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 / 653,037, 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 CR1SPR / 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 agen ts 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 foe present disclosure that may provide, inter alia, improved stability and / or extrahepatic targeting. In certainembodiments, the present disclosure provides a lipid compound of the following formula (Formula (I)):or a pharmaceutically acceptable salt thereof, wherein each of Rland R1' is independently ■■ (C1-C9 aJkylene)-R5; each R2is independently hydrogen or C1-C3 alkyl; each R5is independently - (CH2)p-CH-(R8)R9, -OC(O)- (CH2)q-CH-(R8each R6and R7is independently hydrogen, C7-C12 alkoxy, or Cr-Cn alkenoxy, provided that R6and Rzare not both hydrogen; each R8and R9is independently hydrogen, C1-C42 alkyl, C2-C42 alkenyl, or C1-C12 alkynyl, provided that R8and R9are not both hydrogen; n is 0-5; p is 0-3; q is 0-3; ¥R3or a 3-membered to 8-membered heterocyclyl containing one or more N atoms substituted with C1-C12 alkyl; R3and R4are each independently Ci-Ck alkyl, or R3and R4together with the nitrogen atom to which they are attached form a 5-6 membered heterocycle;wherein eachL is independently -(CH?,)m-C(R2)(R2')-(CH2)t-, --(CHrjm-Cs-Cs cycloalky lene-(CH?,)i-, or - (CH?.)m-(3-6-membered heterocyclylene)-(CH2)t-; R2is hydrogen, Ci-Ci2alkyl, 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 (C1-C4 alkylene)-(optionally substituted Cs-Ce aryl); m is 0-4; and t is 0-4.

[0006] in certain embodiments, R2is hydrogen or methyl; X1is■ p is -(CH2)m-C(R2)(R2")-(CH2)t- or 4CH2)m-C3-C8cycloalkylene-(CH2)t-; R2is hydrogen orC-i-Cn alkyl; R2' is C1-C12 alkyl; m is 2; t is 0 or 2; and n is 2. In certain embodiments, L is - (CH?.)m-C(R2)(R2')-(CH2)t-. In certain embodiments, R2is hydrogen. In certain embodiments, t is 0. In certain embodiments, ¥ is pyrrolidinyl. in certain embodiments, Rlis -(Cj-C'9 alkylene)-R5; R3is -OC(O)-(CH2)q-CH-(R8)R9; and each Rsand R9is independently C1-C12 alkyl. In certain embodiments, R5is w-hexyl; and R9is n-butyl. In certain embodiments, q is 0. In certain embodiments, Rrand R1are the same. In certain embodiments, Rris -(C1-C9 alkylene)-R5; R5is -CH(R6)R7; and each R6and R7is independently C7-C12 alkenoxy. In certain embodiments, Rris -(C1-C9 alkylene)-R3; R5is -CHIR^R7; and each R6and R7is independently C7-C 12 alkoxy.

[0007] In certain embodiments, R2is hydrogen; and X1is O or CH?. In certain embodiments, n is 1-3. In certain embodiments, R1is -(C1-C9 alkylene)-R5; 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, R* and R9are the same. In certain embodiments, R8and R9are n- pentyl. In certain embodiments, p is 2. In certain embodiments, R8and R9are n-heptyl. In certain embodiments, R8and R9are 1 -hepten-7-yl. in certain embodiments, p is 0. in certain embodiments, R1and R1are the same. In certain embodiments, Rris -(C1-C9 alkylene)-R3; R3is -CH(R6)R7; and each R6and R7is independently Cr-Cn alkenoxy . In certainR3I .A R4- 4 embodiments, n is 2 or 3; Y ish; and each R3and R is independently CJ -C3 alkyl, in certain embodiments, Y is dimethylamino, diethylamino, or (methyl)ethylamino. In certain embodiments, n is 1 or 2; and ¥ is pyrrolidinyl or ethyl-substituted piperidinyl.

[0008] In certain embodiments, R1is -(C1-C9 alkylene)-R3; R3is -OC(O)-(CH?.)q-CH-O(R8)R9; each R8and R9is independently C1-C12 alkyl; Xsis;and £ isC3-C8 cycloalkylene, in certain embodiments, n is 2; and Y is pyrrolidinyl. In certain embodiments, q is 0. In certain embodiments, R8is n-hexyl; and R9is w-butyl. In certain embodiments, R1' and R1are the same. L is cyclopentylene or cyclohexylene.

[0009] In certain embodiments,(CHrlt-; R2is hydrogen; R2" is C1-C12 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 C1-C12 alkyl. In certain embodiments, R1is -(C1-C9 alkylene)-R3; 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.

[0010] In certain embodiments, the compound is selected from any one of Compounds 1- 33. In certain embodiments, the compound is selected from any one of Compounds 1-16 and 19-28. In certain embodiments, the compound is Compound 1, 3, 10, 1 1 , 12, 15, 16, 19, 20, 21 , 22, 23, 25, or 28.

[0011] In certain embodiments, R1is -(C1-C9 alkylene)-R3; R3is -C(O)O-(CH?.)p-CH- (R8)R9or -OC(O)-(CH2)q-CH-(Rs)R9; each R8and R9is independently C1-C12 alkyl or C?.-Ci2 o„ / L. U 1Y '0sN ~ alkenyl; n is 1 -3; and R2’ is hydrogen. In certain embodiments, X!is H ;L is -(CH2)m-C(R2)(R2")-(CH2)t-; R2is hydrogen; R2' is C1-C12 alkyl; m is 2; and t is 0. In certain embodiments, Y is pyrrolidinyl. In certain embodiments, R5is -OC(O)-(CH2)q-CH- (R8)R9. In certain embodiments, q is 0; and n is 2. In certain embodiments, R8is n -hexyl; and R9is / ’-butyl. In certain embodiments, R3is -C(O)O-(CH2)p-CH-(R8)R9; and R8and R9are the same. In certain embodiments, R1and R1are the same. In certain embodiments, X1isCH2; Y is pyrrolidinyl; and n is 2. In certain embodiments, X1is O; Y is diethylamino or N- ethylpiperidinyl; and n is 1 or 3.

[0012] 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 20-80 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 the present disclosure, about 30 raol% of phospholipid, about 28.5 mol % of cholesterol, and about 1.5 mol% of PEG lipid.

[0013] 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 nanoparticlecomprises about 47.5 mol% of the compound of fee present disclosure, about 10 mol% of phospholipid, about 40 mol% of cholesterol, and about 2.5 mol% of PEG lipid.

[0014] 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 fee compound of fee present disclosure, about 10 mol% of phospholipid, about 38.5 mol% of cholesterol, and about 1.5 mol% of PEG lipid.

[0015] 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 mol% 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, abou t 10 mol% of phospholipid, about 27.5 mol% of cholesterol, and about 2.5 mol% of PEG lipid.

[0016] 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.

[0017] In certain embodiments, the lipid nanoparticle further comprises a targeting component. In certain embodiments, fee 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.

[0018] 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.

[0019] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle of fee present disclosure, and a pharmaceutically acceptable excipient.

[0020] 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 fee 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, die 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.[002'1] 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.

[0022] 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.

[0023] 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.

[0024] In certain embodiments, the present disclosure provides a lipid nanoparticle or pharmaceutical composition of the present disclosure for use in deli vering an acti ve agent (e.g., one or more polynucleotides) to an extrahepatic cell or tissue in a subject.

[0025] 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 delivering an active agent (e.g., one or more polynucleotides) to an extrahepatic cell or tissue in a subject.

[0026] 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.

[0027] 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

[0028] FIG. I 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 3, or Compound 11.

[0029] 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 or Compound 11.

[0030] 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 1 , Compound 10, or Compound 12.

[0031] FIG. 4 shows mean total flux in several organs from mice, six hours following intravenous administration of LNP formulations comprising MC3, Compound 15, or Compound 16.

[0032] FIG. SA, FIG. SB, and FIG. SC show unique molecular identifier counts in the femur (FIG. SA), spleen (FIG. SB), or muscle (FIG. SC) of cynomolgus monkeys following intravenous administration of LNP formulations comprising MC3, Compound 3, or Compound 11.

[0033] FIG. 6A, FIG. 6B, and FIG. 6C show unique molecular identifier counts in the femur (FIG. 6A), spleen (FIG. 6B), or muscle (FIG. 6C) of cynomolgus monkeys following intravenous administration of LNP formulations comprising MC3, Compound 1, Compound 10, Compound 12, Compound 15, or Compound 16.

[0034] FIG. 7 shows functional deli very of green fluorescent protein (GFP) mRNA to bone marrow aspirate, spleen, and lymph nodes in non-human primates (NHPs) by LNP formulations comprising Compound 11 .DETAILED DESCRIPTIONI. LIPID COMPOUNDS

[0035] 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 tissuesin the subject, when the active agent is encapsulated in an LNP comprising said lipid compound! s).

[0036] In certain embodiments, the present disclosure provides lipid compounds of Formula (I), or a pharmaceutically acceptable salt thereof:wherein each of R1and R1' is independently -(C1-C9 alkylene)-R5; each R2' is independently hydrogen or C1-C3 alkyl; each R5is independently -C(O)O-(CH2)P-CH-(R6)R7, -CH(R6)R7, - C(O)O-(CH2)p-CH-(R8)R9, ■■OC(O)-(CH2)q-CH-(R8)R9, -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, or C1-C12 alkynyl, provided that R8and R9are not bothR13! hydrogen; n is 0-5; p is 0-3; q is 0-3; Y isor a 3 -membered to 8-membered heterocyclyl containing one or more N atoms substituted with Ci-Cn alkyl; R3and R4are each independently Ci-Ce alkyl, or R3and R4together with the nitrogen atom to which they are attached form a 5-6 membered heterocycle; X!is O, CH2, or X2; X2is, and wherein each L is independently -(CH2)m-C(R2)(R2")-(CH2)t-, -(CH?.)m-C3-C8 cycloalkylene-(CH?.)t-, or -(CI-12)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 Cs-Ce aryl, or (C1-C4 alkylene)-(optionally substituted C5-C5 and): m is 0-4; and t is 0-4.O

[0037] In certain embodiments, R2is hydrogen or methyl; X1is; L is ~(CH2)m-C(R2)(R2,)-(CH2)t- or --(CI-Rjm-Cs-Cg cycloalkylene -(CH?.)*.-; R2is hydrogen or C1-C12 alkyl; R2" is C1-C12 alkyl; m is 2; t is 0 or 2; and n is 2.

[0038] In certain embodiments, R2’ is hydrogen. In certain embodiments, R2is methyl.

[0039] In certain embodiments, L is -(CH2)rn-C(R2)(R2')-(CH2)t-.

[0040] In certain embodiments, L is --(CH2)m-Cj-Cs cycloalkylene-iCHr); ■ . In certain embodiments, m and t are 2. In certain embodiments, L. is -(CH2)m-cyclohexylene-(CH2)t-. in certain embodiments, L is ---(CI-RY-cyclohexylene-tCH?.)?.-.

[0041] In certain embodiments, R2is hydrogen.

[0042] In certain embodiments, R2is C1-C12 alkyl. In certain embodiments, R2is C1-C3 alkyl. In certain embodiments, R2is methyl.

[0043] In certain embodiments, t is 0. In certain embodiments, t is 2.R311

[0044] In certain embodiments, Y is, and RJand R , together with the nitrogen atom to which they are attached, form a pyrrolidinyl.

[0045] In certain embodiments, R2’ is C1-C6 alkyl. In certain embodiments, R2" is methyl, ethyl, n-propyl, n-butyl, n-pentyl, or n-hexyl.

[0046] In certain embodiments, R1is -(C1-C9 alkylene)- R3, R5is -OC(O)-(CH2)q-CH- (R8)R9, and each R8and R9is independently C1-C12 alkyl. In certain embodiments, R is -(Ci- C9 alkylene)- R5. In certain embodiments, Rlis -(C4-C7 alkylene)-R3. In certain embodiments, R!is -n-butylene-R5, -n-pentylene-R3, -mhexylene-R3, or -n-heptylene-R5. In certain embodiments, R5is -OC(O)-(CH?.)q-CH-(R8)R9. 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, Rrand R!are the same. In certain embodiments, Rris -(C1-C9 alkylene)- R3, R3is -CH(R6)R7, and each R6and R7is independently C7-C12 alkenoxy. In certain embodiments, each R° and R7is octenoxy. In certain embodiments, R6and R / are crt-5-octen-l-oxy. In certain embodiments, R* is -(C1-C9 alkylene)-R3, R5is -CH(R6)R7, and each R6and R7is independently C7-C12 alkoxy. In certain embodiments, R6and R7are octoxy. In certain embodiments, R1is -(C1-C3 alkylene)-R3. In certain embodiments, R1’ is -ethylene-R3.

[0047] In certain embodiments, R2is hydrogen. In certain embodiments, R2' is hydrogen and X1is O or CH2. In certain embodiments, X1is O. In certain embodiments, X1is CI-I2

[0048] In certain embodiments, n is 1-3. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3.

[0049] In certain embodiments, R1is -(C1-C9 alkylene)- R3, R3is -C(O)O-(CH2)p-CH- (R8)R9, and each R8and R9is independently C1-C12 alkyl. In certain embodiments, R1is •■(<. >■ C7 alkylene)-R3. In certain embodiments, R!is -n-pentylene-R5. In certain embodiments, 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 the same. In certain embodiments, p is 2. In certain embodiments, R1' and R'!are the same.

[0050] In certain embodiments, Rlis -(C1-C9 alkylene)- R5, R3is -C(O)O-(CH2)p-CH- (R8)R9, and each R8and R9is independently 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 C1-C12 alkyl. In certain embodiments, each Rsand R9is independently C3-C7 alkyl. In certain embodiments, R8and R9are n-heptyl. In certain embodiments, each R8and R9is independently C2-C12 alkenyl. In certain embodiments, each R8and R9is independently C3-C7 alkenyl. In certain embodiments, R8and R9are l-hepten-7- yl. In certain embodiments, R5and R9are the same. In certain embodiments, p is 0. In certain embodiments, Rrand R1are the same.

[0051] In certain embodiments, Rris -(C1-C9 alkylene)-R3, R5is -CH(R9)R7, and each R6and R' is independently C7-C12 alkenoxy. In certain embodiments, Rris -(C1-C3 alkylene)- R5. In certain embodiments, R1' is -ethylene-R5. In certain embodiments, each R6and R' is octenoxy. In certain embodiments, R6and R7are cR-5-octen-l-oxy.R3I

[0052] In certain embodiments, n is 2 or 3, Y is, and each R3and R4is independently C1-C3 alkyl.R3i

[0053] In certain embodiments, n is 2, Y is, and each R3and R4is independently C1-C3 alkyl. In certain embodiments, X1is CH2. In certain embodiments, Y is dimethylamino, diethylamino, or (methyl)ethylarnino.

[0054] In certain embodiments, n is 3, Y is, and each R3and R4is independently C1-C3 alkyl. In certain embodiments, X1is O. In certain embodiments, Y is diethylamino.

[0055] In certain embodiments, n is 1 or 2 and Y is pyrrolidinyl or ethyl-substituted piperidinyl.

[0056] In certain embodiments, n is 1 and Y is ethyl-substituted piperidinyl. In certain embodiments, X1is O.

[0057] In certain embodiments, n is 2 and Y is pyrrolidinyl. In certain embodiments, X1is CH2.

[0058] In certain embodiments, R1is -(C1-C9 alkylene)-R3; R5is -OC(O)-(CH2)q-CH-(R8)R9; each R8and R9is independently CJ-C12 alkyl: R1is same as Rl; X1iscycloalkylene. In certain embodiments, R’ is -(C3-C7 alkylene)-R5. In certain embodiments, R1is -n-hexylene-R5. In certain embodiments, n is 2 and Y is 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, R1' and R!are the same. In certain embodiments, L is cyclopentylene or cyclohexylene. In certain embodiments, L. is cyclopentylene. In certain embodiments, L is cyclohexylene.(CH2)t-; R2is hydrogen; R2" is C1-C12 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 C1-C12 alkyl. In certain embodiments, R2is C3-C7 alkyl. In certahi embodiments, R2" is n -hexyl. In certain embodiments, Y is a 5-membered to 6-membered heterocyclyl containing one or more N atoms substituted with C1-C12 alkyl. In certain embodiments, Y is a piperidinyl or pyrrolidinyl, wherein die N atom is substituted with C1-C12 alkyl. In certahi embodiments, Y is a piperidinyl or pyrrolidinyl, wherein the N atom is substituted with C1-C3 alkyl. In certainembodiments, Y is a piperidinyl or pyrrolidinyl, wherein the N atom is substituted with methyl. In certain embodiments, Rz' is hydrogen.

[0060] In certain embodiments, Rlis -(C1-C9 alkylene)-R3, R5is -OC(O)-(CH2)q-CH- (RS)R9, and each R8and R9is independently C1-C12 alkyl. In certain embodiments, R1is -(C3- C7 alkylene)-R5. In certain embodiments, R1is -n-hexylene-R5. In certain embodiments, each R8and R9is independently C3-C7 alkyl. In certain embodiments, R8is / ?-hexyl and R9is n- butyl. In certain embodiments, q is 0. In certain embodiments, Rrand Rlare the same.

[0061] in certain embodiments, R1is -(C1-C9 alkylene)-R5; R5is -C(O)O-(CH2)p-CH- (R8)R9or -OC(O)-(CH2)q-CH-(R8)R9; each R8and R9is independently C1-C12 alkyl or C2-C12 alkenyl: n is 1-3; and R2' is hydrogen.

[0062] In certain embodiments, R* is -(C1-C9 alkylene)-R3; R5is -C(O)O-(CH2)P-CH- (R8)R9; each R® and R9is independently C1-C12 alkyl or C2-C12 alkenyl; n is 1-3; and R2is hydrogen. In certain embodiments, R1is -(C3-C7 alkylene) -R3. In certain embodiments, R1is -n-pentylene-R5. In certain embodiments, each R8and R9is independently C1-C12 alkyl. In certain embodiments, each Rsand R9is independently C3-C7 alkyl. In certain embodiments, R8and R9are the same. In certain embodiments, R8and R9are n-heptyl. In certain embodiments, R8and R9are n -pentyl. In certain embodiments, each R8and R9is independently C2-C12 alkenyl. In certain embodiments, each R8and R9is independently C3- C? alkenyl. In certain embodiments, R8and R9are l-hepten-7-yl. In certain embodiments, R1’ and R!are the same. In certain embodiments, X1is CH2, Y is pyrrolidinyl, and n is 2. In certain embodiments, X1is O, Y is diethylamino or .V-ethylpiperidinyl, and n is 1 or 3. In certain embodiments, X1is O, Y is diethylamino, and n is 3. In certain embodiments, X1is O, Y is A'-ethylpiperidinyl, and n is 1.

[0063] In certain embodiments, Rlis -(C1-C9 alkylene)-R3; R5is -OC(O)-(CH2)q-CH- (RS)R9; each Rsand R9is independently C1-C12 alkyl; n is 2; and R2is hydrogen. In certain embodiments, R1is -(C3-C7 alkylene)-R5. In certain embodiments, R!is -n-hexylene-R5. In certain embodiments, each R8and R9is independently C3-C7 alkyl. In certain embodiments,R8is w-hexyl and R9is / ?-butyl. In certain embodiments,(CH2)m-C(R2)(R2')-(CH2)t-: R2is hydrogen: R2' is C1-C12 alkyl; m is 2; and t is 0. In certain embodiments, R2’ is C3-C7 alkyl. In certain embodiments, R2" is n-hexyl. In certain embodiments, Y is pyrrolidinyl. In certain embodiments, R1' and R1are the same. In certainembodiments, Rris -(C1-C9 alky lene)-R3; R5is -CH(R6)R7; and each R6and R7is independently C7-C12 alkoxy or C7-C12 alkenoxy. In certain embodiments, R6and R7are octoxy. In certain embodiments, R6and R' are octoxy. In certain embodiments, each R6and R' is octenoxy. In certain embodiments, R° and R7are cfs-5-octen-l-oxy. In certain embodiments, q is 0.

[0064] In certain embodiments, Xsis X2and m and t are 2 in L of X2.

[0065] In certain embodiments, one of R6and R' is hydrogen. In certain embodiments, R6is hydrogen. In certain embodiments, R' is hydrogen.

[0066] 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 Compound

[0067] In certain embodiments, the lipid compound is selected from any one ofCompounds 1-32 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is selected from any one of Compounds 1-33 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is selected from any one of Compounds 1-16 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is selected from any one of Compounds 19-28 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 1, 3, 10, 11, 12, 15, 16, 19, 20, 21, 22, 23, 25, or 28 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 1 , 3, 10, 1 1, 12, 15, or 16 from Table 1 , or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound isCompound 19, 20, 21, 22, 23, 25, or 28 from Table I, or a pharmaceutically acceptable salt thereof.

[0068] In certain embodiments, the lipid compound is Compound 1, 2, 3, 4, 11, 17, 18, 19, 20, 21, 24, 25, 26, 27, 28, 29, or 32 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 3 or 4 from Table 1, or a pharmaceutically acceptable salt thereof.

[0069] In certain embodiments, the lipid compound is Compound 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or 16 from Table 1, or a pharmaceutically acceptable salt thereof. in certain embodiments, the lipid compound is Compound 5, 6, 7, 8, 9, 10, 12, 13, or 14 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 5, 6, 7, 8, 9, or 10 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 6, 7, 8, or 9 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 5 or 10 from Table 1, or a pharmaceutically acceptable salt thereof.

[0070] In certain embodiments, the lipid compound is Compound 1, 3, or 11 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 10, 12, 15, or 16 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 10 or 12 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 15 or 16 from Table 1, or a pharmaceutically acceptable salt thereof.

[0071] In certain embodiments, the lipid compound is Compound 22 or 23 from Table 1, or a pharmaceutically acceptable salt thereof.

[0072] In certain embodiments, the lipid compound is Compound 30 or 31 from Table 1, or a pharmaceutically acceptable salt thereof.

[0073] 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 3 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 11 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 15 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 16 from Table I or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 19 from Table 1 or a pharmaceuticallyacceptable salt thereof. In certain embodiments, the lipid compound is Compound 20 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 21 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 22 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 23 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 25 from Table 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound is Compound 28 from Table I or a pharmaceutically acceptable salt thereof.

[0074] 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.10075 ] 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 moiety with 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.IL LIPID COMPOSITIONSLipid Nanoparticles (LNP)

[0076] 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).

[0077] 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)); (ill) 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.

[0078] In certain embodiments, the lipid composition (e.g., LNP) comprises any one of Compounds 1 -16 from Table 1 , or a pharmaceutically acceptable salt thereof. In certain embodimen ts, the lipid composition (e.g., LNP) comprises any one of Compounds 19-28 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises any one of Compounds 1-32 from Table 1, or a pharmaceutically acceptable salt thereof, in certain embodiments, the lipid composition (e.g., LNP) comprises any one of Compounds 1 -33 from Table 1 , or a pharmaceutically acceptable salt thereof.

[0079] 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 3 from Table 1, 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 11 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 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 19 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 20 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 21 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 22 from Table 1 , or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 23 from Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 25 from Table 1 , or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition (e.g., LNP) comprises Compound 28 from Table 1, or a pharmaceutically acceptable salt thereof.

[0080] Phospholipids for use in a lipid composition (e.g., LNP) of the present disclosure can be neutral, uncharged, or zwitterionic phosph olip ids. Examples of phospholipids for use in a lipid composition include, but are not limited to: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn- glycero-3 -phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dirnyristoylphosphatidylcholine (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 phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In certain embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC).

[0081] 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.

[0082] 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 di acylglycerol or diacylglycamide. In certain embodiments, the lipid moiety of the PEG lipid is derived from a dialkylglycerol or dialkylglycamide group. In certain embodiments, the dialkylglycerol 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 dialky lglycerol or dialkylglycamide group comprises one or more substituted alkyl groups.

[0083] 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 1,000 to about 2,000 Da. In certain embodiments, the PEG moiety comprises PEG2000 (having2,000 Da).

[0084] 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 compri ses about 20-80 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipidcomposition (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 moi% 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 (1) 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 (1) or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid composition comprises about 62.5-67.5 mol% (e.g., about 65 mol%) of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0085] 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)).

[0086] 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 certainembodiments, 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).

[0087] 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 glycol-2000 (DMG-PEG 2000, also referred to herein as PEG-DMG).

[0088] 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 embodimen ts, 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).

[0089] 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).

[0090] 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., di stearoylphosphatidylcholine (DSPC)); (hi) 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 rnol% 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).

[0091] 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 40 mol% of a lip id compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (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 (1) or a pharmaceutically acceptable salt thereof;(ii) about 20 mol% of a phospholipid (e.g., di stearoylphosphatidylcholine (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 raol% 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 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 mol% of a PEG lipid (e.g., PEG-DMG).

[0092] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about47.5-52.5 mol% of a lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., di stearoylphosphatidylcholine (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 rnol% of a helper lipid (e.g., cholesterol); and (iv) about 2.5 rnol% of a PEG lipid (e.g., PEG-DMG).

[0093] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about47.5-52.5 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). 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).

[0094] 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 PEGlipid (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 (I) 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).

[0095] 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., di stearoylphosphatidylcholine (DSPC)); (iii) about 37.5-40.5 mol% of a helper lipid (e.g., cholesterol); and (iv) about 3-4 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 (1) or a pharmaceutically acceptable salt thereof; (ii) about 10 mol% of a phospholipid (e.g., distearoylphosphatidylcholine (DSPC)); (iii) about 39 mol% of a helper lipid (e.g., cholesterol); and (iv) about 3.5 mol% of a PEG lipid (e.g., PEG-DMG).

[0096] 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 raol% 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 lip id 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).

[0097] In certain embodiments, the lipid composition (e.g., LNP) comprises: (i) about 20- 35 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 25-38 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) about20 mol% of a lipid compound of Formula (1) or a pharmaceutically acceptable salt thereof; (ii) about 40 mol% of a phospholipid (e.g., di stearoylphosphatidylcholine (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).

[0098] In certain embodiments, the lipid compound of the lipid composition is any one of Compounds 1-33 of Table 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the lipid compound of the lipid composition is any one of Compounds 1-32 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, 3, 10-12, 15, 16, 19-23, 25, and 28 of Table 1, or a pharmaceutically acceptable salt thereof.

[0099] 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 A’-[I -(2,3-dioleyloxy)propyl]-ALV,A’- 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).

[0100] in certain embodiments, the targeting component of a lipid composition (e.g., a lipid nanoparticle (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. Incertain embodiments, the active targeting component comprises a peptide. In certain embodiments, the active targeting component comprises a small molecule.Active Agents

[0101] 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 nanoparticle (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.

[0102] 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.

[0103] 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).

[0104] In certain embodiments, the active agent comprises gRNA. In certain embodiments, the active agent comprises dgRNA or sgRNA.

[0105] 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

[0106] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid composition (e.g., a lipid nanoparticle (LNP)) of the present disclosure. In certain embodiments, the pharmaceutical composition comprises a lipid composition (e.g., a lipid nanoparticle (LNP)) of the presen t 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.

[0107] In certain embodiments, the present disclosure provides a method of deli vering 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 methodcomprises administering to a subject an effective amount of a pharmaceutical composition comprising a lipid composition (e.g., LNP) of the present disclosure.

[0108] 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.

[0109] in certain embodiments, the present disclosure provides 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 1-33 from 'Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid composi tion (e.g., LNP) comprises any one of Compounds 1-32 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid compos ition (e.g., LNP) comprises any one of Compounds 1-16 from Table i, 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 19-28 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 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 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) comprises Compound 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 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) 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) comprises Compound 16 from Table I, 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 19 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 20 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 21 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 2.2 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 23 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 25 from Table I, 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 28 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA).

[0110] In certain embodimen ts, the presen t 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., DN A, RN A) within a lipid composition (e.g., LNP) of the present disclosure.

[0111] In certain embodiments, the lipid composition (e.g., LNP) of the therapeutic composition, prophylactic composi tion, 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 tire therapeutic composition, prophylactic composition, or vaccine comprises any one of Compounds 1-33 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 1-32 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 -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 any one of Compounds 19-28 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 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 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 i 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) 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 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 19 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 20 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 21 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 22 from Table 1, or a pharmaceutically acceptable salt thereof, and encapsulates an active agent (e.g., DNA, RNA). In certain embodiments, the lipid compos ition (e.g., LNP) of the therapeutic composition, prophylactic composition, or vaccine comprises Compound 23 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 25 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 28 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) witha solutiom'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.

[0112] 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

[0113] 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.

[0134] Administering: As used herein, the term "administering" refers to providing a composition to a subject.

[0115] 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.

[0116] 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.

[0117] Alkenylene: As used herein, the term "alkenylene" refers to a divalent alkenyl group. Examples of alkenylene groups include, but are not limited to, ethenylene, propenylene, butenylene, pentenylene, hexenylene, and the like.

[0118] 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.

[0119] 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 beoptionally 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, isopentyl, neopentyl), and the like.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] Approximately / About: As used herein, the terms "approximately" and "about" are used interchangeably and refer to a value that is wi thin + / - 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%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1%, or less of the stated reference value, unless otherwise expressly stated or otherwise clearly evident from the context.

[0124] 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 and groups include, but are not limited to, phenyl and naphthyl.

[0125] Arylene: 'The term "arylene" is a multivalent (e.g., divalent or trivalent) aryl group.

[0126] 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, n-pentyl, n-butyl, n-pentyl, etc.). The number of ring atoms in the cycloalkyl ring can be specified using "Cx-Cy cycloalkyl" 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., fusedbicyclic 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).

[0127] 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.

[0128] 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.

[0129] 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 by standard clinical techniques by those with skill in the art (e.g., extrapolated from dose - response curves derived from testing).

[0130] Halo: As used herein, the term "halo" refers to fluoro, chloro, bromo, and / or iodo.

[0131] 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- ymembered" 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.

[0132] 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, morpholmylene, and the like.

[0133] Hydroxy alkyl: As used herein, the term "hydroxyalkyl" refers to an R-C1-20-OH group. Examples of such groups include, but are not limited to, hydroxymethyl, 2- hydroxy ethyl, 3 -hydroxypropyl, and the like.

[0134] 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 muitilamellar). In other embodiments, lipid nanoparticles are nanospherical vesicles that comprise liposomal lamellar phase lipid bilayers (unilamellar or muitilamellar).

[0135] 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.

[0136] 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.

[0137] Pharmaceutically acceptable excipient: 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.

[0138] 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.

[0139] 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).

[0140] 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.

[0141] Subject: As used herein, the term "subject” refers to any organism to which 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.

[0142] 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 di ffer 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.

[0143] Treating1. 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

[0144] 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. Byway 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.

[0145] 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.

[0146] 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.

[0147] 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 die 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.”

[0148] 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.

[0149] Any- embodiment of the presen t 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.

[0150] 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.

[0151] Section headings, materials, methods, and examples are illustrative only and not intended to be limiting.EXAMPLESExample 1 - General Materials and Methods for Syntheses

[0152] 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 SiOr stationary phase columns with eluent flow-rate ranges of 15 to 200 mL / min, UV detection (254 and 280 ran). Reverse phase purification was carried out using C18 columns, UV detection (214 and 254 nm). The chemical shifts are reported in parts-per- million and are referenced to solvent peaks, which in!H NMR appear at 7.26 ppm for CDCh, 2.50 for DMSO-mj, and 3.31 ppm for CD3OD.

[0153] Terms and abbreviations:4-PPY 4-pyrrolidin- 1 -ylpyridine;Ac acetyl; aq aqueous;Bn benzyl;DB U 1,8 -diazabicyclo [5.4.0] undec- 7 -ene ;DCM dich loro methan e ;DIPEA 7V, / V-diisopropylethy lamin e;DMAP 4-dimethylaminopyridine;EDC1 l-etliyl-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); NaHCOj sodium bicarbonate; Nar.SCU sodium sulfate; PPTS pyridinium p-toluenesulfbnate;Py pyridine;TEA triethylamine;TEMPO 1 -oxidany 1 -2,2,6,6-tetramethyl-piperidme;Tol toluene;NHiCl ammonium chloride ;NMR nuclear magnetic resonance; p-TsOH-HrO para-toluenesulfonic acid monohydrate; sat. saturated;TBS tert-b uty Idimethy 1 si ly 1 ;THF tetrahydrofuran .Example 2 - Synthesis of Compound 1: [7-[3-[4,4"bis[(Z)-oct-5-enoxy]butanoyloxy]-2- [4“(2-pyrrolidiii“l- ylethyfcarbamoyloxy)decanoylamino]propoxy]-7-oxo-heptyl] 2- butyloctanoate

[0154] Step 1:

[0155] 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. The mixture was stirred at 100 °C for 12 hr under N2. 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.

[0156] Step 2: HNT2

[0157] To a solution of 4-hydroxydecanoyloxysodium (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.The mixture was stirred at 25 °C for 5 min under Nc. The reaction mixture was diluted with sat. NaCl (100 mL) and extracted with EtOAc (200 mb) (100 ml, * 2). The combined organic layers were dried over NarSCM, filtered and the fillrate was concentrated under reduced pressure to give a residue. Compound benzyl 4 ■■hydroxy decanoate (12 g, crude) was obtained as a pale yellow oil and it was used in the next step quickly.

[0158] Step 3:

[0159] To a solution of benzyl 4-hydroxydecanoate (12 g, 43.11 mmol, 1 eq) in DCM (120 mL) 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. The mixture was stirred at 25 °C for 1 hr under Nr. 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 (SiOr, Petroleum ether / Ethyl acetate=100 / T to 10 / 1). Compound benzyl 4-(4~nitrophenoxy)carbonyloxydecanoate (10 g, 22.55 mmol, 52.31% yield) was obtained as a colorless oil.lH NMR (400 MHz, CDCb) S ppm 8.30-8.25 (m, 2 H) 7.37-7.27 (m, 7 H) 5.14 (s, 2 H) 4.91-4,85 (m, 1 H) 2.53-2.50 (t, 1=7.6 Hz, 2 H) 2.16-2.07 (m, 1 H) 2.05-1.97 (m, 1 H) 1.77-1.72 (m, 1 H) 1.67-1.60 (m, 1 H) 1.43-1.30 (m, 8 H) 0.91-0.88 (t, J 6.0 Hz, 3 H).

[0161] 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). The mixture was stirred at 2.5 °C for 12hr under N2. 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 (SiOz, Petroleum ether / Ethyl acetate=50 / l to 0 / 1). Compound benzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy) decanoate (7 g, 16.72 mmol, 74.17% yield) was obtained as a colorless oil.

[0162] Step 5:

[0163] To a suspension of Pd / C (2 g, 1.88 mmol, 10% purity, 0.112 eq) in THF (140 mL) was added benzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)decanoate (7 g, 16.72 mmol, 1 eq). The 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 (SiO2, DCM / MeOH=50 / T 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, CDCb) 8 ppm 10.23 (s, 1 H) 6.27 (s, 1 H) 4.77-4.71 (m, 1 H) 3.68-3.60 (m, 1 H) 3.19-3.14 (m, 1 H) 3.08-2.95 (m, 5 H) 2.82- 2.77 (m, 1 H) 2.37-2.21 (m, 2 H) 2.06-2.00 (m, 1 H) 1.96 (s, 4 H) 1.84-1.74 (m, 1 H) 1.64- 1.58 (m, 1 H) 1.52-1.45 (m, 1 H) 1.30-1.26 (m, 8 H) 0.88-0.85 (t, J - 6.8 Hz, 3 H).

[0164] Step 6:SNT7 INT8

[0165] To a solution of heptane- 1,7-diol (98.99 g, 748.81 mmol, 3 eq) in DCM (980 raL) 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 hh 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 NarSOr, 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=l / O to 10 / 1).Compound 7-hydroxyheptyl 2 -butyl octanoate (30 g, 95.39 mmol, 38.22% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 5 ppm 4.08 (t, J ~ 6.6 Hz, 2H), 3.72 - 3.57 (rn, 2H), 2.37 ■■ 2.16 (m, 1H), 1.71 ■■ 1.50 (m, 6H), 1.49 ■■ 1.34 (m, 8H), 1.32 - 1.16 (m, 12.H), 0.95 - 0.81 (m, 6H).

[0166] Step 7:ShSTS ^TS

[0167] To a solution of 7-hydroxyheptyl 2-butyloctanoaie (30 g, 95.39 mmol, 1 eq) in ACN (150 mL) and H2O (150 mL) was added [acetoxy(phenyl)-iodanyl] acetate (76.81 g, 2.38.47 mmol, 2.5 eq) and TEMPO (9.00 g, 57.23 mmol, 0.6 eq) under N2 atmosphere. The reaction mixture was stirred at 2.0 °C for 12 hr under N2 atmosphere. The mixture was diluted with H2O (500 mL) and extracted with DCM 500 mL (250 mL * 2). The combined organic layers were dried over Na2.SO4, 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=4 / 0 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. (H NMR(400 MHz, CDCI3) 5 ppm 4.13 - 4.04 (m, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.36 - 2.28 (m, 1H), 1.76 - 1.55 (m, 6H), 1.51 - 1.36 (m, 6H), 1.34 - 1.19 (m, 12H), 0.98 - 0.83 (m, 6H).

[0168] Step 8:,MT10 iNTH

[0169] 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 Nz atmosphere. The reaction mixture was stirred at 120 °C for 34 hr under Nz 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 mt * 3). The combined organic layers were dried over NazSO-i, 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 1 / 1). Compound 4,4- bis[(Z)-oct-5-enoxy]butanenitrile (10 g, 31.10 mmol, 20.09% yield) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCI3) 8 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), 2.11 ■■ 2.01 (m, 8H), 1.99 - 1.90 (m, 2.H), 1.65 - 1.56 (m, 4H), 1.49 - 1.35 (m. 4H), 0.97 (t, J = 7.4 Hz, 6H).

[0170] Step 9:

[0171] To a solution of 4,4-bis[(Z)-oct-5-enoxy]butanenitrile (10 g, 31.10 mmol, 1 eq) in EtOH (50 mL) and HzO (50 mL) was added KOH (5.24 g, 93.31 mmol, 3 eq) under Nz atmosphere. The reaction mixture was stirred at 1 10 °C for 12 hr under Nz 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 Nz and extracted with EtOAc (500 mL * 3). The combined organic layers were washed with aq. NaCl (300 mL * 2), 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 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.1H NMR (400 MHz, CDCI3) 8 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).

[0173] To a solution of 2-aminopropane- 1,3 -diol (20 g, 219.52 mmol, 1 eq), DMAP(268.18 mg, 2.20 mmol, 0.01 eq) and TEA (88.85 g, 878.07 mmol, 122.22 mL, 4 eq) in DCM(100 mL) was added TBSC1 (82.72 g, 548.80 mmol, 67.52 mL, 2.5 eq) in DCM(50 mL) under N? atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (500 mL) and extracted with DCM 1500 mL (500 mL, * 3). The combined organic layers were dried over NarSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate=20 / l to 0 / 1).Compound l,3-bis[[tert-butyl(dimethyl)silyl]oxy]propan-2 -amine (40 g, 125.14 mmol, 57.01 % yield) was obtained as a colorless oil.lH NMR (400 MHz, CDCI3) 3 ppm 3.62-3.58 (m, 2H), 3.53-3.49(m, 2H), 2.88-2.8.5(m, 1 H), 0.90(s, 18H), 0.05(s, 12H).

[0174] Step 11:

[0175] To a solution of l,3-bis[[tert-butyl(dimetliyl)silyl]oxy]propan-2 -amine (5.84 g, 18.27 mmol, 1 eq) and 4-(2-pyrroiidin-l-yiethylcarbamoyloxy)decanoic acid (6 g, 18.27 mmol, 1 eq) in DCM (60 mL) was added EDCI (4.20 g, 21.92 mmol, 1.2 eq), DMAP (223.18 mg, 1.83 mmol, 0.1 eq) and DIPEA (5.90 g, 45.67 mmol, 7.95 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 ( 100 mL) and extracted with DCM 200 mL ( 100 mL * 2). The combined organic layers were dried over Nar-SCfi, 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 / 1 to 0 / 1). Compound l-[3-[[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tertbutyl(dimethyl)silyl]-oxymethyl]ethyl]amino]-3-oxo- propyl]heptyl N-(2-pyrroIidml-ylethyl-)carbainate (7 g, 11.11 mmol, 60.82% yield) was obtained as a colorless oil.

[0177] To a solution of 1 -[3-[[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymethyl]etliyl]ammo]-3-oxopropyl]heptyl N-(2-pyrrolidin-l- ylethyl)carbamate (7 g, 11.11 mmol, 1 eq) in THE (70 ml) was added tetrab uty (ammonium fluoride trihydrate (1 M, 28.89 ml, 2.6 eq) slowly at 0 °C under N2 atmosphere. The reaction 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 purified by column chromatography (SiOr, DCM / MeOH=200 / l to 10 / 1). Compound l-[3-[[2-hydroxy-l- (hydroxymethyl)ethyl]amino]-3-oxo-propyl]heptyl N-(2-pyrrolidin-l-ylethyl)carbamate (2 g, 4.98 mmol, 44.83% yield) was obtained as a colorless oil.

[0178] Step 13:

[0179] To a solution of 1 -[3-[[2-hydroxy- 1 -(hydroxymethyl)ethyl]amino]-3-oxo- propyljheptyl N-(2-pyrrolidin- 1- ylethyl)carbamate (2 g, 4.98 mmol, 1 eq) in DCM (20 mL) was added EDCI (1.15 g, 5.98 mmol, 1.2 eq), DIPEA (1.61 g, 12.45 mmol, 2.17 mL, 2.5 eq) and DMAP ( 121 .70 mg, 996.17 μmol, 0.2 eq) under N?. atmosphere, then a solution of 7- (2- butyloctanoyloxy)heptanoic acid (1.31 g, 3.98 mmol, 0.8 eq) in DCM (20 mL) was added tothe above reaction mixture dropwise under Nz atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted w'ith H2O (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried overNazSO4, filtered and the filtrate was concentrated under reduced pressure to give a residue.The residue was purified by column chromatography (SiOz, Dichloromethane / Metlianol^SO / l to 5 / 1). Compound [7-[3-hydroxy-2-[4-(2-pyrrolidin-l- ylethylcarbamoyloxy)decanoylamino]propoxy]-7-oxo-heptyl] 2- butyloctanoate (2 g, 2.81 22Compound 1

[0181] To a solution of [7-[3-hydroxy-2-[4-(2-pyrrolidin-l- ylethylcarbamoyloxy)decanoylamino]propoxy]-7-oxo-heptyl] 2-butyloctanoate (0.9 g, 1.26 mmol, 1 eq) in DCM (9 mL) was added EDC1 (290.78 mg, 1.52 mmol, 1 .2 eq), DIPEA (408.41 mg, 3.16 mmol, 550.41 μL, 2.5 eq) and DMAP ( 15.44 mg, 126.40 0.1 eq) μmol, under Nz atmosphere, then 4,4-bis[(Z)- oct-5-enoxy]butanoic acid (430.40 mg, 1.26 mmol, 1 eq) was added to the above reaction mixture. The reaction mixture was stirred at 20 °C for 12 hr under Nz 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 NazSCM, 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 to1 / 1). Compound [7-[3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2-[4-(2-pyrrolidin-l- ylethylcarbamoyloxy)decanoylamino]propoxy]-7-oxo-heptyl] 2-butyloctanoate (0.8 g, 750.13 pmol, 59.34% yield, 97% purity) was obtained as a colorless oil. *H NMR (400 MHz, CDCI3) 5 ppm 7.05 - 6.83 (m, 1H), 5.78 - 5.56 (m, 1 H), 5.46 - 5.26 (m, 4H), 4.70 (d, J - 5.4 Hz, 1H), 4.51 - 4.42 (m, 2H), 4.21 - 4.11 (m, 4H), 4.06 (t, J = 6.6 Hz, 2H), 3.60 - 3.55 (m, 2H), 3.44 - 3.37 (m, 2H), 3.35 ■■ 3.27 (m, 1H), 3.12 ■■ 2.78 (m, 4H), 2.67 (s, 1H), 2.45 - 2.39 (m, 2H), 2.35 - 2.28 (m, 4H), 2.1 1 - 1.91 (m, 14H), 1.89 - 1.71 (m, 2H), 1.69 - 1.52 (m, 12H), 1.51 - 1.19 (m, 34H), 0.99 - 0.85 (m, 15H).Example 3 - Synthesis of Compound 2: [7-[3-[4, 4- bis [(Z) - oct-5-enoxy] butanoyloxy]- 2-[4-(2-pyrrolidm-l-yIethyIcarbamoyIexy) nenaneyiammoJpropoxyl-J-oxe-heptyl] 2- butyloctaaoate

[0182] Step 1: INT18 WH9

[0183] To a solution of 5-pentyltetrahydrofuran-2-one (100 g, 640. 12 mmol, 102.46 mL, 1 eq) in H2O (1000 mL) was added NaOH (26.88 g, 672.12 mmol, 1.05 eq) slowly under N2 atmosphere. The reaction mixture was stirred at 100 °C for 12 hr under N?. atmosphere. The reaction mixture was concentrated under reduced pressure to remove solvent. Compound 4- Hydroxydecanoyloxysodium (100 g, crude) was obtained as a white solid and it was used in next step directly.

[0184] Step 2 !NT19 WT20

[0185] To a solution of 4-hydroxynonanoyloxysodium (10 g, 50.96 mmol, 1 eq) in DMSO (100 mL) was added bromomethylbenzene (8.72 g, 50.96 mmol, 6.05 mL, 1 eq) 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 (100 mL).The combined organic layers were dried over NarSCh, filtered and the filtrate was concentrated under reduced pressure to give a residue. Compound benzyl 4-hydroxynonanoate (8 g, crude) was obtained as a pale yellow oil and it was used in next step quickly.

[0186] Step 3:[01871 To a solution of benzyl 4-hydroxynonanoate (8 g, 30.26 mmol, 1 eq) in DCM (80 mL) was added (4-nitrophenyl) carbonochloridate (12.20 g, 60.52 mmol, 2 eq) and Py. (4.79 g, 60.52 mmol, 4.89 mL, 2 eq) slowly at 0 °C under Nz atmosphere. The reaction mixture was stirred at 25 °C for Ihr 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 (SiOz, Petroleum ether / Ethyl acetate=100 / l to 10 / 1). Compound benzyl 4-(4- nitrophenoxy)carbonyloxynonanoate (4 g, 9.31 mmol, 30.78% yield) was obtained as a colorless oil.

[0189] To a solution of benzyl 4-(4-mtrophenoxy)carbonyloxynonanoate (15 g, 34.93 mmol, 1 eq) in DCM (150 mL) was added DIPEA (13.54 g, 104.78 mmol, 18.25 mL, 3 eq), 2-pyiTolidin-l -ylethanamine (7.98 g, 69.85 mmol, 2 eq) and DMAP (426.70 mg, 3.49 mmol, 0. 1 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under N?. 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 NazSO., filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate^ 100 / 1 to 1 / 1 ). Compound benzyl 4-(2-pyrrolidin- T-ylethylcarbamoyloxy)nonanoate (9.5 g, 23.48 mmol, 67.23% yield) was obtained as a colorless oil.lH NMR (400 MHz, CDCI3) 8 ppm 7.30-7.27(m, 5H), 5.09(s,1H), 5.03(s, 2H), 4.67(s, 1H), 3.24-3. 19(m, 2H), 2.57-2.5(s, 6H), 2.36-2.30(m, 2H), 1.86- 1.7 l(m, 6i ! ). 1.44- 1.35(m, 2H), 1.19-1.13(m, 61- 1), 0.80-0.77(1. J 2.61 ! / . 3H).

[0190] Step s:1NT22 WT23

[0191] To a suspension of Pd / C (12.50 g, 1 1.74 mmol, 10% purity, 0.5 eq) in THF (190 ml) was added benzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)nonanoate (9.5 g, 23.48 mmol, 1 eq) under Ar atmosphere. The suspension was degassed and purged with H2 for 3 times.The reaction mixture was stirred under Hr (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 (SiOr, DCM / MeOH= 100 / 1 to 1 / 1).Compound 4-(2-pyrrolidin-l-ylethylcarbamoyloxy) nonanoic acid (6 g, 19.08 mmol, 81.26% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 5 ppm 12.23 - 1 1.81 (m, 1H), 6.51 (d, J - 2.4 Hz, 1 II ), 4.80 - 4.66 (m, 1H), 3.68 - 3.53 (m, 1H), 3.26 - 3.15 (m, 1H), 3.06 - 2.93 (m, 4H), 2.88 - 2.78 (m, 1H), 2.40 - 2.18 (m, 2H), 2.07 - 1.85 (m, 5H), 1.84 - 1.73 (m. 1H), 1.64 - 1.43 (m, 2H), 1.37 - 1.18 (m, 6H). 0.86 (t, J = 6.8 Hz, 3H).

[0192] Step 6:

[0193] To a solution of 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)nonanoic acid (6 g, 19.08 mmol, 1 eq) in DCM (60 mL) was added EDCI (4.39 g, 22.90 mmol, 1.2 eq), 1 ,3-bis[[tert- butyl(dimethyl)silyI]oxy]propan-2 -amine (6.10 g, 19.08 mmol, 1 eq), DMAP (233.13 mg, 1.91 mmol, 0.1 eq) and DIPEA (6.17 g, 47.71 mmol, 8.31 mL, 2.5 eq) under Nr atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under Nr atmosphere. The reaction mixture was diluted with Hr0 (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over NarSCX filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, DCM / MeOH= 100 / 1 to 10 / 1). Compound l-[3-[[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert-butyl(dimethyl)silyl]oxymethyl]ethyl]amino]-3-oxo- propyl]hexyl N-(2-pyrrolidin-l-ylethyl)carbamate (5 g, 8.12 mmol, 42.53% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCb) 6 ppm 5.96 (d, J = 8.0 Hz, 1H), 5.19 (s, 1H), 4.75 (d, J - 2.2 Hz, 1H), 4.01 - 3.91 (m, 1H), 3.82 - 3.68 (m, 2H), 3.61 - 3.50 (m, 2H), 3.29 (d, J - 5.4 Hz, 2H), 2.67 - 2.48 (m, 6H), 2.30 - 2.14 (m, 2H), 1.95 - 1.76 (m, 6H), 1.63 ■ 1.44 (m, 2H), 1.37 ■■ 1.25 (m, 6H), 0.91 ■■ 0.86 (in, 20H), 0.07 (d, J = 2.4 Hz, 12H).10194] Step 7:MT24 WT25

[0195] To a solution of 1 -[3-[ [2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymethyl] ethyl]amino]-3-oxo-propyl]hexyl N-(2-pyrrolidin-l- ylethyl)carbamate (5 g, 8.12 mmol, 1 eq) in THF (50 ml..) was added pyridine hydrofluoride (2.87 g, 20.29 mmol, 2.61 mL, 70% purity, 2.5 eq) dropwise at 0 °C under Nr atmosphere.Hie reaction mixture was stirred at 25 °C for 12 hr under N? atmosphere. Then NH3.H2O was added to the above reaction mixture to adjust pH >7. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. Compound 1 -[3-[[2- hydroxy-1- (hydroxymethyl)ethyl]arnino] -3 -oxo-propyl] hexyl N-(2-pyrro lidin- 1 -ylethyl) carbamate (2.3 g, 5.94 mmol, 73.13% yield) was obtained as a colorless oil and it was used in next step directly.!H NMR (400 MHz, CDCb) 8 ppm 6.87-6.85(d, J =3.0 Hz, 1 H), 5.86(s, 1H), 4.77-4.75(m, 1H), 4.10-3.89(m, 4H), 3.80-3.72(m, 2H), 3.49(s, 1 H), 3.44-3.36(m, 1H), 3.31-3.21(111, 1H), 2.74-2.55(m, 6H), 2.36-2.25(m, 2H), 2.10-2.01(m, 1H ), 1.84-1.73(m, 5H ), 1.61-1.46(m, 2H), 1.32-1.22(m, 6H), 0.90-0.86(1, J =3.4 Hz, 3H).

[0196] Step 8:

[0197] To a solution of l-[3-[[2-hydroxy-l-(hydroxymethyl)ethyl]amino]-3-oxo- propyljhexyi N-(2-pyrrolidin-1 -ylethyl)carbamate (2.3 g, 5.94 mmol, 1 eq) in DCM (23 mL) was added EDCI (1.37 g, 7.12 mmol, 1.2 eq), 7-(2-butyloctanoyloxy)heptanoic acid (974.83mg, 2.97 mmol, 0.5 eq), DMAP (72.51 mg, 593.53 0.1 eq) anμdm DoIlP, EA (1.92 g, 14.84 mmol, 2.58 mL, 2.5 eq) under N2 atmosphere. The reaction mixture was stirred at 25 °C for12 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 Na?.SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM / MeOH= 100 / 1 to 10 / 1).Compound [7-[3-hydroxy-2-[4-(2-pyrrolidm-l-ylethylcarbamoyloxy) nonanoylamino] propoxy]-7-oxo-heptyl] 2-butyloctanoate (1 g, 1.43 mmol, 24.14% yield) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCI3) 8 ppm 6.76 (d, J - 6.8 Hz, 1H), 5.88 - 5.70 (m, 1H), 4.70 (d, J = 3.4 Hz, 1H), 4.21 ■■ 4.15 (m, 2H), 4.06 (t, J = 6.6 Hz, 2H), 3.92 ■■ 3.75 (m, IH), 3.71 - 3.60 (m, 2H), 3.45 - 3.19 (m, 2H), 2.79 - 2.47 (m, 6H), 2.34 - 2.23 (m, 4H), 2.00 -1,72 (m, 6H), 1.66 - 1.56 (m, 6H), 1.52 - 1.17 (m, 26H), 0.97 - 0.76 (m, 9H).

[0198] Step 9:Compound 2

[0199] To a solution of 4,4-bis[(Z)-oct-5-enoxy]butanoic acid (209.07 mg, 614.01 1 μmol, eq) in THE (6 mL) was added TEA (93.20 mg, 921.02 128.19 pLμ,m 1o.5l, eq) and 2,4,6- trichlorobenzoyl chloride (149.76 mg, 614.01 95.94 uLμ, m 1 o elq,) dropwise at 0 °C underN2 atmosphere. The reaction mixture was stirred at 25 °C for 2 hr under N2 atmosphere. Then[7-[3-hydroxy-2-[4-(2-pyrrolidin-l-ylethylcarbamoyloxy)-nonanoylamino]propoxy]-7-oxo- heptyl] 2-butyloctanoate (300 mg, 429.81 0.7 eq) anμmd o DlM, AP (7.50 mg, 61.40 pmol, 0. 1 eq) was added to the above reaction mixture. The reaction mixture was stirred at 25 °Cfor 12 hr under Nr. atmosphere. The reaction mixture was diluted with HrO (50 mL) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were dried over NarSCE, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM / MeOH= 100 / 1 to 10 / 1). Compound [7-[3-[4, 4- bis [(Z) - oct-5-enoxy] butanoyloxy]-2-[4-(2-pyrrolidin-l- ylethylcarbamoyloxy) nonanoy lamino]propoxy]-7~oxo -heptyl] 2-butyloctanoate (100 mg, 97.99 μmol, 15.96% yield) was obtained as a colorless oil.lH NMR (400 MHz, CDCI3) § ppm 6.67 - 6.56 (m, 1H), 5.41 - 5.27 (m, 4H), 4.80 - 4.65 (m, 1H), 4.55 - 4.40 (m, 2H), 4.25 -3.97 (m, 6H), 3.64 - 3.54 (m, 2H), 3.46 - 3.39 (m, 2H), 3.31 (d, J = 5.4 Hz, 2H), 2.71 - 2.54 (m, 4H), 2.45 ■■ 2.38 (m, 2H), 2.35 ■■ 2.30 (m, 2H), 2.28 - 2.17 (m, 2H), 2.10 - 2.00 (m, 8H),1.97 - 1.78 (m, 12H), 1.68 - 1.49 (m, 12H), 1.45 - 1.24 (m, 28H), 1.00 - 0.82 (m, 15H).Example 4 ■■■• Synthesis of Compound 3: [7-[3-(4,4-dioctoxybutanoyloxy)-2-[4-(2- pyrrolidin-l-ykthykarbanioyIoxy)decaJioyIamino]propoxy]-7- oxo-heptyl] 2- butyloctanoate

[0200] Step 1 :!NTia WT27

[0201] 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 mL) was added OPTS (9.73 g,38.71 mmol, 0.25 eq) under N?_ atmosphere. The reaction mixture was stirred at 110 °C for 36 hr under Nr atmosphere. The reaction mixture 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-dioctoxybutanenitrile (30 g, 92.16 mmol, 59.51 % yield) was obtained as a colorless oil.

[0202] Step 2:

[0203] To a solution of 4,4-dioctoxybutanenitrile (30 g, 92 J 6 mmol, 1 eq) in EtOH (300 mL) and H?.O (300 mL) was added KOH (20.68 g, 368.63 mmol, 4 eq) under N?. atmosphere The reaction mixture was stirred at 110 °C for 12 hr under Nz atmosphere. The reaction mixture was diluted with H2O (1000 mL) and extracted with DCM 2000 mL (1000 mL * 2). The combined organic layers were dried over NaaSCh, 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 1 / 1). Compound 4,4- dioctoxybutanoic acid (19 g, 55.15 mmol, 59.84% yield) was obtained as a colorless oil.

[0204] Step 3:

[0205] To a solution of 4,4-dioctoxybutanoic acid (241.94 mg, 702.24 1 eq) in μmol, DCM (8 mL) was added EDCI (161.54 mg, 842.69 1.2 eq), DμmIPoEl,A (226.90 mg, 1.76 mmol, 305.79 uL, 2.5 eq) and DMAP (17.16 mg, 140.45 umoi, 0.2 eq), then [7-[3-hydroxy- 2-[4-(2-pyrrolidin-l-ylethylcarbamoyloxy)decanoylamino]propoxy]-7-oxoheptyl] 2- butyl octanoate (0.5 g, 702.24 1 eq)μ wmaosl a, dded 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 H?.O (50 mL) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were dried over NazSO-i, 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 / T to 1 / 1). Compound [7-[3-(4,4- dioctoxybutanoy loxy) -2- [4 - (2-pyrrolidin- 1 -y lethy Icarbamoy loxy )decanoyiamino]propoxy] -7 - oxo-heptyl] 2-butyloctanoate (0.5 g, 481.45 umol, 68.56% yield) was obtained as a colorless oil. T-I NMR (400 MHz, CDCI3) 8 ppm 6.91 - 6.64 (m, 1H), 5.64 - 5.35 (m, HI), 4.79 - 4.66 (m, 1H), 4.53 ■ 4.40 (m, 2H), 4.26 ■■ 4.09 (m, 4H), 4.06 (t, J - 6.6 Hz, 2H), 3.62 ■■ 3.51 (m, 2H), 3.47 - 3.36 (m, 3H), 3.34 - 3.22 (m, 1H), 2.79 (s, 4H). 2.54 - 2.18 (m, 8H), 1.98 - 1.87 (m, 6H), 1.84 - 1.68 (m, 2H), 1.67 - 1.51 (m, 12H), 1.47 - 1.41 (m, 2H), 1.39 - 1.22 (m, 46H), 0.91 - 0.85 (m, 15H).Example 5 - Synthesis of Compound 4: [7-[3-(4,4-dioctoxybutanoyloxy)-2-[4-(2- pyrrolidin~l~ylethylcarbamoyloxy)nonanoylamism]propoxy]~7~ oxo-heptyl] 2- butyloctanoate

[0206] Synthetic scheme:

[0207] To a solution of [7-[3-hydroxy-2-[4-(2-pyrrolidin-l - ylethylcarbamoyloxy)nonanoylamino]propoxy] -7 -oxo-heptyl] 2- butyloctanoate (0.3 g, 429.81 umoi, 1 eq) in DCM (3 ml) was added EDCI (98.87 mg, 515.77 nmol, 1.2 eq), DIPEA (194.42 mg, 1.50 mmol, 262.03 uL, 3.5 eq) and 4-pyrro lidin- 1-ylpyridine (6.37 mg, 42.98 μmol 0, . 1 eq) under Nr atmosphere, then 4,4-dioctoxybutanoic acid ( 162.89 mg, 472.79 pmol, 1.1 eq) was added to the above reaction mixture. The reaction mixture was stirred at 20 °C for 12 hr under Nr atmosphere. The reaction mixture was diluted with HrO (50 inL) and extracted with DCM 100 mt (50 ml. * 2). The combined organic layers were dried over NarSOq filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Dichloromethane / Methanol=50 / 1 to 5 / 1). Compound [7-[3-(4,4-dioctoxybutanoyloxy)-2-[4-(2-pyrrolidin-l- ylethylcarbamoyloxy)nonanoylammo]propoxy]-7- oxo-heptyl] 2-butyloctanoate (0.1 g, 97.61 pmol, 22.71% yield) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCb) 5 ppm 6.90 ■■ 6.66 (m, 1H), 5.73 ■■ 5.46 (m, 1H), 4.79 - 4.65 (m, 1H), 4.53 ■■ 4.38 (m, 2H), 4.22 ■■ 4.10 (m, 4H), 4.06 (t, J = 6.6 Hz, 2H), 3.63 - 3.52 (m, 2H), 3.46 - 3.36 (m, 2H), 3.36 - 3.25 (m, 1H), 3.05 - 2.51 (m, 6H), 2.47 - 2.39 (m, 2H), 2.38 - 2.15 (m, 6H), 1.99 - 1.89 (m, 6H), 1.85 - 1.69 (m, 2H), 1.68 - 1.51 (m, 12H), 1.50 - 1.39 (m, 4H), 1.38 - 1.17 (m, 42H), 0.95 - 0.82 (m, 15H).Example 6 - Synthesis of Compound 5: 07-[3-[4,4-bis[(Z)-oct-5-eJioxy]butanoyloxy]“2- (4~pyrrolidisi~l~ylbutanoylamino)propyl] Ol-(3- pentyloctyl) heptanedioate

[0208] Step 1 :INT28

[0209] To a solution of 4-pyrrolidin- 1-ylbutanoic acid (4 g, 20.65 mmol, 1 eq, HC1) in DCM (150 mL) was added D1EA (6.67 g, 51.63 mmol, 8.99 mL, 2.5 eq), EDC1 (4.75 g, 24.78 mmol, 1.2 eq) and DMAP (252.33 mg, 2.07 mmol, 0.1 eq) under N?. atmosphere. Then l,3-bis[[tert-butyl(dimethyl)silyl]oxy]propan-2 -amine (6.60 g, 20.65 mmol, 1 eq) was added to the above reaction mixture under Nr atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under Nr atmosphere. The reaction mixture was poured into HrO (50mL) and extracted with DCM (50 mLx3). The combined organic layers were washed with brine (50 mLx2), dried over NarSCb, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate=3 / 2 to 0 / 1). CompoundN-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymethyl]ethyl]-4-pyrrolidin-l-yl-butanamide (8 g, 17.44 mmol, 42.22% yield) was obtained as a yellow oil.lH NMR (400 MHz, CDCH) 8 ppm 6.08 (d, J = 8.4 Hz, 1H), 4.02 - 3.91 (m, 1H), 3.78 - 3.69 (m, 2.H), 3.59 - 3.51 (m, 2H), 2.54 - 2.42. (m, 6H), 2.24 (t, J- 7.4 Hz, 2H), 1.88 - 1.72 (m, 6H), 0.89 (s, 18H), 0.05 (d, J - 2.2 Hz, 12H).

[0210] Step 2:IMT2S SNT30

[0211] To a solution of N-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyI]oxymethyl]ethyl]-4-pyrrolidin-l-yl-butanamide (8 g, 17.44 mmol, 1 eq) in THF (80 mL) was added pyridine;hydrofluoride (4.94 g, 34.87 mmol, 4.49 mL, 70% purity, 2 eq) at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 2 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO?., DCM: MeOH = 1 / 0 to 0 / 1). Compound N-[2-hydroxy-l-(hydroxymethyl)ethyl]-4-pyrrolidin-l-yl-butanamide (1.8 g,7.82 mmol, 44.78% yield) was obtained as a yellow oil. *H NMR (400 MHz, CDCI3) 6 ppm7.55 (d, J = 7.6 Hz, HI), 5.02 (s, 2H), 4.03 - 3.88 (m, 1H), 3.78 - 3.62 (m, 4H), 2.82 - 2.62 (m, 6H), 2.34 (t, J= 7.0 Hz, 2.H), 2.00 - 1.73 (ra, 6H).

[0212] Step 3: INT31 INT32

[0213] To a solution of undecan-6-one (10 g, 58. / 2 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 N2 atmosphere. The reaction mixture was added sat. NI-LsCl (200 mL) slowly under Nz 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 NazSOr, 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=100 / l to 10 / 1). Tire 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) 5 ppm 5.62 (s, 1 H), 4.14 (q, J-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).

[0214] Step 4:WT33

[0215] To a suspension 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 2.5 °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 (SiOr, Petroleum ether / Ethyl acetate=100 / l to 10 / 1). Compound ethyl 3 ■■pentyloctanoate (8.5 g, 35.07 mmol, 84.29% yield) was obtained as acolorless oil. ‘H NMR (400 MHz, CDCb) 5 ppm 4.16-4.10 (m, 2H), 2.23-2.22(m, 2H), 1.86- 1.84(m, H I ). 1.31 - 1.24(m, 19H), 0.91-0.87(t, J-3.4 Hz, 61 1 ).8NT33

[0216] Step 5: IMT34

[0217] To a solution of LiAlHfi (2.5 M, 28.05 mL, 2 eq) in THF (85 rnL) was added ethyl3-pentyloctenoate (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 N2 after addition. The reaction mixture was added sat. NH4CI (100 mL) slowly at 0 °C under N2 and stirred for another 10 rnin after addition. The reaction mixture was poured into H?.O (100 mL) and extracted with EtOAc 600 mL (3*200 mL). 'The organic phase was evaporated under reduced pressure to get a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate^ 100 / 1 to 0 / 1 ). Compound 3-pentyloctan-l-ol (5 g, 24.96 mmol, 71.17% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCb) 6 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).

[0218] Step 6:SSO34 IHT35

[0219] To a solution of 3-pentyloctan-l-ol (5 g, 24.96 mmol, 1 eq) in DCM (50 mL) was added EDC1 (5.74 g, 29.95 mmol, 1.2 eq), heptenedioic 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 Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate=100 / l 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) 5 ppm 4.09 (t, J - ---- 7.0 Hz, 2H ), 2.37 (t, J --- 1A Hz, 2H), 2.31 (s, 2H), 1.66

[0221] To a solution of N -[2-hydroxy- 1 -(hydroxymethyl)ethyl]-4-pyrro lidin- 1 -yl- butanaraide (0.5 g, 2.17 mmol, 1.6 eq) in DCM (30 mL) was added DIPEA (438.43 mg, 3.39 mmol, 590.87 μL, 2.5 eq), EDCI (312.15 mg, 1.63 mmol, 1.2 eq) and DMAP (16.58 mg, 135.69 μmo 0l,.1 eq) under N2 atmosphere. Then 7-oxo-7-(3-pentyloctoxy)heptanoic acid (464.76 mg, 1.36 mmol, 1 eq) was added to the above reaction mixture under N?. atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under Nz atmosphere. The reaction mixture was poured into H2O (10 ml..) and extracted with DCM (10 mLx3). The combined organic layers were dried over NazSOr, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOz, DCM: MeOH = 6: 1). Compound O7-[3-hydroxy-2-(4-pyrrolidin-l- ylbutanoylamino)propyl] Ol-(3-pentyloctyl) heptanedioate (300 mg, 540.74 9.97% μmol, yield, 100% purity) was obtained as a yellow oil.!H NMR (400 MHz, CDCI3) 8 ppm 7.63 (d, J = 6.6 Hz, 1H), 4.21 - 4.13 (m, 4H), 4.07 (t, J = 7.0 Hz, 2H), 3.72 (d, J = 3.2 Hz, 2H), 3.36 - 3.20 (m, 4H), 3.15 - 3.07 (m, 1 H), 2.72 - 2.60 (m, 1 H), 2.54 - 2.44 (m, 1H), 2.36 - 2.04 (m, 10H), 1.69 - 1.50 (m, 6H), 1.42 - 1.20 (m, 20H), 0.88 (t, J 6.8 Hz, 6H).

[0222] Step 8:Compound 5

[0223] To a solution of O7-[34iydroxy-2.-(4-pyrrolidin-l -ylbutanoylamino)propyl] 01 -(3- pentyloctyl) heptanedioate (400 mg, 720.98 1 eq) in DμCmMol (,7 mL) was added EDCI (165.86 mg, 865.18 μ 1m.2o elq, ), DIPEA (232.95 mg, 1.80 mmol, 313.95 μL, 2.5 eq) and DMAP (8.81 mg, 72.10 nmol, 0.1 eq) under N2 atmosphere. 4,4-bis[(Z)-oct-5- enoxy] butanoic acid (368.2.4 mg, 1.08 mmol, 1.5 eq) was added to the reaction mixture under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was poured into H2O (50 mL) and extracted with DCM (50 mLx3). 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=50 / l to 10 / 1). Compound O7-[3-[4,4-bis[(Z)-oct-5- enoxy]butanoyloxy]-2-(4-pyrrolidin-l-ylbutanoylamino)propyl] 01 -(3- pentyloctyl) heptanedioate (700 mg, 797.92 63.64μ%mo yli,eld) was obtained as a colorless oil.!H NMR (400 MHz, CDC13) 5 ppm 7.39 (d, J- 8.4 Hz, 1H), 5.45 - 5.21 (m, 4H), 4.48 (t, J- 5.4 Hz, 1H), 4.44 - 4.35 (m, 1H), 4.29 - 4.22 (m, 2H), 4.18 - 4.02 (m, 4H), 3.62 - 3.52 (m, 2H ), 3.46 - 3.36 (m, 2H), 3.17 (s, 2H), 3.02 (t, 6.0 Hz, 2H), 2.58 ■■ 2.49 (m, 2H), 2.47 ■■ 2.34 (m, 4H),2.32 - 2.26 (m, 2H), 2.14 - 1.98 (m, 14H), 1.96 - 1.89 (m, 2H), 1.66 - 1.52 (m, 10H), 1.45 - 1.22 (m, 24H), 1 .01 - 0.81 (m, 12H).Example 7 - Synthesis of Compound 6: 07-[3-[4,4-bis[(Z)-oct-5-enoxy]butanoyloxy]-2- [4~(dimethylam~ino)butanoylamino]propyl] Ol-(3- pentyloctyl) heptanedioate

[0224] Step 1:SNT37 !sms

[0225] A mixture of 4-(dimethylamino)butanoic acid (5 g, 38.12 mmol, 1 eq), 1,3- bis[[tert-butyl(dimethyl)silyl]oxy]propan-2-amine (12.18 g, 38.12 mmol, 1 eq), EDCI (8.77 g, 45.74 mmol, 1.2 eq), DMAP (465.68 mg, 3.81 mmol, 0.1 eq), TBAF (25.91 g, 99.11 mmol, 2.6 eq) and DIPEA (12.32 g, 95.29 mmol, 16.60 ml, 2.5 eq) in DCM (200 mL) was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O 150 mL and extracted with EtOAc 150mL (50 mL * 3). 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 (SiOc, Petroleum ether / Ethyl acetate= 100 / 1 to 1 / 1). Compound N-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymethyl]ethyl]-4(dimethylamino)butanamide(25 g, 57.77 mmol, 45.46% yield) was obtained as a colorless oil.H HTBscrTBSO

[0226] Step 2: ™

[0227] To a solution of N-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymethyl]ediyl]-4-(dimethyIammo)butanamide (31 g, 71.63 mmol, 1 eq) hi THF (310 mL) was added pyridine;hydrofluoride (20.28 g, 143.26 mmol, 18.44 mL, 70% purity, 2. eq) dropwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 20 °C for 2 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Dichloromethane / MethanomSO / l to 5 / 1). Compound 4-(dimethy lamino)-N-[2 ■■hydroxy- 1- (hydroxymethyl)ethyl]butanamide (5.5 g, 26.93 mmol, 37.59% yield) was obtained as a colorless oil. ‘H NMR (400 MHz, D2O) 8 ppm 3.95 - 3.84 (m, 1 II ), 3.61 - 3.55 (m, 2H ), 3.54 - 3.42 (m, 2H), 3.09 ■■ 2.97 (m, 2H), 2.78 (s, 6H), 2.31 (t, J = 7.2 Hz, 2H), 1.98 ■■ 1.85 (m, 2H).iNT3§S^T49

[0229] To a solution of 4-(dimethylammo)-N-[2 -hydroxy- 1 ■(hydroxymethyl)ethyl]butanamide (5.5 g, 26.93 mmol, 2.46 eq) in DCM (55 mL) was added EDCI (2.52 g, 13.14 mmol, 1.2 eq), DIPEA (3.54 g, 27.37 mmol, 4.77 mL, 2.5 eq) and DMAP (133.76 mg, 1.09 mmol, 0.1 eq) under N?. atmosphere. Then a solution of 7-oxo-7-(3- pentyloctoxy)heptanoic acid (1.87 g, 5.47 mmol, 0.5 eq) in DCM (55 mL) was added to the above reaction mixture dropwise under Nr 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 (TOO mL * 2). The combined organic layers were dried over NarSCU, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, dichloromethane / methanol=50 / l to 5 / 1). Compound O7-[2-[4-(dimethylamino)butanoylamino]-3-hydroxy-propyl] 01 -(3- pentyloctyl)heptanedioate (1.4 g, 2.65 mmol, 24.18% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 6 ppm 7.64 - 7.53 (m, 1H), 6.49 (s, 1H), 5.63 (br s, 1H), 4.27 - 4.12 (m, 3H), 4.07 (t, J = 7.0 Hz, 2H), 3.76 - 3.63 (m, 2H), 3.38 - 3.27 (m, 4H), 3.24 - 3.14(m, 3H ), 3.06 (t, J - 6.6 Hz, 3H), 3.00 - 2.92 (m, 1H), 2.65 - 2.54 (m, 1H), 2.51 - 2.42 (m, 1H), 2.37 - 2.27 (m, 4H), 2.20 - 2.1 1 (m, 1H), 2.10 - 2.01 (m, 1H). 2.00 - 1.91 (m, 3H), 1.69 -1 .52 (m, 6H), 1.44 - 1.19 (m, 19H), 1.12 (t, J - 7.2 Hz, 4H), 0.89 (t, J - 6.9 Hz, 6H).

[0231] To a solution of O7-[2-[4-(dimethylamino)butanoylammo]-3-hydroxy-propyI] 01- (3-pentyloctyl) heptanedioate (1.3 g, 2.46 mmol, 1 eq) in DCM (13 mL) was added EDCI (565.58 mg, 2.95 mmol, 1.2 eq), DIPEA (794.38 mg, 6.15 mmol, 1.07 mL, 2.5 eq) and DMAP (30.04 mg, 245.86 0μ.m1 o elq,) under N2 atmosphere, then 4,4-bis[(Z)-oct-5- enoxy]butanoic acid (837.13 mg, 2.46 mmol, 1 eq) was added to the above reaction mixture. The reaction mixture was stirred at 20 °C for 12 hr under N 2 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 NarSCti, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO?., Dichloromethane / MethanomSO / l to 5 / 1). Compound O7-[3-[4,4- bis[(Z)-oct-5-enoxy]butanoyloxy]-2-[4-(dimethylam-ino)butanoylamino]propyl] Ol-(3- pentyloctyl) heptanedioate (0. 105 g, 114.71 4.67% yieμldm,o 9l3, % purity) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 6 ppm 7.15 (d, J = 8.4 Hz, 1H), 5.47 ■■ 5.23 (m, 4H), 4.49 (t, J = 5.4 Hz, 1 H), 4.46 - 4.39 (m, 1 H), 4.17 (d, J = 5.2 Hz. 4H), 4.08 (t, J = 7.2 Hz, 2H), 3.62 - 3.52 (m, 2H), 3.46 - 3.35 (m, 2H), 2.62 (t, J - 6.4 Hz, 2H), 2.46 (s, 6H), 2.42 - 2.27 (m, 8H ), 2.10 - 1.99 (m, 8H), 1.96 - 1.86 (m, 4H), 1.68 - 1.55 (m, 10H), 1.45 - 1.37 (m, 6H), 1.35 - 1.18 (m, 18H), 0.96 (t, J = 7.4 Hz, 6H), 0.89 (t, J = 6.8 Hz, 6H).Example 8 - Synthesis of Compound 7: 07“[3“[4,4-bis[(Z)-oct-5“eiioxy]butanoyloxy]-2“ [4-[ethyl(methyI)amino]butanoylamino]propyl] Ol-(3- pentySoctyi) heptanedioate

[0232] Step 1:INT41 !NT42

[0233] To a solution of 2-amino-3-benzyloxy-propanoic acid (20 g, 102.45 mmol, 1 eq) in THF (200 ml..) was added LAH (2.5 M, 61 .47 mL, 1 .5 eq) dropwise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 60 °C for 1.5 hr under N2 atmosphere. The reaction mixture was quenched with sat. NH4CI (100 mL) at 0 °C and diluted with I-I2O (100 mL). Then it was extracted with EtOAc 500 mL (250 mL * 2). The combined organic layers were dried over Na2.SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOi, Dichloromethane / Methanol=50 / 1 to 5 / 1). Compound 2-amino-3-benzyloxy-propan-l-ol (14 g, 77.25 mmol, 75.40% yield) 2-amino-3 -benzyloxy-propan- l-ol (14 g, 77.25 mmol, 75.40%yield) was obtained as a colorless oil. NMR (400 MHz, CDCb) 8 ppm 7.43-7.29 (m, 5H), 4.53 (s, 2H), 3.66-3.59 (m, 1H), 3.54-3.40 (rn, 3H), 3.14-3.04 (m, 1H).

[0234] Step 2: WT42 INT43

[0235] To a solution of 2-amino-3-benzyloxy-propan-l-ol (14 g, 77.25 mmol, 1 eq) inTHF (140 mL) was added TBSO (23.29 g, 154.50 mmol, 19.01 mL, 2 eq) and imidazole(10.52 g, 154.50 mmol, 2 eq) under Nc atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N? 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 bwSO-q 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 l-benzyloxy-3-[tert-butyl(dimethyl)silyl]oxy-propan-2-amine (6.2 g, 20.98 mmol, 27.16% yield) was obtained as a colorless oil.NMR (400 MHz, CDCb) 8 ppm 7.43 - 7.28 (m, 5H), 4.53 (s, 2H), 4.19 - 4.08 (m, 1H), 3.67 - 3.60 (m, 1 H), 3.42 - 3.34(m, 1 H), 3.12 - 3.01 (m, 1 H), 1.27 (t, J - 7.2 Hz, 1H), 0.90 (s, 9H), 0.06 (s, 6H).

[0236] Step 3: INT43 WT44

[0237] To a solution of l-benzyloxy-3-[tert-butyl(dimethyl)silyl]oxy-propan-2 -amine (6.2 g, 20.98 mmol, 1 eq) in DCM (62 mL) was added DIPEA (9.49 g, 73.44 mmol, 12.79 mL,3.5 eq), EDCI (4.83 g, 25.18 mmol, 1.2 eq) and DMAP (256.33 mg, 2.10 mmol, 0.1 eq)Then 4-[ethyl(methyl)amino]butanoic acid (3.35 g, 23.08 mmol, 1.1 eq) was added to the above reaction mixture. The reaction mixture was stirred at 20 °C for 12 hr under Nr. 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 NacSOn filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate:;;:50 / T to 1 / 1 ). Compound N- [l-(benzyloxymethyl)-2-[tert-buh4(dimethyl)silyl]oxy-ethyl]-4- [ethyl(methyl)amino]butanamide (5 g, 11.83 mmol, 56.38 % yield) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCb) 8 ppm 7.42 - 7.27 (m, 5H), 5.31 (s, 2H), 4.52 (s,2H), 4.22 - 4.04 (m, 1H), 3.79 - 3.72 (m, HI), 3.66 - 3.58 (m, 2H), 3.52 - 3.45 (m, 1H), 2.55 - 2.40 (m, 4H ), 2.26 - 2.23 (m, 4H), 1.86 - 1.81 (m, 2H), 1.09 - 1.05 (m, 2H), 0.89 (s, 9H), 0.06 (s, 6H).

[0238] Step 4:

[0239] To a solution of N-[ 1 -(benzyloxymethyl)-2-[tert-butyl(dimethyl)sil yl]oxy-ethyl] -4- [ethyl(methyl)amino]butanamide (4.06 g, 9.61 mmol, 1 eq) in THF (40 mL) was added N,N- diethylethanamine trihydrofluoride (1.55 g, 9.61 mmol, 1.57 mL, 1 eq) dropwise at O °C under Nr 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 purified by column chromatography (SiOr, Dichloromethane / Methanol=50 / 1 to 5 / 1). Compound N-[l-(benzyioxymeth-yl)-2-hydroxy-ethyl]-4- [ethyl(methyl)amino]butanamide (2 g, 6.48 mmol, 67.51% yield) was obtained as a colorless oil.

[0240] Step 5:[024'1] To a solution of N-[l-(benzyloxymethyl)-2-hydroxy-ethyl]-4- [ethyl(methyi)amino]butanamide (2 g, 6.48 mmol, 1 eq) in DCM (20 mL.) was added EDC1 (1.49 g, 7.78 mmol, 1.2 eq), DMAP (79.22 mg, 648.48 umol, 0.1 eq), DIPEA (2.93 g, 22.70 mmol, 3.95 mL, 3.5 eq) under N?. atmosphere. Then 7-oxo-7-(3-pentyloctoxy)heptanoic acid (2.44 g, 7.13 mmol, 1.1 eq) was added to the above reaction mixture. The reaction mixture was stirred at 20 °C for 12 hr under Nr 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 NarSCN, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Dichlorometbane / MethanomoO / T to 5 / 1). Compound O7-[3-benzyioxy-2-[4-[ethyl-(methyl)ammo]butanoylamino]propyl] Ol-(3-pentyloctyl) heptanedioate (1.4 g, 2.21 mmol,34.11% yield) was obtained as a colorless oil.

[0242] Step 6:MT47

[0243] To a suspension of Pd / C (470.80 mg, 442.40 10% puriμtmy,o 0l,.2 eq) in MeOH (14 mL) was added O7-[3-ben2yloxy2-[4-[ethyl(methyl)ammo]butanoylamino]propyl] 01- (3-pentyloctyl)heptanedioate (1.4 g, 2.21 mmol, 1 eq) under Ar atmosphere. The reaction mixture was stirred under H2 (50 Psi) at 80 °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 (S1O2, Dichloromethane / Methano 1=50 / 1 to 5 / 1).Compound O7-[2-[4-[ethyl(methyl)amino]butanoylaraino]-3-hydroxy-propyl] Ol-(3- pentyloctyl) heptanedioate (0.72 g, 1.33 mmol, 59.97% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 5 ppm 7.42 - 7.32 (m, 1H), 4.26 - 4.17 (m, 2H), 4.10 - 4.05 (m, 2H). 3.79 - 3.62 (m, 2H), 3.49 (s, 2H), 2.88 - 2.79 (m, 2H), 2.78 - 2.71 (m, 1H), 2.51 (s, 2H), 2.47 - 2.26 (m, 6H), 2.13 - 1.91 (m, 2H), 1.66 - 1.54 (m, 6H), 1 .38 - 1.23 (m, 22H), 0.89 (t, J = 6.8 Hz, 6H).

[0244] Step 7:Compound 7

[0245] To a solution of O7-[2-[4-[ethyl(methyl)amino]butanoylamino]-3-hydroxy-propyl] Ol-(3-pentyloctyl) heptanedioate (0.72 g, 1.33 mmol, 1 eq) in DCM (7.2 mL) was added EDCI (305.15 mg, 1.59 mmol, 1.2 eq), DIPEA (600.03 mg, 4.64 mmol, 808.67 μL, 3.5 eq), 4-pyrrolidin-l-ylpyridine (19.66 mg, 132.65 0.1 eq)μ umnodle,r N2 atmosphere. Then 4,4- bis[(Z)-oct-5-enoxy]butanoic acid (541.99 mg, 1.59 mmol, 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 Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO?., Dichloromethane / MethanoHsO / l to 5 / 1). Compound O7-[3-[4,4- bis[(Z)-oct-5-enoxy]butanoyloxy]-2-[4-[etiiyl(methyl)amino]butanoylamino]propyl] 01 -(3- pentyloctyl) heptanedioate (0.1 1 g, 127.13 9.58% yieμlmd,ol1,00% purity) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCb) 5 ppm 7.12 (d, .1 - 8.4 Hz, 1H), 5.44 - 5.26 (m, 4H), 4.52 ■■ 4.46 (m, 1H), 4.45 - 4.38 (m, 1H), 4.21 ■■ 4.02 (m, 6H), 3.62 ■■ 3.53 (m, 2H), 3.45 ■■ 3.36 (m, 2H), 2.65 - 2.55 (m, 4H), 2.44 - 2.27 (m, 12H), 2.1 1 - 1.99 (m, 8H), 1.97 - 1.85 (ra, 4H), 1.68 - 1.54 (m, 10H), 1.46 - 1.36 (m, 6H), 1.36 - 1.20 (m, 18H), 1.16 (t, J - 7.2 Hz, 3H), 0.96 (t, J - 7.6 Hz, 6H), 0.89 (t, J - 6.8 Hz, 61 1 ).Example 9 - Synthesis of Compound 8: 07-[3-[4,4-bis[(Z)-oct-5-eJioxy]butanoyloxy]“2- [4- (diethylamino)butanoylamino]propyi]01 -(3~pentylocty!) heptanedioate

[0246] Step 1 :DI PEA, DCMMT48 MT49

[0247] To a solution of 4-(diethylamino)butanoic acid (1.4 g, 8.79 mmol, 1 eq) in DCM (14 mL) was added EDC1 (2.02 g, 10.55 mmol, 1.2 eq), DMAP ( 107.42 mg, 879.26 prnol, 0.1 eq) and DIPEA (2.84 g, 21.98 mmol, 3.83 mL, 2.5 eq) under Nz atmosphere. Then 1,3- bis[[tert-butyl(dimethyi)silyl]oxy]propan-2-amine (2.81 g, 8.79 mmol, 1 eq) was added to the above reaction mixture under Nz 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 Na?.SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate=50 / lto 1 / 1). Compound N-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymeihyl]ethyl]-4(diethylammo)butanamide (1.3 g, 2.82 mmol, 32.08% yield) was obtained as a colorless oil.

[0248] Step 2:MT49 INT50

[0249] To a solution of N-[2-[tert-butyI(dimetfiyl)silyI]oxy-l-[[tert- butyl(dimethyl)silyi]oxymethyl]ethyl]-4-(dietliylamino)butanamide (1.3 g, 2.82 mmol,1 eq) in THF (13 mL) was added pyridinejhydrofluoride (798.78 mg, 5.64 mmol, 726.16 pL, 70% purity, 2 eq) dropwise at 0 °C under Nz atmosphere. The reaction mixture was stirred at 20 °C for 2 hr under Nz atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOz, Dichloromethane / Methano%50 / 1 to 5 / 1 ). Compound 4-(diethylamino)-N-[2-hydroxy-l- (hydroxymethyl)ethyl]butanamide (0.3 g, 1.29 mmol, 45.73 % yield, 99.9% purity) was obtained as a colorless oil.lH NMR (400 MHz, D2O) 8 ppm 4.00 ■■ 3.90 (m, 1H), 3.70 3.60(m, 2H), 3.59 - 3.52 (m, 2H), 2.60 - 2.49 (m, 4H), 2.49 - 2.41 (m, 2H), 2.25 (t, J - 7.2 Hz,2H), 1.83 - 1.64 (m, 2H), 0.98 (t, J = 7.2 Hz, 6H).[025(1] Step 3:

[0251] To a solution of 4-(diethylamino)-N-[2-hydroxy-l-(hydroxymethyr)ethyl]butanamide (0.3 g, 1.29 mmol, 1 eq) in DCM (3 mL) was added EDCI (297.06 mg, 1.55 mmol, 1.2 eq), DIPEA (417.24 mg, 3.23 mmol, 562.32 mL,2.5 eq) and DMAP (15.78 mg, 129.13 0.1 eqμ)m, tohle, n a solution of 7-oxo~7~(3- pentyloctoxy)heptanoic acid (265.38 mg, 774.80 ymol, 0.6 eq) in DCM (3 ml.,) was added to die above reaction mixture dropwise under Nr. atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N?. 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 NarSOr, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography(SiO?., Dichloromethane / Methanol=50 / 1 to 5 / 1). Compound O7-[2-[4-(diethylamino)butanoylami-no]-3-hydroxy-propyl] Ol-(3-pentyloctyl)heptanedioate (0.2 g,359.19 μmol 2,7.82% yield) was obtained as a colorless oil.

[0252] Step 4:

[0253] To a solution of 4,4-bis[(Z)-oct-5-enoxy]butanoic acid (407.67 mg, 1 .20 mmol, 1 eq) in THE ( 6 mL) was added TEA (181.73 mg, 1.80 mmol, 249.97 μL, 1.5 eq) and 2,4,6-trichlorobenzoyl chloride (292.02 mg, 1.20 mmol, 187.07 uL, 1 eq) at 0 °C under Nr atmosphere. The reaction mixture was stirred at 25 °C for 2 h under Nr atmosphere. Then DMAP (14.63 mg, 119.73 0μ.m1 o elq,) and O7-[2-[4- (diethylamino)butanoylamino]-3- hydroxy-propyl] Ol -(3-pentyloctyl) heptanedioate (0.6 g, 1.08 mmol, 0.9 eq) was added to die above reaction under Nr atmosphere. The reaction mixture was stirred at 25 °C for 10 h under Nr atmosphere. The reaction mixture was diluted with HrO (50 mL) and extracted with DCM 200 mL (100 ml., * 2). The combined organic layers were dried over NarSOr, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate=100 / l to 0 / 1).Compound 07■ [3 ~[4,4~bis [(Z)-oct-5 -enoxy] butanoyloxy] -2 ■ [4~ (diethylamino)butanoylamino]propyl]Ol-(3-pentyloctyl) heptanedioate (0.5 g, 568.64 pmol, 23.75% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 8 ppm 7.30 (d, J = 7.6 Hz, 1H), 5.45 - 5.27 (m, 4H), 4.52 ■ 4.47 (m, 1H), 4.46 ■■ 4.38 (m, 1H), 4.28 ■■ 4.20 (in, 2.H), 4.19 - 4.12 (m, 2H), 4.08 (t, J = 7.2 Hz, 2H), 3.61 - 3.54 (m. 2H), 3.45 - 3.37 (m, 2H), 3.08 (s, 4H), 2.98 (s, 2H), 2.54 (t, J - 6.0 Hz, 2H), 2.47 - 2.40 (m, 2H), 2.39 - 2.34 (m, 2H), 2.33 - 2.27 (m, 2H), 2.11 - 2.02 (m, 8H), 1.96 - 1.89 (m, 2H), 1.67 - 1.55 (m, 10H), 1.43 - 1.35 (m, 12H), 1.33 ■■ 1.23 (m, 18H), 0.96 (t, J - 7.6 Hz, 6H), 0.89 (t, J - 7.0 Hz, 6H).Example 10 - Synthesis of Compound 9: 07-[3-[4,4-bis[(Z)-oct-5-enoxy]buta-noyIoxy]- 2-[3-(diethy!amino)propoxycarbony!amino]propyl] Ol~(3~pentyloetyl) heptanedioate

[0254] &iep 1 . MTU MT52

[0255] To a solution of l,3-bis[[tert-butyl(dimethyl)siiyl]oxy]propan-2-amine (50 g, 156.43 mmol, 1 eq) in DCM (500 ml) was added (4-nitrophenyl) carbonochloridate (63.06 g, 312.86 mmol, 2 eq) and Py. (24.75 g, 312.86 mmol, 25.25 mL, 2. eq) at 0 °C under N2 atmosphere. The reaction mixture was stirred at 0 °C for 1 hr under N2 atmosphere. The reaction mixture was poured into H2O (500 mL) and extracted with DCM (200 mLx3). The combined organic layers were washed with brine (200 mLx2), dried over NarSOi, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Dichloromethane / Methanol^SO / l to 5 / 1).Compound (4-nitrophenyl) N-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymethyl]ethyl]carbamate (45 g, 92.83 mmol, 59.34% yield) was obtained as a colorless oil.

[0256] Step 2:

[0257] To a solution of(4-nitrophenyl) N-[2-[tert-butyl(dimethyl)silyl]oxy-l -[[tert- butyl(dimethyi)silyl]oxymethyi]ethyl]carbamate (45 g, 92.83 mmol, 1 eq) in DCM (400 ml..) was added DIPEA (24.00 g, 185.67 mmol, 32.34 mL, 2 eq) and 3-(diethylamino)propan-l-ol (36.54 g, 278.50 mmol, 41.53 mL, 3 eq) under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was poured into H2O (300mL) and extracted with DCM (200 mLx3). The combined organic layers were washed with brine (50 mLx2), dried over Na2.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=3 / l to DCM: MeOH = 10: 1). Compound 3- (diethylamino)propyl N-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymethyl]ethyl]carbamate (15 g, 31.46 mmol, 34.09% yield) was obtained as a yellow oil.

[0258] Step 3:

[0259] To a solution of 3 -(diethyl amino)propyl N-[2-[tert-butyl(dimethyl)silyl]oxy-l- [[tert-butyl(dimethyl)silyl]oxymethyl]etbyl]carbamate (0.5 g, 1.05 mmol, 1 eq) in T HF (50 mL) was added pyridine hydrofluoride (371.14 mg, 2.62 mmol, 337.40 μL, 70% purity,2.5 eq) at 0 °C under bh atmosphere. The reaction mixture was stirred at 25 °C for 1 h under N2 atmosphere. The mixture was evaporated under reduced pressure and the residue was purified by column chromatography (SiOa, Petroleum ether / Ethyl acetate=3 / l to DCM:MeOH 10:1). Compound 3-(diethylamino)propyl N-[2-hydroxy-l-(hydroxymethyl)ethyl]carbamate (100 mg, 402.71 38.46% yielμdm) o wla, s obtained as a yellow oil.

[0260] Step 4:

[0261] To a solution of 3-(dietbylamino)propyl N-[2 -hydroxy- 1 -(hydroxymethyl)ethyl]carbamate (0.1 g, 402.71 1.6 eq) in DμmCMol, (10 mL) was added DIPEA (81.32 mg, 629.23 .nmol, 109.60 μL, 2.5 eq), EDCI (57.90 mg, 302.03 1.2 eq) μmol, and DMAP (3.07 mg, 25.17 0μ.1m eoql), under Nz atmosphere. 7-oxo-7-(3- pentyloctoxy)heptanoic acid (86.21 mg, 251.69 nmol, 1 eq) was added to the above reaction mixture under Nz atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was poured into H?.O (50 mL) and extracted with DCM (50 mLx3). The combined organic layers were washed with brine (50 mLx2), dried over NazSOi, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO?, DCM: MeOH = 3:1). Compound O7-[2-[3- (diethylammo)propoxycarbonylamino] -3 -hydroxy-propyl] 01 -(3 -pentyloctyl) heptanedioate (300 mg, 523.73 μm 60o.l0,0% yield) was obtained as a colorless. ’H NMR (400 MHz, CDC13) 8 ppm 5.84 (d, J - 7.8 Hz, 1 H ), 4.29 - 4.00 (m, 6H), 3.94 - 3.86 (m, 1H), 3.69 (d, J - 3.0 Hz, 2H), 3.48 (s, 1H), 3.06 ■■ 2.94 (m, 5H), 2.41 ■■ 2.26 (m, 4H), 2.06 (s, 2H), 1.72 ■ 1.51 (m, 6H), 1.43 - 1.22 (m, 24H), 0.88 (t, J = 6.8 Hz, 6H).

[0262] Step 5:

[0263] To a solution of O7-[2-[3-(diethylamino)propoxycarbonylamino]-3-hydroxy- propyl] Ol-(3-pentyloctyl) heptanedioate (300 mg, 523.73 1 eq) in DCMμm (1o0l, mL) was added EDCI (120.48 mg, 628.48 nmol, 1.2 eq), DIPEA (169.22 mg, 1.31 mmol, 228.06) pL, 2.5 eq) and DMAP (6.40 mg, 52.37 ymol, 0.1 eq) under N2 atmosphere. Then 4,4-bis[(Z)-oct- 5-enoxy]butanoic acid ( 178.33 mg, 523.73 1 eq) waμsm adodl,ed to the above reaction mixture. The reaction mixture was stirred at 20 °C for 12 hr under N?. 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 NarSOs, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOz, Dichloromethane / MethanomSO / l to 5 / 1). Compound O7-[3-[4,4- bis[(Z)-oct-5-enoxy]buta-noyloxy]-2-[3-(diethylamino)propoxycarbonylamino]propyl] 01- (3-pentyloctyl) heptanedioate (103 mg, 115.05 4.68% yielμdm) wola,s obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 5 ppm 5.44 ■■ 5.27 (m, 4H), 5.10 (s, 1H), 4.49 (t, J = 5.4 Hz, 1H), 4.25 - 4.07 (m, 9H), 3.63 - 3.54 (m, 2H), 3.46 - 3.38 (m, 2H), 2.70 (s, 4H), 2.42 (t, J - 7.4 Hz, 2H), 2.36 - 2.28 (m, 4H), 2.09 - 2.00 (m, 8H), 1.97 - 1.86 (m, 4H), 1.69 - 1.55 (m, 10H), 1.46 - 1.37 (m, 6H), 1.34 - 1.20 (m, 18H), 1.13 (s, 6H), 0.96 (I. J === 7.4 Hz, 6H), 0.89 (t, J = 6.8 Hz, 6H).Example 11 - Synthesis of Compound 10: 07-[3-[4,4-bis[(Z)-oct~5-enoxy]butaBoyloxy]~2~[(l~ethyl-3-piperidyl)methoxycarbonylamino]propyl] Ol~(3~pentyloctyl) heptanedioate

[0264] Step 1: ™

[0265] To a solution of l-benzyloxy-3-[tert-butyl(dimethyl)silyl]oxy-propan-2 -amine (4 g, 13.54 mmol, 1 eq) in DCM (100 mL) was added (4-nitrophenyl) carbonochloridate (5.46 g, 27.07 mmol, 2 eq) and Py. (2.14 g, 27.07 mmol, 2.19 mL, 2 eq) dropwise at 0 °C under Nz atmosphere. The reaction mixture was stirred at 20 °C for 30 min under N?. atmosphere. The mixture was evaporated under reduced pressure. The crude product was poured into petroleum ether (20 mL) at 20 °C and stirred for 30 min. Then it was filtered and the filtrate was evaporated under reduced pressure. Compound (4-nitrophenyl)N-[l-(benzyloxymethyl)-2-[tert-butyl(dimethyl)silyl]oxy-ethyl]carbamate (6 g, 13.03 mmol, 48.12% yield) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCb) 8 ppm 8.33 - 8.13 (m, 2H), 7.42 - 7.25 (m, 7H), 4.57 (s, 2H), 4.02 - 3.90 (m, 1H), 3.87 - 3.81 (m, 1H), 3.78 - 3.67 (m, 2H), 3.65 - 3.55 (m, 1H), 1.00 - 0.81 (m, 9H), 0.21 - 0.03 (m, 6H).

[0266] Step 2:8NT56 !NT57

[0267] A mixture of (4-nitrophenyl) N-[ 1 -(benzyloxymethyl)-2-[tert- butyl(dimethyl)silyl]oxy-ethyl]carbamate (6 g, 13.03 mmol, 1 eq), (l-ethyl-3- piperidyl)methanol (4.66 g, 32.57 mmol, 2.5 eq), DMAP (318.29 mg, 2.61 mmol, 0.2 eq) and Py. (2.06 g, 26.05 mmol, 2.10 mL, 2 eq) in DCM (50 ml) was stirred at 20 °C for 12 hr under Nr 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 NarSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, DCM / MeOH=100 / lto 10 / 1).Compound ( l-ethyl-3-piperidyl)methyl N-[ 1 -(benzyloxymethyl)-2-[tert - butyl(dimethyl)silyl]oxy-ethyl]carbamate (4 g, 8.61 mmol, 66.08% yield) was obtained as a yellow oil.

[0268] Step 3:MT57 SNT58

[0269] A mixture of (l-ethyl-3-piperidyl)methyl N-[l-(benzyloxymethyl)-2-[tert- butyl(dimethyl)silyl]oxyethyl]carbamate (4 g, 8.61 mmol, 1 eq) and N,N-diethylethanamine trihydrofluoride (2.78 g, 17.21 mmol, 2.81 mL, 2 eq) in THF (40 mL) was stirred at 20 °C for 12 hr under Nr atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. Compound (l-ethyl-3-piperidyl)methyl N-[l-(benzyloxymethyl)-2-hydroxy- ethyl]carbamate (2 g, crude) was obtained as a colorless oil.

[0270] Step 4:

[0271] A mixture of (l-ethyl-3-piperidyl)methyl N-[l -(benzyloxymethyl)-2-hydroxy- ethyljcarbamate (2 g, 5.71 mmol, 1 eq), 7-oxo-7-(3-pentyloctoxy)heptanoic acid (2.54 g, 7.42 mmol, 1.3 eq), EDO (1.31 g, 6.85 mmol, 1.2 eq), DIPEA (1.84 g, 14.27 mmol, 2.49 ml, 2.5 eq) and DMAP (84.58 mg, 570.69 jrmol, 0.1 eq) in DCM (80 mL) was stirred at 20 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with H2O (2.00 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 (SiO?., DCM / MeOH= 100 / 1 to 10 / 1).Compound O7-[3-benzyloxy-2-[(l-ethyl-3-piperidyl)methoxycarbonylamino]propyl] Ol-(3-

[0273] To a suspension of Pd / C (315.34 mg, 296.32 10% purμitmy,o 0l,.1 eq) in MeOH(2.0 mL) was added O7-[3-benzyloxy-2.-[(l-etbyi-3-piperidyl)methoxycarbonylamino]propyl]Ol-(3-pentyloctyl)heptanedioate (2 g, 2.96 mmol, 1 eq) under Ar atmosphere. The suspension was degassed under vacuum and purged with H?. several times. The reaction mixture was stirred under H2 (50 Psi) at 80 °C for 24 h. The reaction mixture was filtered andthe filtrate was concentrated to get a residue. The residue was purified by column chromatography (SiCh, DCM / MeOH= 100 / 1 to 10 / 1 ). Compound O7-[2-[(l-ethyl-3- piperidyl)met-hoxycarbonylammo]-3-hydroxy-propyl]Ol-(3-penty4octyl) heptanedioate (0.8 g, 1.37 mmol, 46.16% yield) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCI3) 5 ppm 5.21 (d, J - 6.0 Hz, IH), 4.22 (d, J = 5.8 Hz, 2H), 4.16 - 3.97 (m, 4H), 3.95 - 3.83 (m, 2H), 3.75 ■■ 3.59 (m, 2H), 3.07 - 2.92 (m, 2H), 2.50 (d, J = 6.4 Hz, 2H), 2.39 ■■ 2.30 (in, 4H), 2.12 - 1.94 (m, 3H), 1.85 - 1.54 (m, 10H), 1.45 - 1.25 (m, 18H), 1.14 (t, J = 7.0 Hz, 3H), 1.07 - 0.96 (m, 1 H), 0.89 (t, J - 6.8 Hz, 6H).

[0274] Step 6:Compound 10

[0275] A mixture of O7-[2-[(l -ethyl-3-piperidyl)methoxycarbonylamino]-3-hydroxy- propyl] 01 -(3 -pentyloctyl) heptanedioate (0.5 g, 854.96 1 eq), 4,4-bis[(μZm)-oolc,t-5- enoxy]butanoic acid (349.33 mg, 1.03 mmol, 1.2 eq), EDCI (196.68 mg, 1.03 mmol,1.2 eq), DIPEA (276.24 mg, 2.14 mmol, 372.29 μL, 2.5 eq) and 4-pyrrolidin-l-ylpyridine (25.34 mg, 170.99 μ 0m.2o el,q) in DCM (20 mL) was stirred at 20 °C for 12 hr under N?. atmosphere. The reaction mixture was diluted with HzO (100 mL) and extracted with DCM 200 mL (100 mL * 2). The combined organic layers were dried over NacSOn filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM / MeOH= 100 / 1 to 10 / 1). Compound O7-[3- [4 ,4 -bis [(Z)-oct- 5 -enoxy] butanoyloxy] -2 [( 1 -ethyl - 3 piperidyl)methoxycarbonylamino]propyl] Ol -(3-pentyloctyl) heptanedioate (0.16 g, 176.35 pmol, 20.63% yield) was obtained as a colorless oil. ‘H NMR (400 MHz, MeOD) 6 ppm 5.46 - 5.27 (m, 4H), 5.08 - 4.97 (m, 1H), 4.79 - 4.69 (m, 1H), 4.52 (t, J = 5.4 Hz, 1H), 4.15 - 4.04 (m, 6H), 3.82 - 3.76 (m, 2H), 3.64 ■■ 3.54 (m, 2H), 3.52 - 3.40 (m, 2H), 3.29 - 3.21 (m, 4H),2.86 - 2.79 (m, 1H), 2.60 (d, J - 6.8 Hz, 6H), 2.44 - 2.33 (m, 8H), 2.1 1 - 2.02 (m, 8H), 1.90 (d, J = 5.4 Hz, 2H), 1.83 - 1.80 (m, 4H), 1.67 - 1.62 (m, 4H), 1.57 (d, J - 8.4 Hz, 4H), 1.46 - 1.41 (m, 6H), 1.29 (s, 12H), 0.96 (t, J = 7.6 Hz, 6H), 0.92 - 0.88 (m, 9H).Example 12 - Synthesis of Compound 11: [7-[3~[7-(2”butyloctanoyloxy)heptanoyIoxy]-2- [4"(2-pyrroIidin-l- ylethylcarbamoyloxy)decanoylamino]propoxy]-7-oxo-heptyl] 2- butyloctanoate

[0276] Synthetic scheme:

[0277] To a solution of l-[3-[[2-hydroxy-l-(hydroxymetbyl)ethyl]amino]-3-oxo- propyljheptyl N-(2-pyrrolidin-l-ylethyl)carbamate (2 g, 4.98 mmol, 1 eq) in DCM (20 ml.,) was added EDCI (1.15 g, 5.98 mmol, 1.2 eq), DIPEA (1.61 g, 12.45 mmol, 2.17 mL, 2.5 eq) and DMAP (121.70 mg, 996.17 qmol, 0.2 eq) under N2 atmosphere, then a solution of 7- (2- butyloctanoyloxy)heptanoic acid (1.31 g, 3.98 mmol, 0.8 eq) in DCM (20 mL) was added to the above reaction mixture dropwise under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under bb atmosphere. The reaction mixture was diluted wdth H2O (100 mL) and extracted with DCM 200 mL (100 ml. * 2). The combined organic layers were dried over NaeSO-t, filtered and the filtrate was concentrated under reduced pressure to give a residue.The residue was purified by column chromatography (SiCh, Dichloromethane / Methanol^SO / l to 5 / 1). Compound [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2-[4-(2-pyrrolidin-l~ ylethylcarbamoyloxy)decanoylamino]propoxy]-7-oxo-heptyl] 2 -butyloctanoate (0. 1 g, 98.26% purity) was obtained as a colorless oil. Tl NMR (400 MHz, CDCb) § ppm 6.77 (d, J= 7.6 Hz, HI), 5.70 (s, 1H), 4.72 (d, J 4.4 Hz, 1H), 4.54 - 4.41 (m, 1H), 4.25 - 3.97 (m,8H), 3.48 - 3.22 (m, 2H), 2.81 - 2.53 (m, 5H), 2.39 - 2.14 (m, 8H), 1.97 - 1.75 (m, 5H), 1.72 -1.53 (m, 13H), 1.50 - 1.14 (m, 46H), 0.98 - 0.72 (m, 15H).Example 13 - Synthesis of Compound 12: O'l,Ol-(2-(((3-(diethylamino)propoxy)carbonyl)amino)propane-l,3”diyl) 7-bis(3-peiityloctyl) diheptanedioate

[0278] Synthetic scheme:Compound 12

[0279] T o a solution of 3-(diethylammo)propyl N-[2-hydroxy-l- (hydroxymethyl)ethyl]carbamate (0.1 g, 402.71 1.6 eq) in DμmCMol, (10 ml) was added DIPEA (81.32 mg, 629.23 nmol, 109.60 tiL, 2.5 eq), EDCI (57.90 mg, 302.03 1.2 eq) μmol, and DMAP (3.07 mg, 25.17 0. μ1m eqo)l, under N2 atmosphere. 7-oxo-7-(3- pentyloctoxy)heptanoic acid (86.21 mg, 251.69 1 eq) was aμdmdoeld, to the above reaction mixture under N?. atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N2 atmosphere. 'The reaction mixture was poured into H2O (50 ml.,) and extracted with DCM (50 mLx3). The combined organic layers were washed with brine (50 mL.x2), dried over NazSCb, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO?., DCM: MeOH = 3:1). Compound OT,Ol-(2-(((3- (dietbylamino)propoxy)carbonyl)amino)propane-l,3-diyl) 7 -bis(3 -pentyloctyl) diheptanedioate ( 100 mg, 99.28% purity) was obtained as a colorless oil.!H NMR (400 MHz, CDCb) 5 ppm 5.01 (d, J = 5.0 Hz, HI), 4.23 - 4.18 (m, 2H), 4.15 - 4.05 (m, 8H), 2.63 - 2.47 (m, 6H), 2.36 - 2.28 (m, 8H), 1.86 - 1.73 (m, 2H), 1.70 - 1.55 (m, 12.H), 1.40 - 1.24 (m, 38H),1.04 (t, J - 7.0 Hz, 6H), 0.89 (t, J - 7.0 Hz, 12H).Example 14 - Synthesis of Compound 13: 07-[2-[4-(dimethyIainino)butanoylamiiio]-3“ [7-oxo-7-(3~ pentyloctoxy Jheptanoyl] oxypropyl] Ol~(3-pentyloctyl) heptanedioate

[0280] Synthetic scheme:

[0281] To a solution of 4-(dime±ylamino)-N-[2-hydroxy-l- (hydroxymethyl)ethyl]butanaraide (5.5 g, 26.93 mmol, 2.46 eq) in DCM (55 ml.) was added EDCI (2.52 g, 13.14 mmol, 1.2 eq), DIPEA (3.54 g, 27.37 mmol, 4.77 mb, 2.5 eq) and DMAP (133.76 mg, 1.09 mmol, 0. i eq) under N?. atmosphere. Then a solution of 7-oxo-7-(3- pentyloctoxy)heptanoic acid (1.87 g, 5.47 mmol, 0.5 eq) in DCM (55 mL) was added to the above reaction mixture dropwise under N2 atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N? 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 NaeSCh, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, dichlorometbane / methano>50 / l to 5 / 1 ). Compound O7-[2-[4-(dimethylamino)butanoylamino]-3-[7-oxo-7-(3- pentyloctoxy)heptanoyl]oxypropyl] Ol-(3-pentyloctyl) heptanedioate (0.1 g, 93.81% purity) was obtained as a colorless oil.!H NMR (400 MHz, CDCb) 8 ppm 7.18 (d, J = 8.2 Hz, 1H), 4.48 - 4.39 (m, 1H), 4.25 - 4.14 (m, 4H), 4.08 (t, J - 7.2 Hz, 4H), 2.89 (s, 2H), 2.68 (s, 6H), 2.41 (t, J = 6.6 Hz, 2H), 2.38 ■■ 2.27 (m, 8H), 2.06 - 1.97 (m, 2H), 1.69 ■ 1.54 (m, 12H), 1.40 ■■ 1.22 (m, 38H), 0.89 (t, J = 6.8 Hz, 12H).Example 15 - Synthesis of Compound 14: 07-[3-[7-oxo-7-(3- pentyloctoxy)heptanoyl]oxy-2~(4-pyrroHdin-l~ylbutanoylamino)propyl] Ol~(3~ pentyloctyl) heptanedioate

[0282] Synthetic scheme:Compound 14

[0283] To a solution of N -[2-hydroxy- 1 -(hydroxymethyl)ethyl]-4-pyrro lidin- 1 -yl- butanaraide (0.5 g, 2.17 mmol, 1.6 eq) in DCM (30 mL) was added DIPEA (438.43 mg, 3.39 mmol, 590.87 μL, 2.5 eq), EDCI (312.15 mg, 1.63 mmol, 1.2 eq) and DMAP ( 16.58 mg, 135.69 qmol, 0.1 eq) under Mr atmosphere. Then 7-oxo-7-(3-pentyloctoxy)heptanoic acid (464.76 mg, 1.36 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 poured into H2O (10 mL) and extracted with DCM (10 mLx3). The combined organic layers were dried over NazSCX filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, DCM:MeOH = 6:1). Compound O7-[3-[7-oxo-7-(3-pentyloctoxy)heptanoyl]oxy-2-(4- pyrrolidin-l-ylbutanoylamino)propyl] Ol-(3-pentyloctyl) heptanedioate (100 mg, 98.73% purity) was obtained as a colorless oil.lH NMR (400 MHz, CDCb) 8 ppm 7.40 (d, 8.4Hz, 1H), 4.47 - 4.37 (m, 1H), 4.29 - 4.23 (m, 2H), 4.17 - 4.11 (m, 2H), 4.07 (t, J - 7.2 Hz, 4H), 3.24 (s, 2H), 3.06 (t, J -- 6.6 Hz, 2H), 2.58 - 2.51 (m, 2H), 2.44 - 2.34 (m, 4H), 2.33 - 2.27 (m, 4H), 2.18 ■ 2.07 (m, 6H), 1.68 ■ 1.55 (m, 12H), 1.43 ■■ 1.22 (m, 40H), 0.89 (t, 7.0Hz, 12H).Example 16 - Synthesis of Compound 15: 01-[3-[7-(l-heptyIoctoxy)-7-oxo- heptanoyl]oxy~2-(4-pyrrolidin-l~ylbutanoy!amino)-propyl] O7~(l-hejrtyloetyl) heptanedioate

[0284] Step 1:WT13 ifm4

[0285] To a solution of 2 -aminopropane- 1,3 -diol (20 g, 219.52 mmol, 1 eq), DMAP(268. 18 mg, 2.20 mmol, 0.01 eq) and TEA (88.85 g, 878.07 mmol, 122.22 mL, 4 eq) in DCM (100 mL) was added TBSC1 (82.72 g, 548.80 mmol, 67.52 mL, 2.5 eq) in DCM (50 mL) under Nz atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under Nz atmosphere. The reaction mixture was diluted with H2O ( 1000 mL) and extracted with DCM 2000 mL (1000 mL * 2). The combined organic layers were dried over NazSCri, 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=20 / l to 0 / 1).Compound l,3-bis[[tert-bulyl(dimethyl)silyl]oxy]propan-2 -amine (40 g, 125.14 mmol, 57.01% yield) was obtained as a colorless oil.

[0286] Step 2:INT29

[0287] To a solution of 4-pyrrolidin-l-ylbutanoic acid (4 g, 20.65 mmol, 1 eq, HC1) in DCM (150 mL) was added DIPEA (6.67 g, 51.63 mmol, 8.99 mL, 2.5 eq), EDCi (4.75 g, 24.78 mmol, 1 .2 eq) and DMAP (2.52.33 mg, 2.07 mmol, 0.1 eq) under Nz atmosphere. Then l,3-bis[[tert-butyl(dimethyl)silyl]oxy]propan-2 -amine (6.60 g, 20.65 mmol, 1 eq) was added to the above reaction mixture under Nz atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under Nz atmosphere. The reaction mixture was poured into HzO (50mL) and extracted with DCM (50 mLx3). The combined organic layers were washed with brine (50 mLx2), 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=3 / 2 to 0 / 1). Compound N-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyI]oxymethyl]ethyl]-4-pyrrolidin-l-yl-butanamide (8 g, 17.44 mmol,42.22% yield) was obtained as a yellow oil.lH NMR (400 MHz, CDCI3) 6 ppm 6.08 (d, J 8.4 Hz, 1H), 4.02 - 3.91 (m, 1H), 3.78 - 3.69 (m, 2H), 3.59 - 3.51 (m, 2H), 2.54 - 2.42 (m, 6H), 2.24 (t, J = 7.4 Hz, 2H), 1.88 - 1.72 (m, 6H), 0.89 (s, 18H), 0.05 (d, J = 2.2 Hz, 12H).

[0288] Step 3:

[0289] To a solution of N-[2-[tert-butyl(dimethyl)silyl]oxy-l-[[tert- butyl(dimethyl)silyl]oxymethyl]etiiyl]-4-pyrrolidin-l-yl-butanamide (8 g, 17.44 mmol, 1 eq) in THF (80 mL) was added pyridine hydrofluoride (4.94 g, 34.87 mmol, 4.49 mL, 70% purity, 2 eq) at 0 °C under N?. atmosphere. The reaction mixture was stirred at 25 °C for 2 hr under Nr atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOz, DCM:MeOH =1 / 0 to 0 / 1). Compound N-[2-hydroxy-l-(hydroxymethyl)ethyl]-4-pyrroiidin-l-yi-butanamide (1 .8 g, 7.82 mmol, 44.78% yield) was obtained as a yellow oil. ‘H NMR (400 MHz, CDCI3) 8 ppm 7.55 (d, J = 7.6 Hz, 1 H), 5.02 (s, 2H), 4.03 - 3.88 (m. 1H), 3.78 - 3.62 (m, 4H), 2.82 - 2.62 (m, 6H), 2.34 (t. J = 7.0 Hz, 2H), 2.00 - 1.73 (m, 6H).

[0290] Step 4:

[0291] To a solution of pentadecan-8-one (25 g, 110.43 mmol, 1 eq) in MeOH (250 mL) was added NaBILj (8.36 g, 220.85 mmol, 2 eq) slowly at 0 °C under Nz atmosphere. The reaction mixture was stirred at 20 °C for 2 hr under N?. atmosphere. The reaction mixture was added sat. NH4CI slowly at 0 °C under Nr atmosphere, diluted with H2O (300 ml..) and extracted with EtOAc 600 ml, (200 mL * 3). The combined organic layers were dried over NazSCU, 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 / l to 0 / 1). Compound pentadecan-8-ol (20 g, 87.56 mmol, 79.29% yield) was obtained as a colorless oil.

[0292] Step 5:

[0293] 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 Nr 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 H2O (100 mL) and extracted with DCM 300 mL (100 mL. * 3). The combined organic layers were dried over Na2SCh, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1).Compound 7-(l-heptyloctoxy)-7-oxo-heptanoic acid (2 g, 5.40 mmol, 24.66% yield) was obtained as a colorless oil.

[0294] Step 6:Compound 15

[0295] To a solution of 7-(l-heptyloctoxy)-7-oxo-heptanoic acid (1.5 g, 4.05 mmol,1.50 eq) in DCM (15 mL) was added EDCI (621.52 mg, 3.24 mmol, 1.2 eq), N-[2-hydroxy-l- (hydroxymethyl)ethyl]-4-pyrrolidin-l-yl-butanamide (0.28 g, 1.22 mmol, 0.45 eq), DMAP (4.00 mg, 27.02 umol, 0.01 eq) and DiPEA (872.96 mg, 6.75 mmol, 1.18 mL, 2.5 eq) under Nz atmosphere. The reaction mixture was stirred at 20 °C for 12 hr under N?. 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 NarSCM, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by columnchromatography (SiOz, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound Ol-[3-[7-(l- heplyloctoxy)-7-oxo-heptanoyl]oxy-2-(4-pyrrolidin-l-ylbutanoylamino)-propyl] O7-(l- heptyloctyl) heptanedioate (105 mg, 112.25 4.15% yieμldm)o wl,as obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 5 ppm 7.01 (d, J - 8.4 Hz, 1H), 4.91 - 4.82 (m, 2H), 4.51 - 4.41 (m, 1H), 4.24 - 4.07 (m, 4H), 2.84 - 2.49 (m, 6H), 2.37 - 2.27 (m, 10H), 1.91 - 1.84 (m, 6H), 1.75 ■■ 1.57 (m, 10H), 1.51 (d, J - 5.4 Hz, 8H), 1.42 ■ 1.25 (m, 42H), 0.88 (t, J - 6.8 Hz, 12H).Example 17 - Synthesis of Compound 16: 01-[3-[7-(l-hept-6-enyloct-7-enoxy)-7-oxo- heptanoyl]oxy-2“(4-pyrrolidin-l-ylbutaiioylamino)propyl]07“(l“hept-6-enyloet-7-enyI) heptanedioate

[0296] Step 1:

[0297] To a suspension of Mg (1.44 g, 59.29 mmol, 1.05 eq) in THF (60 mL) was addedI2 ( 143.33 mg, 564.71 prnol, 113.75 μL, 0.01 eq) under N?. atmosphere and then the reaction mixture was stirred at 45 °C for 0.5 h under N2 atmosphere. Then 7-bromohept-l-ene (10 g, 56.47 mmol, 1 eq) was added to the above reaction mixture dropwise at 60 °C for 0.5 h under Nz 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 NazSCL, 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 H?O (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 wzas diluted with H2O (20 mL) and extracted with EtOAc (3* 150 mL). 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 (SiCb, Petroleum ether / Ethyl acetate=l / O to 20 / 1). Compound pentadeca- 1 , 14-dien-8-ol (7.5 g, 33.43 mmol, 19.73% yield) was obtained as a colorless oil.lH NMR (400 MHz, CDCI3) 8 ppm 5.91 - 5.74 (m, 2.H), 5.13 - 4.77 (m, 4H), 3.68 - 3.54 (m, 1 II ), 2.12 - 1.98 (m, 4H), 1.47 - 1.31 (m, 16H).

[0298] Step 2:SNT65 SNT66

[0299] To a solution of pentadeca- l,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, 111.42 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 N2 atmosphere. The reaction mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was diluted with I-I2O (200 mL) and extracted with DCM 500 mL (250 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 (SiCh, Petroleum ether / Ethyl acetate=l / () 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.lH NMR (400 MHz, CDCI3) 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).

[0300] Step 3:Compound 18

[0301] To a solution of 7-( 1 -hept-6-enyloct-7-enoxy)-7-oxo-heptanoic acid (0.8 g, 2. 18 mmol, 5.65e-l eq) in DCM (12 mL) v / as added DIPEA (1.25 g, 9.65 mmol, 1.68 mL, 2.5 eq), N-[2-hydroxy-l-(hydroxymethyl)ethyl]-4-pyrrolidin-l-yl-butanamide (0.4 g, 1.74 mmol, 0.45 eq), 4-pyrrolidin-l-ylpyridine (57.20 mg, 385.96 0.1 eq) aμnmdo El,DCI (887.88 mg, 4.63 mmol, 1.2 eq) under N?. atmosphere. The reaction mixture was stirred at 25 °C for 12 hrunder N? atmosphere. The reaction mixture was diluted with H2O (lOOrnL) and extracted with DCM 150 mb (50 mb * 3). 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 (SiCh, Petroleum ether / Ethyl acetate=l / 0 to 0 / 1 ). Compound 01 -[3- [7 -( 1 -hept-6-enyloc t-7-enoxy)-7 -oxo-heptanoyl]oxy-2-(4-pyrrolidin- l-ylbutanoylamino)propyl]O7-(l-hept-6-enyloct-7-enyl) heptanedioate (0.1 1 g, 118.62 jtmol, 3.07% yield) was obtained as a colorless oil.lH NMR (400 MHz, CDCI3) § ppm 7.31 (d, J = 7.6 Hz, 1H), 5.80 (m, 4H), 5.01 (m, 2H), 5.00 - 4.90 (m, 6H), 4.86 (m, 2H), 4.47 - 4.40 (m, 1H), 4.24 - 4.12 (m, 4H), 3.01 (s, 4H), 2.88 (t, J - 5.8 Hz, 2H), 2.48 (t, J = 6.8 Hz, 2H), 2.36 (in, 4H), 2.29 (t, J = 7.6 Hz, 4H), 2.08 ■■ 1.98 (m, 14H), 1.64 m, 8H), 1.55 ■ 1.48 (m, 8H), 1.40 - 1.34 (m, 12H), 1.33 - 1.25 (m, 16H).Example 18 - Synthesis of Compound 17: [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2- [4“(2“pyrrolidin-l-ylethylcarbainoyloxy)octanoylainino]propoxy]-7-oxo-heptyl] 2- butyloctanoate

[0302] Step 1: !NT67 WT68

[0303] 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 the next step directly without further purification.

[0304] Step 2: WT6§ 9MT69

[0305] To a solution of 4-hydroxyoctanoyloxy sodium (25 g, 147.38 mmol, 1 eq) inDMSO (250 mb) was added BnBr (25.20 g, 147.38 mmol, 18.91 mb, 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 mb) and extracted with ethyl acetate 800 mb(400 mL * 2). The combined organic layers were dried over Na?.SO4, 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.

[0306] Step 3:

[0307] 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 fillrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether / Ethyl acetate=100 / l to 10 / 1). Compound benzyl 4-(4-nitrophenoxy)carbonyloxyoctanoate (12.5 g, 31.81 mmol, 21.94% yield) was obtained as a yellow oil.

[0308] Step 4:W70 SNT71

[0309] A mixture of benzyl 4-(4-nitrophenoxy)carbonyloxyoctanoate (12.5 g, 31.81 mmol, 1 eq), 2-pyrrolidm-l-ylethanamine (7.26 g, 63.62 mmol, 2 eq), DMAP (389 mg, 3.18 mmol, 0.1 eq) and DIPEA (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 (SiCh, Petroleum ether / Ethyl acetate= 100 / 1 to 1 / 1). Compound benzyl 4-(2-pyrro lidin- l-ylethylcarbamoyloxy)octanoate (3.75 g, 9.60 mmol, 31.90% yield) was obtained as a yellow oil.

[0310] Step 5:

[0311] 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 prnol, 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 (SiCh, Dichloromethane / Methanom 100 / 1 to 10 / 1). Compound 4 -(2-pyrrolidin-l- ylethylcarbamoyloxy)octanoic acid (1.25 g, 3.74mmol, 43.43% yield) was obtained as a brown oil. 1 H NMR (400 MHz, CDC13) 6 ppm 7.51 - 7.33 (m, 1 H), 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, 1 H), 1.38 - 1.23 (m, 4H), 0.93 - 0.81 (m, 3H). Boc

[0312] Step 6:^774

[0313] A mixture of tert-butyl N-[2 -hydroxy- 1 -(hydroxymethyl)ethyl]carbamate (10 g,52.29 mmol, 1 eq), KOH (10.86 g, 193.49 mmol, 3.7 eq), BnBr (33.09 g, 193.49 mmol, 22.98 mL, 3.7 eq) in DMF (TOO mL) 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 2.00 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 (S1O2, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound tert - butyl N-[2-benzyloxy-l-(benzyioxymethyl)ethyl]carbamate (6 g, 16.15 mmol, 30.89% yield) was obtained as a white solid.

[0334] Step 7: INT74 1^175

[0315] A mixture of tert-butyl N-[2-benzyloxy-l-(benzyloxymethyl)ethyl]carbamate (6 g,16.15 mmol, 1 eq) in HCl / EtOAc (60 mL) was stirred at 25 °C for 12 h under N?. atmosphereThe reaction mixture was concentrated under reduced pressure to give a residue. Compound l,3-dibenzyloxypropan-2-anrine (3 g, 11.06 mmol, 68.45% yield) was obtained as a white solid and it was used in next step directly without further purification.

[0316] Step 8:WT75 MT78

[0317] A mixture of l,3-dibenzyIoxypropan-2 -amine (3 g, 11.06 mmol, 1 eq), 4-(2- pyrrolidin-l-ylethylcarbamoyloxy)octanoic acid (2.99 g, 9.95 mmol, 0.9 eq), EDCI (2.54 g.13.27 mmol, 1.2. eq), DMAP (135.06 mg, 1.11 mmol, 0.1 eq) and DIPEA (3.57 g, 27.64 mmol, 4.81 mL, 2.5 eq) in DCM (30 mL) was stirred at 25 °C for 12 h 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 NaiSCM, 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^ 100 / 1 to 0 / 1). Compound 1 -[3-[[2- benzyloxy- 1 -(benzy loxymethyl)ethyl]amino] -3 -oxo-propyl]penty 1 N - (2-pyrrolidin- 1 ylethyl)carbamate (3 g, 5.42 mmol, 49.00% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDC13) 5 ppm 7.27 - 7.21 (m, 10H), 6.24 (d, J = 8.2 Hz, 1H), 5.46 (s, 1H), 4.67(d, J = 3.0 Hz, 1H), 4.29 - 4.19 (m, 1H), 4.09 - 4.00 (m, 1H), 3.61 - 3.55 (m, 2H), 3.51 - 3.43 (m, 2H), 3.29 - 3.14 (m, 2H), 2.59 - 2.41 (ra, 6H), 2.13 (d, J = 6.6 Hz, 2H), 1.97 (s, 1H), 1.91- 1.79 (m, 2H), 1.72 (s, 4H), 1.52 - 1.36 (m, 2H), 1.28 - 1.13 (m, 6H), 0.83 - 0.77 (m, 3H).

[0318] Step 9:

[0319] To a suspension of Pd / C (1.73 g, 1.63 mmol, 10% purity, 0.3 eq) in MeOH (60 mL) was added l-[3-[[2-benzyloxy-l-(benzyloxymethyl)ethyl]amino]-3-oxo-propyi]pentyl N-(2-pyrrolidin-l -ylethyl)carbamate (3 g, 5.42 mmol, 1 eq) under Ar atmosphere. Thereaction mixture was stirred at 25 °C for 12 h under H2 (15 Psi). The reaction mixture was filtered and the fillrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Dichlororaethane / Methanol=100 / 1 to 10 / 1). Compound l-[3-[[2-hydroxy-l-(hydroxymethyr)ethyl]amino]-3-oxo-propyl]pentyl N- (2-pyrrolidin-l-yleth-yl)carbamate (0.6 g, 1.61 mmol, 29.65% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 7.27 ■■ 7.16 (m, 1H), 6.20 (s, 1H), 4.79 ■ 4.68 (m, 1H), 4.48 - 4.1 1 (m, 2H), 3.97 - 3.86 (m, 3H), 3.81 - 3.74 (m, 2H). 3.64 - 3.51 (m, 1H), 3.34 - 3.23 (m, 1H), 2.95 (s, 4H), 2.88 - 2.68 (m, 1H), 2.40 - 2.26 (m, 2H), 2.10 - 2.03 (m, 1H), 1.99 ( d. J - 16.2 Hz, 4H), 1.84 - 1.74 (m, 1 H), 1.67 - 1.49 (m, 2H), 1.30 (s, 4H), 0.89 (t, J === 6.8 Hz, 3H).

[0320] Step 10:

[0321] A mixture of l-[3-[[2-hydroxy-l-(hydroxymethyl)ethyl]araino]-3-oxo- propyl]pentyl N-(2-pyrrolidin-l-ylethyl)carbamate (409.32 mg, 1.10 mmol, 0.45 eq), 7-(2- butyloctanoyloxy)heptanoic acid (INT9, 0.8 g, 2.44 mmol, 1 eq), EDCI (560.25 mg, 2.92 mmol, 1.2 eq), DMAP (29.75 mg, 243.54 0.1 eqμ)m anodl, DIPEA (786.90 mg, 6.09 mmol, 1 .06 mL, 2.5 eq) in DCM (8 mL) was stirred at 25 °C for 12 h under Nr atmosphere. The reaction mixture was diluted with HrO (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over NarSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, 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-butyloctanoyloxy)heptanoyfoxy]-2-[4-(2-pyrrolidin- lylethylcarbamoyloxy)octaaoylamino]propoxy]-7-oxo-heptyl] 2 -butyloctanoate (0.15 g, 150.84 μmol 6,.19% yield) was obtained as a colorless oil. 1H NMR (400 MHz, CDCI3) 8 ppm 6.64 - 6.37 (m, 1H), 5.37 - 4.92 (m, 1H), 4.71 - 4.61 (m, 1 H), 4.42 - 4.35 (m, 1 H), 4.13 - 4.02 (m, 4H), 3.99 (t, J = 6.6 Hz, 4H), 3.28 - 3.17 (m, 2H), 2.57 - 2.47 (m, 4H), 2.29 - 2.22 (m, 6H), 2.18 ■ 2.14 (m, 2H), 1.81 ■■ 1.69 (in, 6H), 1.63 ■■ 1.49 (m, 18H), 1.39 ■■ 1.33 (m, 4H), 1.31 - 1.26 (m, 8H), 1.24 - 1.17 (m, 26H), 0.83 - 0.79 (m, 15H).Example 19 - Synthesis of Compound 18: [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2- [4-(2-pyrroIidin-l-ylethyicarbamoyloxy)hexanoylamino]propoxy]-7-oxo-heptyI] 2- butyloctanoate

[0323] A mixture of 5-ethyltetrahydrofuran-2-one (20 g, 175.24 mmol, 20.00 mL, 1 eq) and NaOH (7.36 g, 184.00 mmol, 1.05 eq) in H2O (100 mL) was stirred at 100 °C for 12 h under N?. atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. Compound 4-hydroxyhexanoyloxysodium (2 / g, 175.17 mmol, 99.97% yield) was obtained as a white solid. ‘H NMR (400 MHz, CD3OD) 6 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).

[0324] Step 2:

[0325] To a solution of 4-hydroxyhexanoyloxysodium (27 g, 175.17 mmol, 1 eq) inDMSO (270 mL) was added BnBr (29.97 g, 175.17 mmol, 20.82 mL, 1 eq) under N?. 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 NarSCd, 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 / l to 20 / 1 ). Compound benzyl 4-hydroxyhexanoate (35 g, 125.97 mmol, 71.91% yield, 80% purity) was obtained asa colorless oil. *H NMR (400 MHz, CDCb) 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).

[0326] Step 3:INT80 INT81

[0327] 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 al 0 °C under N? atmosphere. The reaction mixture was stirred at 25 °C for 2 h under N?. 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 (SiO?., Petroleum ether / Ethyl acetate=100 / l 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, CDCb) 3 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, 1H), 1.75 ■■ 1.50 (m, 2H), 1.03 ■ 0.99 (m, 3H).

[0328] Step 4:

[0329] 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 DIPEA (12.5 g, 96.80 mmol, 16.86 mL, 3 eq) and DMAP (394.19 mg, 3.22 mmol, 0. 1 eq) under N?. atmosphere. The reaction mixture was stirred at 25 °C for 12 h under Nr 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 NarSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate= 100 / 1 to 1 / 1). Compoundbenzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)hexaaoate (6.5 g, 14.31 mmol, 53.9% yield, 78% purity) was obtained as a yellow oil.NMR (400 MHz, CDCI3) 6 ppm 7.55-7.15 (m, 5H), 5.12 (s, 2H), 4.77-4.62 (m, 1 H), 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, 1 1.2 Hz, 2H), 0.90 (t, J - 7.2 Hz, 3H).

[0330] Step 5:

[0331] To a solution of benzyl 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)hexanoate (6.5 g, 17.93 mmol, 1 eq) in THF (100 mb) 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 (Sith, Dichloromethane / Methanoml 00 / 1 to 10 / 1). Compound 4-(2-pyrrolidm-l- ylethylcarbamoyloxy)hexanoic acid (5 g, 17.71 mmol, 94.37% yield, 96.436% purity) was obtained as a brown oil.lH NMR (400 MHz, CDCI3) 6 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).

[0332] Step 6:

[0333] To a solution of 1 ,3-dibenzyloxypropan-2 -amine (13.5 g, 49.75 mmol,1.1 eq) in DCM (258 mL) was added EDCI (10.40 g, 54.27 mmol, 1.2 eq), DMAP (552.54 mg, 4.52 mmol, 0.1 eq), 4-(2-pyrrolidin-l-ylethylcarbamoyloxy) hexanoic acid (12.32 g, 45.23 mmol, 1 eq) and DIPEA (14.61 g, 1 13.07 mmol, 19.69 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 H?.O (300 mL) and extracted with DCM 800 mL (400 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 (SiO?., Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound [4-[[2- benzyloxy- 1 -(benzyloxymethyl)etbyl]ammo] - 1 -ethyl -4-oxo-butyl] N -(2-pyrro lidin- 1 - ylethyl)carbamate (10 g, 13.51 mmol, 29.86% yield, 71% purity) was obtained as a colorless oil.1H NMR (400 MHz, CDCb) 6 ppm 7.26 - 7.20 (m, 10H), 6.34 (d, J - ----- 8.0 Hz, 1H), 5.65 (s, 1H), 4.63 ■■ 4.56 (m, 1H), 4.45 (s, 4H), 4.28 ■ 4.22 (m, 1H), 3.60 ■■ 3.56 (in, 2H), 3.51 ■■ 3.47 (m, 2H), 3.39 (s, 4H), 2.67 (s, 6H), 2.19 - 2.1 1 (m, 2H), 1.79 (s, 4H), 1.54 - 1.45 (m, 2H), 0.81 H.. / 7.4 Hz. 3H).

[0334] Step 7:

[0335] To a solution of [4-[[2-benzyloxy- 1 -(benzyloxymethyl)ethyl]aniino]-l -ethyl-4- oxo-butyl] N-(2-pyrrolidin-l-ylethyl)carbamate (2.5 g, 4.76 mmol, 1 eq) in MeOH (50 mL) was added Pd / C (1.52 g, 1.43 mmol, 10% purity, 0.3 eq) under Ar atmosphere. The reaction mixture was stirred at 80 °C for 12 h under H2 (50 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 (SiO?., Petroleum ether / Ethyl acetate= 100 / 1 to 10 / 1). Compound [ 1 -ethyl -4- [ [2 -hydroxy- 1 -(hydroxymethy l)ethyl ] amino] - 4-oxo-butyl]N-(2-pyrrolidin-i-ylethyl)carbamate (1 g, 2.89 mmol, 60.87% yield) was obtained as a colorless oil.

[0336] Step 8:

[0337] To a solution of 7-(2-butyloctanoyloxy)heptanoic acid (0.8 g, 2.44 mmol,1 eq) in DCM (11 mL) was added EDCI (560.25 mg, 2.92 mmol, 1.2 eq), DMAP (29.75 mg, 243.54 umol, 0.1 eq), [l-ethyl-4-[[2-hydroxy-l-(hydroxymethyl)ethyl]amino]-4-oxo-butyl] N-(2-pyrrolidin-1 -ylethyl)carbamate (336.51 mg, 974.17 gmol, 0.4 eq) and DIPEA (786.90 mg, 6.09 mmol, 1.06 mL, 2.5 eq) under N?. atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N?. atmosphere. The reaction mixture was diluted with HzO (50 mL) and extracted with DCM 80 mL (40 ml., * 2). The combined organic layers were dried over NarSOr, 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= 100 / 1 to 0 / 1). Then it was purified by prep-HPLC (column: X-Select CSH Phenyl- Hexyl 100*30 5u; mobile phase: [H2O (0.04%HCl)-ACN: THF~1:1J; gradient: 30%-70% B over 12.0 min). Compound [7-[3-[7-(2 -butyloctanoy loxy)heptanoyloxy]-2-[4-(2-pyrrolidin- 1- ylethylcarbamoyloxy)hexanoylamino]propoxy]-7-oxo-heptyl] 2- butyloctanoate (0.15 g, 413.92 μmol 1,7.00% yield, HC1 salt) was obtained as a colorless oil.lH NMR (400 MHz, CDCh) 6 ppm 11.90 (s, 1H), 7.44 (d, J- 8.4 Hz, 1H), 6.28 (s, 1H), 4.64 (d, J- 5.8 Hz, TH), 4.48 (d, J - ---- 7.6 Hz, 1H), 4.19 - 4.12 (m, 4H), 4.08 - 4.04 (m, 4H), 3.95 - 3.76 (m, 3H), 3.46 - 3.19 (m, 3H), 2.93 ■ 2.81 (m, 2H), 2.42 ■ 2.25 (m, 121- 1), 2.14 - 1.98 (m, 3H), 1.81 ■■ 1.74 (m, 1H), 1.66 - 1.57 (m, 14H). 1.47 - 1.35 (m, 12H). 1.32 - 1.23 (m, 24H). 0.91 - 0.86 (m, 15H).Example 20 -- Synthesis of Compound 19: -[6-(2-butyloctanoyIoxy)hexanoyloxy]-2-[4-(2“ pyrrolidin-lylethylcarbamoyloxy)decanoylamino]propoxy]“6-oxo-hexyl] 2~ butyloctanoate

[0338] Step l :IINT86 INT87

[0339] 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 EDCI (21.5 g, 112.32 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 Na?.SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by columnchromatography (SiO?., Petroleum ether / Ethyl acetate= 100 / 1 to 5 / 1). Compound 6- hydroxyhexyl 2-butyloctanoate (15.2 g, 50.58 mmol, 67% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 6 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, 611 ). 1.31 - 1.21 (m, 1211 ). 0.93 - 0.83 (m, 6H).

[0340] Step 2:1NT87 55088

[0341] To a solution of 6-hydroxyhexyl 2-butyloctanoate (15.2 g, 50.58 mmol, 1.0 eq) in MeCN (75 mL) and H2O (75 mL) was added TEMPO (791 mg, 5.06 mmol, 0.1 eq) and (acetoxy(phenyl)-iodanyl] 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 N? 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 (SiO?., Petroleum ether / Ethyl acetate=100 / l to 8 / 1). Compound 6-(2- butyl octanoy loxy)hexanoic acid (16.0 g, 50.88 mmol, 72% yield) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 6 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, 12H), 0.93 ■■ 0.84 (m, 6H).Compound 19

[0343] A mixture of 6-(2-butyioctanoyloxy)hexanoic acid (0.8 g, 2.54 mmol, 1 eq), l-[3- [[2 -hydroxy- 1 -(hydroxymethyl)ethyl]ammo]-3-oxo-propyl]heptyl N -(2-pyrrolidm- 1 - ylethyl)carbamate (408.61 mg, 1.02 mmol, 0.4 eq), EDCI (585.24 mg, 3.05 mmol,1.2 eq), DMAP (31.08 mg, 254.41 0.1 μ emq)o aln, d DIPEA (822.00 mg, 6.36 mmol, 1.11 mL, 2.5 eq) in DCM (8 ml.,) was stirred at 25 °C for 12 h under Nc 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 NazSCX 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 it was purified by prep-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H2O (0.04%HCl)-ACN: THF-l:!]; gradient: 35%-75% B over 14.0 min). Compound [6-[3-[6-(2- butyIoctanoyloxy)hexanoyloxy]-2-[4-(2-pyrrolidin-1 ylethylcarbamoyloxy)decanoylammo]propoxy]-6-oxo-hexyl] 2-butyloctanoate (0.15 g, 144.05 umoi, 5.66% yield, 99% purity, HC1 salt) was obtained as a colorless oil. ^-I NMR (400 MHz, CDCI3) 6 ppm 12.40 ■■ 12.30 (m, 1H), 6.34 (t, J- 5.4 Hz, 1H), 5.85 (d, J - 8.4 Hz, 1H), 4.61 - 4.54 (m, 1H), 4.47 - 4.43 (m, 1H), 4.2.3 - 4.19 (m, 2H), 4.07 (t, J = 6.8 Hz, 6H), 3.90 - 3.85 (m, 2H), 3.69 - 3.65 (m, 2H), 3.24 (d, J - 5.4 Hz, 2H), 2.84 (d, J - 9.6 Hz, 2H), 2.36 - 2.31 (m, 6H), 2.30 ■■ 2.25 (m, 2H), 2.20 ■■ 2. 15 (m, 2H), 2.10 (d, J - 4.6 Hz, 2H), 2.02 - 1 .97 (m, 1H), 1.89 (d, J = 4.0 Hz, 1H), 1 .79 (d, J = 5.4 Hz, 2H), 1.71 (d, J = 1.2 Hz, 2H), 1 .65- 1.61 (m, 10H), 1.57 (d, J- 8.2 Hz, 4H), 1.47 - 1 .43 (m, 4H), 1.38 - 1.36 (m, 8H), 1.26 (d, . / - 3.4 Hz, 22H ), 0.91 - 0.86 (m, 15 H ).Example 21 - Synthesis of Compound 20: [8-[3-[8“(2-butyIoctaiioyIoxy)octanoyloxy]-2- [4-(2-pyrroHdin-l-yk,thykarbam0yloxy)decan0y-!amino]propoxy]-8~oxo-octyI] 2- butyloctanoate

[0344] Step 1 :

[0345] 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 Nr. atmosphere. The reaction mixture was stirred at 25 °C for 12 h under N?. 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 (SiOa, Petroleum ether / Ethyl acetate=100 / l 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.

[0346] Step 2:

[0347] To a solution of 8-hydroxyoctyl 2-butyloctanoate (15 g, 44.72 mmol, 1 eq) in MeCN (75 mL) and H2O (75 mL) 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 NarSOr, 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^ 100 / 1 to 1 / 1). Compound 8-(2- butyloctanoyloxyjoctanoic acid (7.5 g, 19.91 mmol, 43.61% yield, 90.94% purity) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCI3) 5 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 (rn, 20H), 0.92 - 0.85 (rn, 6H).

[0349] A mixture of 8-(2-butyioctanoyloxy)octanoic acid (0.8 g, 2.34 mmol, 1 eq), l-[3- [[2 -hydroxy- 1 -(hydroxymethyl)ethyl]ammo]-3-oxo-propyl]heptyl N -(2-pyrrolidm- 1 - ylethyl)carbamate (375.15 mg, 934.27 0.4 eq),μ EmDoCl,I (537.30 mg, 2.80 mmol,1.2 eq), DMAP (28.53 mg, 233.57 pmol, 0.1 eq) and DIPEA (754.67 mg, 5.84 mmol, 1.02 mL, 2.5 eq) in DCM (8 ml) was stirred at 25 °C for 12 h under Nr atmosphere. The reaction mixture was diluted with H?O (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over NazSCX filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, 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: [HrO (0.04%HCl)-ACN: THF=1:1]; gradient: 35%-75% B over 14.0 min). Compound [8-[3-[8-(2- butyloctanoyloxy)octanoyloxy]-2-[4-(2-pyrrolidin-l-yiethylcarbamoyloxy)decanoy- lamino]propoxy]-8-oxo-octyl] 2-butyloctanoate (0.15 g, 142.78 grnol, 6.1 1% yield, HC1 salt) was obtained as a colorless oil.SH NMR (400 MHz, CDCI3) 8 ppm 12.04 (s, 1H), 7.25 (d, J = 8.6 Hz, 1H), 6.26 (d, J = 4.4 Hz, 1H), 4.75 - 4.66 (m, 1H), 4.53 - 4.45 (m, 1H), 4.20 - 4.13 (m, 4H), 4.08 (t, J - 6.8 Hz, 4H), 3.91 (d, J = 7.0 Hz, 2H), 3.84 - 3.76 (m, 1H), 3.43 (d, J ----- 15.0 Hz, 1H), 3.37 - 3.28 (m, 1H), 3.22 (s, 1H), 2.93 - 2.81 (m, 2H), 2.39 - 2.26 (m, 10H), 2.15 ■ 2.09 (m, 2H), 2.02 ■■ 1.97 (m, 1H), 1.85 ■■ 1.75 (m, 2H), 1.65 ■■ 1.59 (m, 12H), 1.48 ■■ 1.43 (m, 4H), 1.37 - 1.25 (m, 44H), 0.92 - 0.87 (m, 15H).Example 22 - Synthesis of Compound 21: [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2-[methy !-[4-(2-pyrrolidin-l -yleth ylcar-bamoyloxy)deeanoyl] amino] propoxy] -7-oxo- heptyl] 2-butyloctanoate

[0350] Step 1: §NT74 WT92

[0351] To a solution of NaOH (538.36 mg, 13.46 mmol, 1 eq) in THF (50 mL) was added a solution of tert-butyl N-[2-benzyloxy-l-(benzyloxymethyl)ethyl]carbamate (5 g, 13.46 mmol, 1 eq) in THF (160 mL) dropwise at 0 °C under Nr atmosphere. After addition, the reaction mixture was added Mel (2.10 g, 14.81 mmol, 921.73 μL, 1.1 eq) dropwise at 0 °C under Nr atmosphere. The reaction mixture was stirred at 25 °C for 12 h under Nr atmosphere. The reaction mixture was diluted with HrO (TOO mL) and extracted with Ethyl acetate 200 mL (100 mL * 2). The combined organic layers were dried over NarSCL, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate=100 / 1 to 10 / 1). Compound tert-butyl N-[2-benzyloxy-l-(benzyloxy-methyl)ethyl]-N-methyl-carbamate (2.5 g, 6.49 mmol, 48.18% yield) was obtained as a white solid.

[0352] Step 2’ 8HTS2 1NT93

[0353] A mixture of tert-butyl N-[2-benzyloxy-l-(ben2yloxymethyl)ethyl]-N-methyl- carbamate (2.5 g, 6.49 mmol, 1 eq) in HCl / EtOAc (50 mL) was stirred at 25 °C for 5 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate=20 / l to 1 / 1). Compound l,3-dibenzyloxy-N-methyl-propan-2-amine (1.7 g, 5.96 mmol, 91.86% yield) was obtained as a white solid.

[0354] Step 3:

[0355] A mixture of 4-(2-pyrrolidin-l-ylethylcarbamoyloxy)decanoic acid (2.53 g, 7.71 mmol, 1.1 eq), l,3-dibenzyloxy-N-methyl-propan-2-amine (2 g, 7.01 mmol, 1 eq), EDCI (1.61 g, 8.41 mmol, 1.2 eq), DIPEA (4.53 g, 35.04 mmol, 6.10 mL, 5 eq) and DMAP (171.23 mg, 1.40 mmol, 0.2 eq) in DCM (40 ml) was stirred at 25 °C for 12 h under bh 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 (SiCh, Petroleum ether / Ethyl acetate^ 100 / 1 to 0 / 1 ). Compound l-[3-[[2- benzyloxy-l-(benzyloxymethyl)ethyl]-methyl-amino]-3-oxo-propyl]heptyl N-(2-pyrrolidin- l-ylethyl)carbamate (2 g, 3.36 mmol, 47.90% yield) was obtained as a colorless oil.

[0356] Step 4:

[0357] A suspension of l-[3-[[2-benzyloxy-l -(benzyloxymethyl)ethyl]-methyl-amino]-3- oxo-propyl]heptyl N-(2-pyrrolidin-l-ylethyl)carbamate (2 g, 3.36 mmol, 1 eq), Pd / C (3.57 g, 3.36 mmol, 10% purity, 1 eq) in MeOH (20 mL) was stirred at 25 °C for 24 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 (SiCh, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound l-[3-[[2-hydroxy-l- (hydroxymethyl)ethyl]-methyl-ammo]-3-oxo-propyl]heptyl N-(2-pyrro lidin- 1- ylethyl)carbamate (1 g, 2.41 mmol, 71.69% yield) was obtained as a colorless oil.fH NMR (400 MHz, CDCI3) 8 ppm 4.85 ■■ 4.72 (m, 1H), 4.11 ■■ 3.67 (m, 8H), 3.43 ■■ 3.21 (m, 2H), 3.03 (s, 2H), 2.84 (s, 1H), 2.77 (s, 1H), 2.70 - 2.56 (m, 4H), 2.51 - 2.43 (m, 1 H), 2.35 - 2.25 (m.H i ). 2.11 - 2.02 (m, 1H), 1.90 - 1.69 (m, 5H), 1.62 - 1.49 (m, 2H), 1.28 (d, J - 9.4 Hz, 8H),0.87 (t, J - 6.4 Hz, 3H).

[0359] A mixture of 7-(2-butyloctanoyloxy)heptanoic acid (1.41 g, 4.28 mmol, 1 eq), 1- [3-[[2 -hydroxy- l~(hydroxymethyl)ethyl]~methyl-amino]-3-oxo-propyl]heptyl N-(2- pyrrolidin-l-ylethyl)carbamate (0.8 g, 1.93 mmol, 0.45 eq), EDC1 (984.10 mg, 5.13 mmol, 1 .2 eq), DIPEA (1 .38 g, 10.69 mmol, 1.86 mL, 2.5 eq) and DMAP (52.26 mg, 427.80 gmol, 0. 1 eq) in DCM (20 mL) was stirred at 25 °C for 12 h under Nz 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 NazSOr, 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 / 1 to 0 / 1). Then it was purified by prep-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [HzO (0.04%HCl)-ACN: THF= 1: 1]; gradient: 35%-75% B over 14.0 min). Compound [7-[3-[7-(2- butyloctanoyloxy)heptanoyloxy]-2-[methyl-[4-(2-pyrrolidin-l-ylethylcar- bamoyloxy)decanoyl]amino]propoxy]-7-oxo-heptyl] 2-butyloctanoate (0.15 g, 144.72 pmol, 3.38% yield, HC1 salt) was obtained as a colorless oil.!H NMR (400 MHz, CDCb) 8 ppm 12.50 - 11.61 (m, 1 H), 6.69 - 6.08 (rm 1H), 4.98 - 4.76 (m, 1H), 4.38 - 4.11 (m, 4H), 4.08 - 4.04 (m, 4H), 3.88 (d, J - 4.6 Hz, 2H), 3.71 ■■ 3.61 (m, 1H), 3.26 (s, 2H), 2.97 (s, 1H), 2.83 (s, 2H), 2.48 - 2.21 (m, 15H), 2.15 - 1.91 (m, 3H), 1.85 - 1.74 (m, 1H), 1.66 - 1.55 (m, 14H), 1 .45 - 1 .24 (m, 46H), 0.90 - 0.86 (m, 15H).Example 23 - Synthesis of Compound 22: [7-[3-[7-(2-butyloctanoyloxy)heptanoyloxy]-2- [ [3-(2-pyrrondin-1 -ylethykar bamoyloxy)cydo-pentanecarbon yl] amino] propoxy] -7-oxo- heptyl] 2-butyloctanoateINT96

[0360] Step 1 :

[0361] To a solution of benzyl 3 -oxocyclopentanecarboxylate (10 g, 45.82 mmol, 1 eq) inTHF (100 mL), toluene (25 mL) and H2O (50 mL) was added NaBIL (8.67 g, 229.10 mmol, 5 eq) portion wise at 0 °C under N2 atmosphere. The reaction mixture was stirred at 0 °C for1 h under N2 atmosphere. The reaction mixture was quenched by addition of HC1 (IM lOOmL) slowly at 0 °C under N? 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 (S1O2, Petroleum ether / Ethyl 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.

[0362]

[0363] 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 (SiOr, 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.lH NMR (400 MHz,CDCb) 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).

[0364] Step 3:

[0365] A mixture of benzyl 3-(4-nitrophenoxy)carbonyloxycyclopentanecarboxylate (10 g, 25.94 mmol, 1 eq), 2-pyrrolidin-l-ylethanamine (5.92 g, 51.90 mmol, 2 eq), DIPEA (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 (TOO mL * 2). The combined organic layers were dried over NazSCX filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate^lOO / l to 1 / 1). Compound benzyl 3-(2-pyrrolidin-l - ylethylcarbamoyloxyjcyclopentanecarboxylate (8.1 g, 22.24 mmol, 85.73% yield, 99% purity') was obtained as a yellow oil.lH NMR (400 MHz, CDCb) 8 ppm 7.45-7.27 (m, 5H), 5.13 (s, 2H), 5.10 - 5.02 (m, 1H), 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).

[0366] Step 4:

[0367] To a solution of benzyl 3-(2-pyrrol.idm-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 Ha (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 (SiCh, 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, CDCb) 8 ppm7.11-6.82 (m, 1H), 5.13 - 4.88 (m, 1 H), 3.60 - 3.32 (m, 2H), 3.29 - 2.82 (m, 6H), 2.78 - 2.54(m, 1H), 2.41 - 1.51 (m, 10H).

[0368] Step 5:

[0369] A mixture of l,3-dibenzyloxypropan-2-atnine (1.5 g, 5.53 mmol, 1 eq), 3-(2- pyrro lidin- 1 -ylethylcarbamoyloxy)cyclopentanecarboxylic acid (1.49 g, 5.53 mmol,1 eq), EDO (1.27 g, 6.63 mmol, 1 .2 eq), DMAP (67.53 mg, 552.78 0. 1 eq) and DμiPmEoAl, (1.79 g, 13.82 mmol, 2.41 mL, 2.5 eq) in DCM (15 mL) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with HrO (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 (S1O2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1).Compound [3-[[2-benzyloxy-l-(benzyloxymethyl)ethyl]carbamoyl]cyclopentyl] N-(2- pyrrolidin-l-ylethyl)carbamate (0.7 g, 1.34 mmol, 24.18% yield) was obtained as a colorless oil.SNT191 ^T162

[0371] To a suspension of Pd / C (548.70 mg, 515.60 10% purμitym,o 0l,.3 eq) in MeOH (18 mL) was added [3-[[2-benzyloxy-l-(benzyloxymethyl)ethyl]carbamoyl]cyclopentyl] N- (2-pyrroIidin-l-yIethyl)carbamate (0.9 g, 1.72 mmol, 1 eq) under Ar atmosphere. The rnixtur was stirred at 80 °C for 12 h under H2 (50 Psi). The reaction mixture was filtered and thefiltrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Dichloromethane / MethanoM 100 / 1 to 1 / 1). Compound [3- [[2 -hydroxy- 1 -(hydroxymethyl)ethyl]carbanioyl]cyclopentyl] N-(2-pyrrolidin- 1 -ylethyl)car- bamate (0.45 g, 1.31 mmol, 76.24% yield) was obtained as a coiorless oil.

[0372] Step 7:

[0373] A mixture of 7-(2-butyloctanoyloxy)heptanoic acid (0.8 g, 2.44 mmol, 1 eq), [3- [[2-hydroxy-l-(hydroxymethyl)ethyl]carbamoyl]cyclopentyl] N-(2-pyrrolidin-l- ylethyl)carbamate (334.55 mg, 974.17 prnol, 0.4 eq), EDC1 (560.25 mg, 2.92 mmol,1 .2 eq), DMAP (29.75 mg, 243.54 ymol, 0.1 eq) and DIPEA (786.90 mg, 6.09 mmol, 1 .06 mL, 2.5 eq) in DCM (8 ml) was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H?O (50 mL) and extracted with DCM 80 mL (40 mL * 2). The combined organic layers were dried over NazSO-g filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, 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 (0.04%HCl)-ACN: THF=1 : 1J: gradient: 20%-70% B over 12.0 min). Compound [7-[3-[7-(2- butylocianoyloxy)heptanoyloxy]-2-[[3-(2-pyrrolidm-l-ylethylcarbamoyloxy)cyclo- pentanecarbonyl]amino]propoxy]-7-oxo-heptyl] 2-butyloctanoate (0.15 g, 148.38 μmol, 6.09% yield, 99% purity, HC1 salt) was obtained as a colorless oil.fH NMR (400 MHz, CDCh) 8 ppm 12.22. (s, 1H), 6.38 (s, 1H), 6.17 (d, J= 8.2 Hz, 1H), 5.15 - 5.05 (m, 1 H), 4.52. - 4.43 (m, IH), 4.33 - 4.27 (m, 2H), 4.17 - 4.10 (m, 2H), 4.07 (t, J - 6.8 Hz, 4H), 3.89 (d, J- 3.4 Hz, 2H), 3.64 (d, 5.2 Hz, 2H), 3.24 (s, 2H), 2.91 - 2.78 (m, 2H), 2.72 - 2.65 (m, 1H),2.36 - 2.30 (m, 6H), 2.28 - 2.23 (m, 2H), 2.08 (s, 2H), 1.98 - 1.94 (m, 2H), 1.84 (d, J -- 11.2 Hz, 4H), 1.66 - 1.57 (m, 12H), 1.45 (d, J= 2.6 Hz, 4H), 1.39 - 1.34 (m, 8H), 1.31 - 1.22 (m, 24H), 0.91 - 0.86 (m, 12H).Example 24 - Synthesis of Compound 23: [7-[3-[7-(2-butyloctanoyIoxy)heptanoyloxy]-2- [[3-(2~pyrroiidin~l~yIethyicarbamoyioxy)eycIO"hex4mecarbonyT]amisio]propoxy]~7~oxo- heptyl] 2-butyloctanoate

[0374] Step

[0375] To a solution of 3 -oxocyclohexanecarboxylic acid (10 g, 35.17 mmol, 1 eq) in MeCN (100 mL) was added DBU (22.48 g, 147.72 mmol, 22.28 mL, 2 eq) and BnBr (13.24 g, 77.38 mmol, 9.2raL, 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 H?.O ( 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 (Sith, Petroleum ether / Ethyl acetate= 100 / 1 to 5 / 1 ). Compound benzyl 3 -oxocyclohexanecarboxylate (16 g, 68.88 mmol, 97.92% yield) was obtained as a yellow oil. fi-l NMR (400 MHz, CDCI3) 6 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).

[0377] To a solution of benzyl 3-oxocyclohexanecarboxylate (16 g, 68.88 mmol, 1 eq) in THE (160 mL), toluene (40 mL) and H2O (80 mL) was added NaBIL (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 of HC1 (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 NarSOn filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethylacetate= 100 / 1 to 3 / 1 ). Compound benzyl 3-hydroxycyclohexanecarboxylate (12.6 g, 53.78 mmol, 63.00% yield) was obtained as a yellow oil.SH NMR (400 MHz, CDCI3) 5 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).

[0378] Step 3: !NTW5 SNT106

[0379] 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 Nz 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 (SiO?., Petroleum ether / Ethyl acetate^ 100 / 1 to 3 / 1). Compound benzyl 3-(4-nitrophenoxy)carbonyloxycyclohexanecarboxylate (20 g, 50.08 mmol, 80.00% yield) was obtained as a yellow oil.!H NMR (400 MHz, CDCb) 5 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 (rn, 1 H), 1.92 ■■ 2.04 (m, 2 H), 1.37 ■■ 1.61 (in, 4 H).

[0380] Step 4:

[0381] 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 layerswere dried over Na?.SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate=100 / l 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.

[0382] Step 5:

[0383] To a solution of benzyl 3-(2-pyrrolidm-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 li under Hr (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 (SiOr, Dichloromethane / Methanol=100 / 1 to 1 / 1). Compound 3- (2-pyrrolidin-l-ylethylcarbamoyloxy)cycl-ohexanecarboxylic acid (11 g, 37.83 mmol, 83.34% yield, 97.8% purity) was obtained as a brown oil.!H NMR (400 MHz, CDCI3) 6 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- 40.8, 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, 1=11.6 Hz, 1 H), 1.16 ■■ 1.47 (in, 3 H).

[0384] Step 6:

[0385] A mixture of l,3-dibenzyIoxypropan-2 -amine (1.5 g, 5.53 mmol, 1 eq), 3-(2- pyrro lidin- 1-ylethylcarbamoyloxyjcyclohexanecarboxy lie acid (1.57 g, 5.53 mmol, 1 eq), EDCI (1.27 g, 6.63 mmol, 1.2 eq), DMAP (67.53 mg, 552.78 0,1 eq) and μ DmIPoEl,A (1.79 g, 13.82 mmol, 2.41 mL, 2.5 eq) in DCM (15 mL) was stirred at 25 °C for 12 h under Nr. atmosphere. The reaction mixture was diluted with HrO (TOO mL) and extracted with DCM 200 mL (100 mL. * 2). The combined organic layers were dried over NaiSCh, filtered and thefiltrate 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). Compound [3-[[2-benzyloxy-l-(benzyloxymethyl)ethyl]carbamoyl]cyclohexyl] N-(2-pyrrolidin-l - ylethyl)carbamate (0.8 g, 1.49 mmol, 26.92% yield) was obtained as a colorless oil.

[0386] Step 7:

[0387] To a suspension of Pd / C (593.76 mg, 557.94 prnol, 10% purity, 0.3 eq) in MeOH(20 mL) was added [3-[[2 -benzyloxy- l-(benzyloxymethyl)ethyl]carbamoyl]cyclohexyl] N- (2-pyrrolidin-l -ylethyl)carbamate (1 g, 1.86 mmol, 1 eq) under Ar atmosphere. The mixture was stirred at 80 °C for 12 h under Hr (50 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 (SiOz, Petroleum ether / Ethyl acetate=100 / l to 0 / 1). Compound[3-[[2-hydroxy-l-(hydroxymethyl)ethyl]carbamoyl]cyclohexyl] N-(2-pyrrolidin-1 - ylethyl)carb-amate (0.5 g, 1.40 mmol, 75.21% yield) was obtained as a colorless oil.

[0388] Step 8:

[0389] A mixture of 7-(2-butyloctanoyloxy) heptanoic acid (0.9 g, 2.74 mmol, 1 eq), [3- [[2 -hydroxy- 1 -(hydroxymethyl)ethyl]carbamoyl]cyclohexyl] N -(2-pyrro lidin- 1 - ylethyl)carbamate (391.74 mg, 1.10 mmol, 0.4 eq), EDCI (630.28 mg, 3.29 mmol, 1.2 eq), DIPEA (885.26 mg, 6.85 mmol, 1.19 mL, 2.5 eq) and DMAP (33.47 mg, 273.98 nmol, 0.1 eq) in DCM (9 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 NazSCL, filtered and the filtrate was concentrated underreduced pressure to give a residue. The residue was purified by column chromatography (SiO?., 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 (0.04%HCl)-ACN : THF=1:1]; gradient: 40%-80% B over 15.0 min). Compound [7-[3-[7-(2- butyloctanoyloxy)heptanoyloxy]-2-[[3-(2-pyrrolidin-l-ylethylcarbamoyloxy)cyclo- hexanecarbonyl]amino]propoxy]-7-oxo-heptyl] 2-butyloctanoate (0.15 g, 146.33 prnol, 5.34% yield, 99% purity, HC1 salt) was obtained as a colorless oil.!H NMR (400 MHz, CDCh) 5 ppm 12.27 (d, J - 1.2 Hz, 1H), 6.36 (s, 1H), 5.89 (d, J = 8.4 Hz, 1 H), 4.57 (s, 1H), 4.48 > 4.41 (m, 1H), 4.24 - 4.18 (m, 2H), 4.10 - 4.05 (m, 6H), 3.92 - 3.84 (rn, 2H), 3.69 - 3.63 (m, 2H), 3.28 ■■ 3.20 (m, 2H), 2.85 (d, J = 7.2 Hz, 2H), 2.36 - 2.30 (m, 6H), 2.29 ■ 2.24 (m, 2H), 2.21 - 2.14 (m, 2H), 2.09 (d, J - 4.2 Hz, 2H), 2.00 (s, 2H), 1.89 - 1.78 (m, 6H), 1 .65 - 1.56 (m, 12H), 1.39 - 1.34 (m, 10H), 1.31 - 1.22 (m, 24H), 0.90 - 0.86 (m, 12H).Example 25 - Synthesis of Compound 24: [7-[3~[7-(2-butyloetaiioyloxy) heptanoyloxy]- 2-[4-[2-(2-pyiTolidiii”l-ylethylcarbamoyloxy) ethyl] octanoyl amino] propoxy] -7-oxo- heptyl] 2-butyloctanoateDess-Martin1NT112

[0390] Step l :

[0391] A solution of 4-butyicyclohexanol (25 g, 160.00 mmol, 1 eq) in DCM (250 ml) was degassed and purged with N?. 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 (SiO?., 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.!H NMR (400 MHz, CDCb) 6 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).

[0392] Step

[0393] To a solution of 4-butylcyclohexanone (25 g, 162.10 mmol, 1 eq) in DCM (250 mL) was added rn-CPBA (46.05 g, 226.9 mmol, 85% purity, 1.4 eq) slowly at 0 °C. Themixture was stirred at 25 °C for 2 h under N2 atmosphere. The reaction mixture was quenched by addition of aq. NazSCh (100 mL). It was extracted with DCM 450 mL (150 mL* 3). The combined organic layers were dried over anhydrous NazSCL, 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 of8—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.lH NMR (400 MHz, CDCh) 5 ppm 4.34 - 4.23 (m, 1H), 4.20 - 4.12 (m, 1 H), 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).

[0394]

[0395] To a solution of 5-butyloxepan-2-one (27 g, 158.58 mmol, 1 eq) in H?.O (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) octanoy loxy sodium (30 g, crude) was obtained as a white solid.

[0396] Step

[0397] To a solution of 4-(2-hydroxyethyl) octanoyloxysodium (30 g, 142.68 mmol, 1 eq) in DMSO (150 mL) 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 N?. 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 NazSOq filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (1SCO®: 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, 1 14.82 mmol, 80.47% yield, 99.89% purity) was obtained as a colorless oil.!H NMR (400 MHz, CDCb) 0 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).

[0398] Step s:

[0399] 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 Nr for 3 times. 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 Nr 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 was purified 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) carbonyloxyethyl] octanoate (28 g, 61.87 mmol, 78.29% yield, 98% purity) was obtained as a colorless oil.lH NMR (400 MHz, CDCb) 8 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 (m, 2H), 1.79 ■ 1.66 (m, 4H), 1.58 - 1.46 (m, 1H), 1.40 ■■ 1.23 (in, 6H), 0.95 ■■ 0.85 (m, 3H).

[0400] Step 6:

[0401] To a mixture of benzyl 4-[2-(4-mtrophenoxy) carbonyloxyethyl] octanoate (28 g, 63.12 mmol, 1 eq) and 2-pyrrolidm-l-ylethanamine (14.4 g, 12.6.28 mmol, 2 eq) in DCM (300 mL) was added DIPEA (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 Nr 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 NarSOq 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% Ethylacetate / Petroleum ether gradient at 120 mL / rnm). Compound benzyl 4-[2-(2-pyrrolidin-l- ylethylcarbamoyloxy) ethyl] octanoate (26 g, 61.74 mmol, 97.80% yield, 99.40% purity) was obtained as a pale yellow oil.

[0402] Step 7:

[0403] To a solution of benzyl 4-[2-(2-pyrrolidin-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 to give a residue. The residue was purified by column chromatography (SiCh, DCM / MeOH =20 / 1 to 5 / 1). 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. (H NMR (400 MHz, CDCb) 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).

[0404] Step 8:

[0405] A mixture of 4-[2-(2-pyrrolidin-l-ylethylcarbamoyloxy) ethyl] octanoic acid (2 g, 6.09 mmol, 1 eq), l,3-dibenzyloxypropan-2-amine (1.69 g, 5.48 mmol, 0.9 eq, HCl salt), EDCI (1.40 g, 7.31 mmol, 1.2 eq), DIPEA (2.75 g, 21.31 mmol, 3.71 mL, 3.5 eq) and DMAP (74.39 mg, 608.93 μ 0m.1o el,q) in DCM (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 quenched by addition of water (100 mL) and extracted with DCM 150 rnL (50 mL * 3). The combined organic layers were dried over Na2SO4, filtered and tire filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate^lO / l to 0 / 1). Compound 3-[3-[[2- benzyloxy-1 -(benzyloxy methyl) ethyl] amino]-3-oxo-propyl] heptyl N-(2 -pyrro lidin- 1- ylethyl) carbamate (1.5 g, 2.58 mmol, 42.34% yield) was obtained as a colorless oil.

[0406] Step 9:

[0407] To a solution of 3-[3-[[2-benzyloxy-l -(benzyloxymethyl) ethyl] amino]-3-oxo- propyl] heptyl N-(2-pyrrolidin-l-ylethyl) carbamate (1.5 g, 2.58 mmol, 1 eq) in MeOH (30 mL) was added Pd(OH)2 (72.42 mg, 515.66 0.2 eq) uμnmdeorl, Nr atmosphere. The suspension was degassed under vacuum and purged with Hz several times. The mixture was stirred at 80 °C for 24 hours under Hr (50 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 (SiOr, Petroleum ether / Ethyl acetate^! 0 / 1 to 0 / 1). Compound 3-[3-[[2-hydroxy-l-(hydroxymethyl) ethyl] amino]-3-oxo-propyl] heptyl N-(2- pyrrolidin-l-ylethyl) carbamate (0.66 g, 1.64 mmol, 63.75% yield) was obtained as a colorless oil.

[0408] Step 10:

[0409] A mixture of 3-[3-[[2-hydroxy-l-(hydroxymethyl) ethyl] amino] -3 -oxo -propyl] heptyl N-(2-pyrrolidin-l-ylethyl) carbamate (440.06 mg, 1.10 mmol, 0.45 eq), 7-(2- butyloctanoyloxy) heptanoic acid (0.8 g, 2.44 mmol, 1 eq), EDC1 (560.25 mg, 2.92 mmol, 1.2 eq), DIPEA (786.90 mg, 6.09 mmol, 1.06 mL, 2.5 eq) and DMAP (59.51 mg, 487.08 μmol, 0.2 eq) in DCM (15 mL) was degassed and purged with Nr for 3 times. The mixture was stirred at 25 °C for 12 hr under Nr atmosphere. The reaction mixture was quenched byaddition of water (50 mL) and extracted with DCM 150 mL (50 mL * 3). The combined organic layers were dried over NazSCX 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=10 / l to 0 / 1 ) and prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um; mobile phase: [HzO(0.04%HCl)-ACN:THF=l:l]; gradient: 30 %- 70% B over 16.0 min). Compound [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-[4-[2-(2- pyrro lidin- 1 -ylethylcarbamoyloxy) ethyl] octanoyl amino] propoxy] -7 -oxo-heptyl] 2- butyloctanoate (160 mg, HC1 salt, 95.61% purity) was obtained as a colorless oil.lH NMR (400 MHz, CDCI3) 6 ppm 12.04 (s, III), 6.79 (s, III), 6.66 (d, J S.4 Hz, 1 H), 4.49 (d, >7.9 Hz, 1 H), 4.25 ■■ 3.98 (m, 10H), 3.89 (d, >4.88 Hz, 2H), 3.72 ■■ 3.54 (m, 2H), 3.24 (q, >5.17 Hz, 2H), 2.91 - 2.78 (m, 2H), 2.39 - 2.17 (m, 10H), 2.16 - 2.04 (m, 2H), 1.76 - 1 .52 (m, 22H), 1.49 - 1.11 (m, 45H), 0.90 - 0.86 (m, 15 H).Example 26 - Synthesis of Compound 25: [7-[3~[7-(2-butyloetanoyloxy) heptanoyloxy]- 2-[[4,4-dimethyi-6-(2-pyrroIidm-l-yIethyIcarbamoyIexy) hexanoyl] amino] propoxyJ-7- oxo-heptyl] 2-butyloctanoate

[0410] Step

[0411] To a solution of 4,4-dimethylcyclohexanone (40 g, 316.97 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 of aq. Na?.SOs (800 mL) slowly. It was extracted with DCM 500 ml (100 mL *5). The combined organic layers were dried over NazSCL, 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 / 1 to 0 / 1). Compound 5,5- dimethyloxepan-2-one (18 g, 126.59 mmol, 39.94% yield) was obtained as a colorless oil.

[0412] Step

[0413] 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 H?.O (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.

[0414] Step 3: SNT122 SNT123

[0415] 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). The mixture 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 NarSOn 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.

[0416] Step 4:

[0417] To a solution of benzyl 6-hydroxy-4,4-dimethyl-hexanoate (12 g, 47.94 mmol, 1 eq), (4-nitrophenyI) 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 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 (SiCh, Petroleum ether / Ethyl acetate=100 / l to 5 / 1). Compound benzyl 4,4-dimethyl-6-(4-mtrophenoxy) carbonyloxy-hexanoate (10 g, 24.07 mmol, 50.21% yield) was obtained as a colorless oil.

[0418] Step 5:

[0419] A mixture of benzyl 4, 4-dimethyl-6-(4-nitrophenoxy) carbonyloxy-hexanoate (10 g, 24.07 mmol, 1 eq), 2-pyrrolidm-l-ylethanamme (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 mt, 3 eq) in DCM (100 mL) was degassed and purged with N?. for 3 times. The mixture was stirred at 25 °C for 12 hr under Nr 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 NarSO-n filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, 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.

[0420] Step 6:

[0421] To a suspension of Pd / C (4.91 g, 4.61 mmol, 10% purity, 0.3 eq) in THE (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 (SiOr, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1). Compound 4, 4-dimethyi-6-(2-pyrrolidin-l- ylethylcarbamoyloxy) hexanoic acid (3 g, 9.99 mmol, 65.00% yield) was obtained as a colorless oil.10422 ] Step 7:

[0423] A mixture of l,3-dibenzyioxypropan-2 -amine (1.33 g, 4.91 mmol, 0.74 eq), 4,4- dimethyl-6-(2-pyrrolidin-l-ylethylcarbamoyloxy) hexanoic acid (2 g, 6.66 mmol, 1 eq), EDC1 (1.53 g, 7.99 mmol, 1.2 eq), DMAP (81.34 mg, 665.80 0.1 eq) and DμImPoElA, (2.58 g, 19.97 mmol, 3.48 mL, 3 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 NarSO-i, 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= 100 / 1 to 0 / 1). Compound [6-[[2- benzyloxy-l-(benzyloxymethyl) ethyl] amino]-3,3-dimethyl-6-oxo-hexyl] N -(2-pyrro lidin- 1- ylethyl) carbamate (1.5 g, 2.71 mmol, 40.69% yield) was obtained as a colorless oil. ’H NMR (400 MHz, CDCI3) 6 ppm 7.40 - 7.27 (m, 10H), 6.06 - 5.89 (m, 1H), 5.69 (s, 1 H), 4.52 (s, 4H), 4.36 ■■ 4.25 (m, 1H) 4.09 (t, J=7.2 Hz, 2H), 3.85 ■■ 3.69 (m, 1H), 3.67 ■■ 3.63 (m, 2H), 3.56 - 3.52 (m, 2.H), 3.33 - 3.20 (m, 2H), 2.69 - 2.39 (m. 6H), 2.17 - 2.09 (m, 2 H), 1.80 (s, 4H), 1 .60 - 1 .51 (m, 4H), 0.92 - 0.89 (m, 6H).

[0424] Step 8:

[0425] To a suspension of Pd / C (864.84 mg, 812.67 10% purμitmy,o 0l,.3 eq) in MeOH (30 mL) was added [6-[[2-benzyloxy-l-(benzyloxymeihyl) ethyl] amino]-3,3-dimethyl-6- oxo-hexyl] N-(2-pyrrolidin-l-ylethyl) carbamate (1.5 g, 2.71 mmol, 1 eq). The mixture was stirred at 80 °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 (SiOr, Petroleum ether / Ethyl acetate=100 / 1 to 0 / 1).Compound [6-[[2-hydroxy-l-(hydroxymethyl) ethyl] amino]-3,3-dimethyl-6-oxo-hexyl] N- (2-pyrrolidin-l-ylethyl) carbamate (0.8 g, 2.14 mmol, 79.07% yield) was obtained as a yellow oil.

[0426] Step 9:

[0427] A mixture of [6-[[2 -hydroxy- 1 -(hydroxymethyl) ethyl] ammo] -3, 3 -dimethyl -6- oxo-hexyl] N-(2-pyrrolidin-l-ylethyl) carbamate (363.84 mg, 974.17 umol, 0.4 eq), 7-(2- butyloctanoyloxy) heptanoic acid (0.8 g, 2.44 mmol, 1 eq), EDO (560.25 mg, 2.92 mmol, 1.2 eq), DMAP (29.75 mg, 243.54 0.1μm eqo)l, and DIPEA (786.90 mg, 6.09 mmol, 1.06 mL, 2.5 eq) in DCM (8 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 25 °C for 12 hr under N?. 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 (S1O2, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1 ) and p-HPLC (column: X-Select CSH Pheny 1-HexyI 100*30 5u: mobile phase: [H20(0.05%HC1)-ACN:THF=1:1]; gradient:40%-80% B over 14.0 min). Compound [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-[[4,4-dimethyl-6-(2-pyrrolidin-l- ylethylcarbamoyloxy) hexanoyl] amino] propoxy] -7-oxo-heptyl] 2-butyloctanoate (0.15 g, HC1 salt, 93% purity) was obtained as a colorless oil.fH NMR (400 MHz, CDCI3) 8 ppm 12.04 s, 1H), 6.84 (s, 1H). 6.67 (d. 1=5.4 Hz, 1 H), 4.49 (d, 1=5.0 Hz. 1 H), 4.25 - 4.19 (m, 2H), 4.17 - 4.00 (m, 8H), 3.89 (d, .1=5.3 Hz, 2H), 3.63 (d, J=4.4 Hz, 2H), 3.24 (q, 1=5.29 Hz, 2H), 2.88 - 2.83 (m, 2H), 2.38 - 2.19 (m, 10 H), 2.15 - 2.06 (m, 2 H), 1.94 (s, 3H), 1.66 - 1.52 (in, 16H), 1.48 ■■ 1.23 (in, 36H), 0.93 (s, 6 H), 0.90 ■ 0.86 (m, 12H).Example 27 - Synthesis of Compound 26: [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]- 2-[3~[l-[2~(2~ ylethylearbamoyloxy)ethyl]cyclohexyl]propanoylamino] propoxy]-7~oxo- heptyl] 2-butyloctanoate

[0428] Step l:sm30

[0429] To a solution of spiro [5.5] imdecan-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 N?. atmosphere. The reaction mixture was quenched by addition of aq. NaaSOs (20 mL). It was diluted with H2O (40 ml) and extracted with ethyl acetate 120 mL (40 mLx3). The combined organic layers were dried over NaiSOq filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate= 100 / 1 to 5 / 1). Compound 9-oxaspiro [5.6] dodecan- 10-one (7 g, 38.41 mmol, 93% yield) was obtained as a yellow oil.!H NMR (400 MHz, CDCI3) 8 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).

[0430]

[0431] 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 N?. atmosphere. The reaction mixture was concentrated to give a crude product. Compound 3-[l-(2-hydroxyethyl) cyclohexyl] propanoyloxysodium (8 g crude) was obtained as a white solid.

[0432]

[0433] To a solution of 3-[1 -(2-hydroxyethyl) 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 x 3). 'The combined organic layers were dried over NarSO-i, filtered and the filtrate was concentratedunder reduced pressure to give a residue. The residue was purified by column chromatography (SiO?., 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. ’H NMR (400 MHz, CDCI3) 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).

[0434] Step 4:

[0435] To a solution of benzyl 3-[l-(2-hydroxyethyl) cyclohexyl] propanoate (5.5 g, 18.94 mmol, 1 eq) and (4-nitrophenyl) carbon ochloridate (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 2.5 °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 (SiO?, Petroleum ether / Ethyl acetate= 100 / 1 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. 'H NMR (400 MHz, CDCI3) 6 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

[0437] A mixture of benzyl 3-[l-[2-(4-nitrophenoxy) carbonyloxyethyl] cyclohexyl] propanoate (6.8 g, 14.92 mmol, 1 eq), 2 -pyrro lidin- 1-yle thanamine (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 N?_ atmosphere. The mixture was concentrated under reduced pressure togive 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 (SiCh, 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.10438] Step 6:

[0439] To a solution of benzyl 3-[l-[2-(2-pyrrolidin- 1 -ylethy 1 carbamoyl oxy) ethyl] cyclohexyl] propanoate (5.3 g, 12.31 mmol, 1 eq) in THE (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 (SiOr, DCM / MeOH =100 / 1 to 5 / 1). Compound 3-[l-[2-(2-pyrrolidin-l-ylethyicarbamoyioxy) ethyl] cyclohexyl] propanoic acid (3 g, 8.22 mmol, 66.81% yield) was obtained as a yellow oil.1H NMR (400 MHz, CDCI3) 8 ppm 6.70 (s, 1H), 6.00 (s, HI), 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).

[0440] Step 7:

[0441] A mixture of l,3-dibenzyloxypropan-2 -amine (1.5 g, 4.42 mmol, 1 eq), 3-[l-[2-(2- pyrrolidin-l-ylethylcarbamoyloxy) ethyl] cyclohexyl] propanoic acid (1.51 g, 4.42 mmol, 1 eq), EDCI (2.12 g, 1 1.06 mmol, 2.5 eq), DIPEA (685.86 mg, 5.31 mmol, 924.33 p.L, 1.2 eq) and DMAP (54.03 mg, 442.23 qmol, 0.1 eq) in DCM (12 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 wasquenched by addition of H?.O ( 15 mL) and extracted with ethyl acetate 45 mL ( 15 mlx3).The combined organic layers were dried over NarSO4, filtered and die filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate^ 100 / 1 to 5 / 1). Compound 2-[l -[3-[[2- benzyloxy-1 -(benzyloxy methyl) ethyl] amino]-3-oxo-propyl] cyclohexyl] ethyl N-(2- pyrrolidin-l-ylethyl) carbamate (1.8 g, 3.03 mmol, 68.55% yield) was obtained as a yellow oil. 1 H NMR (400 MHz, CDCI3) 5 ppm 7.38 - 7.27 (m, 10H), 6.07 (d, J = 8.4 Hz, 1 H), 5.51 (s, 1 H), 4.52 (s, 4H), 4.35 - 4.27 (m, 1H), 4.07 (t, J - 6.8 Hz, 2H), 3.66 - 3.63 (m, 2H), 3.58 - 3.53 (m, 2H ), 3.26 (d, J - 5.6 Hz, 2H), 2.60 - 2.46 (m, 6H), 2.16 - 2.07 (m, 2H), 1.79 - 1.74 (m, 4H), 1.67 ■■ 1.55 (m, 4H), 1.42 (d, J - 13.6 Hz, 6H), 1.28 (d, J - 4.4 Hz, 4H).

[0442] Step 8:

[0443] To a solution of 2-[l-[3-[[2-benzyloxy-l-(benzyloxymethyl) ethyl] amino]-3-oxo- propyl] cyclohexyl] ethyl N-(2-pyrrolidin-l-ylethyl) carbamate (1.8 g, 3.03 mmol, 1 eq) in MeOH (36 mL) was added Pd / C (161.30 mg, 151.57 nmol, 10% purity, 0.05 eq) and Pd(OH)?. (106.43 mg, 151 .57 imiol, 20% purity, 0.05 eq) under Nr atmosphere. The suspension was degassed and purged with Hr for 3 times. The mixture was stirred at 60 °C for 36 hr under H2 (50 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 (SiOr, DCM / MeOH =100 / 1 to 5 / 1). Compound 2-[l-[3-[[2-hydroxy-l- (hydroxymethyl) ethyl] amino] -3 -oxo-propyl] cyclohexyl] ethyl N-(2-pyrro lidin- 1-ylethyl) carbamate (900 mg, 2.18 mmol, 56.25% yield) was obtained as a yellow oil.

[0444] Step 9:

[0445] A mixture of 2-[l-[3-[[2-hydroxy-l-(hydroxymethyl) ethyl] amino]-3-oxo~propyl] cyclohexyl] ethyl N-(2-pyrrolidin-l-ylethyl) carbamate (800 mg, 2.18 mmol, 1 eq), 7-(2- butyloctanoyloxy) heptanoic acid (1.43 g, 4.35 mmol, 2 eq), EDCI (1.00 g, 5.22 mmol, 2.4 eq), DMAP (53. 17 mg, 435.26 0.2μm eqo)l, and DIPEA (843.80 mg, 6.53 mmol, 1.14 mL, 3 eq) in DCM (8 mL) was degassed and purged with Nz for 3 times. The mixture was stirred at 25 °C for 36 hr under Nz atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with HzO (100 mL) and extracted with ethyl acetate 300 mL (100 mLx3). The combined organic layers were dried over Na?SOi, filtered and the filtrate was concentrated under reduced pressure to get a residue. The residue was purified by column chromatography (SiOz, Petroleum ether / Ethyl acetate= 100 / 1 to 1 / 4). Compound [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-[3-[l-[2-(2- ylethylcarbamoyloxy)ethyl]cyclohexyl]propanoylamino] propoxy]-7-oxo-heptyl] 2- butyloctanoate (150 mg, 90.46% purity , HC1 salt) was obtained as a yellow oil.lH NMR (400 MHz, CDCh) 8 ppm 11.98 (s, 1H), 6.91 (s, 2H), 4.49 (5, 1H), 4.26 - 4.04 (m, 10H), 3.89 (s, 2H), 3.63 (s, 2H), 3.24 (s, 2H), 2.86 (s, 2H), 2.37 - 2.18 (m, 10H), 2.10 (s, 2H), 1.95 (s, 4H), 1.67 - 1.53 (m, 16H ), 1.50 - 1.19 (m, 48H), 0.90 - 0.86 (m, 12H).Example 28 - Synthesis of Compound 27: f7-[3-[7-(2-butyloctanovloxv) heptanovloxv]- ino] propoxy]-7-

[0446] Step 1 : 5NT138 !NT138

[0447] 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 HzO (50 mL) was degassed and purged with Nz for 3 times. The mixture was stirred at 100 °C for 12 hr under Nz 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.

[0448] Step 2: SNT139

[0449] A mixture of (4-hydroxy-4-methyl-pentanoyl) oxysodium (12 g, / 7.85 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 Nz for 3 times. The mixture was stirred at 25 °C for 5 min under N?_ atmosphere. The mixture was quenched by addition of HzO (500 mL). It was extracted with ethyl acetate (100 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and die 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.10450] Step 3:

[0451] To a mixture of benzyl 4-hydroxy-4-methyl-pentanoate (14.8 g, 66.58 mmol, 1 eq) and (4-mtrophenyl) 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 °C. The mixture was warmed to 25 °C and stirred for 1 hr under Nz 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.!H NMR (400 MHz, CDCb) 5 = 8.33 - 8.19 (m, 2H), 7.41 - 7.30 (m, 7H), 5.15 (s, 2H), 2.58 - 2.48 (m, 2H), 2.26 - 2.15 (ra, 2H), 1.58 (s, 6H).

[0452] Step 4:INT141 1^1142

[0453] A mixture of benzyl 4-methyl-4-(4-nitrophenoxy) carbonyloxy-pentanoate (19 g, 49.05 mmol, 1 eq), 2-pyrrolidin-l-ylethanamine (11.20 g, 98.09 mmol, 2 eq), DIPEA (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 of 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 (S1O2, 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.lH NMR (400 MHz, CDCb) 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), 2.1 1 - 2.05 (ra, 2H), 1.77 (t, J = 3.2 Hz, 4H), 1.45 (s, 6H).

[0454] Step 5:8NT142 iN"H43

[0455] To a solution of benzyl 4-methyI-4-(2-pyrrolidin-l-ylethylcarbamoyloxy) pentanoate (15 g, 44.14 mmol, 1 eq) in THF (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.

[0456] Step 6:

[0457] A mixture of 1 ,3-dibenzyloxypropan-2-amine (2 g, 6.50 mmol, 1 eq, HC1 salt), 4- methyl-4-(2-pyrrolidin-l- ylethylcarbamoyloxy) pentanoic acid (2 g, 7.80 mmol, 1.2 eq), EDCI (1.87 g, 9.75 mmol, 1.5 eq), DMAP (79.38 mg, 649.74 prnol, 0.1 eq) and DIPEA (3.36 g, 25.99 mmol, 4.53 mL, 4 eq) in DCM (20 mL) was degassed and purged with N?_ for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The mixture was quenched by addition of H2O (100 ml.,). It was extracted with 300 mL DCM (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 silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 5 to 1 / 1). Compound [4-[[2-benzyloxy- 1 -(benzyloxymethyl) ethyl] amino]- 1 , 1 -dimethyl-4-oxo-butyl] N-(2- pyrrolidin-1- ylethyl) carbamate (2.2 g, 4.10 mmol, 64% yield, 98% purity) obtained as a yellow oil. 1H NMR (400 MHz, CDCb) 6 = 7.39 - 7.27 (m, 10H), 5.09 (s, 1H), 4.53 (s, 4H), 4.37 - 4.25 (rn, 1H), 3.69 - 3.61 (m, 2H), 3.59 - 3.51 (m, 2H), 3.21 (d, J - 5.6 Hz, 2H), 2.58 - 2.44 (m, 6H), 2.30 ■■ 2.19 (m, 2H), 2.09 ■■ 2.00 (m, 2H), 1.77 (s, 4H), 1.70 (s, 1H), 1.45 (s, 6H).

[0458] Step 7:

[0459] To a solution of [4-[[2-benzyloxy-l-(benzyloxymethyl) ethyl] amino]-! ,1- dimethyl-4-oxo-butyl] N-(2-pyrrolidin-l -ylethyl) carbamate (2.2 g, 4.10 mmol, 1 eq) inMeOH (22 mL) was added Pd / C (364.40 mg, 410 10% puriμtmy,o 0l.,1 eq) and Pd(OH)2 (480.87 mg, 342.42 umol, 10% purity, 0.1 eq) under N2 atmosphere. The suspension was degassed and purged with H2 for 3 times. The suspension was stirred at 60 °C for 12 hr under H2 (50 Psi) atmosphere. The suspension was filtered and the filtrate was concentrated to giveresidue. The residue was purified by silica gel column chromatography (DCM / MeOH = 0 / 1 to 1 / 5). Compound [4-[[2-hydroxy-l-(hydroxymethyl) ethyl] amino]-l,l-dimethyl-4-oxo- butyl] N-(2-pyrrolidin-l-ylethyl) carbamate (1.1 g, 2.79 mmol, 81.47% yield, 87.6% purity) obtained as a brown oil.

[0460] Step 8:

[0461] A mixture of [4- [[2 ■■hydroxy- 1 ■•(hydroxymethyl) ethyl] amino] -1,1 -dimethy 1-4- oxo-butyl] N-(2-pyrrolidin-l-ylethyl) carbamate (1 g, 3 mmol, 1 eq), 7-(2-butyloctanoyloxy) heptanoic acid (2.25 g, 6.6 mmol, 2.2 eq), EDCi (1.44 g, 7.5 mmol, 2.5 eq), DMAP (73 mg, 0.6 p.mol, 0.2 eq) and DIEA (1.55 g, 12 mmol, 1.98 mL, 4 eq) in DCM (20 mL) was degassed and purged with N2 for 3 times. The mixture was stirred at 2.5 °C for 12 hr under b% atmosphere. The mixture was quenched by addition of H2O (100 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 silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 5 to 1 / 1) and p-HPLC (column: X-Select CSH Phenyi-Hexyi 100*30 5u; mobile phase: [H?.O(().05%HCl)-ACN:THF = 1:1]; gradient:40%-80% B over 14.0 min). Compound [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-[[4-methyl-4-(2-pyrrolidin-l- ylethylcarbamoyloxy)pentanoyl] amino] propoxy] -7 -oxo-heptyl] 2-butyloctanoate (150 mg, HC1 salt, 92% purity) was obtained as a colorless oil.NMR (400 MHz, CDCI3) 8 ppm 11.90 (s, 1 H), 7.12 (d, J=8.5 Hz, 1 H), 6.58 ■■ 6.34 (m, 1H), 4.51 ■ 4.45 (m, 1H), 4.24 - 4.12 (m, 4H), 4.08 - 4.04 (m, 4H), 3.88 (d, 1=5.0 Hz, 2H), 3.55 (d, J=4.4 Hz, 2H), 3.23 (d, 1=4.6 Hz, 2H), 2.87 - 2.83 (m, 2H), 2.44 - 2.39 (m, 2 H), 2.37 - 2.24 (m, 8H), 2.16 - 2.07 (m, 2H),2.01 (t, J=7.4 Hz, 2H), 1.68 - 1.57 (m, 20H), 1.47 (s, 6H), 1.38 - 1.33 (m, 8H), 1.29 - 1.24 (rn,20H), 0.90 - 0.86 (m, 12H).Example 29 - Synthesis of Compound 28: 06-[3~[6-oxo~6~(3-pentyloetoxy) hexanoyl] oxy-2~[4~(2-pyrrolidin-l"ylethylcarbamoyloxy) decanoylamino] propyl] 01-(3- pentyioctyl) hexanedioate

[0462] Step 1: WT146 SNT147

[0463] To a solution of undecan-6-one (70 g, 41 1.06 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 reaction mixture was quenched by addition of H2O (2000 mL) at 0 °C. It was extracted with ethyl acetate 4500 mL (1500 mL3). The combined organic layers were dried over Nar-SCfi, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, 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.

[0464] Step 2: INT147

[0465] To a suspension of Pd / C (17.71 g. 16.64 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 Hi atmosphere. The reaction mixture 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=100 / l to 0 / 1 ). Compound ethyl 3 -pentyloctanoate (20 g, 82.51 mmol, 49.58% yield) was obtained as a colorless oil.

[0466] Step 3: INT148 INT149

[0467] 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 of 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 Na?.SO4, 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 / l to 1 / 1). Compound 3-pentyloctan-l-ol (8 g, 39.93 mmol, 48.39% yield) was obtained as a white solid.

[0468] Step 4:

[0469] A mixture of 3-pentyloctan-l-ol (8 g, 39.93 mmol, 1 eq), adipic acid (29.18 g, 199.64 mmol, 33.15 rnL, 5 eq), EDC1 (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 THF (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 rnL (40 mL * 3). The combined organic layers were dried over NarSCE, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate= 100 / 1 to 0 / 1 ). Compound 6-oxo-6-(3-pentyloctoxy) hexanoic acid (4 g, 12.18 mmol, 30.50% yield) was obtained as a colorless oil.

[0470] Step 5:Compound 3S

[0471] A mixture of 6-oxo-6-(3-pentyloctoxy) hexanoic acid (0.8 g, 2.44 mmol, 1 eq), 1- [3 - [[2-hydroxy- 1 -(hydroxymethyl ) ethyl] amino] -3 -oxo-propyl] heptyl -N-(2-pyrrolidin- 1 ■ ylethyl) carbamate (391.17 mg, 974.17 0.4 eq),μ EmDoCl,I (560.25 mg, 2.92 mmol, 1.2 eq), DMAP (29.75 mg, 243.54 gmol, 0. 1 eq) and DIPEA (786.90 mg, 6.09 mmol, 1 .06 mL, 2.5 eq) in DCM (8 mL) was degassed and purged with N?_ for 3 times. The mixture was stirred at 25 °C for 12 hr under Nr 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 NarSOr, 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 / 1 to 0 / 1) and p-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H?.O(0.05%HCl)-ACN:THF=l:l]; gradient:40%-80% B over 14.0 min). Compound O6-[3-[6-oxo-6-(3-pentyloctoxy) hexanoyl] oxy-2-[4-(2-pyrrolidin-l-ylethylcarbamoyloxy) decanoylamino] propyl] Ol-(3-pentyloctyl) hexanedioate (0.15 g, HC1 salt, 90% purity) was obtained as a colorless oil. ‘H NMR (400 MHz, CDCI3) 8 ppm 1 1.77 (s, 1 H), 7.67 (d, >7.0 Hz, 1 H), 6.47 - 6.02 (m, 1H), 4.69 (d, >5.4 Hz, H I ). 4.48 (d, J -6.0 Hz, 1 H i. 4.26 - 4.12 (m, 4H), 4.06 ■■ 4.05 (m, 1H), 4.11 ■■ 4.05 (m, 4H), 3.91 (s, 2H), 3.79 (d, >7.0 Hz, 2 H), 3.46 ■■ 3.31 (m, 2H), 3.26 - 3.07 (m. 4H), 2.92 - 2.80 (m, 2H), 2.45 - 2.30 (m, 10H), 2.14 - 1.99 (m, 3H), 1.80 - 1.74 (m, 1 H), 1.71 - 1.64 (m, 8H), 1.60 - 1.55 (m, 4H), 1.53 - 1.45 (m, 1H), 1.40 (s, 2H), 1.34 - 1.23 (m, 44H), 0.92 - 0.87 (m, 15H).Example 30 - Synthesis of Compound 29: 01-[3-[6-(l-heptyIoctoxy)-6-oxo-hexanoylj oxy~2-[4-(2~pyrrolidin~l~ylethykarbamoyloxy) decanoylamino] propyl] O6~(l~ heptyloctyl) hexanedioate

[0472] ‘Step 1 :

[0473] 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), EDC1 (6.04 g, 31.52 mmol, 1.2 eq), DMAP (320.91 mg, 2.63 mmol, 0.1 eq) and DIPEA (8.49 g, 65.67 mmol, 1 1.44 mL, 2.5 eq) in DCM (251 mL) was degassed and purged with Nz for 3 times. The mixture was stirred at 25 °C for 12 hr under Nz atmosphere. The reaction mixture was diluted with HzO (200 mL) and extracted with ethyl acetate 600 mL. (200 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 (SiOz, 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.

[0474] Step 2:

[0475] A mixture of 6-(l-heptyloctoxy)-6-oxo-hexanoic acid (0.9 g, 2.52 mmol, 1 eq), 1- [3 - [[2-hydroxy- 1 ■•(hydroxymethyl) ethyl] amino] -3 -oxo-propyl] heptyl N -(2 -pyrrolidin- 1 ■■ ylethyl) carbamate (405.44 mg, 1.01 mmol, 0.4 eq), EDCI (580.69 mg, 3.03 mmol, 1.2 eq),DMAP (30.84 mg, 252.43 0μ.m1 o elq,) and DIPEA (815.61 mg, 6.31 mmol, 1.10 mL, 2.5 eq) in DCM (34mL) was degassed and purged with N?. for 3 times. The mixture was stirred at 25 °C for 12 hr under N2 atmosphere. 'The reaction mixture was diluted with H2O (2.0 mL) and extracted with ethyl acetate 60 mL (20 mL * 3). The combined organic layers were dried over NarSCM, filtered and the filtrate was concentrated under reduced pressure to give a residue. Hie residue was purified by column chromatography (SiOr, Petroleum ether / Ethyl acetate=100 / l to 0 / 1) and p-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H20(0.04%HC1)-ACN:THF=;1 :1]; gradient:40%-90% B over 15.0 min). Compound Ol-[3-[6-(l-heptyloctoxy)-6-oxo-hexanoyl] oxy-2-[4-(2-pyrrolidin-l-ylethylcarbamoyloxy) decanoylamino] propyl] O6-(l -heptyloctyl) hexanedioate (0.15 g, 133.18 5.28% yield, μmol, 99% purity, HC1 salt) was obtained as a colorless oil. 1H NMR (400 MHz, CDCb) b ppm 12.00 (s, 1 H), 7.35 (d, J-8.63 Hz, 1 H), 6.24 (s, 1 H), 4.90 - 4.82 (m, 2H), 4.69 (d, J-5.8 Hz, 1H), 4.54 ■■ 4.41 (m, 1H), 4.26 - 4.08 (m, 4H), 3.90 (s, 2 H), 3.77 (d, 3=9.2 Hz, 1H), 3.50 ■ 3.26 (m, H), 3.2.1 (s, 1 H), 2.97 - 2.72 (m, 2H), 2.41 - 2.28 (m, 10H), 2.16 - 1.94 (m, 4H), 1.82 - 1.60 (m, 1H), 1.51 (d, .1=5.63 Hz, 8H), 1.26 (s, 50H), 1.00 - 0.78 (m, 15H).Example 31 - Synthesis of Compound 30: l-[3-[[2"[7-(2-butyIoctanoyloxy) heptanoyloxy]-l-[7-(2-butyIoctanoyloxy) heptanoyloxymethyl] ethyl] amiaoJ-3- oxopropylj heptyl l-inethyIpiperidine-4-carboxyIate10476] Step 1:10477] To a solution of 4-hydroxydecanoyloxysodium (15 g, 71.35 mmol, 1 eq) in H2O(75 mL) was added NaHzPCL (1.76 g, 14.7 mmol, 2.06e-l eq), NarHPCL (8.46 g, 39.78 mmol, 8.36e-l eq) and NaQO (127.47 g, 171.24 mmol, 106 mL, 10% purity, 2.4 eq) at 25 oC. The mixture was stirred at 25 oC for 12 h under N2 atmosphere. The reaction mixture was quenched by addition of IM HC1 (200 mL). It was extracted with ethyl acetate 900 mL, (300 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 (petroleum ether / ethyl acetate= 8 / 1 to 6 / 1). 4-oxodecanoic acid (9 g, 48.32 mmol, 67.73% yield) was obtained as a white solid.!H NMR (400 MHz, CDCb) 8 -2.74 - 2.70 (m, 2H ), 2.67 - 2.60 (m, 2H), 2.45 (t, J - 7.2 Hz, 2H), 1.57 - 1.60 (m, 2H), 1.41 - 1.20 (m, 6H), 1.01 - 0.82 (m, 3H).

[0478] Step 2:!HT1 S3 ISm54

[0479] To a mixture of 4-oxodecanoic acid (6 g, 32.22 mmol, 1 eq) and 1,3- dibenzyioxypropan-2-amine (9.90 g, 32.22 mmol, 1 eq, HC1 salt) in DCM (60 mL) was added DMAP (396 mg, 3.24 mmol, 0.1 eq), EDO (15.42 g, 80.52 mmol, 2.5 eq) and DIPEA (12.51 g, 96.66 mmol, 16.83 mL, 3 eq). The mixture was stirred at 25 oC for 12 h under Nz atmosphere. The mixture was quenched by addition HzO (500 mL). It was extracted with ethyl acetate 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 silica gel column chromatography (petroleum ether / ethyl acetate- 8 / 1 to 3 / 1). Product N-[2-benzyloxy-l -(benzyloxymethyl) ethyl]-4-oxo-decanamide (7.1 g, 16.15 mmol, 50.14% yield, 100% purity) was obtained as a colorless oil. ’H NMR (400 MHz, CDCI3) 5 - 7.39 - 7.27 (m, 10H), 5.98 (d, J - 8.4 Hz, 1H), 4.55 - 4.47 (m, 4H), 4.33 ■■ 4.22 (m, 1H), 3.66 - 3.62 (m, 2H), 3.56 -3.52 (m, 2H), 2.74 (t, J = 6.4 Hz, 2H), 2.48 ■■ 2.37 (m, 4H), 1.59 - 1.55 (m, 2H), 1.34 - 1.26 (m, 6H), 0.89 (t, J = 6.8 Hz, 3H).[0481 J To a solution of N-[2-benzyloxy-l-(benzyloxymethyl) ethyl]-4-oxo-decanamide(6.6 g, 15.00 mmol, 1 eq) in THF (66 mL) was added Pd / C (699 mg, 656.82 10% μmol, purity, 4.37e-2 eq). The suspension was heated to 60 °C and stirred for 4 h under Hz (50 Psi) atmosphere. The suspension was filtered and the filtrate was concentrated to give a crude product. The crude product was purified by triturated with ethyl acetate (20 ml..) at 0 °C for 0.5 h. Then it was filtered and the filter cake was dried under reduced pressure. Product N-[2- hydroxy-1 -(hydroxymethyl) ethylJ-4-oxo-decanamide (3.3 g, 12.49 mmol, 83.17% yield, 98. 14% purity) was obtained as a white solid.lH NMR (400 MHz, MeOD) S = 3.94 - 3.85(m, 1H), 3.60 (d, J - 5.6 Hz, 4H), 2.75 (t, J - 6.8 Hz, 2H), 2.55 - 2.41 (m, 4H), 1.63 - 1.49(m, 2H), 1.38 - 1.24 (m, 6H), 0.97 - 0.84 (m, 3H).

[0482] Step 4:

[0483] To a mixture of 7-(2-butyloctanoyloxy) heptanoic acid (2 g, 6.09 mmol, 1 eq) and N-[2-hydroxy-l-(hydroxymethyl) ethyl] -4-oxo-decanamide (790.00 mg, 3.05 mmol, 0.5 eq) in DCM (20 mL) was added EDCI (2.92 g, 15.22 mmol, 2.5 eq), DMAP (75 mg, 613.91 pmol, 0. 1 eq) and DIPEA (2.36 g, 18.27 mmol, 3.18 mL, 3 eq). The mixture was stirred at 25 °C for 12 h under N2 atmosphere. The reaction mixture quenched by addition of H2O (200 mL). It was extracted with ethyl acetate 150 mL (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 (petroleum ether / ethyl acetate^ 3 / 1 to 2 / 1). Product [7-[3-[7-(2- butyloctanoyloxy) heptanoyloxy]-2-(4-oxodecanoylamino) propoxy]-7-oxo-heptyl] 2- butyloctanoate (2.6 g, 2.95 mmol, 96.85% yield, 99.82% purity) was obtained as a colorless oil.

[0484] Step 5:

[0485] To a solution of [7-[3-[7-(2- butyloctanoyloxy)heptanoyloxy]-2-(4 oxodecanoylamino)propoxy]-7-oxo-heptyl] 2 -butyloctanoate (2.6 g, 2.95 mmol. 1 eq) in THF (16 mL), toluene (4 mL) and H2O (8 mL) was added NaBH? (782.00 mg, 20.67 mmol, 7 eq) at 0 °C. Then the reaction mixture was allowed to warm to 25 °C and stirred for 12 h. The mixture was quenched by addition of IM HC1 (30 mL). Then it was diluted with H2O (100 mL) and extracted with ethyl acetate 90 mL (30 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 (petroleum ether / ethyl acetate= 0: 1 to 3:1). Product [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy] -2-(4-hydroxydecanoylamino) propoxy] -7 -oxo-heptyl] 2 -butyl octanoate (2.4 g, 2.53 mmol, 85.68% yield, 93.03 % purity) was obtained as a colorless oil.1H NMR (400 MHz, CDCh) 5 - 6.04 (d, J - 8.4 Hz, 1 H), 4.53 ■■ 4.39 (m, 1H), 4.22 (dd, J = 5.6, 11.2 Hz, 2.H), 4.14 - 4.03 (m, 6H), 3.61 (s, 1H), 2.44 - 2.26 (m, 9H). 1.95 - 1 .86 (m, 1H), 1.70 - 1.59 (m, 12H), 1.49 - 1.23 (m, 46H), 0.96 - 0.81 (m, 15H).

[0486] Step 6:Compound 30

[0487] To a mixture of [7-[3-[7-(2- butyloctanoyloxy)heptanoyloxy]-2-(4- hydroxydecanoylamino)propoxy]-7-oxo-heptyl] 2-butyloctanoate (1 g, 1.13 mmol, 1 eq) and 1 -methylpiperidine -4-carboxylic acid (244 mg, 1.70 mmol, 1.5 eq) in DCM (10 mL) was added EDC1 (543 rag, 2.83 mmol, 2.5 eq), DMAP (14 mg, 114.60 0,1 eq) and DIPμEmAol, (439 mg, 3.40 mmol, 591.64 μL, 3 eq). The mixture was stirred at 25 °C for 12 h under N? atmosphere. The mixture was concentrated under reduced pressure to give a residue. The residue was quenched by addition of H2O (100 mL). It was extracted with ethyl acetate 150 mL (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 (petroleum ether / ethyl acetate = 3 / 1 to 2 / 1). Product l -[3-[[2-[7-(2-butyloctanoyloxy) heptanoyloxy]-i-[7-(2- butyloctanoyloxy) heptanoyloxymethyl] ethyl] amino] -3 -oxopropyl] heptyl 1- methylpiperidine- 4 -carboxylate (150 mg, 96.33% purity) was obtained as a colorless oil. *H NMR (400 MHz, CDCI3) 6 ppm 6.09 (d, J=8.4 Hz, IH), 4.93 - 4.82 (m, 1H), 4.51 - 4.40 (m, 1H), 4.26 - 4.04 (m, 8H), 2.83 (d, J-1 1 .4 Hz, 2 H), 2.36 - 2.29 (m, 6H), 2.27 (s, 3H), 2.26 - 2.07 (m, 3H), 2.03 - 1.88 (m, 6H), 1.86 - 1.74 (m, 3H), 1.67 - 1.51 (m, 14H), 1.49 - 1.33 (m, 14H), 1.33 ■■ 1.21 (m, 32H), 0.90 ■■ 0.86 (m, 15H).Example 32 - Synthesis of Compound 31: l-[3-[[2-[7-(2-butyloctanoyloxy) heptanoyloxy]~l~[7-(2~butyloctanoyloxy) heptanoyloxymethyl] ethyl] amino]-3- oxopropyl] heptyl 1 -methyIpyrrolidine-3-carboxylate

[0488] Synthetic scheme:Compound 31

[0489] To a mixture of [7-[3~[7-(2- butyloctanoyloxy)heptanoyloxy]-2-(4- hydroxydecanoylamino)propoxy]-7-oxo-heptyl] 2 -butyloctanoate (1 g, 1.13 mmol, 1 eq) and l-methylpyrrolidine-3-carboxylic acid (293 mg, 2.27 mmol, 2 eq) in DCM (10 mL) was added EDCI (543 rag, 2.83 mmol, 2.5 eq), DMAP (14 mg, 114.60 0.1 eq) and DIμPmEoAl, (439 mg, 3.40 mmol, 591.64 p.L, 3 eq) at 25 °C. The mixture was stirred at 25 °C for 12 h under N? atmosphere. The mixture was quenched by addition of I-fcO (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 60% to 80%). Product l-[3-[[2-[7-(2-butyloctanoyloxy) heptanoyloxy]-l-[7-(2- butyloctanoyloxy) heptanoyloxymethyl] ethyl] amino]-3-oxopropyl] heptyl 1- methylpyrrolidine-3-carboxylate (150 mg, 98.06% purity) was obtained as a colorless oil. 1H NMR (400 MHz, CDCI3) 8 ppm 6.37- 6.11 (m, 1H), 4.94 - 4.81 (m, 1H), 4.53 - 4.40 (m, 1 H), 4.23 ■■ 4.04 (m, 8H), 3.10 ■■ 2.94 (m, 1H), 2. 86 ■■ 2.51 (rn, 4H), 2.41 ■■ 2.31 (m, 8H), 2.25 ■■ 2.07 (ra, 4H), 2.00 - 1.90 (m, 1H), 1.84 - 1.69 (m, 4H), 1.68 - 1.53 (m, 14H), 1.49 - 1.21 (m, 46H), 0.90 - 0.86 (m, 15 H).Example 33 - Synthesis of Compound 32: [7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]- 2-[4~(2-pyrrolidin-l -ylethykarbamoyloxy) heptanoylamino] propoxy]-7-oxo-heptyl] 2- butyloctanoate

[0490] Step 1 : SNT158 1NT159

[0491] A mixture of 5 -propyl tetTahydrofuran-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 Nz 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- hydroxyheptanoyloxysodium (22 g, crude) was obtained as a white solid.

[0493] 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 of H2O (500 mL). 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, 1 14 mmol, 88% yield) obtained as a colorless oil.NMR (400 MHz, CDCI3) 6 = 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, 1 H), 1.50 - 1.40 (m, 3H), 1.39 - 1.29 (m, 1 H), 0.98 - 0.89 (m, 3H).

[0494] Step 3:

[0495] 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 al 0 °C. The mixture was allowed to warm to 25 °C and stirred for 1 h under N2 atmosphere. Hie 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. Hl NMR (400 MHz, CDCh) 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, 1H), 1.80 - 1.68 (m, 1H), 1.67 - 1.58 (m, 1H), 1.52 - 1.36 (m, 2H), 0.97 (t, J - 7.2 Hz, 3H).

[0496] Step 4:

[0497] A mixture of benzyl 4-(4-nitrophenoxy) carbonyloxyheptanoate (16.6 g, 41.35 mmol, 1 eq), 2-pyrrolidin- 1 - ylethanamine (9.44 g, 82.71 mmol, 2 eq), DMAP (505.23 mg, 4.14 mmol, 0.1 eq) and DIPEA (16.03 g, 124.06 mmol, 21.61 mL, 3 eq) in DCM (150 mL) was degassed and purged with Nz for 3 times. The mixture was stirred at 25 °C for 12 hr under Nr 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.!H NMR (400 MHz, CDCI3) 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, 1H), 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).

[0498] Step 5:

[0499] To a solution of benzyl 4-(2-pyrrolidm-l-yletiylcarbamoyloxy) 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 / 1 to 1 / 8). Compound 4-(2-pyrrolidin-l-ylethylcarbamoyloxy) heptanoic acid (6 g, 20.95 mmol, 65.74% yield) obtained as a brown oil.lH NMR (400 MHz, CDCI3) § = 6.84 (s, 1 H), 6.60 (d, J - 4.8 Hz, 1H), 4.82 - 4.67 (m, 1H), 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).

[0500] Step 6:

[0501] To a mixture of 4-(2-pyrrolidin-1 -ylethylcarbamoyloxy) heptanoic acid (1.5 g, 5.24 mmol, 1.0 eq) and l,3-dibenzyloxypropan-2-amine (1.93 g, 6.29 mmol, 1.2 eq, HC1 salt) in DCM (15 mL) was added EDO (1.2 g, 6.29 mmol, 1.2 eq), DMAP (192 mg, 1.57 mmol, 0.3 eq) and DIPEA (2.7 g, 20.96 mmol, 4.0 eq) 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 flash silica gel chromatography (ISCO®; 40 g Sepa Flash ® Silica Flash Column, eluent of 0-90% EtOAc / PE, gradient at 200 mL / min). Compound [4-[[2-benzyloxy-l-(benzyloxymethyl) ethyl] amino] -4 -oxo- 1- propyl-butyl] N-(2-pyrrolidin-l-ylethyl) carbamate (1.6 g, 2.88 mmol, 56% yield) was obtained as a colorless oil.1H NMR (400 MHz, DMSO-d6) 8 - 7.82 (d, J - 8.0 Hz, 1H), 7.42 ■■ 7.19 (m, 10H), 6.89 (t, J - 5.6 Hz, 1H), 4.63 ■ 4.57 (m, 1H), 4.46 (s, 4H), 4.13 ■■ 4.08 (in, 1H), 3.45 (d, J = 5.2 Hz, 4H), 3.11 - 2.99 (m, 2H), 2.47 - 2.35 (ra, 6H), 2.10 (t, J = 8.0 Hz, 2H), 1.76 - 1.58 (m, 6H), 1.42 (q, J - 7.2 Hz, 2H), 1.34 - 1.18 (m, 2H), 0.85 (t, J - 7.2 Hz, 3H).

[0502] Step 7:

[0503] To a solution of [4-[[2-benzyloxy-l-(benzyIoxymethyl) etliyl] amino]-4-oxo~l- propyl-butyl] N-(2-pyrrolidin-l-ylethyl) carbamate (1.6 g, 2.96 mmol, 1.0 eq) in MeOH (32 mL) was added Pd / C (315 mg, 296 10%μ pmuroilt,y, 0.1 eq) and Pd(OH)2 / 'C (208 mg, 296 pmol, 20% purity, 1.0 eq). The suspension was degassed and purged with Hr for 3 times. Then the suspension was stirred at 60 °C for 12 h under H?. (50 psi) atmosphere. The suspension was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (I SCO®; 20 g Sepa Flash® Silica Flash Column, Eluent of 0~15% MeOH / DCM at 100 mL / min). Compound [4- [ [2 -hydroxy- 1 ■(hydroxymethyl) etliyl] amino] -4-oxo- 1 -propyl- butyl] N-(2-pyrrolidin- 1 ■■ yletbyl) carbamate (1 .0 g, 2.67 mmol, 90% yield) was obtained as a colorless oil.{H NMR (400 MHz, DMSO-d6) 6 ~ 7.46 (d, J ~ 8.0 Hz, 1H), 6.89 (t, J = 5.2 Hz, 1H), 4.74 - 4.35 (m,3H), 3.70 - 3.66 (m, 1H), 3.37 (d, J - 5.2 Hz, 4H), 3.14 - 2.99 (m, 2H), 2.48 - 2.36 (m, 6H),2.09 (t, J = 8.0 Hz, 2H), 1.78 ■■ 1.59 (m, 6H), 1.43 (q, J - 7.2 Hz, 2H), 1.34 - 1.21 (m, 2H),0.86 (t. J = 7.2 Hz, 3H).

[0504] Step 8:Compound 32

[0505] To a mixture of [4 ■ [[2 -hydroxy- 1 ■■(hydroxymethyl) ethyl] amino]-4-oxo-l -propyl - butyl] N-(2-pyrrolidin-1 -ylethyl) carbamate (713 mg, 1.99 mmol, 1.0 eq) and 7-(2- butyloctanoyloxy) heptanoic acid (1.5 g, 4.57 mmol, 2.3 eq) in DCM (30 mL) was added EDCI (875 mg, 4.57 mmol, 2.3 eq), DMAP (73 mg, 596 umol, 0.3 eq) and DIPEA (1.03 g,7.94 mmol, 4.0 eq). The mixture was stirred at 25 °C for 12 h under N? 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 TsteSOa, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g Sepa Flash® Silica Flash Column, Eluent of 0-70% EtOAc / PE, gradient at 2.00 mL / min) and p-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H20(0.04%HCl)-ACN:THF=i:l]; gradient:40%-80% B over 16.0 min). Compound (7-[3-[7-(2-butyloctanoyloxy) heptanoyloxy]-2-[4-(2-pyrrolidin-l- ylethylcarbamoyloxy) heptanoylamino] propoxy] -7 -oxo-heptyl] 2-butyloctanoate (150 mg, HO salt, 93.06% purity) was obtained as a colorless oil.!H NMR (400 MHz, CDCI3) 5 ppm 11.76 (s, IH), 7.69 (d, J-7.40 Hz, I H), 6.29 (s, 1H), 4.72 (d, J=6.0 Hz, IH), 4.48 (d, >5.8 Hz, IH), 4.24 - 4.10 (m, 4H), 4.06 (t, J=6.6 Hz, 4H), 3.91 (s, 2H), 3.78 (d, 1=12.8 Hz, IH), 3.45 - 3.2.7 (m, 2H), 3.23 (s, IH), 3.01 - 2.79 (m, 4H), 2.50 - 2.39 (m, 2.H), 2.39 - 2.22 (m, 8H), 2.15 - 1.98 (m, 3H), 1.80 - 1.72 (m, IH), 1.72 - 1.52 (m, 14H), 1.18 - 1.50 (m, 41H), 0.95 - 0.84 (m, 15 H).Example 34 - Synthesis of Compound 33: [7~[3-[7~(2-butyloctanoyloxy)hep- tanoyloxyxy]"2~[4-(4"pyrrolidisi"l”yibutanoyloxy)decaiioylamino]propoxy]-7"oxo“ heptyl] 2-butyloctanoate

[0506] Step

[0507] A mixture of benzyl 4-hydroxydecanoate (5 g, 14.37 mmol, 1 eq), 4-pyrrolidin-l- 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 Nr for 3 times, and then the reaction mixture was stirred at 25 °C for 12 h under N?. atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was added H2O (50 ml.,) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine (100 mL x 2), dried over Na?.SO4, filtered and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiCh, 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. HI NMR (400 MHz, CDCb) 8 ppm 7.30 - 7.40 (m, 5 H), 5.12 (s, 2 II ), 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 (ra, 3 H).

[0508] Step 2:

[0509] To a suspension of benzyl 4-(4-pyrrolidin-l-ylbutanoyloxy)decanoate (4.2 g, 10.06 mmol, 1 eq) in THE (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. Hie 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, CDCb) 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).

[0510] Step

[0511] A mixture of 1 ,3-dibenzyloxypropan-2-amine (1.4 g, 5.16 mmol, 1 eq), 4-(4- pyrrolidin- 1 -ylbutanoyloxy)decanoic acid (2.03 g, 6.19 mmol, 1.2 eq), EDCI (1.19 g, 6.19 mmol, 1.2 eq), DIPEA (1.67 g, 12.90 mmol, 2.25 mL, 2.5 eq) and DMAP (63.03 mg, 515.93 pmol, 0. 1 eq) in DCM (14 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 (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over NaeSOa, 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 = 100 / 1 to 1 / 4). Compound l-[3-[[2-benzyloxy-l-(benzyIoxyrnethyl)ethyl]amino]-3-oxo-propyI]heptyl 4-pyrrolidin-l-ylbutanoate (2.1 g, 3.62 mmol, 70.08% yield) was obtained as yellow oil. 1H NMR (400 MHz, CDCb) 5 ppm 7.27 - 7.37 (m, 10 H), 6.02 (d, J = 8.4 Hz, 1 H), 4.88 (s, 1 H), 4.52 (s, 4 H), 4.31 (s, 1 H), 3.64 (d, J - 4.4 Hz, 2 H), 3.55 (d, J = 4.0 Hz, 2 H), 2.41 - 2.54 (m, 6 H), 2.30 - 2.38 (m, 2 H), 2.08 - 2.21 (m, 2 H), 1.73 - 1.89 (m, 8 H), 1.46 - 1.58 (m, 2 H), 1.25 (s, 8 H), 0.81 - 0.92 (m, 3 H).

[0512] Step 4:

[0513] To a suspension of l-[3-[[2-benzyloxy-l-(benzyIoxymethyl)ethyl]amino]-3-oxo- propyl]heptyl 4-pyrrolidin-l -ylbutanoate (2.1 g, 3.62 mmol, 1 eq) in MeOH (21 mL) was added Pd / 'C (384.79 mg, 361.57 nmol, 10% purity, 0.1 eq) and Pd(OH)?. (253.89 mg, 361.57 pmol, 20% purity, 0.1 eq) under Ar atmosphere. The suspension was degassed and purged with Hr for 3 times. The reaction mixture was stirred under H2 (50 Psi) at 60 °C for 12 h. The mixture reaction was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOr, DCM: MeOH = 100 / 1 to 3 / 1). Compound l-[3-[[2-hydroxy-l-(hydroxymethyl)ethyl]amino]-3-oxo-propyl]heptyI 4- pyrro lidin- 1 -ylbutanoate (900 mg, 2.25 mmol, 62.14% yield) was obtained as yellow oil.A mixture of l-[3-[[2-hydroxy-l-(hydroxymethyl)etbyl]amino]-3-oxo-propyl]heptyl 4- pyrrolidin- 1 -ylbutanoate (800 mg, 1.75 mmol, 1 eq), 7-(2-butyloctanoyloxy)heptanoic acid (1.15 g, 3.50 mmol, 2 eq) , DIPEA (1.13 g, 8.74 mmol, 1.52 mL, 5 eq), DMAP (42.70 mg,349.52 μmol 0,.2 eq) and EDCI (804.04 mg, 4.19 mmol, 2.4 eq) in DCM (7 ml) was degassed and purged with N?. for 3 times, and then the reaction mixture was stirred at 25 °C for 12 h under Nr atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was added H2O (10 mt) and extracted with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine (10 ml * 2), dried over NarSCU, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiOi, Petroleum ether / Ethyl acetate = 100 / 1 to 1 / 4).Compound [7-[3-[7-(2-butyloctanoyioxy)hep-tanoyloxyxy]-2-[4-(4-pyrrolidin-1- ylbutanoyloxy)decanoylamino]propoxy]-7-oxo-heptyl] 2-butyloctanoate (150 mg, 94% purity) was obtained as yellow oil. ‘H NMR (400 MHz, CDCb) 8 ppm 6.17 (m, 1 H), 4.81 - 4.94 (m, 1 H), 4.41 - 4.52 (m, 1 H), 4.17 - 4.25 (m, 2 H), 4.05 - 4.15 (m, 6 H), 2.48 - 2.54 (m, 4 H), 2.29 - 2.40 (m, 8 H), 2.11 (s, 2 H) 1.77 - 1.90 (m, 7 H), 1.52 - 1 .67 (m, 16 H), 1.42 - 1.48 (m, 4 H), 1.34 - 1.40 (m, 8 H), 1.22 - 1.32 (m, 32 H), 0.88 (m, 15 H).Example 35 - Preparation and analysis of Lipid Nanopartides (LNPs) a. LNP Formulations

[0515] Formulations comprising lipid compounds of the present disclosure were prepared.Thirteen general formulations were used, according to Table 2:Table 2. General Formulations

[0516] LNP Formulations were produced using lipid compounds from Table 1 :Compounds 1. 3, 10-12, 15, 16, 19-23, 25, and 28. The formulations produced for each compound are shown in 'Fable 3 :Table 3. LNP Formulationsb. LNP Preparation

[0517] Lipids and active ingredients (e.g., RNA, DNA, etc.) were assembled into LNPs using micro fluidic 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 raL / 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 36 - In vivo Activity in Mee

[0518] 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 post - injection), the animals were anesthetized via isofl urane and optionally subjected to in-lifeimaging 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 / anirnal. 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.

[0519] 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 isofl urane 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.

[0520] All IVIS images were processed with computer software to identify the regions of interest 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.

[0521] Organ mean total flux results are shown in FIG. 1 , FIG. 2, FIG. 3, FIG. 4, and Table 4. LNPs comprising MC3 (in formulation Fl) were used as a control. LNPs comprising Compound 11 have formulation F 1.Table 4. Organ Mean Total FhsxExample 37 - / « vivo Biodistribution in Non-Human Primates

[0522] Non-human primate (NHP) biodistribution studies were performed in cynomolgus monkeys (e.g., Macaca fascicularis), aged 4-6 years. Animals were premedicated withdexamethasone, 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.

[0523] 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.

[0524] To determine the unique LNPs distributed in the tissues, samples were homogenized by a tissue homogenizer (e.g., TissueLyser). Total RN A 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 (UM1) 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.

[0525] Biodistribution results are shown in FIG. 5A, FIG. SB, FIG. SC, FIG. 6A, FIG. 6B, FIG. 6C, Table 5, and 'Fable 6. LNPs comprising MC3 (in formulation Fl) were used as a control. LNPs comprising Compound 1 1 have formulation Fl .Table 5. Mean Normalized Unique Molecular Identifier CountsTable 6. Mean Normalized Unique Molecular Identifier Counts

[0526] Further biodistribution results are shown in Table 7 and Table 8. LNPs comprisingMC3 (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 39 - Functional delivery in Nun-Human PrimatesGeneral Protocols

[0527] Non-human primate (NHP) expression studies were performed in cynomoigus 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 doshig, 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.

[0528] At the predetermined necropsy time point, study animals were anesthetized with a combination of Ketamine (10 mg / kg, IM) and Dexmedetomidine (0.01 - 0.03 mg / kg, IM), a single dose of buprenorphine (0.03 mg / kg, IM) was administered. Then bone marrow aspirate samples will be collected as indicated below. After sample collection, the animals were euthanized with Euthasol® (>50 mg / kg, IV) and spleen and lymph node samples were collected. This method of euthanasia is in accordance with American Veterinary Medical Association (AVMA) guidelines.Bone marrow aspirate collection

[0529] 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 CRl (0.3 -0.6 mg / kg / min) may be used as needed. A non-medicated lubricant was applied to the eyes.

[0530] 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' collec tion was clipped. Any loose hair was removed (by vacuum as appropriate). The sites of collection were 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 / 8tbsof 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 die 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 marrow 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 K2EDTA tubes and stored on wet ice. 'The samples were then analyzed by standard flow cytometry for GFP expression.Spleesi ami lymph node collection

[0531] Following euthanasia as described above, spleen and lymph node samples were collected and analyzed by standard flow cytometry for GFP expression. Briefly, lymph node membranes were torn open in a petri dish, after which lymph nodes were pressed through a 70 pm strainer using the plunger of a 5 mF syringe. The strainer was washed with complete medium from the petri dish, followed by 5 ml of fresh medium. Strained samples were centrifuged at 300 RCF for 10 minutes at 4°C and resuspended in 10 mL bench medium. Yield and dissociated cell viability were recorded. Cell suspension should be kept on ice during the cell counting step. An aliquot of 1 * 10A6 isolated cells was stained with a viability stain. Stained samples were centrifuged at 350 RCF for 5 minutes at 4°C and washed with cell staining buffer. Stained cells were centrifuged again at 350 RCF for 5 minutes at 4°C and resuspended in 200 yL of cell staining buffer to be analyzed on a flow cytometer for viability and GFP expression.

[0532] The spleen samples were placed in a petri dish and cut into smaller pieces, making sure the capsule was only present on one side of each piece. Spleen pieces were pressed through a 70 pm strainer using the plunger of a 5 mL syringe. The strainer was washed with 15 mL of bench medi um from die petri dish and 10 mL of fresh bench medium. Cell suspension was centrifuged at 300 x g for 5 min at 4°C and resuspended in 15 mL ACK lysing buffer for 3 minutes. After incubation, the bench media was added to the tubes containing cells and lysing buffer. Samples were centrifuged at 300 x g for 5 min at 4°C and resuspended in bench medium for cell counting. Yield and dissociated cell viability were recorded. Cell suspension should be kept on ice during the cell counting step. An aliquot of 1 x 10A6 isolated cells was stained with a viability stain. Stained samples were centrifuged at 350 RCF for 5 minutes at 4°C and washed with cell staining buffer. Stained cells were centrifuged again at 350 RCF for 5 minutes at 4°C and resuspended in 200 uL of cell staining buffer to be analyzed on a flow cytometer for viability and GFP expression.

[0533] Results are shown in FIG. 7.

Claims

CLAIMSWe claim:

1. A compound of Formula (I),(I), or a pharmaceutically acceptable salt thereof, wherein each of R1and Rris independently -(C1-C9 alkylene)-R5; each R2' is independently hydrogen or C1-C3 alkyl; each R5is independently - CH-(R8)R9, -OC(O)-(CH2)q-Ceach R6and R7is independently hydrogen, C7-C12 alkoxy, or C7-C12 alkenoxy, provided that R6and Rzare not both hydrogen; each R® and R9is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, or C1-C12 alkynyl, provided that R8and R9are not both hydrogen; n is 0-5; p is 0-3; q is 0-3;R3or a 3-membered to 8-membered heterocyclyl containing one or more N atoms substituted with C1-C12 alkyl;R3and R4are each independently Cj-Co alkyl, or R3and R4together with the nitrogen atom to which they are attached form a 5-6 membered heterocycle;X1is O, CH?., or X2:wherein each L is independently -(CH2)m-C(R2)(R2")-(CH2)t-, --(CH2)m-C3-C8 cycloalkylene -(CH?)-,-, 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 Cs-Cs aryl, or (C1-C4 alkylene)-(optionally substituted C5-C6 aryl): m is 0-4; and is 0-4.The compound of claim 1, whereinR2' is hydrogen or methyl;cycloalkylene-(CH?.)t-;R2is hydrogen or C1-C12 alkyl;R2is Ca~(-i2 alkyl; m is 2; t is 0 or 2; and n is 2.

3. The compound of claim 2, wherein L is ~(CH2)m-C(R2)(R2')-(CH2)t-.

4. The compound of claim 2 or 3, wherein R2is hydrogen.

5. The compound of any one of claims 2-4, wherein t is 0.

6. The compound of any one of claims 2-5, wherein Y is pyrrolidinyl.

7. The compound of any one of claims 2-6, whereinR1is -(C1-C9 alkylene)-^;R5is -0C(0)-(CH2)q-CH-(Rs)R9; and each R8and R9is independently C1-C12 alky].

8. The compound of claim 7, whereinR8is n-hexyl; andR9is n-butyl.

9. The compound of any one of claims 2-8, wherein q is 0.

10. The compound of any one of claims 2-9, wherein R1' and R1are the same.

11. 'The compound of any one of claims 2-9, whereinRris -(Ci-Cy alkylene)-R5;R5is -CH( R6)R7: and each R6and R7is independently C7-C12 alkenoxy.

12. The compound of any one of claims 2-9, whereinR!is -(C1-C9 alkylene)-R3;R“ is -CH(R6)R7; and each R° and R7is independently C7-C12 alkoxy.

13. The compound of claim 1 , whereinR2' is hydrogen; andX1is O or CH?.

14. The compound of claim 13, wherein n is 1-3.

15. The compound of claim 13 or claim 14, whereinR1is -(C1-C9 alkyiene)-R5;R5is -C(O)O-(CH2)P-CH-(R8)R9; and each R8and R9is independently C1-C12 alkyl or C2-C12 alkenyl.

16. The compound of claim 15, wherein R8and R9are the same.

17. The compound of claim 15 or claim 16, wherein R8and R9are n -pentyl.

18. The compound of any one of claims 15-17, wherein p is 2.

19. The compound of claim 15 or claim 16, wherein R8and R9are n-heptyl.

20. The compound of claim 15 or claim 16, wherein R8and R9are l-hepten-7-yl.

21. 'The compound of claim 19 or claim 20, wherein p is 0.

22. 'The compound of any one of claims 15-21, wherein Rrand Rlare the same.

23. The compound of any one of claims 15-18, whereinR1' is -(C1-C9 alkylene)-R5;R5is -CH(R6)R7; and each R6and R' is independently C7-C12 alkenoxy.

24. The compound of claim 23, wherein n is 2 or 3;each RJand R4is independently C1-C3 alkyl.

25. The compound of claim 24, wherein Y is dimethylamino, diethylamino, or (methyl )ethy lamino .

26. The compound of claim 23, wherein n is 1 or 2; andY is pyrrolidinyl or ethyl-substituted piperidinyl.

27. The compound of claim 1, whereinR1is -(C1-C9 alkylene)-R5;R5is -OC(O)-(CH?.)q-CH-(R8)R9; each R8and R9is independently CJ-C12 alkyl:L. is Cj-Cs cycloalkylene.

28. The compound of claim 27, wherein n is 2; andY is pyrrolidinyl.

29. The compound of claim 27 or claim 28, wherein q is 0.

30. The compound of any one of claims 27-29, whereinR8is n-hexyl; and R9is n-butyl.

31. The compound of any one of claims 27-30, wherein Rrand R1are the same.

32. The compound of any one of claims 27-31, wherein L is cyclopenty lene or cyclohexylene.

33. The compound of claim 1, whereinR2is hydrogen;R2" is Ci-Crz 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.

34. The compound of claim 33, whereinR1is -(C1-C9 alkylene)-R5;R5is -OC(O)-(CH2)q-CH-(R8)R9; and each Rsand R9is independently C1-C12 alkyl.

35. The compound of claim 33 or 34, whereinRsis n-hexyl; andR9is n -butyl.

36. The compound of any one of claims 33-35, wherein q is 0.

37. The compound of any one of claims 33-36, wherein Rrand R1are the same.

38. The compound of claim 1, wherein the compound is selected from any one ofCompounds 1 -33.

39. The compound of claim 1, wherein the compound is selected from any one of Compounds 1-16 and 19-28, optionally wherein the compound is Compound 1, 3, 10, 11, 12, 15, 16, 19, 20, 21, 22, 23, 25, or 28.

40. The compound of claim 1, whereinR1is -(C1-C9 alkylene)-R5;R5is -C(O)O-(CH2)p-CH-(R8)R9or -OC(O)-(CH2)q-CH-(R8)R9; each P.” and R9is independently C1-C12 alkyl or C2-C12 alkenyl; n is 1-3; andR2is hydrogen.

41. 'The compound of claim 40, where inR2is hydrogen;R2" is Ci- Cs 2 alkyl; m is 2; and t is 0.

42. The compound of claim 41, wherein Y is pyrrolidinyl.

43. The compound of claim 41 or claim 42, wherein R5is -OC(O)-(CH2)q-CH-(R8)R9.

44. The compound of any one of claims 41-43, wherein q is 0; and n is 2.

45. The compound of any one of claims 41 -44, whereinR8is / j-hexyl; andR9is M-butyl.

46. The compound of claim 40, whereinR5is -C ’(O)O-(CI I2)p-CH-( R8)R9; andRsand R9are the same.

47. The compound of claim 46, wherein Rrand R!are the same.

48. The compound of claim 46 or claim 47, whereinX1is CH.-:Y is pyrrolidinyl; and49. The compound of claim 46 or claim 47, whereinX1is O;Y is diethylamino or .V-ethylpiperidinyl; and n is 1 or 3.

50. A lipid nanoparticle comprising the compound of any one of claims 1 -49.

51. A lipid nanoparticle comprising the compound of any one of claims 1-49; a phospholipid; a cholesterol; and a polyethylene glycol lipid.

52. A lipid nanoparticle comprising: about 20-80 mol% of the compound of any one of claims 1-49, about 7.5-40 mol% of phospholipid, about 6-50 mol% of cholesterol, and about 1-4 mol% of PEG lipid.

53. The lipid nanoparticle of claim 52, comprising: about 40 mol% of the compound of any one of claims 1-49, about 30 mol% of phospholipid, about 28.5 mol% of cholesterol, and about 1.5 mol% of PEG lipid.

54. A lipid nanoparticle comprising: about 45-50 mol% of the compound of any one of claims 1-49, about 10 mol% of phospholipid, about 38-42 mol% of cholesterol, and about 2-3 mol% of PEG lipid.

55. The lipid nanoparticle of claim 54, comprising: about 47.5 mol% of the compound of any one of claims 1-49, about 40 mol% of cholesterol, and about 2.5 mol% of PEG lipid.

56. A lipid nanoparticle comprising: about 47.5-52.5 mol% of the compound of any one of claims 1-49, about 10 mol% of phospholipid, about 37-40 mol% of cholesterol, and about1-2 mol% of PEG lipid.

57. The lipid nanoparticle of claim 56, comprising: about 50 mol% of the compound of any one of claims 1-49, about 38.5 mol% of cholesterol, and about 1.5 mol% of PEG lipid.

58. A lipid nanoparticle comprising: about 57.5-62.5 mol% of the compound of any one of claims 1-49, about 10 mol% of phospholipid, about 26-29 mol% of cholesterol, and about2-3 mol% of PEG lipid.

59. The lipid nanoparticle of claim 58, comprising: about 60 mol% of the compound of any one of claims 1-49, about 27.5 mol% of cholesterol, and about 2.5 mol% of PEG lipid.

60. A lipid nanoparticle comprising: about 45-50 mol% of the compound of any one of claims 1 -49, about 10 mol% of phospholipid, about 37.5-40.5 mol% of cholesterol, and about3-4 mol% of PEG lipid.61 . The lipid nanoparticle of claim 59, comprising: about 47.5 mol% of the compound of any one of claims 1 -49, about 39 mol% of cholesterol, and about 3.5 mol% of PEG lipid.

62. The lipid nanoparticle of any one of claims 51-61, further comprising a targeting component.

63. The lipid nanoparticle of claim 62, wherein the targeting component is a targeting lipid.

64. The lipid nanoparticle of claim 62, wherein the targeting component is an active targeting component.The lipid nanoparticle of claim 64, wherein the active targeting component is an antibody, an antigen-binding fragment of an antibody, a protein, a peptide, or a small molecule.

66. 'The lipid nanoparticle of any one of claims 50-65, further comprising one or more polynucleotides encapsulated within the lipid nanoparticle.

67. The lipid nanoparticle of claim 66, wherein the one or more polynucleotides comprises RNA.

68. The lipid nanoparticle of claim 66, wherein the one or more polynucleotides comprises DNA.

69. The lipid nanoparticle of claim 66, wherein the one or more polynucleotides comprises DNA and RN A.

70. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 50-69, and a pharmaceutically acceptable excipient.71 . A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 66-69, and a pharmaceutically acceptable excipient.

72. 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 50-69 or the pharmaceutical composition of claim 70 or claim 71 .

73. The method of claim 72, wherein the extrahepatic cell or tissue comprises a brain cell or tissue.

74. The method of claim 72, wherein the extrahepatic cell or tissue comprises a lung cell or tissue.

75. The method of claim 72, wherein the extrahepatic cell or tissue comprises a bone marrow cell or tissue.

76. The method of claim 72, wherein the extrahepatic cell or tissue comprises a spleen cell or tissue.

77. The method of claim 72, wherein the extrahepatic cell or tissue comprises a kidney cell or tissue.

78. The method of claim 72, wherein the extrahepatic cell or tissue comprises a heart cell or tissue.

79. The method of claim 72, wherein the extrahepatic cell or tissue comprises a pancreatic cell or tissue.

80. The method of claim 72, wherein the extrahepatic cell or tissue comprises a muscle cell or tissue.81 . The method of claim 72, wherein the extrahepatic cell or tissue comprises an immune cell or tissue.

82. A method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 70 or claim 71.

83. 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 -49.

84. 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 1-49.

85. The method of claim 83 or claim 84, wherein the active agent comprises DMA.

86. The method of claim 83 or claim 84, wherein the active agent comprises RNA.

87. The method of claim 83 or claim 84, wherein the active agent comprises DNA andRNA.

88. The lipid nanoparticie of any one of clams 50-69 or the pharmaceutical composition of claim 70 or claim 71 tor use in delivering an active agent (e.g., one or more polynucleotides) to an extrahepatic cell or tissue in a subject.

89. Use of the lipid nanoparticie of any one of clams 50-69 or the pharmaceutical composition of claim 70 or claim 71 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.

90. The lipid nanoparticie of any one of clams 50-69 or the pharmaceutical composition of claim 70 or claim 71 for use in treating a disease in a subject.

91. Use of the lipid nanoparticie of any one of elams 50-69 or the pharmaceutical composition of claim 70 or claim 71 in the manufacture of a medicament for treating a disease in a subject.

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