m, 4H), 2.06 - 1.86 (m, 4H), 1.81 (s, 4H), 1.67 - 1.54 (m, 12H), 1.43 - 1.20 (m, 40H), 0.91 - 0.85 (m, 9H).Example 32 - Synthesis of Compound 31: [l~[2"[6-(3"heptylsulfanyl-2~methyl- propaswyl)oxyhexanoyfoxy]~l~[6"(3"heptylsulfanyl”2~me" thylpropanoyl)oxyhexanoyloxymethyl] ethyl] azetidiHe-3-yI] 4»(2»pyrroIidiB”l » ylethykarbamoyloxy)decanoate

[0479] Step 1 :INT153 INT154

[0480] A mixture of bromomethylbenzene (12.06 g, 70.49 mmol, 8.37 ml, 1.2 eq) and potassium;6-hydroxyhexanoate (10 g, 58.74 mmol, 1 eq) in DMSO (150 ml) was stirred at25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with Ethyl acetate 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate:::50 / l to 5 / 1). Compound benzyl 6-hydroxyhexanoate (6 g, 26.99 mmol, 45.96% yield) was obtained as a colorless oil.

[0481] Step 2:

[0482] A mixture of 3-heptylsulfanyl-2-methyl-propanoic acid (7.07 g, 32.39 mmol, 1 .2 eq), benzyl 6-hydroxyhexa-noate (6 g, 26.99 mmol, 1 eq), EDCI (6.21 g, 32.39 mmol, 1.2 eq), DIPEA (8.72 g, 67.48 mmol, 11.75 ml, 2.5 eq) and DMAP (329.77 mg, 2.70 mmol, 0.1 eq) in DCM (100 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound benzyl 6-(3- heptylsulfanyl-2-methyl-propanoyl)oxyhexanoate (5 g, 11.83 mmol, 43.83% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 7.45 - 7.31 (m, 5H), 5.12 (s, 2H), 4.09 (t, J = 6.6 Hz, 2H), 2.87 - 2.75 (m, 1H), 2.72 - 2.61 (m, 1H), 2.60 - 2.47 (m, 3H), 2.38 (t, J - 7.4 Hz, 2H), 1.76 - 1.52 (m, 7H), 1.46 - 1 .22 (rn, 13H), 0.92 - 0.85 (m, 3H)

[0483] Step 3 :

[0484] To a solution of benzyl 6-(3-heptylsulfanyl-2-methyl-propanoyl)oxyhexanoate (5 g, 11.83 mmol, 1 eq) in THF (140 mL) was added Pd / C (6.30 g, 5.92 mmol, 10% purity, 0.5 eq) under Ar atmosphere. The reaction mixture was stirred at 25 °C for 12 h under H2 (30 Psi). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=50 / l to 5 / 1). Compound 6-(3 -heptylsulfanyl- 2 -methyl-propanoyl)ox-yhexanoic acid (3 g, 9.02 mmol, 76.26% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 4.11 (t, J = 6.6 Hz, 2H), 2.88 ■■ 2.78 (m, 1H), 2.70 ■■ 2.62(m, 1 H), 2.60 - 2.48 (m, 3H), 2.38 (t, J - 7.4 Hz, 2H), 1.74 - 1.64 (m, 4H), 1 .61 - 1 .51 (m,2H), 1.49 - 1.22 (m, 13H), 0.94 ■■ 0.82 (m, 3H).

[0485] Step 4:

[0486] A mixture of 6-(3-heptylsulfanyl-2miethyl-propanoyl)oxyhexanoic acid (4 g, 12.03 mmol, 1 eq), l,3-dihydroxypropan-2-one (487.64 mg, 5.41 mmol, 0.45 eq), EDCI (3.46 g, 18.05 mmol, 1.5 eq), DIPEA (4.66 g, 36.09 mmol, 6.29 ml, 3 eq) and DMAP (293.94 mg, 2.41 mmol, 0.2 eq) in DCM (50 ml.) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound [3-[6-(3- heptylsulfanyl-2-methyl-propanoyl)oxyhexanoyloxy]-2-oxo-propyl] 6-(3-heptyTlsulfanyl-2- methyl-propanoyl)oxyhexanoate (3 g, 4.17 mmol, 77.07% yield) was obtained as a colorless oil.

[0487] Step 5:

[0488] A mixture of [3-[6-(3-heptylsulfanyl-2-methyl-propanoyl)oxyhexanoyloxy]-2-oxo- propyl] 6-(3-heptylsulfanyl-2-methyl-propanoyl)oxyhexanoate (3 g, 4.17 mmol, 1 eq), azetidin-3-ol;hydrochloride (914.17 mg, 8.34 mmol, 2 eq) and TEA (928.80 mg, 9.18 mmol, 1.28 mL, 2.2 eq) in DCM (42 mL) and MeOH (10.5 mL) was stirred at 25 °C for 0.5 h under N2 atmosphere. Then NaBH(OAc)3 (1.77 g, 8.34 mmol, 2 eq) was added to the above reaction mixture and the reaction mixture was stirred at 25 °C for 11.5 h under N2 atmosphere. The reaction mixture was quenched by slowly addition of H2O (100 mL) at 0 °C under N2 atmosphere and extracted with DCM 200 mL (100 mL. * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reducedpressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyi acetate=100 / T to 0 / 1). Compound [3~[6-(3-heptylsulfanyL2-methyl- propanoyl)oxyhexanoyloxy]-2-(3-hydroxyazetidin-1 -yl)propyl] 6-(3-heptylsulfanyl-2- methyl”prop-anoyl)oxyhexanoate (1.5 g, 1.93 mmol, 30.88% yield) was obtained as a colorless oil.^0489^ Step 6:Compound 31A mixture of 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)decanoic acid (423.18 mg, 1.29 mmol, 1 eq), [3-[6-(3-heptylsulfanyl-2-methyl-propanoyl)oxyhexanoyloxy]-2-(3-hydroxyazetidin-l- yl)propyl] 6-(3-heptylsulfan-yl-2-methyl-propanoyl)oxyhexanoate (1 g, 1.29 mmol, 1 eq), EDO (370.49 mg, 1.93 mmol, 1.5 eq), DIPEA (499.56 mg, 3.87 mmol, 673.26 pL, 3 eq) and DMAP (31.48 mg, 257.69 nmol, 0.2 eq) in DCM (20 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyi acetate=100 / l to 0 / 1) to give the compound. Then the compound was filtered with EtOAc(5 ml) and EtOH(5 ml) respectively and the filtrate was concentrated under reduced pressure to give the final product. Compound [ l-[2-[6-(3-heptylsulfanyl-2-methyl-propanoyl)oxyhexanoyloxy]- 1 -[6- (3-heptylsulfanyl-2-me-thylpropanoyl)oxyhexanoyloxymethyl]ethyl]azetidine-3-yl] 4-(2- pyrrolidin-l-ylethylcarbamoyloxy)decanoate (0.15 g, 138.05 pmol, 2.50% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 8 ppm 5.13 (s, 1 H), 5.10 - 4.98 (m, 1H), 4.74 (s, 1H), 4.14 - 3.99 (m, 8H), 3.82 ■■ 3.71 (m, 2H), 3.35 ■ 3.16 (m, 4H), 2.86 ■■ 2.78(m, 2H), 2.72 - 2.63 (m, 3H), 2.62 - 2.46 (m, 12H), 2.43 - 2.28 (m, 6H), 1 .98 - 1 .72 (m, 9H), 1.70 - 1.61 (m, 8H), 1.60 ■■ 1.52 (m, 5H), 1.44 ■■ 1.23 (m, 34H), 0.95 ■■ 0.83 (m, 9H).Example 33 - Preparation and analysis of Lipid Nanoparticles (LNPs) a. LNP Formulations

[0490] Formulations comprising lipid compounds of the present disclosure were prepared.Two general formulations were used, according to Table 2:Table 2. General Formulations

[0491] LNP Formulations were produced using lipid compounds from Table 1:Compounds 1-7, 10, and 12-18. The formulations produced for each compound are shown inTable 3:Table 3. LNP Formulationsb. LNP Preparation

[0492] Lipids and active ingredients (e.g., RNA, DNA, etc.) were assembled into LNPs using microfluidic mixing. An ethanol phase was prepared by solubilizing ionizable lipids (i.e., lipid compounds), phospholipids (DSPC), cholesterol, and PEG lipids (PEG-DMG) in ethanol, at predetermined mol% ratios given in Table 2. An aqueous phase was prepared by diluting a nucleic acid cargo (e.g., firefly luciferase mRNA) in an acidified buffer (pH 4.0 citrate buffer, 50 mM), A chip with microfluidic mixing architecture (e.g., toroidal mixer) was used to mix the two phases at predetermined flow rate ratios (e.g., 9 mL / min for the aqueous phase and 3 ml / min for the ethanol phase). Resulting LNPs comprising the lipids and encapsulating the nucleic acid cargo were dialyzed against PBS pH 7.4 for 2 hours at room temperature in a 20 kDa MWCO dialysis membrane. Then, PBS was refreshed to continue dialysis overnight at 2-8 °C. Upon completion of the buffer exchange, LNPs were optionally concentrated using an ultracentrifugal unit with a 10-100 kDa MWCO membrane.Example 34 - In vivo Activity is Mice

[0493] Small rodent biodistribution studies were performed in mice (e.g., C57BL / 6J, Balb-c, CD-I , etc.). For evaluating LNP biodistribution and tissue activity upon systemic administration, C57BL / 6J mice received dose administration by single intravenous injection via tail vein with LNP formulations (individual or pooled) in PBS, at different dose (0.5-1 mg / kg) levels of firefly luciferase mRNA. At predetermined time points (4-6 hours postinjection), the animals were anesthetized via isoflurane and optionally subjected to in-life imaging sessions for bioluminescence using an In Vivo Imaging System (IVIS). All animals were dosed with D-Luciferin at 15 mg / mL via subcutaneous (SC) injection at 0.2 mL / animal. Animals had their abdomen hair shaved using an animal trimmer. They were then placed so their shaved belly faced up toward the IVIS camera. Whole body imaging sessions were performed 10-15 minutes following D-Luciferin administration.

[0494] For in detail organ distribution and activity of LNP formulations, ex vivo imaging sessions were also performed. All animals received SC D-Luciferin and were then euthanized by isoflurane overdose, followed by cardiac perfusion with saline. Following perfusion, the organs were collected and subjected to IVIS imaging for luminescence within 10-15 minutes of D-Luciferin injection.

[0495] All IVIS images were processed with a computer software to identify the regions of interests for individual organs to detect the total flux (p / s) values as luminescence quantification. Then the total flux values were graphed to evaluate the in vivo activity in each collected organ.

[0496] Organ mean total flux results are shown in FIG. 1, FIG. 2, FIG. 3, and Table 4. LNPs comprising MC3 (in formulation Fl) were used as a control. LNPs comprising Compound 10 have formulation F 1.Table 4. Organ Mean Total FluxExample 35 - In viva Biodistribution in Nou-Haman Primates

[0497] Non-human primate (NHP) biodistribution studies were performed in cynomolgus monkeys (e.g., Macaca fascicularis), aged 4-6 years. Animals were premedicated with dexamethasone, famotidine and diphenhydramine (at 1 , 0.5 and 5 mg / kg, respectively) -2 and -1 day prior to test material dosing. LNP formulations were prepared with ionizable lipids as described above so that each LNP formulation encapsulates one or more mRNAs with predetermined unique barcode sequences as their cargo. LNPs in PBS, identified by their unique barcoded mRNA cargo, are then pooled together at predetermined ratios. To evaluate LNP biodistribution into tissues upon systemic administration, NHPs received the dose administration by single intravenous infusion via peripheral vein (cephalic, saphenous, or another available) for 30 minutes with LNP dosing material (individual or pooled) in PBS at different total dose (0.5-1 mg / kg) levels.

[0498] At predetermined time points (4-6 hours after pooled test material administration), animals were sacrificed by exsanguination via the femoral artery’ after the intravenous injection of ketamine (10 mg / kg) and Euthasol® (0.25 mL / kg). Then, all animals underwent whole-body perfusion with PBS prior to tissue collection. 20-25 mg samples for biodistribution analysis were collected from all treatment group animals and then placed in sample tubes containing RNALater™ solution. All tissue samples were stored at room temperature for 24h, then the supernatants were removed, and samples were frozen.

[0499] To determine the unique LNPs distributed in the tissues, samples were homogenized by a tissue homogenizer (e.g., TissueLyser). Total RNA from the tissue homogenates were extracted by standard methods. Then, normalized amounts of RNA were sequenced using a targeted RNA sequencing approach with unique molecular identifiers (UMI) to ensure accurate RNA molecule counting. For each tissue, the resulting RNA molecule counts were normalized by pooled test article RNA counts. The results were analyzed and graphed using the internal bioinformatics pipeline.

[0500] Biodistribution results are shown in FIG. 4A, FIG. 4B, FIG. 4C, FIG. 5A, FIG. SB, FIG. 5C, Table 5, and Table 6. LNPs comprising MC3 (in formulation Fl) were used as a control. LNPs comprising Compound 10 have formulation Fl.Table 5. Mean Normalized Unique Molecular Identifier CountsTable 6. Mean Normalized Unique Molecular Identifier Counts

[0501] Further biodistribution results are shown in Table 7 and Table 8. LNPs comprising MC3 (in formulation Fl) and SM102 (in formulation Fl) were used as controls. NA = data not available.Table 7. Mean Normalized Unique Molecular Identifier CountsTable 8. Mean Normalized Unique Molecular Identifier CountsExample 36 - Functional delivery in Nou-Hwmao PrimatesGeneral Protocols

[0502] Non-human primate (NHP) expression studies were performed in cynomolgus monkeys (e.g. Macaca fascicularis), aged 4-6 years. Animals were premedicated intramuscularly (IM) with famotidine (0.5 mg / kg), diphenhydramine (5 mg / kg), and dexamethasone (1 mg / kg) the day before the dosing, and the day of dosing at 0.5 to 1 hour before LNP administration. Individual LNP formulations were prepared using ionizable lipids as described earlier, using GFP mRNA as the reporter protein cargo. To determine the reporter protein expression in the bone marrow and circulating PBMCs, NHPs received the dose administration by single intravenous infusion over 1 hour via a peripheral vein (cephalic, saphenous, or another available), with the individual LNP dosing material in PBS at a dose level of 0.5 mg / kg.PBMC analysis

[0503] At predetermined time points (predose, 6 hours, and 48 hours after LNP administration), the blood samples were collected from an appropriate peripheral vein (not the vein used for dosing) in EDTA treated tubes. Whole blood will be processed at the Testing Facility to PBMCs using SepMate reagents, tubes, and instructions (Stem Cell Technologies). PBMCs will be washed and counted. Following aliquoting the PBMC samples for the mentioned time points, the samples were stained with specific antibodies against the predetermined targets, identifying the cell types of interest. Stained cells were analyzed by a flow cytometer against the target cell type population and GFP expression. Bone marrow aspirate collection

[0504] Bone marrow aspirate collection was performed 12 to 18 hours post-dose administration. Prior to procedures, animals were administered Cerenia (1 .0 mg / kg SC or 2 mg / kg PO) and a single dose of Simbadol (0.50 mg / kg or as recommended, SC). Meloxicam (0.2 mg / kg, SC) was administered, followed by two days of Meloxicam (0.1 mg / kg, PO). As an alternative to Simbadol, buprenorphine may be given (0.03 mg / kg, IM) BID x 2 days. Animals were sedated using a combination of Ketamine (5-10 mg / kg, IM) and Dexmedetomidine (0.01-0.03 mg / kg, IM). Supplemental isoflurane (3-5%, via face mask) or Propofol CRI (0.3-0.6 mg / kg / min) may be used as needed. A non-medicated lubricant was applied to the eyes.

[0505] The animals were maintained on a circulating warm water blanket and / or forced warm air blanket during the procedure. Hair at the site of bone marrow collection was clipped. Any loose hair was removed (by vacuum as appropriate). The sites of collectionwere prepared aseptically utilizing 3 alternating scrubs of' either povidone iodine or chlorhexidine scrub solution and sponges soaked in 70% Isopropyl Alcohol. The animals were moved to the operating table and positioned as appropriate to facilitate the procedures. A local analgesic (i.e., Lidocaine 2.0%, up to 2-4 mg / kg) ~0.2±0.5 mL / SC was administered at the site of collection. A final prep of ChloraPrep™ or appropriate antimicrobial was applied and allowed to dry at the aspiration site. Animals were re -positioned and re-prepped as needed. A small skin incision may be made with a scalpel blade at the selected collection site. The point of needle insertion was just lateral to the ridge of the greater tubercle for the humerus or at the distal or proximal femur if using an alternative site. The bone marrow biopsy needle with the stylet in place was inserted into the incision. Holding the needle firmly, it was driven through soft tissue into the bone. Using a clockwise / counterclockwise motion, the needle was advanced into the medullary cavity to the preset depth stop that comes with the device (approximately 7 / 8thsof an inch). The stylet was removed and an appropriately sized syringe (pre-rinsed with approximately 1 mL. of sodium heparin solution [1000 usp / mL or equivalent]) was attached to the bore of the needle, and bone marrow was aspirated into the syringe. Suction was released as soon as marrow was noted in the hub of the syringe to minimize contamination of the sample with blood. A target volume of 4.0 to 6.0 mL was collected (which may be obtained by using two sites [e.g. right or left humerus and right or left femur]). If no bone marrovr is obtained, the needle may be repositioned for a second attempt. Total volume of bone marrow was recorded. After sample collection, the bone marrow biopsy needle was withdrawn, and the skin incision closed in an appropriate manner (e.g., with tissue adhesive). The bone marrow aspirate sample was divided into aliquots, each placed into K.2EDTA tubes and stored on wet ice.

[0506] The samples were then analyzed by standard flow cytometry for GFP expression. Cells were isolated from whole blood, collected in EDTA-treated tubes, and treated for red blood cell lysis. An entire sample was transferred into a tube containing 1 mL of room temperature Gibco™ ACK Lysing Buffer for every 1 mL of whole blood. The blood and lysing buffer were incubated at room temperature for 3-5 minutes, then the sample was centrifuged at 300 x g for 5 minutes at room temperature. The supernatant was decanted, and the pellet was washed with phosphate buffered saline (PBS) and resuspended in 5 mL of cold PBS to perform a cell count. Yield and dissociated cell viability were recorded. Samples should be kept on ice during the counting step. An aliquot of 1 x 10A7 isolated cells was incubated with Fc block and subsequently stained according to the panel, which includes CD45 and CD14 antibodies. Stained samples were centrifuged at 350 RCF for 5 minutes at4°C and washed with cell staining buffer. Stained cells were centrifuged again at 350 RCF for 5 minutes at 4°C and resuspended in lOOuL of cell staining buffer to be analyzed on a SONY flow cytometer, for panel markers, and GFP expression. Cells were then sorted according to GFP expression, with a GFP+(positive) and GFP-(negative) pool. Once sorted, these cells were then sequenced by standard 10X Genomics single-cell RNA sequencing protocols, including adding pools to lanes of the 10X chip, which partitions cells and converts mRNA into cDNA. cDNA is then amplified with a cell-specific barcode to generate a sequencing library. This library was then sequenced using standard NGS methodology. Samples were then analyzed using Loupe browser and known cell transcripts.

[0507] Results are shown in FIG. 6 and FIG. 7.

Claims

CLAIMSWe claim:A compound of Formula (I),or a pharmaceutically acceptable salt thereof, wherein each of R‘ and R1is independently -(C1-C9 alkylene)-R5; each R5is independently C2-C12 alkyl, C2-C12 alkenyl, -CH(R6)R / , -C(O)O-(CH2)p- CH-(RS)R9, -OC(O)-(CH2)q-CH-(R8)R9, or -OC(O)O-(CH2)P-CH(R8)R9; each R6and R7is independently hydrogen, C7-C12 alkoxy, or C7-C12 alkenoxy, provided that R6and R7are not both hydrogen; each R8and R9is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or Cf-Ci2 alkylsulfide, provided that R8and R9are not both hydrogen and not both C1-C12 alkylsulfide; n is 0-5; p is 0-3; q is 0-3;Xsis O, CH2, NH, N-(CI-C3 alkyl), or X2;wherein each L is independentlycycloalky lene-(CH2)t-, or -(CH2)m-(3-6-membered heterocyclylene)-(CH2)t is hydrogen, C1-C12 alkyl, or C1-C12 alkoxy:R‘- is optionally substituted C1-C12 alkyl, C1-C12 alkoxy, optionally substituted Ca- C12 cycioalkyl, (C1-C4 alkylene)-(optionally substituted C3-C12 cycloalkyl), optionally substituted C5-C6 aryl, or (C1-C4 alkylene)-(optionally substituted C5-C6 aryl); and m and t are each independently selected from 0-4; and wherein-membered to 8-membered heterocyclyl containing one or moreN atoms substituted with C1-C12 alkyl;R;and R4are each independently C1-C6 alkyl; orR3and R4, together with the nitrogen atom to which they are attached, form a 5-6 membered heterocycle comprising one or more N atoms.

2. A compound of Formula (I),(I), or a pharmaceutically acceptable salt thereof, wherein each of R1and Rris independently -(C1-C9 alkylene)-R5; each R is independently C2-C12 alkyl, C2-C12 alkenyl, -CH(R6)R7, -C(O)O-(CH2)p- CH-(R8)R9, -OC(O)-(CH2)q-CH-(Rs)R9, or -OC(O)O-(CH2)p-CH(Rs)R9; each R.° and Rzis independently hydrogen, C7-C12 alkoxy, or C7-C12 alkenoxy, provided that R° and R'7are not both hydrogen; each R8and R9is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 alkylsulfide, provided that Rsand R9are not both hydrogen and not both Ci -Ci 2 alkylsulfide; n is 0-5; p is 0-3; q is 0-3;X!is O, CH2, NH, N-(CI-C3alkyl), or X2;wherein each L is independently -(CH2)m-C(R2)(R2")-(CH2)t-, or ~(Q h)m-(A-Cs cycloalkylene-(CH2)t-, or -{CH2)m-(3-6-membered heterocyclylene)-((Ah):.-;Rzis hydrogen, C1-C12 alkyl, or C1-C12 alkoxy:R2is optionally substituted Ci-C’12 alkyl, C1-C12 alkoxy, optionally substituted C3- Ci2 cycloalkyl, (C1-C4 alkylene)-(optionally substituted C3-C12 cycloalkyl), optionally substituted C5-C6 aryl, or (C1-C4 alkylene)-(optionally substituted C5-C6 aryl); and m and t are each independently selected from 0-4: and whereinR!3IAf ' AY is or a 3-membered to 8-membered heterocyclyl containing one or moreN atoms substituted with C1-C12 alkyl;R3and R4are each independently Ci-Ce alkyl; orR3and R4, together with the nitrogen atom to which they are attached, form a 5-6 membered heterocycle comprising one or more N atoms.The compound of claim 1 or claim 2, whereinL is -(CH2)m-C(R2)(R2''')-(CH2)t- or -(CH2)m-C3-C8 cycloalkylene-fClh h-;Rzis hydrogen or Ch -Ci 2 alkyl;R2" is C1-C12 alkyl; m is 2; t is 0 or 2; n is 2; and4. The compound of claim 3, wherein L is -(CH2)HI-C(R2)(RZ)-(CH2)t-.

5. The compound of claim 3 or 4, wherein R2is hydrogen.

6. The compound of any one of claims 3-5, wherein t is 0.

7. The compound of any one of claims 3-6, wherein R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyL8. The compound of any one of claims 3-7, whereinR‘ is -(C1-C9 alkylene)-R5;R5is -OC(O)-(CH2)q-CH-(R8)R9; and each R8and R9is independently C1-C12 alkyl.

9. The compound of claim 8, whereinR8is w-hexyl; andR9is w-butyl.

10. The compound of any one of claims 3-7, whereinR!is -(C1-C9 alkylene)-R:‘’;R5is -OC(O)-(CH2)q-CH-(R8)R9; and each R8is Cs-Ci? alkylsulfide; and R9is C1-C4 alkyl.

11. The compound of claim 10, whereinR8is C4-C8 alkylsulfide; andR9is C1 -C4 alkyl.

12. The compound of claim 10, whereinR8is C5, C6, or C8 alkylsulfide; andR9is methyl.

13. The compound of any one of claims 3-12, wherein q is 0.

14. The compound of any one of claims 3-13, wherein R1and R1are the same.

15. The compound of any one of claims 3-13, whereineach R° and R'7is independently C7-C12 alkenoxy.

16. The compound of claim 1 or claim 2, whereinX1is O or CH2;R3and R4are each independently C1-C6 alkyl, or R’ and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyL17. The compound of claim 16, wherein n is 2-3.

18. The compound of claim 16, wherein X? is O, n is 2-3, and R~ and RT are each independently C1-C6 aikyi; optionally C1-C2 alkyl.

19. The compound of claim 16, wherein X1is CH2, n is 1-3, and R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyi.

20. The compound of claim 16, whereinR1is -(C1-C9 alkylene)-R5;R5is -C(O)O-(CH2)P-CH-(R8)R9; and each R8and R9is independently C1-C12 alkyl or C2-C12 alkenyl.

21. The compound of claim 20, wherein R8and R9are the same.

22. The compound of claim 20, wherein R8and R9are / / -pentyl.

23. The compound of any one of claims 20-22, wherein p is 2.

24. The compound of claim 20, wherein Rsand R9are n-heptyl.

25. The compound of claim 20, wherein R8and R9are 1 -hepten-7-yl.

26. The compound of claim 24 or claim 25, wherein p is 0.

27. The compound of claim 16, whereinR!is -(C1-C9 alkylene)-R5;R5is -OC(O)-(CH2)irCH-(R8)R9; and each R8is C1-C12 alkylsulfide; and R9is C1-C4 alkyl.

28. The compound of claim 27, whereinR8is C4-C8 alkylsulfide; andR9is C1-C4 alkyl.

29. The compound of claim 28, whereinR8is Cs, C,6, or (5s alkylsulfide; and R9is methyl.

30. The compound of any one of claims 20-29, wherein R1and R1are the same.

31. The compound of any one of claims 20-23, whereinR ' is -(C1-C9 alkylene)-R5;R5is -CH(R6)R7; and each R6and R7is independently C7-C12 alkenoxy.

32. The compound of claim 16, whereinR‘ is -(C1-C9 alkylene)-R3; andR5is C4-C12 dienyl.

33. The compound of claim 32, whereinRris -(C1-C9 alkylene)- R5;R5is -( H(R!')R : and each R6and R7is independently C7-CJ2 alkoxy.

34. The compound of claim 32, wherein R!and R!are the same.

35. The compound of claim 1 , whereinR‘ is -(C1-C9 alkylene)-Ri;R5is -OC(O)-(CH2)q-CH-(R8)R9; each R8and R9is independently C1-C12 alkyl or C1-C12 alkylsulfide, but not both C1-C12 alkylsulfide;L is ~(CH2)fn-C3-C8 cycloalkylene-(CH2)t; m is 0; and t is 0.

36. The compound of claim 35, wherein n is 2;R’ and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl.

37. The compound of claim 35 or claim 36, wherein q is 0.

38. The compound of any one of claims 35-37, whereinR8is w-hexyl; andR9is n-butyl.

39. The compound of any one of claims 35-37, whereinRsis Cs, Ce, or Cs alkylsulfide; andR9is methyl.

40. The compound of any one of claims 35-39, wherein R1and IT are the same.

41. The compound of any one of claims 35-40, wherein L is cyclopentylene or cyclo hexylene.

42. The compound of claim 1 or claim 2, whereinR2is hydrogen;R2" is C1-C12 alkyl; m is 2; t is 0; n is 0: andY is a 3-membered to 8-membered heterocyclyl containing one or more N atoms substituted with C1-C12 alkyl.

43. The compound of claim 42, whereinR’ is -(C1-C9 alkylene)-R5;R5is -OC(O)-(CH2)q"CH-(R8)R9; and each Rsand R9is independently C1-C12 alkyl.

44. The compound of claim 42 or claim 43, whereinR8is w-hexyl; andR9is «-butyl.

45. The compound of any one of claims 42-44, wherein q is 0.

46. The compound of any one of claims 42-45, wherein Rf' and R1are the same.

47. The compound of claim 1 or claim 2, wherein the compound is selected from any one of Compounds 1 -31.

48. The compound of claim 1 or claim 2, wherein the compound is selected from any one of Compounds 1-18, optionally wherein the compound is Compound 1 , 2, 3, 4, 5, 6, 7, 10, 12, 13, 14, 15, 16, 17, or 18.

49. The compound of claim 1 or claim 2, whereinR1is -(C1-C9 alkylene)-R5;R5is -CH( R6)R7, -C(O)O-(CH2)P-CH-(R8)R9, or -OC(O)-(CH2)q-CH-(R8)R9; n is 2 or 3; and50. The compound of claim 49, whereinR5is -CH(R6)R7; andeach R6and Rzis independently C7-C12 alkoxy or C7-C12 alkenoxy.

51. The compound of claim 50, whereinR'!' is -(C1-C9 alkylene)- R5;R5is ■■C(O)O-(CH2)p-CH-(Rs)R9or -OC(O)-(CH2)q-CH-(R8)R9; and each R8and R9is independently Ci-Ci2 alkyl.

52. The compound of claim 50, whereinR!' is -(Ci-C9 alkylene)-R5; andR is C2-C12 alkenyl.

53. The compound of claim 49, whereinR5is -C(O)O-(CH2)P-CH-(R8)R9; and each R8and R9is independently CI-CJ2 alkyl or C2-C12 alkenyl.

54. The compound of claim 49, whereinR is "OC(O)"(CH2)q-CH-(R8)R9; and each R8and R9is independently C1-C12 alkyl.

55. The compound of claim 53 or claim 54, wherein R1’ and R? are the same.

56. The compound of any one of claims 53-55, wherein n is 2.

57. The compound of any one of claims 53-56, wherein R3and R4, together with the nitrogen atom to which they are attached, form a pyrrolidinyl.

58. A lipid nanoparticle comprising the compound of any one of claims 1-57.

59. A lipid nanoparticle comprising the compound of any one of claims 1-57: a phospholipid; a cholesterol; and a polyethylene glycol lipid.

60. A lipid nanoparticle comprising: about 20-80 mol% of the compound of any one of claims 1-57, about 7.5-40 mol% of phospholipid, about 6-50 mol% of cholesterol, and about 1-4 mol% of PEG lipid.

61. The lipid nanoparticle of claim 60, comprising: about 40 mol% of the compound of any one of claims 1-57, about 30 mol% of phospholipid, about 28.5 mol% of cholesterol, and about 1.5 mol% of PEG lipid.

62. A lipid nanoparticle comprising: about 45-50 mol% of the compound of any one of claims 1-57, about 10 mol% of phospholipid, about 38-42 mol% of cholesterol, and about 2-3 mol% of PEG lipid.

63. The lipid nanoparticle of claim 62, comprising: about 47.5 mol% of the compound of any one of claims 1-57, about 40 mol% of cholesterol, and about 2.5 mol% of PEG lipid.

64. A lipid nanoparticle comprising: about 47.5-52.5 mol% of the compound of any one of claims 1--57, about 10 mol% of phospholipid, about 37 -40 mol% of cholesterol, and about 1-2 mol% of PEG lipid.

65. The lipid nanoparticle of claim 64, comprising: about 50 mol% of the compound of any one of claims 1-57, about 38.5 mol% of cholesterol, and about 1.5 mol% of PEG lipid.

66. A lipid nanoparticle comprising: about 57.5-62.5 mol% of the compound of any one of claims 1 -57, about 10 mol% of phospholipid, about 26-29 mol% of cholesterol, and about 2-3 mol% of PEG lipid.

67. The lipid nanoparticle of claim 66, comprising: about 60 mol% of the compound of any one of claims 1-57, about 27.5 mol% of cholesterol, and about 2.5 mol% of PEG lipid.

68. A lipid nanoparticle comprising: about 45-50 mol% of the compound of any one of claims 1-57, about 10 mol% of phospholipid, about 37.5-40.5 mol% of cholesterol, and about 3-4 mol% of P EG lipid.

69. The lipid nanoparticle of claim 68, comprising: about 47.5 mol% of the compound of any one of claims 1 -57, about 39 mol% of cholesterol, and about 3.5 mol% of PEG lipid.

70. The lipid nanoparticle of any one of claims 59-69, further comprising a targeting component.71 . The lipid nanoparticle of claim 70, wherein the targeting component is a targeting lipid.

72. The lipid nanoparticle of claim 70, wherein the targeting component is an active targeting component.

73. The lipid nanoparticle of claim 72, wherein the active targeting component is an antibody, an antigen-binding fragment of an antibody, a protein, a peptide, or a small molecule.

74. The lipid nanoparticle of any one of claims 58-73, further comprising one or more polynucleotides encapsulated within the lipid nanoparticle.

75. The lipid nanoparticle of claim 74, wherein the one or more polynucleotides comprises RNA.

76. The lipid nanoparticle of claim 74, wherein the one or more polynucleotides comprises DNA.

77. The lipid nanoparticle of claim 74, wherein the one or more polynucleotides comprises DNA and R.\ A.

78. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 58-77, and a pharmaceutically acceptable excipient.

79. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 74-77, and a pharmaceutically acceptable excipient.

80. A method of delivering a polynucleotide to an extrahepatic cell or tissue in a subject, comprising administering to the subject an effective amount of the lipid nanoparticle of any one of claims 58-77 or the pharmaceutical composition of claim 78 or claim 79.

81. The method of claim 80, wherein the extrahepatic cell or tissue comprises a brain cell or tissue.

82. The method of claim 80, wherein the extrahepatic cell or tissue comprises a lung cell or tissue.

83. The method of claim 80, wherein the extrahepatic cell or tissue comprises a bone marrow cell or tissue.

84. The method of claim 80, wherein the extrahepatic cell or tissue comprises a spleen cell or tissue.

85. The method of claim 80, wherein the extrahepatic cell or tissue comprises a kidney cell or tissue.

86. The method of claim 80, wherein the extrahepatic cell or tissue comprises a heart cell or tissue.

87. The method of claim 80, wherein the extrahepatic cell or tissue comprises a pancreatic cell or tissue.

88. The method of claim 80, wherein the extrahepatic cell or tissue comprises a muscle cell or tissue.

89. The method of claim 80, wherein the extrahepatic cell or tissue comprises an immune cell or tissue.

90. A method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 78 or claim 79.

91. A method of producing a therapeutic composition, comprising encapsulating an active agent within a lipid nanoparticle, wherein the lipid nanoparticle comprises the compound of any one of claims 1-57.

92. A method of producing a vaccine or prophylactic composition, comprising encapsulating an active agent within a lipid nanoparticle, wherein the lipid nanoparticle comprises the compound of any one of claims 1 -57.

93. The method of claim 91 or claim 92, wherein the active agent comprises DNA.

94. The method of claim 91 or claim 92, wherein the active agent comprises RNA.

95. The method of claim 91 or claim 92, wherein the active agent comprises DNA andRNA.

96. The lipid nanoparticle of any one of claims 58-77 or the pharmaceutical composition of claim 78 or claim 79 for use in delivering an active agent (e.g., one or more polynucleotides) to an extrahepatic cell or tissue in a subject.

97. Use of the lipid nanoparticle of any one of claims 58-77 or the pharmaceutical composition of claim 78 or claim 79 in the manufacture of a medicament for delivering an active agent (e.g., one or more polynucleotides) to an extrahepatic cell or tissue in a subject.

98. The lipid nanoparticle of any one of claims 58-77 or the pharmaceutical composition of claim 78 or claim 79 for use in treating a disease in a subject.

99. Use of the lipid nanoparticle of any one of claims 58-77 or the pharmaceutical composition of claim 78 or claim 79 in the manufacture of a medicament for treating a disease in a subject.

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