Modified amine lipids

Abstract

The present disclosure provides ionizable amine lipids and salts thereof (e.g., pharmaceutically acceptable salts thereof) that are useful in the delivery of bioactive agents, such as the delivery of bioactive agents to cells to prepare engineered cells. The ionizable amine lipids disclosed herein can be used as ionizable lipids in the formulation of lipid nanoparticle-based compositions.

Description

[0001] This invention is a divisional application of Chinese Patent Application No. 201980086265.9, filed on December 5, 2019, whose original invention was entitled "Modified Amine Lipids".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 775,783, filed December 5, 2018, the entire contents of which are incorporated herein by reference. Background Technology

[0004] Lipid nanoparticles formulated with ionizable amine-containing lipids can serve as cargo mediators for delivering bioactive agents, particularly polynucleotides such as RNA, mRNA, and guide RNA, into cells. LNP compositions containing ionizable lipids can facilitate the delivery of oligonucleotide agents across cell membranes and can be used to introduce components and compositions for gene editing into living cells. Bioactive agents that are particularly difficult to deliver into cells include proteins, nucleic acid-based drugs and their derivatives, especially drugs containing relatively large oligonucleotides such as mRNA. Compositions for delivering promising gene editing technologies into cells, such as those for delivering components of the CRISPR / Cas9 system (e.g., mRNA encoding nucleases and associated guide RNA (gRNA)), are of particular interest.

[0005] There is a need for compositions for delivering CRISPR / Cas protein and nucleic acid components into cells, such as cells in patients. Specifically, compositions for delivering mRNA encoding CRISPR protein components and compositions for delivering CRISPR guide RNA are of particular interest. Compositions with useful properties for in vitro and in vivo delivery, and compositions that can stabilize and deliver RNA components, are also of particular interest. Summary of the Invention

[0006] This disclosure provides amine-containing lipids that can be used to formulate lipid nanoparticle (LNP) compositions. Such LNP compositions may have properties that facilitate the delivery of nucleic acid cargoes, such as CRISPR / Cas gene-editing components, into cells.

[0007] In some embodiments, this disclosure relates to a compound having the structure of formula (IA):

[0008]

[0009] Among them, each occurrence is independent.

[0010] X 1 C 1-3 alkylene or

[0011] X 2 Selected from O, NH, NMe and bonds,

[0012] X 3 C 2-4 Alkylene

[0013] X 4 It is a C1 alkylene group or bond.

[0014] X 5 It is a C1 alkylene group or bond.

[0015] R 1 C 1-3 alkyl,

[0016] R 2 C 1-3 Alkyl, or

[0017] R 2 With nitrogen atoms and R 1 or X 3 Carbon atoms together form 4-, 5-, or 6-membered rings.

[0018] Y 1 Selected from the following: -CH=CH-, -(C=O)O-, and -O(C=O)-.

[0019] Y 2 Selected from -CH2-CH=CH- and C3-C4 alkylene groups,

[0020] R 3 Selected from H, C 5-7 cycloalkyl, C8-C 10 alkenyl and C 3-18 alkyl,

[0021] R 4 C 4-8 alkyl,

[0022] or its salt,

[0023] The condition is that when R 2 For Me, R 3 For straight chain C 12 Alkyl, X 1 It is a C2 alkylene group, X 2 For O, X 3 It is a C3 alkylene group, X 4 As the key, X 5 It is a C1 alkylene group, Y 1 For cis -CH = CH-, and -Y 2 –R 4When it is cis-CH2-CH=CH-(C5-alkyl), then R 1 C 2-3 alkyl.

[0024] In some embodiments, this disclosure relates to a compound having the structure of formula (I):

[0025] Among them, each occurrence is independent.

[0026] X 1 C 2-3 Alkylene

[0027] X 2 Selected from O, NH, NMe and bonds,

[0028] X 3 C 2-4 Alkylene

[0029] X 4 It is a C1 alkylene group or bond.

[0030] X 5 It is a C1 alkylene group or bond.

[0031] R 1 C 1-2 alkyl,

[0032] R 2 C 1-2 Alkyl, or

[0033] R 2 With nitrogen atoms and R 1 or X 3 Carbon atoms together form 5- or 6-membered rings.

[0034] Y 1 Selected from -CH=CH-, -(C=O)O-, and -O(C=O)-.

[0035] R 3 Selected from H, C 5-7 cycloalkyl and C 3-18 alkyl,

[0036] R 4 C 4-7 alkyl,

[0037] or its salt,

[0038] The condition is that when R 2 For Me, R 3 For straight chain C 12 Alkyl, X 1 It is a C2 alkylene group, X 2 For O, X3 It is a C3 alkylene group, X 4 As the key, X 5 It is a C1 alkylene group, Y 1 For cis-CH=CH-, and R 4 When it is a C5 alkyl group, then R 1 It is a C2 alkyl group, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0039] In some embodiments, the compound is a compound of formula (I), wherein the condition is that R 2 For Me, R 3 For straight chain C 12 Alkyl, X 1 It is a C2 alkylene group, X 2 For O, X 3 It is a C3 alkylene group, X 4 As the key, X 5 It is a C1 alkylene group, and Y 1 When it is cis-CH=CH-, then R 1 It is a C2 alkyl group.

[0040] In some implementations, X 1 It is a straight-chain C2-alkylene group. In other embodiments, X 1 It is a C1 alkylene group. In some embodiments, X 1 It is a straight-chain C3 alkylene or a branched C3 alkylene.

[0041] In some other implementations, X 2 It is NH or O. In some preferred embodiments, X 2 For O and X 3 C 3-4 Alkylene. In some embodiments, X 3 C 2-3 Alkylene. In some embodiments, X 2 For NMe or key.

[0042] In some implementations, R 2 With X 3 The carbon atoms together form a four-membered ring. In some implementations, R 2 With X 3 The carbon atoms together form a 5-membered ring. In some implementations, R 2 With X 3 The carbon atoms together form a 6-membered ring. Alternatively, R 2 It can be a C2 alkyl group. In some cases, R 2 It is a C1 alkyl group. In some embodiments, R 2 For example, replaced by a hydroxyl group, R 2Examples include hydroxylated C2 alkyl groups.

[0043] In some implementations, R 1 It is a C1 alkyl group. Alternatively, R 1 It can be a C2 alkyl group. In some embodiments, R 1 For example, replaced by a hydroxyl group, R 1 Examples include hydroxyl-substituted C2 alkyl groups. In other embodiments, R 1 With R 2 Together they form a 4-membered ring. In other implementations, R 1 With R 2 Together they form a 5-membered ring. In other implementations, R 1 With R 2 Together they form a 6-membered ring.

[0044] In some implementation schemes, R 3 For linear C 8-16 Alkyl groups, such as linear C 7-12 Alkyl groups, such as linear C 7-9 Alkyl group. Alternatively, R 3 It can be a straight chain C 3-6 Alkyl group. In some embodiments, R 3 For branch C 6-10 Alkyl group. In other embodiments, R 3 C 5-7 Cycloalkyl, such as C6 cycloalkyl. In some embodiments, R 3 For H. In some implementations, R 3 C 8-10 Alkenyl group.

[0045] In some implementations, R 4 For straight chain C 5-6 alkyl.

[0046] In some implementation schemes, X 4 For key.

[0047] In some implementations, X 5 It is a C1 alkylene group.

[0048] In some implementation schemes, Y 2 –R 4 –CH2-CH=CH-R 4 .

[0049] In other implementations, Y 2 It is a straight-chain C3 alkylene group.

[0050] In some implementation schemes, Y 1Selected from the bond, -CH=CH- and -O(C=O)-, for example -CH=CH- and -O(C=O)-.

[0051] In some embodiments, the compound is a compound of formula II:

[0052] The variables are defined as shown in equation (IA).

[0053] In some embodiments, the compound is a compound of formula III;

[0054]

[0055] (III), where the variables are defined as in equation (IA).

[0056] Representative compounds of formula (IA) or (I) include:

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Or its salt, preferably a pharmaceutically acceptable salt.

[0086] In some embodiments, the compound is selected from:

[0087]

[0088]

[0089]

[0090] Or its salts. In other embodiments, the compound is selected from:

[0091]

[0092]

[0093]

[0094] Or its salts. In other embodiments, the compound is selected from:

[0095]

[0096]

[0097]

[0098] Or its salt.

[0099] In some embodiments, this disclosure relates to any of the compounds described herein, wherein the pKa of the protonated form of said compound is from about 5.1 to about 8.0, for example, from about 5.6 to about 7.2, from about 5.7 to about 6.5, or from about 5.8 to about 6.2. In some embodiments, the pKa of the protonated form of said compound is from about 5.5 to about 6.0. In some embodiments, the pKa of the protonated form of said compound is from about 6.1 to about 6.3.

[0100] In some embodiments, this disclosure relates to a composition comprising any of the compounds described herein.

[0101] In some embodiments, this disclosure relates to a composition comprising any of the compounds described herein and a lipid component, such as comprising about 50% by weight of the compounds described herein and a lipid component, such as an amine lipid, preferably a compound of formula (IA), (I), (II), or (III).

[0102] In some embodiments, this disclosure relates to any composition described herein, wherein said composition is an LNP composition. For example, this disclosure relates to an LNP composition comprising any of the compounds described herein and a lipid component. In some embodiments, this disclosure relates to any LNP composition described herein, wherein said lipid component comprises accessory lipids and PEG lipids. In some embodiments, this disclosure relates to any LNP composition described herein, wherein said lipid component comprises accessory lipids, PEG lipids, and neutral lipids. In some embodiments, this disclosure relates to any LNP composition described herein that further comprises a cryoprotectant. In some embodiments, this disclosure relates to any LNP composition described herein that further comprises a buffer.

[0103] In some embodiments, this disclosure relates to any LNP composition described herein that further comprises a nucleic acid component. In some embodiments, this disclosure relates to any LNP composition described herein that further comprises an RNA or DNA component. In some embodiments, this disclosure relates to any LNP composition described herein where the N / P ratio of the LNP composition is about 3-10, for example, an N / P ratio of about 6 ± 1, or an N / P ratio of about 6 ± 0.5. In some embodiments, this disclosure relates to any LNP composition described herein where the N / P ratio of the LNP composition is about 6.

[0104] In some embodiments, this disclosure relates to any LNP composition described herein, wherein the RNA component comprises mRNA. In some embodiments, this disclosure relates to any LNP composition described herein, wherein the RNA component comprises an RNA-directed DNA binder, such as Cas nuclease mRNA, such as class 2 Cas nuclease mRNA or Cas9 nuclease mRNA.

[0105] In some embodiments, this disclosure relates to any LNP composition described herein, wherein the mRNA is a modified mRNA. In some embodiments, this disclosure relates to any LNP composition described herein, wherein the RNA component comprises gRNA nucleic acid. In some embodiments, this disclosure relates to any LNP composition described herein, wherein the gRNA nucleic acid is gRNA.

[0106] In some embodiments, this disclosure relates to the LNP compositions described herein, wherein the RNA component comprises two types of Cas nuclease mRNA and gRNA. In some embodiments, this disclosure relates to any LNP composition described herein, wherein the gRNA nucleic acid is or encodes a bidirectional guide RNA (dgRNA). In some embodiments, this disclosure relates to any LNP composition described herein, wherein the gRNA nucleic acid is or encodes a unidirectional guide RNA (sgRNA).

[0107] In some embodiments, this disclosure relates to any LNP composition described herein, wherein the gRNA is a modified gRNA. In some embodiments, this disclosure relates to any LNP composition described herein, wherein the modified gRNA contains a modification at one or more of the first five nucleotides at the 5' end. In some embodiments, this disclosure relates to any LNP composition described herein, wherein the modified gRNA contains a modification at one or more of the last five nucleotides at the 3' end.

[0108] In some embodiments, this disclosure relates to any LNP composition described herein that further comprises at least one template nucleic acid.

[0109] In some embodiments, this disclosure relates to a method of gene editing that includes contacting cells with LNPs. In some embodiments, this disclosure relates to any method of gene editing described herein that includes cleaving DNA.

[0110] In some embodiments, this disclosure relates to a method of lysing DNA, which includes contacting cells with an LNP composition. In some embodiments, this disclosure relates to any method of lysing DNA described herein, wherein the lysis step includes introducing single-stranded DNA nicks. In some embodiments, this disclosure relates to any method of lysing DNA described herein, wherein the lysis step includes introducing double-stranded DNA breaks. In some embodiments, this disclosure relates to any method of lysing DNA described herein, wherein the LNP composition comprises two types of Cas mRNA and guide RNA nucleic acids. In some embodiments, this disclosure relates to any method of lysing DNA described herein, which further includes introducing at least one template nucleic acid into the cells. In some embodiments, this disclosure relates to any method of lysing DNA described herein, which includes contacting the cells with an LNP composition containing template nucleic acids.

[0111] In some embodiments, this disclosure relates to any method of gene editing described herein, wherein said method includes administering the LNP composition to an animal, such as a human. In some embodiments, this disclosure relates to any method of gene editing described herein, wherein said method includes administering the LNP composition to a cell, such as a eukaryotic cell.

[0112] In some embodiments, this disclosure relates to any method of gene editing described herein, wherein the method includes administering mRNA formulated in a first LNP composition and a second LNP composition, the second LNP composition comprising one or more of mRNA, gRNA, gRNA nucleic acid, and template nucleic acid. In some embodiments, this disclosure relates to any method of gene editing described herein, wherein the first LNP composition and the second LNP composition are administered simultaneously. In some embodiments, this disclosure relates to any method of gene editing described herein, wherein the first LNP composition and the second LNP composition are administered sequentially. In some embodiments, this disclosure relates to any method of gene editing described herein, wherein the method includes administering mRNA and guide RNA nucleic acid formulated in a single LNP composition.

[0113] In some embodiments, this disclosure relates to any of the gene editing methods described herein, wherein the gene editing produces gene knockout.

[0114] In some embodiments, this disclosure relates to any of the gene editing methods described herein, wherein the gene editing produces gene correction. Attached Figure Description

[0115] Figures 1A-1C The in vivo editing at the TTR locus and the compounds tested in the assay are shown. Figure 1AThe chemical structure of the tested compound is shown. Figure 1B The percentage of TTR edited in mouse hepatocytes after delivery is shown. Figure 1C The serum TTR level (μg / mL) after delivery is shown.

[0116] Figure 2 This is a graph showing the percentage of TTR editing in mouse hepatocytes after LNP delivery with either the inclusion (IA) compound or the control compound. Dose-response data are also shown.

[0117] Figures 3A-3B The results of LNP delivery using the inclusion (IA) compound are shown. Figure 3A This is a graph showing the percentage of TTR editing in mouse hepatocytes after delivery. Figure 3B This is a graph showing serum TTR (μg / mL) after delivery.

[0118] Figures 4A-4C The in vivo editing at the TTR locus and the compounds tested in the assay are shown. Figure 4A The chemical structure of the tested compound is shown. Figure 4B The percentage of TTR edited in mouse hepatocytes after delivery is shown. Figure 4C The serum TTR level (μg / mL) after delivery is shown.

[0119] Figures 5A-5C The compound of formula (IA) and the delivery results using said compound are shown. Figure 5A The chemical structure of the tested compound is shown. Figure 5B The percentage of TTR edited in mouse hepatocytes after delivery is shown. Figure 5C The serum TTR level (μg / mL) after delivery is shown.

[0120] Figures 6A-6C The compound of formula (IA) and the delivery results using said compound are shown. Figure 6A The chemical structure of the tested compound is shown. Figure 6B The percentage of TTR edited in mouse hepatocytes after delivery is shown. Figure 6C The serum TTR level (μg / mL) after delivery is shown.

[0121] Figures 7A-7C The compound of formula (IA) and the delivery results using said compound are shown. Figure 7A The chemical structure of the tested compound is shown. Figure 7B The percentage of TTR edited in mouse hepatocytes after delivery is shown. Figure 7C The serum TTR level (μg / mL) after delivery is shown.

[0122] Figures 8A-8C The compound of formula (IA) and the delivery results using said compound are shown. Figure 8A The chemical structures of the tested compounds are shown, where n = 1, 2, or 3. Figure 8B The percentage of TTR edited in mouse hepatocytes after delivery is shown. Figure 8C The serum TTR level (μg / mL) after delivery is shown.

[0123] Figures 9A-9C The compound of formula (IA) and the delivery results using said compound are shown. Figure 9A The chemical structure of the tested compound is shown. Figure 9B The percentage of TTR edited in mouse hepatocytes after delivery is shown. Figure 9C The serum TTR level (μg / mL) after delivery is shown.

[0124] Figures 10A-10C The compound of formula (IA) and the delivery results using said compound are shown. Figure 10A The chemical structure of the tested compound is shown. Figure 10B The percentage of TTR edited in mouse hepatocytes after delivery is shown. Figure 10C The serum TTR level (μg / mL) after delivery is shown.

[0125] Figures 11A-11D The compound of formula (IA) and the delivery results using said compound are shown. Figure 11A and Figure 11B The chemical structure of the tested compound is shown. Figure 11C The percentage of TTR edited in mouse hepatocytes after delivery is shown. Figure 11D The serum TTR level (μg / mL) after delivery is shown.

[0126] Figures 12A-12B The compound of formula (IA) and the delivery results using said compound are shown. Figure 12A The percentage of TTR edited in rat hepatocytes after delivery is shown. Figure 12B The serum TTR level (μg / mL) after delivery is shown.

[0127] Figures 13A-13B The compound of formula (IA) and the delivery results using said compound are shown. Figure 13A The chemical structure of the tested compound is shown. Figure 13B The percentage of TTR edited in HUH7 cells after delivery is shown. Detailed Implementation

[0128] This disclosure provides lipids, particularly ionizable lipids, that can be used to deliver bioactive agents to cells, and methods for preparing and using such compositions, the bioactive agents including nucleic acids, such as CRISPR / Cas component RNA (“cargo”). The lipids and pharmaceutically acceptable salts thereof are provided, optionally as compositions comprising the lipids, including LNP compositions. In some embodiments, the LNP composition may comprise a bioactive agent, such as an RNA component, and a lipid component comprising compounds of formula (IA), (I), (II), or (III) as defined herein. In some embodiments, the RNA component comprises nucleic acids. In some embodiments, the lipids are used to deliver bioactive agents, such as nucleic acids such as mRNA, to cells such as hepatocytes. In some embodiments, the RNA component comprises gRNA and, optionally, mRNA encoding class 2 Cas nucleases, and optionally gRNA or nucleic acids encoding gRNA. Gene editing methods using these compositions and methods for preparing engineered cells are also provided.

[0129] Lipid nanoparticle composition

[0130] This document discloses various LNP compositions for delivering bioactive agents, such as nucleic acids, e.g., mRNA and gRNA, including CRISPR / Cas cargo. These LNP compositions include “ionizable amine lipids” as well as neutral lipids, PEG lipids, and cofactor lipids. “Lipid nanoparticles” or “LNPs” refers to, but is not limited to, particles comprising multiple (i.e., more than one) LNP components physically associated with each other by intermolecular forces.

[0131] lipids

[0132] This disclosure provides lipids that can be used in LNP compositions.

[0133] In some embodiments, this disclosure relates to a compound having the structure of formula (IA):

[0134]

[0135] Among them, each occurrence is independent.

[0136] X 1 C 1-3 alkylene or

[0137] X 2 Selected from O, NH, NMe and bonds,

[0138] X 3 C 2-4 Alkylene

[0139] X 4It is a C1 alkylene group or bond.

[0140] X 5 It is a C1 alkylene group or bond.

[0141] R 1 C 1-3 alkyl,

[0142] R 2 C 1-3 Alkyl, or

[0143] R 2 With nitrogen atoms and R 1 or X 3 Carbon atoms together form 4-, 5-, or 6-membered rings.

[0144] Each Y 1 Independently selected from the bonds, -CH=CH-, -(C=O)O-, and -O(C=O)-.

[0145] Each Y 2 Independently selected from -CH2-CH=CH- and C3-C4 alkylene groups.

[0146] R 3 Selected from H, C 5-7 cycloalkyl, C8-C 10 alkenyl and C 3-18 alkyl,

[0147] Each R 4 Independently for C 4-8 alkyl,

[0148] or its salt,

[0149] The condition is that when R 2 For Me, R 3 For straight chain C 12 Alkyl, X 1 It is a C2 alkylene group, X 2 For O, X 3 It is a C3 alkylene group, X 4 As the key, X 5 It is a C1 alkylene group, Y 1 For cis -CH = CH-, and -Y 2 –R 4 When it is cis-CH2-CH=CH-(C5-alkyl), then R 1 C 2-3 alkyl.

[0150] In some embodiments, the compound is a compound of formula (IA), wherein the condition is that R 2 When it is Me, R 1 C 2-3alkyl.

[0151] In some embodiments, the compound is a compound of formula (IA), wherein R 2 Not Me.

[0152] In some embodiments, the compound is a compound of formula (IA), wherein R 1 Not Me.

[0153] In some embodiments, the compound is a compound of formula (IA), wherein R 3 Not a straight chain C 12 alkyl.

[0154] In some embodiments, the compound is a compound of formula (IA), wherein X 1 It is not a C2 alkylene group.

[0155] In some embodiments, the compound is a compound of formula (IA), wherein X 2 It's not O.

[0156] In some embodiments, the compound is a compound of formula (IA), wherein X 3 It is not a C3 alkylene group.

[0157] In some embodiments, the compound is a compound of formula (IA), wherein X 4 It's not a key.

[0158] In some embodiments, the compound is a compound of formula (IA), wherein X 5 It is not a C1 alkylene group.

[0159] In some embodiments, the compound is a compound of formula (IA), wherein Y 1 It is not cis-CH=CH-.

[0160] In some embodiments, the compound is a compound of formula (IA), wherein -Y 2 –R 4 It is not cis-CH2-CH=CH-(C5-alkyl).

[0161] In some embodiments, this disclosure relates to a compound of formula (I):

[0162] Among them, each occurrence is independent.

[0163] X 1 C 2-3 Alkylene

[0164] X 2Selected from O, NH, NMe and bonds,

[0165] X 3 C 2-4 Alkylene

[0166] X 4 It is a C1 alkylene group or bond.

[0167] X 5 It is a C1 alkylene group or bond.

[0168] R 1 C 1-2 alkyl,

[0169] R 2 C 1-2 Alkyl, or

[0170] R 2 With nitrogen atoms and R 1 or X 3 Carbon atoms together form 5- or 6-membered rings.

[0171] Y 1 Selected from -CH=CH-, -(C=O)O-, and -O(C=O)-.

[0172] R 3 Selected from H, C 5-7 cycloalkyl and C 3-18 alkyl,

[0173] R 4 C 4-7 alkyl,

[0174] or its salt,

[0175] The condition is that when R 2 For Me, R 3 For straight chain C 12 Alkyl, X 1 It is a C2 alkylene group, X 2 For O, X 3 It is a C3 alkylene group, X 4 As the key, X 5 It is a C1 alkylene group, Y 1 For cis-CH=CH-, and R 4 When it is a C5 alkyl group, then R 1 It is a C2 alkyl group, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0176] In some embodiments, the compound is a compound of formula (I), wherein the condition is that R 2 For Me, R 3 For straight chain C 12 Alkyl, X 1It is a C2 alkylene group, X 2 For O, X 3 It is a C3 alkylene group, X 4 As the key, X 5 It is a C1 alkylene group, and Y 1 When it is cis-CH=CH-, then R 1 It is a C2 alkyl group.

[0177] In some embodiments, the compound is a compound of formula (I), wherein the condition is that R 2 When it is Me, R 1 It is a C2 alkyl group.

[0178] In some embodiments, the compound is a compound of formula (I), wherein R 2 Not Me.

[0179] In some embodiments, the compound is a compound of formula (I), wherein R 1 Not Me.

[0180] In some embodiments, the compound is a compound of formula (I), wherein R 3 Not a straight chain C 12 alkyl.

[0181] In some embodiments, the compound is a compound of formula (I), wherein X 1 It is not a C2 alkylene group.

[0182] In some embodiments, the compound is a compound of formula (I), wherein X 2 It's not O.

[0183] In some embodiments, the compound is a compound of formula (I), wherein X 3 It is not a C3 alkylene group.

[0184] In some embodiments, the compound is a compound of formula (I), wherein X 4 It's not a key.

[0185] In some embodiments, the compound is a compound of formula (I), wherein X 5 It is not a C1 alkylene group.

[0186] In some embodiments, the compound is a compound of formula (I), wherein Y 1 It is not cis-CH=CH-.

[0187] In some embodiments, the compound is enriched enantiomerically or diastereomerically; for example, the compound is enriched enantiomerically.

[0188] In some implementations, X 1 It is a straight-chain C2-alkylene group. In other embodiments, X 1 It is a C1 alkylene group. In some embodiments, X 1 It is a straight-chain C3 alkylene or a branched C3 alkylene.

[0189] In some other implementations, X 2 It is NH or O. In some preferred embodiments, X 2 For O and X 3 C 3-4 Alkylene. In some embodiments, X 3 C 2-3 Alkylene. In some embodiments, X 2 For NMe or key.

[0190] In some implementations, R 2 With X 3 The carbon atoms together form a four-membered ring. In some implementations, R 2 With X 3 The carbon atoms together form a 5-membered ring. In some implementations, R 2 With X 3 The carbon atoms together form a 6-membered ring. Alternatively, R 2 It can be a C2 alkyl group. In some cases, R 2 It is a C1 alkyl group. In some embodiments, R 2 For example, replaced by a hydroxyl group, R 2 Examples include hydroxylated C2 alkyl groups.

[0191] In some implementations, R 1 It is a C1 alkyl group. Alternatively, R 1 It can be a C2 alkyl group. In some embodiments, R 1 For example, replaced by a hydroxyl group, R 1 Examples include hydroxyl-substituted C2 alkyl groups. In some embodiments, R 1 It is not replaced. In some implementations, R 2 It is not replaced. In the preferred embodiment, R 1 and R 2 All are unreplaced. In other implementations, R 1 With R 2 Together they form a 4-membered ring. In other implementations, R 1 With R 2 Together they form a 5-membered ring. In other implementations, R 1 With R 2 Together they form a 6-membered ring.

[0192] In some implementation schemes, R 3 For linear C 8-16 Alkyl groups, such as linear C 7-12 Alkyl groups, such as linear C 7-9 Alkyl group. Alternatively, R 3 It can be a straight chain C 3-6 Alkyl group. In some embodiments, R 3 For branch C 6-10 Alkyl group. In other embodiments, R 3 C 5-7 Cycloalkyl, such as C6 cycloalkyl. In some embodiments, R 3 For H. In some implementations, R 3 C 8-10 Alkenyl group.

[0193] In some implementations, R 4 For straight chain C 5-6 alkyl.

[0194] In some implementation schemes, X 4 For key.

[0195] In some implementations, X 5 It is a C1 alkylene group.

[0196] In some implementation schemes, Y 2 –R 4 –CH2-CH=CH-R 4 .

[0197] In other implementations, Y 2 It is a straight-chain C3 alkylene group.

[0198] In some implementation schemes, Y 1 Selected from the bond, -CH=CH- and -O(C=O)-, for example -CH=CH- and -O(C=O)-.

[0199] In some embodiments, the compound is a compound of formula II:

[0200] The variables are defined as shown in equation (IA).

[0201] In some implementations, X 1 It is a straight-chain C2-alkylene group. In other embodiments, X 1 It is a C1 alkylene group. In some embodiments, X 1 It is a straight-chain C3 alkylene or a branched C3 alkylene.

[0202] In some implementation schemes, R 3 For linear C 8-16Alkyl groups, such as linear C 7-12 Alkyl groups, such as linear C 7-9 Alkyl group. Alternatively, R 3 It can be a straight chain C 3-6 Alkyl group. In some embodiments, R 3 For branch C 6-10 Alkyl group. In other embodiments, R 3 C 5-7 Cycloalkyl, such as C6 cycloalkyl. In some embodiments, R 3 For H. In some implementations, R 3 C 8-10 Alkenyl group.

[0203] In some implementations, R 4 For straight chain C 5-6 alkyl.

[0204] In some implementation schemes, X 4 For key.

[0205] In some implementations, X 5 It is a C1 alkylene group.

[0206] In some implementation schemes, Y 2 –R 4 –CH2-CH=CH-R 4 .

[0207] In other implementations, Y 2 It is a straight-chain C3 alkylene group.

[0208] In some implementation schemes, Y 1 Selected from the bond, -CH=CH- and -O(C=O)-, for example -CH=CH- and -O(C=O)-.

[0209] In some embodiments, the compound is selected from:

[0210]

[0211]

[0212] Or its salt.

[0213] In some embodiments, the compound is a compound of formula III;

[0214]

[0215] (III), where the variables are defined as in equation (IA).

[0216] In some implementations, X1 It is a straight-chain C2-alkylene group. In other embodiments, X 1 It is a C1 alkylene group. In some embodiments, X 1 It is a straight-chain C3 alkylene or a branched C3 alkylene.

[0217] In some other implementations, X 2 It is NH or O. In some preferred embodiments, X 2 For O and X 3 C 3-4 Alkylene. In some embodiments, X 3 C 2-3 Alkylene. In some embodiments, X 2 For NMe or key.

[0218] In some implementations, R 2 With X 3 The carbon atoms together form a four-membered ring. In some implementations, R 2 With X 3 The carbon atoms together form a 5-membered ring. In some implementations, R 2 With X 3 The carbon atoms together form a 6-membered ring. Alternatively, R 2 It can be a C2 alkyl group. In some cases, R 2 It is a C1 alkyl group. In some embodiments, R 2 For example, replaced by a hydroxyl group, R 2 Examples include hydroxylated C2 alkyl groups.

[0219] In some implementations, R 1 It is a C1 alkyl group. Alternatively, R 1 It can be a C2 alkyl group. In some embodiments, R 1 For example, replaced by a hydroxyl group, R 1 Examples include hydroxyl-substituted C2 alkyl groups. In some embodiments, R 1 It is not replaced. In some implementations, R 2 It is not replaced. In the preferred embodiment, R 1 and R 2 All are unreplaced. In other implementations, R 1 With R 2 Together they form a 4-membered ring. In other implementations, R 1 With R 2 Together they form a 5-membered ring. In other implementations, R 1 With R 2 Together they form a 6-membered ring.

[0220] In some implementation schemes, R 3For straight chain C 8-16 Alkyl groups, such as straight-chain C 7-12 Alkyl groups, such as straight-chain C 7-9 Alkyl group. Alternatively, R 3 It can be a straight chain C 3-6 Alkyl group. In some embodiments, R 3 For branch C 6-10 Alkyl group. In other embodiments, R 3 C 5-7 Cycloalkyl, such as C6 cycloalkyl. In some embodiments, R 3 For H. In some implementations, R 3 C 8-10 Alkenyl group.

[0221] In some implementation schemes, X 4 For key.

[0222] In some implementations, X 5 It is a C1 alkylene group.

[0223] Representative compounds of formula (IA) or (I) include:

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252] Or its salts, such as its pharmaceutically acceptable salts.

[0253] Representative compounds of formula (IA) or (I) include:

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267] In some embodiments, the compound is selected from:

[0268]

[0269]

[0270]

[0271] Or its salts. In other embodiments, the compound is selected from:

[0272]

[0273]

[0274] Or its salts. In other embodiments, the compound is selected from:

[0275]

[0276]

[0277] Or its salt.

[0278] In some embodiments, at least 75% of the compound of formula (IA), (I), (II), or (III) in the lipid composition formulated as disclosed herein is cleared from the subject's plasma within 8, 10, 12, 24, or 48 hours or within 3, 4, 5, 6, 7, or 10 days after administration. In some embodiments, at least 50% of the lipid composition comprising a compound of formula (IA), (I), (II), or (III) as disclosed herein is cleared from the subject's plasma within 8, 10, 12, 24, or 48 hours or within 3, 4, 5, 6, 7, or 10 days after administration, which may be determined, for example, by measuring lipids (e.g., compounds of formula (IA), (I), (II), or (III)), RNA (e.g., mRNA), or other components in the plasma. In some embodiments, the encapsulated lipids and free lipids, RNA, or nucleic acid components of the lipid composition are measured.

[0279] Lipid clearance can be measured as described in the literature. See Maier, MA et al., Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAiTherapeutics. Mol. Ther. 2013, 21(8), 1570-78 (“Maier”). For example, in Maier, a LNP-siRNA system containing siRNA targeting luciferase was administered to six- to eight-week-old male C57Bl / 6 mice via intravenous bolus injection via the lateral tail vein at a dose of 0.3 mg / kg. Blood, liver, and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 hours after administration. Mice were perfused with saline prior to tissue collection, and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. In addition, Maier describes the procedures used to assess the toxicity following administration of the LNP-siRNA composition. For example, male Sprague-Dawley rats were administered luciferase-targeting siRNA via a single intravenous bolus at dose volumes of 5 mL / kg, at doses of 0, 1, 3, 5, and 10 mg / kg (5 animals / group). Approximately 1 mL of blood was obtained from the jugular vein of conscious animals 24 hours later, and serum was separated. All animals were euthanized and autopsied 72 hours after administration. Clinical signs, body weight, serum chemistry, organ weight, and histopathology were assessed. Although Maier describes methods for evaluating siRNA-LNP compositions, these methods can be used to evaluate the clearance, pharmacokinetics, and toxicity of lipid compositions of this disclosure, such as LNP compositions.

[0280] In some embodiments, lipid compositions using compounds of formulas (IA), (I), (II), or (III) disclosed herein exhibit increased clearance relative to alternative ionizable amine lipids. In some such embodiments, clearance is a lipid clearance rate, such as the rate at which compounds of formulas (IA), (I), (II), or (III) are cleared from blood, serum, or plasma. In some embodiments, clearance is a cargo (e.g., a bioactive agent) clearance rate, such as the rate at which cargo components are cleared from blood, serum, or plasma. In some embodiments, clearance is an RNA clearance rate, such as the rate at which mRNA or gRNA is cleared from blood, serum, or plasma. In some embodiments, clearance is the rate at which LNPs are cleared from blood, serum, or plasma. In some embodiments, clearance is the rate at which LNPs are cleared from tissues such as liver or spleen tissue. Ideally, high clearance rates can result in a safety profile with no substantial side effects and / or reduced LNP accumulation in circulation and / or tissues.

[0281] The compounds of formulas (IA), (I), (II), or (III) of this disclosure may form salts depending on the pH of the medium in which they are present. For example, in a weakly acidic medium, the compounds of formulas (IA), (I), (II), or (III) may be protonated and thus carry a positive charge. Conversely, in a weakly alkaline medium, such as blood with a pH of about 7.35, the compounds of formulas (IA), (I), (II), or (III) may be unprotonated and thus uncharged. In some embodiments, the compounds of formulas (IA), (I), (II), or (III) of this disclosure may be protonated primarily at a pH of at least about 9. In some embodiments, the compounds of formulas (IA), (I), (II), or (III) of this disclosure may be protonated primarily at a pH of at least about 10.

[0282] The pH at which compounds of formula (IA), (I), (II), or (III) are primarily protonated is related to their inherent pKa. In a preferred embodiment, the pKa of salts of compounds of formula (IA), (I), (II), or (III) of this disclosure is in the range of about 5.1 to about 8.0, more preferably about 5.5 to about 7.5, for example, about 6.1 to about 6.3. In other preferred embodiments, the pKa of salts of compounds of formula (IA), (I), (II), or (III) of this disclosure is in the range of about 5.3 to about 8.0, for example, about 5.6 to about 7.2, about 5.7 to about 6.5. In other embodiments, the pKa of salts of compounds of formula (IA), (I), (II), or (III) of this disclosure is in the range of about 5.7 to about 6.4, for example, about 5.8 to about 6.2. In other preferred embodiments, the pKa of salts of compounds of formula (IA), (I), (II), or (III) of this disclosure is in the range of about 5.7 to about 6.5, for example, about 5.8 to about 6.4. Alternatively, the salts of the compounds of formula (IA), (I), (II), or (III) of this disclosure have a pKa in the range of about 5.8 to about 6.5. In some embodiments, the pKa of the protonated form of the compounds of formula (IA), (I), (II), or (III) is about 5.5 to about 6.0. The salts of the compounds of formula (IA), (I), (II), or (III) of this disclosure may have a pKa in the range of about 6.0 to about 8.0, preferably about 6.0 to about 7.5. The pKa of the salts of the compounds of formula (IA), (I), (II), or (III) can be an important consideration in the formulation of LNPs, as LNPs formulated with certain lipids in the range of about 5.5 to about 7.0 have been found to be effective for in vivo delivery of cargo, for example, to the liver. Furthermore, LNPs formulated with certain lipids in the range of about 5.3 to about 6.4 have been found to be effective for in vivo delivery, for example, to tumors. See, for example, WO 2014 / 136086.

[0283] In some embodiments, this disclosure relates to an enantiomerically enriched compound of formula (IA), (I), (II), or (III). The enantiomer enrichment level of a compound can be expressed as an enantiomer excess (ee). The ee of a compound can be measured by dividing the difference in enantiomer fractions by the sum of the enantiomer fractions. For example, if a compound is found to contain 98% (S)-enantiomers and 2% (R)-enantiomers, then the ee of said compound is (98-2) / (98+2), or 96%. In some embodiments, the compounds of formula (IA), (I), (II), or (III) have about 30%ee or greater, 40%ee or greater, 50%ee or greater, 60%ee or greater, 70%ee or greater, about 80%ee, about 85%ee, about 88%ee, about 90%ee, about 91%ee, about 92%ee, about 93%ee, about 94%ee, about 95%ee, about 96%ee, about 97%ee, about 98%ee, about 99%ee, or greater than about 99%ee, even if this %ee is greater than the %ee of the starting material, such as 0%ee (racemic). In some embodiments, the compounds of formula (IA), (I), (II), or (III) are enantiomerically enriched. In some embodiments, the compounds of formula (IA), (I), (II), or (III) are enantiomerically pure. In embodiments where the compound has more than one stereocenter, the stereocenter can be enriched substantially independently of the stereochemical distribution / enrichment of any other stereocenter in the molecule. For example, the compounds described herein may have 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or even 98% or more at a particular stereocenter.

[0284] Additional lipids

[0285] The term "neutral lipid" as used in the lipid compositions of this disclosure includes, for example, various neutral lipids, uncharged lipids, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in this disclosure include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), lecithinylcholine (EPC), dilaurylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl- 2-Stearylphosphatidylcholine (PSPC), 1,2-disarachido-sn-glycerol-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-eicosenoyl-sn-glycerol-3-phosphocholine (DEPC), palmitoyloleylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In some embodiments, the neutral phospholipid may be selected from distearylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE), preferably distearylphosphatidylcholine (DSPC).

[0286] "Assisted lipids" include steroids, sterols, and alkylresorcinols. Assisted lipids suitable for use in this disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In some embodiments, the assisted lipid may be cholesterol or a derivative thereof, such as cholesterol hemisuccinate.

[0287] PEG lipids can influence the length of time nanoparticles can remain in vivo (e.g., in the blood). PEG lipids can aid in formulation processes, for example, by reducing particle aggregation and controlling particle size. The PEG lipids used herein modulate the pharmacokinetic properties of LNPs. Typically, PEG lipids comprise a lipid moiety and a polymer moiety based on PEG (sometimes referred to as poly(ethylene oxide))(PEG moiety). Information on PEG lipids suitable for lipid compositions having compounds of formulas (IA), (I), (II), or (III) of this disclosure, as well as biochemical information on such lipids, can be found in Romberg et al., Pharmaceutical Research 25(1), 2008, pp. 55–71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41–52. Additional suitable PEG lipids are disclosed, for example, in WO 2015 / 095340 (page 31, line 14 to page 37, line 6), WO2006 / 007712 and WO 2011 / 076807 (“Hidden Lipids”).

[0288] In some embodiments, the lipid moiety may be derived from diacylglycerol or diacylglycerol amide, including those comprising a dialkylglycerol or dialkylglycerol amide group having an alkyl chain length independently comprising about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups, such as amides or esters. In some embodiments, the alkyl chain length comprises about C10 to C20. The dialkylglycerol or dialkylglycerol amide group may also comprise one or more substituted alkyl groups. The chain length may be symmetrical or asymmetrical.

[0289] Unless otherwise indicated, the term "PEG" as used herein means any polyethylene glycol or other polyalkylene ether polymer, such as a linear or branched polymer of optionally substituted ethylene glycol or ethylene oxide. In some embodiments, the PEG moiety is unsubstituted. Alternatively, the PEG moiety may be substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxyl, or aryl groups. For example, the PEG moiety may comprise a PEG copolymer, such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety may be a PEG homopolymer. In some embodiments, the PEG moiety has a molecular weight of about 130 to about 50,000, such as about 150 to about 30,000, or even about 150 to about 20,000. Similarly, the PEG moiety may have a molecular weight of about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, or even about 150 to about 5,000. In some preferred embodiments, the PEG moiety has a molecular weight of about 150 to about 4,000, about 150 to about 3,000, about 300 to about 3,000, about 1,000 to about 3,000, or about 1,500 to about 2,500.

[0290] In some preferred embodiments, the PEG portion is “PEG-2K”, also known as “PEG 2000”, with an average molecular weight of about 2,000 Daltons. PEG-2K is represented herein by formula (II), where n is 45, meaning that the number-average degree of polymerization comprises about 45 subunits. However, other PEG embodiments known in the art can be used, including, for example, those in which the number-average degree of polymerization comprises about 23 subunits (n = 23) and / or 68 subunits (n = 68). In some embodiments, n may be in the range of about 30 to about 60. In some embodiments, n may be in the range of about 35 to about 55. In some embodiments, n may be in the range of about 40 to about 50. In some embodiments, n may be in the range of about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl groups, and unsubstituted alkyl groups. In some embodiments, R may be an unsubstituted alkyl group, such as methyl.

[0291] In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroyl glycerol, PEG-dimyristoyl glycerol (PEG-DMG) (catalog number GM-020, obtained from NOF, Tokyo, Japan), PEG-dipalmitoyl glycerol, PEG-distearyl glycerol (PEG-DSPE) (catalog number DSPE-020CN, NOF, Tokyo, Japan), PEG-dilauryl glyceramide, PEG-dimyristoyl glyceramide, PEG-dipalmitoyl glyceramide, and PEG - Distearate glyceryl amide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-di-tetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (catalog number 880150P, obtained from Avanti) Polar Lipids, Alabaster, Alabama, USA), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](PEG2k-DSPE) (catalog number 880120C, obtained from Avanti PolarLipids, Alabaster, Alabama, USA), 1,2-distearyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF) The PEG lipids are PEG2k-DMA (poly(ethylene glycol)-2000-dimethacrylate) and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In some such embodiments, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In other embodiments, the PEG lipid may be PEG2k-DSPE. In some embodiments, the PEG lipid may be PEG2k-DMA. In other embodiments, the PEG lipid may be PEG2k-C-DMA. In some embodiments, the PEG lipid may be compound S027, which is disclosed in WO2016 / 010840 (paragraphs

[00240] to

[00244] ). In some embodiments, the PEG lipid may be PEG2k-DSA. In other embodiments, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16.In some implementations, the PEG lipid may be PEG2k-C18.

[0292] The cationic lipids suitable for the lipid compositions of this disclosure include, but are not limited to, N,N-diolenoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(1-(2,3-diolenoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoylcarbamoyl-3-dimethylammonium-propane (DOCDAP), 1,2-dilinolenoyl-3-dimethylammonium-propane (DLINDAP), dilauryl(C12:0)trimethylammonium-propane (DLTAP), di-octadecylamidoglycylsperylsperylamine (DOGS), DC-Choi, dioleoyloxy-N-[ 2-(Sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propanetrifluoroacetate ammonium (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), N,N-dimethyl-2,3-dioleenoxy The formulations include DODMA (5'-(cholest-5-en-3[β]-oxy)-3'-oxaproxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-diolenooxybenzylamine (DMOBA), and 1,2-N,N'-diolenoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP). In one embodiment, the cationic lipid is DOTAP or DLTAP.

[0293] The anionic lipids applicable to this disclosure include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine cholesterol hemisuccinate (CHEMS), and lysylphosphatidylglycerol.

[0294] lipid compositions

[0295] This disclosure provides a lipid composition comprising at least one compound of formula (IA), (I), (II), or (III) or a salt thereof (e.g., a pharmaceutically acceptable salt thereof) and at least one other lipid component. Such compositions may also contain a bioactive agent, optionally in combination with one or more other lipid components. In some embodiments, the lipid composition comprises a lipid component and an aqueous component containing a bioactive agent.

[0296] In one embodiment, the lipid composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, and at least one other lipid component. In another embodiment, the lipid composition further comprises a bioactive agent, optionally in combination with one or more other lipid components. In another embodiment, the lipid composition is in the form of liposomes. In another embodiment, the lipid composition is in the form of lipid nanoparticles (LNPs). In another embodiment, the lipid composition is suitable for delivery to the liver.

[0297] In one embodiment, the lipid composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, and another lipid component. Such other lipid components include, but are not limited to, neutral lipids, cofactor lipids, PEG lipids, cationic lipids, and anionic lipids. In some embodiments, the lipid composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, and a neutral lipid such as DSPC, optionally having one or more additional lipid components. In another embodiment, the lipid composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, and a cofactor lipid such as cholesterol, optionally having one or more additional lipid components. In other embodiments, the lipid composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, and a PEG lipid, optionally having one or more additional lipid components. In other embodiments, the lipid composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, and a cationic lipid, optionally having one or more additional lipid components. In other embodiments, the lipid composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, and an anionic lipid, optionally having one or more additional lipid components. In a sub-implementation, the lipid composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, an auxiliary lipid, and a PEG lipid, optionally having a neutral lipid. In other sub-implementations, the lipid composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, an auxiliary lipid, a PEG lipid, and a neutral lipid.

[0298] Compositions containing lipids of formula (IA), (I), (II), or (III) or pharmaceutically acceptable salts thereof, or lipid compositions thereof, may be in various forms, including but not limited to particle-forming delivery agents for delivering various molecules to cells, said particle-forming delivery agents including microparticles, nanoparticles, and transfection agents. Specific compositions are effective in transfection or delivery of bioactive agents. Preferred bioactive agents are RNA and DNA. In other embodiments, the bioactive agent is selected from mRNA, gRNA, and DNA. The gRNA may be dgRNA or sgRNA. In some embodiments, the cargo comprises mRNA, gRNA, or nucleic acid encoding gRNA, or a combination of mRNA and gRNA, encoding an RNA-directed DNA binder (e.g., Cas nuclease, class 2 Cas nuclease, or Cas9).

[0299] Exemplary compounds of formula (IA), (I), (II) or (III) for use in the above lipid compositions are given in Examples 2-48, 50-97, 99-103 and 105-113.

[0300] In some embodiments, compound (IA) is compound 2. In some embodiments, compound (IA) is compound 3. In some embodiments, compound (IA) is compound 4. In some embodiments, compound (IA) is compound 5. In some embodiments, compound (IA) is compound 6. In some embodiments, compound (IA) is compound 7. In some embodiments, compound (IA) is compound 8. In some embodiments, compound (IA) is compound 9. In some embodiments, compound (IA) is compound 10.

[0301] In some embodiments, compound (IA) is compound 11. In some embodiments, compound (IA) is compound 12. In some embodiments, compound (IA) is compound 13. In some embodiments, compound (IA) is compound 14. In some embodiments, compound (IA) is compound 15. In some embodiments, compound (IA) is compound 16. In some embodiments, compound (IA) is compound 17. In some embodiments, compound (IA) is compound 18. In some embodiments, compound (IA) is compound 19. In some embodiments, compound (IA) is compound 20.

[0302] In some embodiments, compound (IA) is compound 21. In some embodiments, compound (IA) is compound 22. In some embodiments, compound (IA) is compound 23. In some embodiments, compound (IA) is compound 24. In some embodiments, compound (IA) is compound 25. In some embodiments, compound (IA) is compound 26. In some embodiments, compound (IA) is compound 27. In some embodiments, compound (IA) is compound 28. In some embodiments, compound (IA) is compound 29. In some embodiments, compound (IA) is compound 30.

[0303] In some embodiments, compound (IA) is compound 31. In some embodiments, compound (IA) is compound 32. In some embodiments, compound (IA) is compound 33. In some embodiments, compound (IA) is compound 34. In some embodiments, compound (IA) is compound 35. In some embodiments, compound (IA) is compound 36. In some embodiments, compound (IA) is compound 37. In some embodiments, compound (IA) is compound 38. In some embodiments, compound (IA) is compound 39. In some embodiments, compound (IA) is compound 40.

[0304] In some embodiments, compound (IA) is compound 41. In some embodiments, compound (IA) is compound 42. In some embodiments, compound (IA) is compound 43. In some embodiments, compound (IA) is compound 44. In some embodiments, compound (IA) is compound 45. In some embodiments, compound (IA) is compound 46. In some embodiments, compound (IA) is compound 47. In some embodiments, compound (IA) is compound 48. In some embodiments, compound (IA) is compound 50.

[0305] In some embodiments, compound (IA) is compound 51. In some embodiments, compound (IA) is compound 52. In some embodiments, compound (IA) is compound 53. In some embodiments, compound (IA) is compound 54. In some embodiments, compound (IA) is compound 55. In some embodiments, compound (IA) is compound 56. In some embodiments, compound (IA) is compound 57. In some embodiments, compound (IA) is compound 58. In some embodiments, compound (IA) is compound 59. In some embodiments, compound (IA) is compound 60.

[0306] In some embodiments, compound (IA) is compound 61. In some embodiments, compound (IA) is compound 62. In some embodiments, compound (IA) is compound 63. In some embodiments, compound (IA) is compound 64. In some embodiments, compound (IA) is compound 65. In some embodiments, compound (IA) is compound 66. In some embodiments, compound (IA) is compound 67. In some embodiments, compound (IA) is compound 68. In some embodiments, compound (IA) is compound 69. In some embodiments, compound (IA) is compound 70.

[0307] In some embodiments, compound (IA) is compound 71. In some embodiments, compound (IA) is compound 72. In some embodiments, compound (IA) is compound 73. In some embodiments, compound (IA) is compound 74. In some embodiments, compound (IA) is compound 75. In some embodiments, compound (IA) is compound 76. In some embodiments, compound (IA) is compound 77. In some embodiments, compound (IA) is compound 78. In some embodiments, compound (IA) is compound 79. In some embodiments, compound (IA) is compound 80.

[0308] In some embodiments, compound (IA) is compound 81. In some embodiments, compound (IA) is compound 82. In some embodiments, compound (IA) is compound 83. In some embodiments, compound (IA) is compound 84. In some embodiments, compound (IA) is compound 85. In some embodiments, compound (IA) is compound 86. In some embodiments, compound (IA) is compound 87. In some embodiments, compound (IA) is compound 88. In some embodiments, compound (IA) is compound 89. In some embodiments, compound (IA) is compound 90.

[0309] In some embodiments, compound (IA) is compound 91. In some embodiments, compound (IA) is compound 92. In some embodiments, compound (IA) is compound 93. In some embodiments, compound (IA) is compound 94. In some embodiments, compound (IA) is compound 95. In some embodiments, compound (IA) is compound 96. In some embodiments, compound (IA) is compound 97. In some embodiments, compound (IA) is compound 99. In some embodiments, compound (IA) is compound 100.

[0310] In some embodiments, compound (IA) is compound 101. In some embodiments, compound (IA) is compound 102. In some embodiments, compound (IA) is compound 103. In some embodiments, compound (IA) is compound 105. In some embodiments, compound (IA) is compound 106. In some embodiments, compound (IA) is compound 107. In some embodiments, compound (IA) is compound 108. In some embodiments, compound (IA) is compound 109. In some embodiments, compound (IA) is compound 110.

[0311] In some embodiments, the compound of formula (IA) is compound 111. In some embodiments, the compound of formula (IA) is compound 112. In some embodiments, the compound of formula (IA) is compound 113.

[0312] In some embodiments, the compound is not compound 1. In some embodiments, the compound is not compound 49. In some embodiments, the compound is not compound 114.

[0313] LNP Composition

[0314] Lipid compositions can be provided as LNP compositions. Lipid nanoparticles can be, for example, microspheres (including monolayer and multilayer vesicles, such as "liposomes"—in some embodiments essentially spherical layered lipid bilayers, and in more specific embodiments may contain an aqueous core, such as an RNA molecule containing a substantial portion), dispersed phases in emulsions, micelles, or internal phases in suspensions.

[0315] The size of the LNP is about 1 nm to about 1,000 nm, about 10 nm to about 500 nm, about 20 nm to about 500 nm, about 50 nm to about 400 nm in one sub-implementation, about 50 nm to about 300 nm in another sub-implementation, about 50 nm to about 200 nm in another sub-implementation, about 50 nm to about 150 nm in another sub-implementation, and about 60 nm to about 120 nm in yet another sub-implementation. Preferably, the LNP size is about 60 nm to about 100 nm. The average size (diameter) of the fully formed LNP can be measured by dynamic light scattering on a Malvern Zetasizer. The LNP sample is diluted in phosphate-buffered saline (PBS) such that the counting rate is about 200–400 kcps. The data are expressed as a weighted average of intensity measures.

[0316] Embodiments of this disclosure provide lipid compositions described according to the corresponding molar ratios of the component lipids in the composition. All molar percentages are given as a fraction of the lipid components in the lipid composition, or more specifically, the LNP composition. In some embodiments, the molar percentage of the compound of formula (IA), (I), (II), or (III) may be from about 30 mol% to about 70 mol%. In some embodiments, the molar percentage of the compound of formula (IA), (I), (II), or (III) may be at least 30 mol%, at least 40 mol%, at least 50 mol%, or at least 60 mol%.

[0317] In some embodiments, the mole percentage of neutral lipids can be from about 0 mol% to about 30 mol%. In some embodiments, the mole percentage of neutral lipids can be from about 0 mol% to about 20 mol%. In some embodiments, the mole percentage of neutral lipids can be about 10 mol%. In some embodiments, the mole percentage of neutral lipids can be about 9 mol%.

[0318] In some embodiments, the molar percentage of the cofactor lipid can be from about 0 mol% to about 80 mol%. In some embodiments, the molar percentage of the cofactor lipid can be from about 20 mol% to about 60 mol%. In some embodiments, the molar percentage of the cofactor lipid can be from about 30 mol% to about 50 mol%. In some embodiments, the molar percentage of the cofactor lipid can be from about 30 mol% to about 40 mol% or from about 35 mol% to about 45 mol%. In some embodiments, the molar percentage of the cofactor lipid is adjusted based on the concentration of compounds of formula (IA), (I), (II), or (III), neutral lipids, and / or PEG lipids to bring the lipid component to 100 mol%.

[0319] In some embodiments, the molar percentage of PEG lipid can be from about 1 mol% to about 10 mol%. In some embodiments, the molar percentage of PEG lipid can be from about 1 mol% to about 4 mol%. In some embodiments, the molar percentage of PEG lipid can be from about 1 mol% to about 2 mol%. In some embodiments, the molar percentage of PEG lipid can be about 1.5 mol%.

[0320] In various embodiments, the LNP composition comprises a compound of formula (IA), (I), (II), or (III) or a salt thereof (such as a pharmaceutically acceptable salt thereof, e.g., as disclosed herein), a neutral lipid (e.g., DSPC), an accessory lipid (e.g., cholesterol), and a PEG lipid (e.g., PEG2k-DMG). In some embodiments, the LNP composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof (e.g., as disclosed herein), DSPC, cholesterol, and a PEG lipid. In some such embodiments, the LNP composition comprises a PEG lipid comprising DMG, such as PEG2k-DMG. In some preferred embodiments, the LNP composition comprises a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, cholesterol, DSPC, and PEG2k-DMG.

[0321] In some embodiments, the lipid composition (such as the LNP composition) comprises a lipid component and a nucleic acid component, such as an RNA component, and the molar ratio of a compound of formula (IA), (I), (II), or (III) to a nucleic acid can be measured. Embodiments of this disclosure also provide lipid compositions having a defined molar ratio between a positively charged amino group (N) of a pharmaceutically acceptable salt of a compound of formula (IA), (I), (II), or (III) and a negatively charged phosphate group (P) of the nucleic acid to be encapsulated. This can be mathematically represented as the equation N / P. In some embodiments, the lipid composition, such as the LNP composition, may comprise a lipid component comprising a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof; and a nucleic acid component, wherein the N / P ratio is about 3 to 10. In some embodiments, the LNP composition may comprise a lipid component comprising a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof; and an RNA component, wherein the N / P ratio is about 3 to 10. For example, the N / P ratio may be about 4 to 7. Alternatively, the N / P ratio can be about 6, for example 6 ± 1 or 6 ± 0.5.

[0322] In some embodiments, the aqueous component comprises a bioactive agent. In some embodiments, the aqueous component comprises a polypeptide, optionally combined with a nucleic acid. In some embodiments, the aqueous component comprises a nucleic acid, such as RNA. In some embodiments, the aqueous component is a nucleic acid component. In some embodiments, the nucleic acid component comprises DNA, and may be referred to as a DNA component. In some embodiments, the nucleic acid component comprises RNA, and may be referred to as an RNA component. In some embodiments, the aqueous component, such as the RNA component, may comprise mRNA, such as mRNA encoding an RNA-directed DNA binder. In some embodiments, the RNA-directed DNA binder is a Cas nuclease. In some embodiments, the aqueous component may comprise mRNA encoding Cas9. In some embodiments, the aqueous component may comprise gRNA. In some compositions comprising mRNA encoding an RNA-directed DNA binder, the composition also comprises a gRNA nucleic acid, such as gRNA. In some embodiments, the aqueous component comprises an RNA-directed DNA binder and gRNA. In some embodiments, the aqueous component comprises Cas nuclease mRNA and gRNA. In some embodiments, the aqueous component comprises two types of Cas nuclease mRNA and gRNA.

[0323] In some embodiments, lipid compositions such as LNP compositions may comprise mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, an accessory lipid, optionally a neutral lipid, and a PEG lipid. In some compositions comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), the accessory lipid is cholesterol. In other compositions comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), the neutral lipid is DSPC. In additional embodiments comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease, e.g., Cas9), the PEG lipid is PEG2k-DMG. In specific compositions, the composition comprises mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease) and a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof. In some compositions, the composition also comprises gRNA, such as dgRNA or sgRNA.

[0324] In some embodiments, the lipid composition, such as the LNP composition, may contain gRNA. In some embodiments, the composition may contain a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, gRNA, an accessory lipid, optionally a neutral lipid, and a PEG lipid. In some LNP compositions containing gRNA, the accessory lipid is cholesterol. In some compositions containing gRNA, the neutral lipid is DSPC. In additional embodiments containing gRNA, the PEG lipid is PEG2k-DMG. In some compositions, the gRNA is selected from dgRNA and sgRNA.

[0325] In some embodiments, the lipid composition, such as the LNP composition, comprises mRNA and gRNA encoding an RNA-directed DNA binder in an aqueous component and a compound of formula (IA), (I), (II), or (III) in the lipid component, wherein the gRNA may be sgRNA. For example, the LNP composition may comprise a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, mRNA encoding a Cas nuclease, gRNA, an accessory lipid, a neutral lipid, and a PEG lipid. In some compositions comprising mRNA and gRNA encoding a Cas nuclease, the accessory lipid is cholesterol. In some compositions comprising mRNA and gRNA encoding a Cas nuclease, the neutral lipid is DSPC. In an additional embodiment comprising mRNA and gRNA encoding a Cas nuclease, the PEG lipid is PEG2k-DMG.

[0326] In some embodiments, the lipid composition, such as the LNP composition, comprises an RNA-directed DNA binder (such as class 2 Cas nuclease mRNA) and at least one gRNA. In some embodiments, the LNP composition comprises a ratio of gRNA to RNA-directed DNA binder mRNA (such as class 2 Cas nuclease mRNA) of about 1:1 or about 1:2. In some embodiments, the ratio is about 25:1 to about 1:25, about 10:1 to about 1:10, about 8:1 to about 1:8, about 4:1 to about 1:4, or about 2:1 to about 1:2.

[0327] The lipid compositions disclosed herein, such as LNP compositions, may contain a template nucleic acid, such as a DNA template. The template nucleic acid may be delivered together with or alone with a lipid composition containing a compound of formula (IA), (I), (II), or (III) or a pharmaceutically acceptable salt thereof, said lipid composition containing the LNP composition. In some embodiments, the template nucleic acid may be single-stranded or double-stranded, depending on the desired repair mechanism. The template may have regions homologous to the target DNA, such as within the target DNA sequence, and / or adjacent to the target DNA sequence.

[0328] In some embodiments, LNPs are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. For example, the organic solvent may be 100% ethanol. Pharmaceutically acceptable buffers, such as those used for in vivo administration of LNPs, may be used. In some embodiments, a buffer is used to maintain the pH of the LNP-containing composition at or above pH 6.5. In some embodiments, a buffer is used to maintain the pH of the LNP-containing composition at or above pH 7.0. In some embodiments, the pH of the composition is in the range of about 7.2 to about 7.7. In additional embodiments, the pH of the composition is in the range of about 7.3 to about 7.7 or about 7.4 to about 7.6. In other embodiments, the pH of the composition is about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of the composition can be measured using a micropH probe. In some embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may contain up to 10% cryoprotectant, such as sucrose. In some embodiments, the composition may contain tris-saline sucrose (TSS). In some embodiments, the LNP composition may contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% cryoprotectant. In some embodiments, the LNP composition may contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% sucrose. In some embodiments, the LNP composition may contain a buffer. In some embodiments, the buffer may contain phosphate-buffered saline (PBS), Tris buffer, citrate buffer, and mixtures thereof. In some exemplary embodiments, the buffer contains NaCl. In some embodiments, the buffer is NaCl-free. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl can range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Exemplary amounts of Tris can range from about 20 mM to about 60 mM. Exemplary amounts of Tris can range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary embodiments of the LNP composition comprise 5% sucrose and 45 mM NaCl in a Tris buffer. In other exemplary embodiments, the composition comprises sucrose in an amount of about 5% w / v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5.The amounts of salt, buffer, and cryoprotectant can be varied to maintain the overall osmotic pressure of the composition. For example, the final osmotic pressure can be maintained below 450 mOsm / L. In other embodiments, the osmotic pressure is between 350 mOsm / L and 250 mOsm / L. In some embodiments, the final osmotic pressure is 300 ± 20 mOsm / L or 310 ± 40 mOsm / L.

[0329] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing of aqueous RNA and lipid solutions in organic solvents is used. In some aspects, flow rate, adapter size, adapter geometry, adapter shape, tube diameter, and solution and / or RNA and lipid concentrations may vary. LNPs or LNP compositions may be concentrated or purified, for example, via dialysis, centrifugal filtration, tangential flow filtration, or chromatographic concentration. LNPs may be stored, for example, in the form of suspensions, emulsions, or lyophilized powders. In some embodiments, LNP compositions are stored at 2-8°C; in some aspects, LNP compositions are stored at room temperature. In additional embodiments, LNP compositions are frozen, for example, at -20°C or -80°C. In other embodiments, LNP compositions are stored at temperatures ranging from about 0°C to about -80°C. Frozen LNP compositions may be thawed before use, for example, on ice, at room temperature, or at 25°C.

[0330] LNPs can be, for example, microspheres (including monolayer and multilayer vesicles, such as "liposomes"—in some embodiments essentially spherical layered lipid bilayers—and, in more specific embodiments, may contain an aqueous core, such as an RNA molecule containing a substantial portion), a dispersed phase in an emulsion, a micelle, or an inner phase in a suspension.

[0331] Preferred lipid compositions, such as LNP compositions, are biodegradable because they do not accumulate in vivo to cytotoxic levels at therapeutically effective doses. In some embodiments, the compositions do not induce innate immune responses that cause substantial side effects at therapeutic dose levels. In some embodiments, the compositions provided herein do not cause toxicity at therapeutic dose levels.

[0332] In some embodiments, the LNP disclosed herein has a polydispersity index (PDI) ranging from about 0.005 to about 0.75. In some embodiments, the LNP has a PDI ranging from about 0.01 to about 0.5. In some embodiments, the LNP has a PDI ranging from about zero to about 0.4. In some embodiments, the LNP has a PDI ranging from about zero to about 0.35. In some embodiments, the LNP has a PDI ranging from about zero to about 0.35. In some embodiments, the LNP PDI is ranging from about zero to about 0.3. In some embodiments, the LNP PDI is ranging from about zero to about 0.25. In some embodiments, the LNP PDI is ranging from about zero to about 0.2. In some embodiments, the LNP has a PDI less than about 0.08, 0.1, 0.15, 0.2, or 0.4.

[0333] The sizes (e.g., Z-average diameters) of the LNPs disclosed herein are from about 1 nm to about 250 nm. In some embodiments, the LNP size is from about 10 nm to about 200 nm. In other embodiments, the LNP size is from about 20 nm to about 150 nm. In some embodiments, the LNP size is from about 50 nm to about 150 nm. In some embodiments, the LNP size is from about 50 nm to about 100 nm. In some embodiments, the LNP size is from about 50 nm to about 120 nm. In some embodiments, the LNP size is from about 60 nm to about 100 nm. In some embodiments, the LNP size is from about 75 nm to about 150 nm. In some embodiments, the LNP size is from about 75 nm to about 120 nm. In some embodiments, the LNP size is from about 75 nm to about 100 nm. Unless otherwise noted, all sizes mentioned herein are average sizes (diameters) of fully formed nanoparticles, as measured by dynamic light scattering on a Malvern Zetasizer. The nanoparticle samples were diluted in phosphate-buffered saline (PBS) to achieve a counting rate of approximately 200–400 kcps. The data are presented as a weighted average of the intensity measure (Z-mean diameter).

[0334] In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 50% to about 95%. In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 75% to about 95%.

[0335] goods

[0336] The cargo delivered via the LNP composition may be a bioactive agent. In some embodiments, the cargo is or comprises one or more bioactive agents, such as mRNA, guide RNA, nucleic acid, expression vector, template nucleic acid, RNA-directed DNA binder, antibody (e.g., monoclonal, chimeric, humanized, nanobody and fragment thereof), cholesterol, hormone, peptide, protein, chemotherapeutic agents and other types of antitumor agents, low molecular weight drugs, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzyme nucleic acids, antisense nucleic acids, triple-helical oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, homoenzymes, aptamers, ribozymes, decoys and analogues. Plasmids and other types of vectors, as well as small nucleic acid molecules, RNAi agents, short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and "self-replicating RNA" molecules (encoding replicase activity and capable of guiding its own replication or amplification in vivo), peptide nucleic acids (PNA), locked ribonucleotides (LNA), morpholinonucleotides, threonine nucleic acids (TNA), glycol nucleic acids (GNA), sisiRNA (small internally fragmented interfering RNA), and iRNA (asymmetric interfering RNA). The above list of bioactive agents is merely exemplary and is not intended to be limiting. Such compounds may be purified or partially purified, may be naturally occurring or synthetic, and may be chemically modified.

[0337] The cargo delivered via the LNP composition may be RNA, such as an mRNA molecule encoding a target protein. For example, it may include mRNA for expressing proteins such as green fluorescent protein (GFP), RNA-directed DNA binders, or Cas nucleases. LNP compositions are provided that contain Cas nuclease mRNA, such as class 2 Cas nuclease mRNA that allows expression of class 2 Cas nucleases (such as Cas9 or Cpf1 proteins) in cells. Furthermore, the cargo may contain one or more guide RNAs or nucleic acids encoding guide RNAs. For example, template nucleic acids for repair or recombination may also be included in the composition, or the template nucleic acids may be used in the methods described herein. In one sub-implementation, the cargo contains mRNA encoding Streptococcus pyogenes Cas9, optionally and Streptococcus pyogenes gRNA. In another sub-implementation, the cargo contains mRNA encoding Neisseria meningitidis Cas9, optionally and nme gRNA.

[0338] “mRNA” refers to a polynucleotide and contains an open reading frame that can be translated into a polypeptide (i.e., can be used as a substrate for translation by ribosomes and aminoacylated tRNA). mRNA may contain a phosphate-sugar backbone comprising ribose residues or analogues thereof, such as 2'-methoxyribose residues. In some embodiments, the sugars in the mRNA phosphate-sugar backbone are substantially composed of ribose residues, 2'-methoxyribose residues, or combinations thereof. Typically, mRNA does not contain substantial amounts of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). mRNA may contain modified uridine at some or all of its uridine sites.

[0339] CRISPR / Cas cargo

[0340] In some embodiments, the disclosed compositions comprise mRNA encoding an RNA-directed DNA binder such as a Cas nuclease. In a specific embodiment, the disclosed compositions comprise mRNA encoding a class 2 Cas nuclease such as Streptococcus pyogenes Cas9.

[0341] As used herein, “RNA-directed DNA binding agent” means a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the RNA sequence. Exemplary RNA-directed DNA binding agents include Cas lysins / nicking enzymes and their inactivated forms (“dCas DNA binding agents”). As used herein, “Cas nuclease” encompasses Cas lysins, Cas nicking enzymes, and dCas DNA binding agents. Cas lysins / nicking enzymes and dCas DNA binding agents include the Csm or Cmr complex of a type III CRISPR system, its Cas10, Csm1, or Cmr2 subunit; the cascade complex of a type I CRISPR system, its Cas3 subunit; and class 2 Cas nucleases. As used herein, “class 2 Cas nuclease” is a single-stranded polypeptide having RNA-directed DNA binding activity. Class 2 Cas nucleases include two classes of Cas lysin / nicking enzymes (e.g., H840A, D10A, or N863A variants) that also possess RNA-directed DNA lysin or nicking enzyme activity; and two classes of dCas DNA binders in which the lysin / nicking enzyme activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants) and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and their modified forms. The Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated in its entirety by reference. See, for example, Zetsche, Tables S1 and S3. See, for example, Makarova et al., Nat RevMicrobiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0342] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a guide RNA accompanied by an RNA-directed DNA binding agent such as a Cas nuclease, for example, a Cas lyase, a Cas nickase, or a dCas DNA binding agent (e.g., Cas9). In some embodiments, the guide RNA directs the RNA-directed DNA binding agent (such as Cas9) to a target sequence, and the guide RNA hybridizes to the target sequence and the agent binds to the target sequence; where said agent is a lyase or nickase, the binding can be cleavage or nicking.

[0343] In some embodiments of this disclosure, the cargo for the LNP composition comprises at least one guide RNA containing a guide sequence that directs an RNA-directed DNA binder to target DNA, the RNA-directed DNA binder being a nuclease (e.g., a Cas nuclease, such as Cas9). The gRNA can direct a Cas nuclease or a class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to a class 2 Cas nuclease and provides specificity for cleavage by the class 2 Cas nuclease. In some embodiments, the gRNA and the Cas nuclease can form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex, such as a CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex can be a type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex can be a type V CRISPR / Cas complex, such as a Cpf1 / guide RNA complex. The Cas nuclease and homologous gRNA can pair. The gRNA scaffold structure paired with each class 2 Cas nuclease varies depending on the specific CRISPR / Cas system.

[0344] The terms “guide RNA,” “gRNA,” and simply “guide” are used interchangeably throughout this document to refer to homologous guide nucleic acids used as RNA-directed DNA binders. gRNA can be crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). Guide RNA may include RNAs modified as described herein. crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. trRNA can be a naturally occurring sequence or a trRNA sequence modified or mutated compared to a naturally occurring sequence.

[0345] As used herein, a “guide sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to guide the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-directed DNA binder. A “guide sequence” may also be referred to as a “target sequence” or a “spacer sequence.” The guide sequence can be 20 base pairs long, for example, for Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences may also be used as guides, for example, sequences of 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the target sequence is, for example, located in a gene or on a chromosome and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between the guide sequence and its corresponding target sequence can be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical in regions of at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, wherein the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches, wherein the guide sequence contains at least 17, 18, 19, 20, or more nucleotides. In some embodiments, when the guide sequence contains 20 nucleotides, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches.

[0346] The target sequences of RNA-guided DNA-binding proteins (such as Cas proteins) include both the positive and negative strands of genomic DNA (i.e., a given sequence and its inverse complement), because the nucleic acid substrate of Cas proteins is a double-stranded nucleic acid. Therefore, when a guide sequence is referred to as "complementary to the target sequence," it should be understood that the guide sequence directs gRNA to bind to the inverse complement of the target sequence. Thus, in some embodiments, in the case where the guide sequence binds to the inverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., a target sequence excluding PAM) except that U replaces T in the guide sequence.

[0347] The length of the target sequence can depend on the CRISPR / Cas system and the components used. For example, different classes of Cas nucleases from different bacterial species have different optimal target sequence lengths. Therefore, the length of the target sequence can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides. In some embodiments, the target sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of the naturally occurring CRISPR / Cas system. In some embodiments, the Cas nuclease and the gRNA scaffold will be derived from the same CRISPR / Cas system. In some embodiments, the target sequence may contain 18-24 nucleotides or consist of them. In some embodiments, the target sequence may contain 19-21 nucleotides or consist of them. In some implementations, the target sequence may contain or consist of 20 nucleotides.

[0348] In some embodiments, the sgRNA is a "Cas9 sgRNA" capable of mediating RNA-directed DNA cleavage by the Cas9 protein. In some embodiments, the sgRNA is a "Cpf1 sgRNA" capable of mediating RNA-directed DNA cleavage by the Cpf1 protein. In some embodiments, the gRNA comprises crRNA and tracrRNA sufficient to form an active complex with the Cas9 protein and mediate RNA-directed DNA cleavage. In some embodiments, the gRNA comprises crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-directed DNA cleavage. See Zetsche 2015.

[0349] Some embodiments of this disclosure also provide nucleic acids encoding the gRNAs described herein, such as expression cassettes. The term "guide RNA nucleic acid" is used herein to refer to guide RNAs (e.g., sgRNAs or dgRNAs) and guide RNA expression cassettes, which are nucleic acids encoding one or more guide RNAs.

[0350] Modified RNA

[0351] In some embodiments, the lipid composition, such as the LNP composition, contains modified nucleic acids, including modified RNA.

[0352] Modified nucleosides or nucleotides may be present in RNA, such as gRNA or mRNA. For example, gRNA or mRNA containing one or more modified nucleosides or nucleotides is referred to as “modified” RNA to describe the presence of one or more non-natural and / or naturally occurring components or conformations that are substituted for or appended to the canonical A, G, C, and U residues. In some embodiments, modified RNA is synthesized using non-canonical nucleosides or nucleotides (referred to herein as “modified”).

[0353] The modified nucleosides and nucleotides may include one or more of the following: (i) alterations, such as replacing one or more of the non-linked phosphate groups and / or the linked phosphate groups in the main chain phosphodiester bonds (exemplary main chain modification); (ii) alterations, such as replacing components of the ribose, such as the 2' hydroxyl group on the ribose (exemplary sugar modification); (iii) large-scale replacement of the phosphate moiety with a "dephosphorylation" linker (exemplary main chain modification); (iv) modification or replacement of naturally occurring nucleobases, including modification or replacement with non-canonical nucleobases (exemplary base modification); (v) replacement or modification of the ribose-phosphate main chain (exemplary main chain modification); (vi) modification of the 3' or 5' end of the oligonucleotide, such as removing, modifying, or replacing the terminal phosphate group or conjugation portion, cap, or linker (e.g., 3' or 5' cap modification may include sugar and / or main chain modification); and (vii) modification or replacement of sugars (exemplary sugar modification).

[0354] Some embodiments include modifications to mRNA, gRNA, or nucleic acid. Some embodiments include 5' end modifications to mRNA, gRNA, or nucleic acid. Some embodiments include 3' end modifications to mRNA, gRNA, or nucleic acid. The modified RNA may contain both 5' and 3' end modifications. The modified RNA may contain one or more modified residues at non-terminal positions. In some embodiments, the gRNA includes at least one modified residue. In some embodiments, the mRNA includes at least one modified residue.

[0355] Unmodified nucleic acids are prone to degradation by enzymes such as intracellular nucleases or those found in serum. For example, nucleases can hydrolyze the phosphodiester bonds of nucleic acids. Therefore, in one aspect, the RNA (e.g., mRNA, gRNA) described herein may contain one or more modified nucleosides or nucleotides, for example, to introduce stability against intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein may exhibit a reduced innate immune response when introduced into a cell population in vivo and in vitro. The term "innate immune response" includes cellular responses to exogenous nucleic acids comprising single-stranded nucleic acids, involving cytokine expression and release (particularly interferons) and the induction of cell death.

[0356] Therefore, in some embodiments, the RNA or nucleic acid in the disclosed LNP compositions comprises at least one modification that confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to digestion by nucleases in vivo. As used herein, the terms “modified” and “modified” as referring to the nucleic acids provided herein include at least one alteration that preferably enhances stability and makes the RNA or nucleic acid more stable than wild-type or naturally occurring RNA or nucleic acid (e.g., resistant to nuclease digestion). As used herein, the terms “stable” and “stability” as referring to the nucleic acids of this disclosure and particularly with respect to RNA, mean increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that are typically capable of degrading such RNA. Increased stability may include, for example, lower sensitivity to hydrolysis or other disruption by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within target cells or tissues, thereby increasing or enhancing the residence of such RNA or nucleic acid in target cells, tissues, subjects, and / or cytoplasm. The stable RNA or nucleic acid molecules presented herein exhibit a longer half-life relative to their naturally occurring unmodified counterparts (e.g., wild-type forms of mRNA). The terms “modified” and “modified” as used with respect to mRNAs involving the LNP compositions disclosed herein also encompass alterations that improve or enhance the translation of mRNA nucleic acids, including, for example, sequences that play a role in protein translation initiation (e.g., Kozak concordant sequences). (Kozak, M., Nucleic Acids Res 15(20):8125-48(1987)).

[0357] In some embodiments, the RNA or nucleic acid of the LNP compositions disclosed herein has undergone chemical or biological modifications to make it more stable. Exemplary modifications to RNA (such as mRNA) include the deletion of bases (e.g., by deletion or by substitution of one nucleotide for another) or modifications of bases, such as chemical modifications of bases. As used herein, the phrase “chemical modification” includes modifications that introduce chemical properties that differ from those seen in naturally occurring RNA or nucleic acids, such as covalent modifications, such as the introduction of modified nucleotides (e.g., nucleotide analogs, or side groups not naturally present in such RNA or nucleic acid molecules).

[0358] In some embodiments of main-chain modification, the phosphate groups of the modified residues can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, the modified residues, such as those present in the modified nucleic acid, can include large-scale replacement of unmodified phosphate moieties with the modified phosphate groups described herein. In some embodiments, main-chain modification of the phosphate backbone can include alterations resulting in uncharged linkers or charged linkers with an asymmetric charge distribution.

[0359] Examples of modified phosphate groups include thiophosphates, selenophosphates, boroalkyl phosphates, hydrophosphonates, aminophosphates, alkyl or aryl phosphonates, and phosphate triesters. The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the aforementioned atoms or groups can make the phosphorus atom chiral. The stereophosphorus atom can have an "R" configuration (Rp herein) or an "S" configuration (Sp herein). The main chain can also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate to the nucleoside) with nitrogen (bridging aminophosphates), sulfur (bridging thiophosphates), and carbon (bridging methylene phosphonates). Substitution can occur at the linking oxygen or both linking oxygens. In some main chain modifications, the phosphate group can be replaced by a phosphorus-free linker. In some embodiments, the charged phosphate group can be replaced by a neutral portion. Examples of phosphate group substitutes may include, but are not limited to, methylphosphonates, hydroxyamino, siloxanes, carbonates, carboxymethyl, carbamates, amides, thioethers, ethylene oxide connectors, sulfonates, sulfonamides, thiomethyl acetals, methyl acetals, oximes, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino.

[0360] mRNA

[0361] In some embodiments, the compositions or formulations disclosed herein comprise mRNA containing an open reading frame (ORF) encoding an RNA-directed DNA binding agent such as a Cas nuclease or a class 2 Cas nuclease as described herein. In some embodiments, mRNA comprising an ORF encoding an RNA-directed DNA binding agent, such as a Cas nuclease or a class 2 Cas nuclease, is provided, used, or administered. The mRNA may contain one or more of a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenylated tail. The mRNA may contain modified open reading frames, such as those encoding nuclear localization sequences or using alternative codons to encode proteins.

[0362] The mRNA in the disclosed LNP composition may encode, for example, a secreted hormone, enzyme, receptor, polypeptide, peptide, or other normally secreted target protein. In one embodiment, the mRNA may optionally have chemical or biological modifications that, for example, improve the stability and / or half-life of such mRNA or improve or otherwise promote protein production.

[0363] In addition, suitable modifications include changes to one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable than the codon seen in the wild-type form of mRNA. For example, an inverse relationship has been demonstrated between RNA stability and a higher number of cytidine (C) and / or uridine (U) residues, and RNA lacking C and U residues has been found to be stable to most RNases (Heidenreich et al., J Biol Chem 269, 2131-8 (1994)). In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by replacing one codon encoding the same or related amino acid with one codon encoding a specific amino acid. Desired modifications to mRNA nucleic acids also include the incorporation of pseudouridine. Incorporation of pseudouridine into mRNA nucleic acids can enhance stability and translational capacity, as well as reduce immunogenicity in vivo. See, for example, Karikó, K. et al., Molecular Therapy 16(11):1833-1840 (2008). Substitution and modification of mRNA can be performed using methods readily known to those skilled in the art.

[0364] Compared to untranslated regions, the limitations on reducing the number of C and U residues in a sequence may be greater within the coding regions of mRNA (i.e., it may not be possible to eliminate all C and U residues present in the messenger while still preserving the ability of the messenger to encode the required amino acid sequence). However, the degeneracy of the genetic code offers the opportunity to allow for a reduction in the number of C and / or U residues present in a sequence while maintaining the same coding capacity (i.e., depending on the amino acids encoded by the codons, there may be several different possibilities for modifying the RNA sequence).

[0365] The term "modification" also includes, for example, incorporating non-nucleotide bonds or modified nucleotides into the mRNA sequence (e.g., modification of one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secretory protein or enzyme). Such modifications include adding bases to the mRNA sequence (e.g., including a poly A tail or a longer poly A tail), altering the 3' or 5' UTR, complexing the mRNA with an agent (e.g., a protein or complementary nucleic acid molecule), and including elements that alter the structure of the mRNA molecule (e.g., its formation of secondary structures).

[0366] Poly A tails are considered to stabilize natural messengers. Therefore, in one embodiment, a long poly A tail can be added to the mRNA molecule, thus making the mRNA more stable. A variety of techniques recognized in the art can be used to add a poly A tail. For example, a long poly A tail can be added to synthetic or in vitro transcribed mRNA using a poly A polymerase (Yokoe et al., Nature Biotechnology. 1996; 14:1252-1256). Transcription vectors can also encode long poly A tails. Alternatively, a poly A tail can be added by transcription directly from PCR products. In one embodiment, the poly A tail is at least about 90, 200, 300, 400, or at least 500 nucleotides long. In one embodiment, the length of the poly A tail is modulated to control the stability of the modified mRNA molecule, and thus control protein transcription. For example, since the length of the poly A tail can affect the half-life of the mRNA molecule, the length of the poly A tail can be modulated to alter the level of mRNA resistance to nucleases, and thus control the timing of protein expression in the cell. In one implementation, stable mRNA molecules are sufficiently resistant to in vivo degradation (e.g., by nucleases) to allow them to be delivered to target cells without a transfer medium.

[0367] In one embodiment, the mRNA can be modified by incorporating non-naturally present 3' and / or 5' untranslated (UTR) sequences from wild-type mRNA. In one embodiment, the 3' and / or 5' flanking sequences of naturally flanked mRNAs encoding a second unrelated protein can be incorporated into the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein to modify it. For example, the 3' or 5' sequence of a stable mRNA molecule (e.g., globulin, actin, GAPDH, tubulin, histone, or citrate cycle enzyme) can be incorporated into the 3' and / or 5' region of a sense mRNA molecule to increase the stability of the sense mRNA molecule. See, for example, US2003 / 0083272.

[0368] A more detailed description of mRNA modifications can be found on pages 57-68 of US2017 / 0210698A1, which is incorporated herein by reference.

[0369] Template Nucleic Acid

[0370] The compositions and methods disclosed herein may comprise a template nucleic acid. The template can be used to alter or insert a nucleic acid sequence at or near a target site of an RNA-directed DNA-binding protein such as a Cas nuclease, for example, a class 2 Cas nuclease. In some embodiments, the method includes introducing the template into a cell. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided so that editing can occur at two or more target sites. For example, different templates may be provided to edit a single gene or two different genes in a cell.

[0371] In some embodiments, the template can be used for homologous recombination. In some embodiments, homologous recombination can result in the integration of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In other embodiments, the template can be used for homologous directed repair, which involves DNA strand invasion at a cleavage site in the nucleic acid. In some embodiments, homologous directed repair can result in the inclusion of the template sequence in the edited target nucleic acid molecule. In other embodiments, the template can be used for gene editing mediated by non-homologous end joining. In some embodiments, the template sequence is not similar to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is incorporated. In some embodiments, the template includes a side-joined inverted terminal repeat (ITR) sequence.

[0372] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. It may also, or alternatively, correspond to, comprise, or consist of an exogenous sequence of the target cell. As used herein, the term "endogenous sequence" refers to a sequence native to the cell. The term "exogenous sequence" refers to a sequence not native to the cell, or a sequence located in a different position from its native position in the cell's genome. In some embodiments, the endogenous sequence may be a genomic sequence of the cell. In some embodiments, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell.

[0373] In some embodiments, the template comprises ssDNA or dsDNA containing side-joined inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, plasmid, microloop, nanoloop, or PCR product.

[0374] In some embodiments, nucleic acids are purified. In some embodiments, nucleic acids are purified using precipitation methods (e.g., LiCl precipitation, alcohol precipitation, or equivalent methods, such as those described herein). In some embodiments, nucleic acids are purified using chromatographic methods, such as HPLC-based methods, or equivalent methods (e.g., those described herein). In some embodiments, nucleic acids are purified using both precipitation methods (e.g., LiCl precipitation) and HPLC-based methods. In some embodiments, nucleic acids are purified by tangential flow filtration (TFF).

[0375] Compounds or compositions typically, but not necessarily, contain one or more pharmaceutically acceptable excipients. The term "excipient" includes any component other than one or more compounds, one or more other lipid components, and bioactive agents disclosed herein. Excipients may impart functional (e.g., drug release rate control) and / or non-functional (e.g., processing aids or diluents) characteristics to the composition. The selection of excipients will depend extensively on factors such as the specific route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0376] Parenteral preparations are typically aqueous or oily solutions or suspensions. When the preparation is aqueous, excipients such as sugars (including but not limited to glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffers (preferably with a pH of 3 to 9) can be used. However, for some applications, the preparation may be more appropriately formulated as a sterile non-aqueous solution or in a dried form to be used in conjunction with a suitable medium such as sterile pyrogen-free water (WFI).

[0377] While the invention has been described in conjunction with the illustrated embodiments, it should be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents, including equivalents of specific features, which may be included within the invention as defined by the appended claims.

[0378] Both the foregoing general description and detailed embodiments, as well as the following examples, are exemplary and illustrative only and do not limit the teachings. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. In the event of any conflict between any terminology defined in this specification and any reference incorporated herein by reference, this specification shall prevail. Unless otherwise stated, all scopes given herein cover endpoints.

[0379] definition

[0380] It should be noted that, unless the context clearly specifies otherwise, as used herein, the singular forms “a / an” and “the” include plural references. Thus, for example, a reference to “composition” includes multiple compositions and a reference to “cell” includes multiple cells, etc. Unless otherwise stated, the word “or” as used is inclusive and means “and / or”.

[0381] Unless expressly stated in the foregoing description, embodiments in this specification that "comprise" various components are also contemplated as "composed of" or "substantially composed of" the components; embodiments in this specification that "composed of" various components are also contemplated as "comprising" or "substantially composed of" the components; embodiments in this specification that "about" various components are also contemplated as "being" the components; and embodiments in this specification that "substantially composed of" various components are also contemplated as "composed of" or "comprising" the components (this interchangeability does not apply to the use of these terms in the claims).

[0382] Numerical ranges include numbers that define a range. Taking into account significant figures and errors associated with measurement, measured and measurable values ​​should be understood as approximate. As used herein, the terms “about” and “approximately” have their meanings as understood in the art; use of one does not necessarily imply a different range than another. Unless otherwise stated, numbers used herein with or without modifiers such as “about” or “approximately” should be understood to cover normal divergence and / or fluctuations, as understood by one of ordinary skill in the art. In some embodiments, the terms “about” or “approximately” refer to a range of values ​​of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less than) of the reference value, unless otherwise stated or otherwise apparent from the context (except where such numbers would exceed 100% of possible values).

[0383] As used herein, the term "contact" means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticles share a physical connection. Methods for contacting cells with external entities in vivo and in vitro are well known in the biological field. For example, contacting a nanoparticle composition with mammalian cells placed in a mammalian body can be performed via different routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve different amounts of the nanoparticle composition. Furthermore, more than one type of mammalian cell can be contacted by the nanoparticle composition.

[0384] As used herein, the term “delivery” means providing an entity to a destination. For example, delivering a therapeutic and / or preventative agent to a subject may involve administering a nanoparticle composition comprising the therapeutic and / or preventative agent to the subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes). Applying a nanoparticle composition to a mammal or mammalian cells may involve contacting one or more cells with the nanoparticle composition.

[0385] As used herein, “encapsulation efficiency” refers to the amount of therapeutic and / or preventive agents that become part of the nanoparticle composition relative to the initial total amount of therapeutic and / or preventive agents used to prepare the nanoparticle composition. For example, if 97 mg of therapeutic and / or preventive agents are encapsulated in a nanoparticle composition initially provided with a total of 100 mg of therapeutic and / or preventive agents, the encapsulation efficiency may be given as 97%. As used herein, “encapsulation” may mean complete, substantial, or partial encapsulation, restriction, surrounding, or enclosing.

[0386] As used herein, the term "biodegradable" refers to a material that, when introduced into cells, is broken down into components that are reusable or disposable by the cells without one or more significant toxic effects on the cells. In some embodiments, the components resulting from the breakdown of the biodegradable material do not induce inflammation and / or other side effects in vivo. In some embodiments, the biodegradable material is enzymatically broken down. Alternatively or additionally, in some embodiments, the biodegradable material is broken down by hydrolysis.

[0387] As used herein, “N / P ratio” is, for example, in a nanoparticle composition comprising a lipid component and RNA, the molar ratio of ionizable (within the physiological pH range) nitrogen atoms in the lipid (e.g., compound of formula I) to phosphate groups in the RNA.

[0388] The composition may also comprise a salt of one or more compounds. The salt may be a pharmaceutically acceptable salt. As used herein, a “pharmaceutically acceptable salt” refers to a derivative of the disclosed compound in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting the free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (such as amines); alkali metal or organic salts of acidic residues (such as carboxylic acids); and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, diglucuronate, dodecyl sulfate, ethanesulfonate, fumarate, glucohepanoate, glucohepanoate, hemisulfate, heptaate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Pharmaceutically acceptable salts of this disclosure include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of this disclosure can be synthesized from parent compounds containing a basic or acidic moiety using conventional chemical methods. Typically, such salts are prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, in an organic solvent, or in a mixture of both; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use; P.Stahl and C.G.Wermuth (eds.), Wiley-VCH, 2008; and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0389] As used herein, the "polydispersity index" is a ratio that describes the uniformity of the particle size distribution of a system. For example, a small value less than 0.3 indicates a narrow particle size distribution. In some implementations, the polydispersity index may be less than 0.1.

[0390] As used in this article, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, outside the body, or in vivo.

[0391] As used herein, the term "alkyl" is a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Alkyl groups can be cyclic or acyclic. Alkyl groups can be branched or unbranched (i.e., straight-chain). Alkyl groups can also be substituted or unsubstituted (preferably unsubstituted). For example, an alkyl group can be substituted with one or more groups, including but not limited to alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfoxide, sulfonate, carboxylic acid ester, or thiol, as described herein. "Lower alkyl" is an alkyl group containing one to six (e.g., one to four) carbon atoms.

[0392] As used herein, the term "alkenyl" refers to an aliphatic group comprising at least one carbon-carbon double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter referring to an alkenyl moiety having a substituent on one or more carbons replacing the alkenyl group. Such substituents may be present on one or more carbons, including or not included in one or more double bonds. Furthermore, such substituents include all those substituents contemplated for alkyl groups, as discussed below, unless stability is prohibited. For example, it is contemplated that alkenyl groups may be substituted with one or more alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups. Exemplary alkenyl groups include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

[0393] "Alkylene" refers to a divalent alkyl group, which can be branched or unbranched (i.e., straight-chain). Any monovalent alkyl group described above can be converted into an alkylene group by abstracting a second hydrogen atom from the alkyl group. Representative alkylene groups include C14 and C24. 2-4 Alkylene and C 2-3Alkylenes. Typical alkylenes include, but are not limited to, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc. Alkylenes can also be substituted or unsubstituted. For example, an alkylene may be substituted with one or more groups, including but not limited to alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfoxide, sulfonate, sulfonamide, urea, amide, carbamate, ester, carboxylic acid ester, or thiol, as described herein.

[0394] The term "alkenyl" includes divalent, straight-chain or branched, unsaturated, acyclic hydrocarbon groups having at least one carbon-carbon double bond, and in one embodiment, no carbon-carbon triple bond. Any of the above-described monovalent alkenyl groups can be converted into an alkenyl group by abstracting a second hydrogen atom from the alkenyl group. Representative alkenyl groups include C1... 2-6 Alkenyl group.

[0395] Term "C" x-y When used in conjunction with chemical moieties such as alkyl or alkylene groups, it means to include groups containing x to y carbons in the chain. For example, the term "C x-y "Alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain and branched alkyl and alkylene groups containing x to y carbons in the chain.

[0396] Reference merge

[0397] The contents of any articles, patents, and patent applications mentioned or cited herein, as well as all other documents and electronically available information, are incorporated herein by reference in their entirety, to the extent that each individual publication is specifically and individually indicated as incorporated by reference. The applicant reserves the right to physically incorporate any and all material and information from any such articles, patents, patent applications, or other physical and electronic documents into this application.

[0398] Example

[0399] General Information

[0400] All reagents and solvents were purchased from commercial suppliers and used as is, or synthesized according to the cited procedures. All intermediates and final compounds were purified by rapid column chromatography on silica gel. NMR spectra were recorded on a Bruker or Varian 400 MHz spectrometer, and NMR data were collected in CDCl3 at ambient temperature. Chemical shifts are reported in parts per million (ppm) relative to CDCl3 (7.26). 1The 1H NMR data are reported as follows: chemical shifts, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, dd = doubletuplet, dt = doubletuplet, q = quartet, m = multiplet, ddd = doubletuplet, td = tripletuplet, tt = tripletuplet, tdd = tripletuplet, dddd = doubletuplet), coupling constants, and integrals. MS data were recorded on a Waters SQD2 mass spectrometer equipped with an electrospray ionization (ESI) source. The purity of the final compounds was determined by UPLC-MS-ELS using a Waters Acquity H-class liquid chromatograph equipped with a SQD2 mass spectrometer, which has a photodiode array (PDA) and an evaporative light scattering (ELS) detector.

[0401] The pKa of each amine lipid was determined using the following adaptation, following the method described by Jayaraman et al. (Angewandte Chemie, 2012). The pKa of unprepared amine lipids in ethanol was determined. Lipid stock solutions (2.94 mM) were diluted in sodium phosphate buffer (0.1 M, Boston Bioproducts) at different pH values ​​(pH range: 4.5–9.0) to obtain a final lipid concentration of approximately 100 μM. Test samples were supplemented with TNS {sodium 6-(p-toluidine)-2-naphthalenesulfonate}, incubated, and fluorescence intensity was measured using excitation and emission wavelengths of 321 nm and 448 nm, respectively. The recorded data were normalized, and the corresponding pKa values ​​were derived from sigmoid fitting.

[0402] Table 1. Compounds

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432] Example 1 – Comparative Compound 1

[0433] Intermediate 1a: 4-oxohexadecanoic acid

[0434]

[0435] A solution of succinic anhydride (3.0 g, 1.0 equivalent) in THF (0.1 M) was cooled to -78 °C, and then magnesium dodecyl bromide (1.0 M solution in hexane, 0.5 equivalent) was added dropwise over 5 min. The reaction was maintained at -78 °C for at least 18 h, and then quenched by adding water and 1 M HCl. The reaction mixture was concentrated under vacuum, and the resulting aqueous layer was extracted three times with DCM. The combined DCM layers were then extracted twice with 2 M NaOH, and the combined aqueous layers were acidified to pH 1 with concentrated HCl. The mixture was then extracted three times with DCM. The combined DCM layers were dried over Na2SO4, filtered, and concentrated under vacuum to obtain a crude material. The crude material was ground three times with hexane, and the resulting precipitate was filtered to give a product as a white solid (1.31 g, 33%). 1 H NMR(400MHz, CDCl3)δ2.72(ddd,J=7.0,5.8,1.1Hz,2H),2.67–2.60(m,2H),2.44( t, J=7.5Hz, 2H), 1.59 (q, J=7.4Hz, 2H), 1.26 (d, J=2.7Hz, 18H), 0.91–0.85 (m, 3H).

[0436] Intermediate 1b: 2-oxopropane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0437]

[0438] Linoleic acid (2.05 equivalents), DMAP (0.2 equivalents), DIPEA (2.4 equivalents), and EDCI (2.4 equivalents) were added to a solution of dihydroxyacetone (1.55 g, 1.0 equivalents) in DCM (0.1 M). The reaction was stirred at room temperature for at least 18 h. After completion, the mixture was quenched by adding water, and the organic layer was washed once with 1 M HCl and once with 5% NaHCO3. The organic layer was then dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (EtOAc / hexane) to a product (8.0 g, 75%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.44–5.28(m,8H),4.75(s,4H),2.82–2.74(m,4H),2.42(t,J=7.5Hz ,4H),2.05(q,J=6.7Hz,8H),1.67(q,J=7.4Hz,4H),1.40–1.24(m,28H),0.95–0.86(m,6H).

[0439] Intermediate 1c: 2-hydroxy-3-[(9Z,12Z)-octadec-9,12-dienoyloxy]propyl(9Z,12Z)-octadec-9,12-dienoate

[0440]

[0441] A solution of intermediate 1b (8.1 g, 1.0 equivalent) in 4:2:1 THF / water / toluene (0.05–0.1 M) was cooled to 0–5 °C, and then NaBH4 (1.7 equivalent) was added. The reaction was stirred at 0–25 °C for at least 1 h. After completion, the mixture was quenched by adding water and EtOAc, and the mixture was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (EtOAc / hexane) to give a product (5.9 g, 73%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.46–5.26(m,8H),4.23–4.10(m,4H),2.81–2.73(m,4H),2.42(d,J=4.8Hz,1H) ,2.38–2.31(m,4H),2.05(q,J=6.7Hz,8H),1.68–1.57(m,4H),1.42–1.23(m,29H),0.93–0.85(m,6H).

[0442] Intermediate 1d: 2-((4-oxohexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0443]

[0444] Intermediate 1a (1.0–1.2 equivalents), Et3N or DIPEA (1.5–4.5 equivalents), DMAP (0.1–0.2 equivalents), and EDCI (1–2 equivalents) were added to a solution of intermediate 1c (1.0 equivalents) in DCM (0.2 M). The reaction was stirred at room temperature for at least 12 h, and then quenched by adding water. The mixture was washed once with 1 M HCl and once with 5% NaHCO3. The organic layer was then dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (EtOAc / hexane) to give a product (780 mg, 79%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ5.45–5.28(m,8H),5.24(tt,J=5.9,4.3Hz,1H),4.29(dd,J=11. 9,4.3Hz,2H),4.15(dd,J=11.9,5.8Hz,2H),2.84–2.74(m,4H),2.71(t,J=6.8Hz,2H) ,2.59(t,J=6.3Hz,2H),2.43(t,J=7.5Hz,2H),2.32(t,J=7.6Hz,4H),2.05(q,J=6.8H z,8H),1.60(dd,J=14.7,7.3Hz,6H),1.44–1.20(m,49H),0.88(td,J=6.9,4.3Hz,9H).

[0445] Intermediate 1e: 2-((4-oxohexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0446]

[0447] A solution of intermediate 1d (1.43 g, 1.0 equivalent) in 4:2:1 THF / water / toluene (0.05–0.1 M) was cooled to 0–5 °C, and then NaBH4 (5.0 equivalent) was added. The reaction was maintained at 0–25 °C for at least 2 h. The reaction mixture was then diluted with water and EtOAc, and the resulting two-phase mixture was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (EtOAc / hexane) to give the intermediate alcohol as a colorless oil.

[0448] The resulting alcohol was immediately dissolved in DCM (0.1 M), followed by the addition of pyridine (2-3 equivalents) and 4-nitrobenzene chloroformate (1-1.5 equivalents). The reaction was stirred at room temperature for at least 1 h. The mixture was quenched by adding water and extracted three times with DCM. The combined DCM layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (EtOAc / hexane) to give a product as a colorless oil (910 mg, 54%). 1H NMR (400MHz, CDCl3) δ8.37–8.25(m,2H),7.48–7.40(m,2H),5.49–5.25(m,9H),4.96–4.84( m,1H),4.34(dd,J=12.0,4.3Hz,2H),4.18(dd,J=11.9,5.7Hz,2H),2.79(t,J=6.4Hz,4H),2. 49(dd,J=8.4,6.7Hz,2H),2.33(tt,J=7.6,4.2Hz,4H),2.16–1.95(m,10H),1.81–1.71(m,1H ), 1.65 (ddq, J=15.2, 7.5, 4.8, 4.2Hz, 6H), 1.50–1.13 (m, 50H), 0.90 (td, J=6.8, 3.7Hz, 9H).

[0449] Compound 1: 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0450]

[0451] To a solution of intermediate 1e (250 mg, 1.0 equivalent) in MeCN (0.1 M), pyridine (3.0 equivalent), 3-(dimethylamino)prop-1-ol (1.5–3.0 equivalent), and DMAP (1.0 equivalent) were added. The reaction was stirred at room temperature for at least 2 hours. Heptane (0.1 M) was then added to the reaction mixture, and the heptane layer was washed three times with MeCN. The combined MeCN layers were back-extracted three times with heptane, and the combined heptane layers were washed once more with MeCN. The heptane was then removed under vacuum, and the crude material was purified by column chromatography (MeOH / DCM) to give a product (100 mg, 41%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.46–5.22(m,9H),4.72(tdd,J=7.5,5.3,4.0Hz,1H),4.29(dd,J=12.0,4.4Hz,2H),4.23–4.10(m,4H),2.84–2.73( m,4H),2.62–2.27(m,13H),2.05(q,J=6.9Hz,8H),2.01–1.84(m,4H),1.68–1.50(m,7H),1.43–1.21(m,50H),0.88(td,J=6.9,4.1Hz,9H). MS: 1001.2m / z[M+H].

[0452] Example 2 – Compound 2

[0453] Compound 2: 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0454]

[0455] Compound 2 was synthesized from intermediate 1e and 3-(diethylamino)prop-1-ol using the method employed in Example 1, with a yield of 64%. 1 H NMR(400MHz, CDCl3)δ5.43–5.20(m,9H),4.72(tdd,J=7.5,5.3,4.0Hz,1H),4.29( dd,J=11.9,4.4Hz,2H),4.23–4.10(m,4H),2.81–2.73(m,4H),2.57–2.36(m,8H),2 .31(td,J=7.6,1.3Hz,4H),2.05(q,J=6.8Hz,8H),1.97–1.77(m,4H),1.61(t,J=7 .4Hz, 6H), 1.40–1.21 (m, 49H), 1.01 (t, J = 7.1Hz, 6H), 0.88 (td, J = 6.9, 4.1Hz, 9H). MS: 1029.7m / z[M+H].

[0456] Example 3 – Compound 3

[0457] Compound 3: 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0458]

[0459] Compound 3 was synthesized from intermediate 1e and 3-((methyl)ethylamino)prop-1-ol using the method employed in Example 1, with a yield of 79%. 1H NMR (400MHz, CDCl3) δ5.49–5.20(m,9H),4.77–4.67(m,1H),4.29(dd,J=11.9,4.4Hz,2H),4.16(tdd,J=11.9,6.2,2.5Hz,4H),2.77(t,J=6.4Hz, 4H),2.70–2.37(m,7H),2.37–2.25(m,7H),2.13–1.82(m,12H),1.69–1. 48(m,7H),1.42–1.20(m,46H),1.14(s,2H),0.88(td,J=6.8,3.9Hz,7H). MS: 1015.7m / z[M+H].

[0460] Example 4 – Compound 4

[0461] Compound 4: 2-((4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0462]

[0463] Compound 4 was synthesized from intermediate 1e and 2-dimethylaminoethanol using the method employed in Example 1, with a yield of 93%. 1 H NMR (400MHz, CDCl3) δ5.42–5.31(m,8H),5.25(tt,J=5.8,4.3Hz,1H),4.73(tdd,J=7.6, 5.5,4.0Hz,1H),4.29(dd,J=11.9,4.4Hz,4H),4.14(ddd,J=11.9,5.8,3.4Hz,2H),2.77( td,J=6.0,1.1Hz,4H),2.70(d,J=16.4Hz,1H),2.49–2.29(m,11H),2.05(q,J=6.8Hz,8H) ,1.99–1.81(m,2H),1.67–1.50(m,8H),1.40–1.21(m,48H),0.88(td,J=7.0,4.2Hz,9H). MS: 987.6m / z[M+H].

[0464] Example 5 – Compound 5

[0465] Compound 5: 2-((4-(((3-(pyrrolidone-1-yl)propoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0466]

[0467] Compound 5 was synthesized from intermediate 1e and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 54%. 1 H NMR (400MHz, CDCl3) δ5.45–5.21(m,9H),4.72(tdd,J=7.3,5.5,4.2Hz,1H),4.29(dd,J=11.9,4 .4Hz,2H),4.17(dddd,J=15.3,9.1,7.3,3.7Hz,4H),2.77(td,J=6.0,1.1Hz,4H),2.57(d,J=8. 8Hz, 6H), 2.40 (dt, J=8.7, 6.5Hz, 2H), 2.31 (td, J=7.6, 1.2Hz, 4H), 2.05 (q, J=6.8Hz, 8H), 1.98 –1.83(m,5H),1.80(p,J=3.2Hz,4H),1.68–1.50(m,7H),1.41–1.18(m,48H),0.96–0.81(m,9H). MS: 1027.6m / z[M+H].

[0468] Example 6 – Compound 6

[0469] Example 6: 2-((4-((((1-ethylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0470]

[0471] Compound 6 was synthesized from intermediate 1e and 1-ethylpiperidine-4-ol using the method employed in Example 1, with a yield of 53%. 1H NMR (400MHz, CDCl3) δ5.42–5.21(m,9H),4.72(tdd,J=7.6,5.3,4.0Hz,1H),4.64(d,J=5.1Hz,1H ),4.29(dd,J=11.9,4.4Hz,2H),4.14(ddd,J=11.9,5.8,4.1Hz,2H),2.84–2.70(m,6H),2.51–2.3 6(m,4H),2.31(td,J=7.6,1.4Hz,4H),2.24(d,J=23.1Hz,2H),2.11–1.93(m,11H),1.92–1.73(m ,4H),1.70–1.52(m,8H),1.43–1.21(m,48H),1.09(t,J=7.2Hz,3H),0.88(td,J=7.0,4.2Hz,9H). MS: 1028.2m / z[M+H].

[0472] Example 7 – Compound 7

[0473] Compound 7: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0474]

[0475] Compound 7 was synthesized from intermediate 1e and N1-ethyl-N1-methylethane-1,2-diamine using the method employed in Example 1, with a yield of 66%. 1 H NMR (400MHz, CDCl3) δ5.47–5.08(m,10H),4.76(s,1H),4.28(ddd,J=11.9,4.4,1. 4Hz,2H),4.15(ddd,J=11.9,5.9,3.3Hz,2H),3.24(q,J=6.0Hz,2H),2.82–2.74(m ,4H),2.53–2.26(m,10H),2.21(s,3H),2.05(q,J=6.8Hz,8H),1.98–1.74(m,2H), 1.68–1.41(m,8H),1.41–1.19(m,49H),1.04(t,J=7.2Hz,3H),0.95–0.82(m,9H). MS: 1001.1m / z [M+H].

[0476] Example 8 – Compound 8

[0477] Compound 8: 2-((4-(((3-(dimethylamino)propyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0478]

[0479] Compound 8 was synthesized from intermediate 1e and N1,N1-dimethylpropane-1,3-diamine using the method employed in Example 1, with a yield of 55%. 1 H NMR (400MHz, CDCl3) δ5.46(t,J=5.8Hz,1H),5.43–5.28(m,8H),5.24(tt,J=5.8,4. 4Hz,1H),4.75(s,1H),4.28(ddd,J=11.9,4.5,2.2Hz,2H),4.15(ddd,J=11.9,5.8, 1.6Hz,2H),3.23(h,J=6.4Hz,2H),2.82–2.73(m,4H),2.48–2.20(m,14H),2.05(q, J=6.8Hz,8H),1.97–1.42(m,12H),1.42–1.20(m,48H),0.88(td,J=6.9,4.1Hz,9H). MS: 1000.6m / z[M+H].

[0480] Example 9 – Compound 9

[0481] Compound 9: 2-((4-((((1-methylpyrrolidone-2-yl)methyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0482]

[0483] Compound 9 was synthesized from intermediate 1e and (1-methylpyrrolidone-2-yl)methylamine using the method employed in Example 1, with a yield of 65%. 1¹H NMR (400MHz, chloroform-d) δ 5.45–5.28 (m, 8H), 5.25 (ddd, J = 10.2, 5.8, 4.4Hz, 1H), 5.19–5.11 (m, 1H), 4.76 (s, 1H), 4.28 (dd, J = 11.9, 4.4Hz, 2H), 4.14 (ddd, J = 11.9, 5.9, 3.0Hz, 2H), 3.44–3 .31(m,1H),3.19–3.02(m,2H),2.84–2.72(m,4H),2.46–2.16(m,12H),2.05(q,J=6.9Hz,8 H),1.97–1.66(m,6H),1.66–1.42(m,8H),1.42–1.20(m,49H),0.88(td,J=7.0,4.2Hz,9H). MS: 1013.1m / z[M+H].

[0484] Example 10 – Compound 10

[0485] Compound 10: 2-((4-((1,4-dimethylpiperidin-4-carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0486]

[0487] A solution of intermediate 1d (360 mg, 1.0 equivalent) in 4:2:1 THF / water / toluene (0.05 M) was cooled to 0 °C, and then NaBH4 (5.0 equivalent) was added. The reaction was maintained at 0 °C for 18 h. The reaction mixture was then diluted with water and EtOAc, and the resulting two-phase mixture was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (EtOAc / hexane) to give the intermediate alcohol as a colorless oil.

[0488] The crude alcohol was immediately dissolved in DCM (0.1 M), followed by the addition of 1,4-dimethylpiperidine-4-carboxylic acid (1.0 equivalent), DMAP (0.1 equivalent), DIPEA (4.0 equivalent), and EDCI (1.5 equivalent). The reaction was stirred at room temperature. After 16 h, additional 1,4-dimethylpiperidine-4-carboxylic acid (1.0 equivalent), DMAP (0.05 equivalent), DIPEA (2.0 equivalent), and EDCI (1.5 equivalent) were added, and the reaction was heated to 40 °C. After another two hours, the reaction was quenched by adding water and DCM, and the organic layer was washed once with 1 M HCl, once with 5% NaHCO3, and once with brine. The resulting organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (MeOH / DCM) to give a product (94 mg, 22%) as a pink oil. 1 H NMR (400MHz, CDCl3) δ5.46–5.22(m,9H),4.92(ddd,J=11.6,7.5,4.7Hz,1H),4.30 (dd,J=11.9,4.4Hz,2H),4.14(ddd,J=12.0,5.8,1.9Hz,2H),2.82–2.74(m,4H),2. 67(s,2H),2.41–2.25(m,9H),2.14(d,J=14.2Hz,4H),2.05(q,J=6.8Hz,8H),1.98 –1.78(m,3H),1.66–1.49(m,9H),1.43–1.18(m,50H),0.88(td,J=6.9,4.3Hz,9H).

[0489] Example 11 – Compound 11

[0490] Compound 11: 2-((4-((4-(diethylamino)butyryl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0491]

[0492] Compound 11 was synthesized from intermediate 1d and 3-(diethylamino)butyric acid using the method employed in Example 10, with a yield of 17%. 1H NMR (400MHz, CDCl3) δ5.46(t,J=5.8Hz,1H),5.43–5.28(m,8H),5.24(tt,J=5.8,4. 4Hz,1H),4.75(s,1H),4.28(ddd,J=11.9,4.5,2.2Hz,2H),4.15(ddd,J=11.9,5.8, 1.6Hz,2H),3.23(h,J=6.4Hz,2H),2.82–2.73(m,4H),2.48–2.20(m,14H),2.05(q, J=6.8Hz,8H),1.97–1.42(m,12H),1.42–1.20(m,48H),0.88(td,J=6.9,4.1Hz,9H). MS: 1013.9m / z[M+H].

[0493] Example 12 – Compound 12

[0494] Compound 12: 2-((4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0495]

[0496] Compound 12 was synthesized from intermediate 1e and N1,N1-dimethylethane-1,2-diamine using the method employed in Example 1, with a yield of 67%. 1 H NMR (400MHz, CDCl3) δ5.44–5.29(m,8H),5.29–5.18(m,2H),4.75(s,1H),4.34–4.24( m,2H),4.15(ddd,J=11.9,5.8,3.6Hz,2H),3.25(q,J=5.8Hz,2H),2.77(td,J=6.2,1. 2Hz,4H),2.47–2.35(m,4H),2.35–2.28(m,4H),2.24(s,6H),2.05(q,J=7.1Hz,8H),1 .98–1.73(m,4H),1.65–1.42(m,7H),1.40–1.20(m,48H),0.88(td,J=7.0,4.3Hz,9H). MS: 987.2m / z[M+H].

[0497] Example 13 – Compound 13

[0498] Compound 13: 2-((4-(((3-(diethylamino)propyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0499]

[0500] Compound 13 was synthesized from intermediate 1e and N1,N1-diethylpropane-1,3-diamine using the method employed in Example 1, with a yield of 62%. 1 H NMR (400MHz, CDCl3) δ5.93(s,1H),5.44–5.28(m,8H),5.24(ddd,J=5.8,4.5,1.3Hz,1H),4.74(s,1H),4.28(ddd,J=11.9 ,4.5,2.7Hz,2H),4.14(ddd,J=11.9,5.8,1.2Hz,2H),3.24(tt,J=13.2,6.6Hz,2H),2.81–2.71(m,4H),2.57(d,J=7.7Hz, 6H),2.38(ddd,J=8.9,6.8,3.2Hz,2H),2.35–2.28(m,4H),2.05(q,J=6.6Hz,8H),1.94–1.85(m,2H),1.79(dq,J=14.8,8 .1Hz,2H),1.73–1.56(m,7H),1.56–1.43(m,3H),1.40–1.20(m,47H),1.06(t,J=7.1Hz,6H),0.88(td,J=7.0,4.3Hz,9H). MS: 1029.2m / z[M+H].

[0501] Example 14 – Compound 14

[0502] Compound 14: 2-((4-(((4-(diethylamino)butoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0503]

[0504] Compound 14 was synthesized from intermediate 1e and N1,N1-dimethylethane-1,2-diamine using the method employed in Example 1, with a yield of 60%. 1H NMR(400MHz, CDCl3)δ5.45–5.30(m,9H),5.28(tt,J=5.8,4.3Hz,1H),4.78–4.69(m,1H),4.31 (dd,J=11.9,4.4Hz,2H),4.22–4.09(m,4H),2.79(td,J=6.1,1.1Hz,4H),2.63(q,J=7.2Hz,4H ),2.55(t,J=7.6Hz,2H),2.47–2.38(m,2H),2.38–2.30(m,4H),2.07(q,J=7.1Hz,8H),2.02–1 .85(m,2H),1.76–1.51(m,11H),1.45–1.21(m,49H),1.08(t,J=7.2Hz,6H),0.95–0.82(m,9H). MS: 1044.1m / z[M+H].

[0505] Example 15 – Compound 15

[0506] Compound 15: 2-((4-(((2-(diethylamino)ethyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0507]

[0508] Compound 15 was synthesized from intermediate 1e and N1,N1-diethylethane-1,2-diamine using the method employed in Example 1, with a yield of 75%. 1H NMR (400MHz, CDCl3) δ5.44–5.29(m,9H),5.25(ddd,J=5.8,4.4,1.4Hz,1H),4.75(s,1H),4.28(ddd,J=11.9,4.4 ,1.2Hz,2H),4.14(ddd,J=11.9,5.8,3.0Hz,2H),3.23(q,J=5.7Hz,2H),2.80–2.73(m,4H),2.57(q,J=7.0Hz,6H) ,2.38(ddd,J=9.0,6.6,4.7Hz,2H),2.34–2.28(m,4H),2.05(q,J=6.8Hz,8H),1.90(h,J=9.0,7.9Hz,1H),1.80( p,J=7.3,6.5Hz,1H),1.66–1.44(m,7H),1.40–1.20(m,53H),1.03(t,J=7.1Hz,6H),0.88(td,J=6.9,4.3Hz,9H). MS: 1014.9m / z[M+H].

[0509] Example 16 – Compound 16

[0510] Compound 16: 2-((4-(((2-(1-methylpyrrolidone-2-yl)ethoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0511]

[0512] Compound 16 was synthesized from intermediate 1e and 2-(1-methylpyrrolidone-2-yl)ethanol using the method employed in Example 1, with a yield of 78%. 1 H NMR (400MHz, CDCl3) δ5.43–5.28(m,9H),5.25(tt,J=5.8,4.3Hz,1H),4.72(qd,J=7 .6,4.6Hz,1H),4.29(dd,J=11.9,4.4Hz,2H),4.26–4.09(m,4H),3.14(t,J=8.8Hz,1 H),2.82–2.74(m,4H),2.45–2.28(m,9H),2.22(td,J=11.5,10.9,6.0Hz,2H),2.15 –1.91(m,12H),1.69–1.48(m,9H),1.42–1.20(m,50H),0.88(td,J=6.9,4.1Hz,9H). MS: 1027.9m / z[M+H].

[0513] Example 17 – Compound 17

[0514] Intermediate 17a: Hexadecyl magnesium bromide

[0515]

[0516] I₂ (0.01 equivalent) was added to a solution of Mg (15.92 g, 2 equivalents) in THF (300 mL), and then 1-bromohexadecane (1.0 equivalent) in THF (100 mL) was added dropwise to the solution at 80 °C. The mixture was stirred at 80 °C for 6 h. A product in the form of a brown liquid was obtained in quantitative yield and immediately used in the next step.

[0517] Intermediate 17b: 4-oxoeicosanoic acid

[0518]

[0519] A solution of succinic anhydride (54 g, 1.0 equivalent) in THF (1.2 M) was cooled to -78 °C, and then intermediate 17a was added. The reaction was maintained at -78 °C for 12 h, then saturated NH4Cl was added and the mixture was extracted three times with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated. Purification by column chromatography gave the product as a white solid (4 g, 2%). 1 H NMR(400MHz, CDCl3)δ4.11(t,J=6.7Hz,1H),2.80–2.62(m,4H),2.46(t,J=7.5H z,1H),1.63(dq,J=14.7,7.1Hz,2H),1.49–1.20(m,27H),0.90(t,J=6.7Hz,3H).

[0520] Intermediate 17c: 2-((4-oxoeicosanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0521]

[0522] Using the method employed with intermediate 1d, intermediate 17c was synthesized from intermediate 17b and intermediate 1c with a yield of 53%. 1H NMR (400MHz, CDCl3) δ5.43–5.21(m,9H),4.29(dt,J=11.9,4.1Hz,2H),4.14(dd ,J=11.9,5.9Hz,2H),4.06(t,J=6.8Hz,1H),2.73(dt,J=22.5,6.5Hz,6H),2.67 –2.54(m,4H),2.42(t,J=7.5Hz,2H),2.31(t,J=7.6Hz,4H),2.04(q,J=6.9Hz,9 H), 1.61 (q, J=7.9, 7.3Hz, 8H), 1.42–1.15 (m, 55H), 0.88 (td, J=6.7, 4.0Hz, 9H).

[0523] Intermediate 17d: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)eicosyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0524]

[0525] Using the method employed with intermediate 1e, intermediate 17d was synthesized from intermediate 17c with a yield of 29%. 1 H NMR (400MHz, CDCl3) δ8.33–8.24(m,2H),7.43–7.34(m,2H),5.45–5.23(m,8H),4.92 –4.82(m,1H),4.31(dd,J=12.0,4.3Hz,2H),4.20–4.09(m,2H),2.76(t,J=6.3Hz,4H ),2.47(dd,J=8.3,6.7Hz,2H),2.30(td,J=7.6,4.2Hz,4H),2.04(q,J=7.2Hz,9H),1 .61(ddt,J=11.5,7.4,3.7Hz,4H),1.43–1.22(m,49H),0.88(td,J=6.9,3.8Hz,9H).

[0526] Compound 17: 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)eicosyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0527]

[0528] Compound 17 was synthesized from intermediate 17d and 3-(dimethylamino)prop-1-ol using the method employed in Example 1, with a yield of 74%.1 H NMR (400MHz, CDCl3) δ5.42–5.22(m,10H),4.72(tdd,J=7.5,5.4,4.0Hz,1H),4.36–4.25(m,2H),4.23–4.08(m,4H),2.82–2.71(m,4H ),2.50–2.28(m,9H),2.25(s,6H),2.09–1.97(m,9H),1.91–1.80(m,3H),1.70–1.47(m,7H),1.40–1.20(m,57H),0.96–0.82(m,9H). MS: 1058.1m / z[M+H].

[0529] Example 18 – Compound 18

[0530] Compound 18: 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)eicosyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0531]

[0532] Compound 18 was synthesized from intermediate 17d and 3-((methyl)ethylamino)prop-1-ol using the method employed in Example 1, with a yield of 77%. 1 H NMR (400MHz, CDCl3) δ5.45–5.22(m,10H),4.72(qd,J=7.7,4.7Hz,1H),4.29(dd ,J=12.0,4.4Hz,2H),4.20–4.10(m,4H),2.83–2.73(m,4H),2.51–2.36(m,6H),2 .35–2.28(m,4H),2.24(s,3H),2.10–1.99(m,9H),1.91–1.82(m,3H),1.66–1.52 (m,6H),1.42–1.19(m,60H),1.06(t,J=7.1Hz,3H),0.88(td,J=6.9,4.1Hz,9H). MS: 1072.3 m / z [M+H].

[0533] Example 19 – Compound 19

[0534] Compound 19: 2-((4-(((3-(pyrrolidone-1-yl)propoxy)carbonyl)oxy)eicosyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0535]

[0536] Compound 19 was synthesized from intermediate 17d and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 67%. 1 H NMR (400MHz, CDCl3) δ5.44–5.22(m,9H),4.72(tt,J=7.5,4.5Hz,1H),4.29(dd,J=11.9,4.4H z,2H),4.24–4.10(m,4H),2.83–2.72(m,4H),2.57(d,J=8.3Hz,6H),2.40(dt,J=8.6,6.7Hz, 2H),2.31(td,J=7.6,1.2Hz,4H),2.04(dd,J=8.5,5.3Hz,8H),2.00–1.86(m,4H),1.86–1.78 (m,4H),1.53(ddt,J=14.1,8.3,4.7Hz,7H),1.44–1.20(m,56H),0.88(td,J=6.9,4.2Hz,9H). MS: 1084.2m / z[M+H].

[0537] Example 20 – Compound 20

[0538] Compound 20: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)eicosyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0539]

[0540] Compound 20 was synthesized from intermediate 17d and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 73%. 1H NMR (400MHz, CDCl3) δ5.44–5.17(m,10H),4.75(s,1H),4.28(ddd,J=11.9,4.4,1.3 Hz,2H),4.15(ddd,J=11.9,5.8,3.4Hz,2H),3.25(d,J=6.2Hz,2H),2.81–2.73(m,4H ),2.50–2.36(m,6H),2.36–2.28(m,4H),2.22(s,3H),2.05(q,J=6.8Hz,8H),1.94–1 .71(m,4H),1.66–1.20(m,63H),1.05(t,J=7.1Hz,3H),0.88(td,J=6.9,4.2Hz,9H). MS: 1056.6m / z[M+H].

[0541] Example 21 – Compound 21

[0542] Compound 21: 2-((4-((((1-methylpyrrolidone-2-yl)methyl)carbamoyl)oxy)eicosyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0543]

[0544] Compound 21 was synthesized from intermediate 17d and (1-methylpyrrolidone-2-yl)methylamine using the method employed in Example 1, with a yield of 85%. 1 H NMR (400MHz, CDCl3) δ5.43–5.30(m,8H),5.29–5.18(m,2H),4.76(s,1H),4.28(dd,J=11.9, 4.4Hz,2H),4.21–4.07(m,2H),3.46–3.30(m,1H),3.11(ddt,J=22.3,8.9,4.3Hz,2H),2.80– 2.72(m,4H),2.45–2.15(m,12H),2.04(dd,J=8.5,5.3Hz,9H),1.96–1.77(m,4H),1.71(tq, J=8.4,5.1,4.0Hz,2H),1.65–1.45(m,8H),1.39–1.20(m,60H),0.88(td,J=6.9,4.4Hz,9H). MS: 1068.8m / z[M+H].

[0545] Example 22 – Compound 22

[0546] Intermediate 22a: (Z)-9-(non-2-en-1-yloxy)-9-oxonanoic acid

[0547]

[0548] Oxaloyl chloride (1.0 equivalent) was added dropwise to a solution of azelaic acid (25 g, 1.3 equivalents) in THF (100 mL) over one minute at 0 °C. Next, DMF (0.1 equivalent) was added dropwise over one minute. The reaction mixture was stirred at 0 °C for 10 min, then at room temperature for 10 min. The reaction was then cooled to 0 °C, and cis-2-nonen-1-ol (1.0 equivalent) was added to THF (100 mL). The reaction was stirred at 0 °C for 5 min, then at room temperature for 16 h. After completion, the reaction was concentrated under vacuum, slurried with DCM, and filtered. The filtrate was concentrated and purified by column chromatography (80 g, MeOH / DCM) to give a product (15 g, 47%) as a pale yellow oil, which was used directly in subsequent steps.

[0549] Intermediate 22b: 9,9'-Di((Z)-non-2-en-1-yl)O'1,O1-(2-oxopropane-1,3-diyl)di(azelate)

[0550]

[0551] Intermediate 22b was synthesized using the same method as intermediate 1b by combining dihydroxyacetone and intermediate 22a, with a yield of 73%.

[0552] Intermediate 22c: O'1,O1-(2-hydroxypropane-1,3-diyl)9,9'-di((Z)-non-2-en-1-yl)di(azelate)

[0553]

[0554] Intermediate 22c was synthesized from intermediate 22b using the same method as intermediate 1c (except that the reaction was completed after 45 min), with a yield of 71%. 1H NMR(400MHz, CDCl3)δ5.64(dtt,J=11.0,7.3,1.3Hz,2H),5.52(dtt,J=10.9,6.8,1.5Hz,2H),4.67–4.58(m,4H),4.23–4.05(m,5H),2.51 (d,J=4.6Hz,1H),2.32(dt,J=18.1,7.6Hz,8H),2.15–2.00(m,4H),1.61(td,J=10.0,2.7Hz,8H),1.42–1.20(m,28H),0.93–0.83(m,6H).

[0555] Intermediate 22d: 9,9'-Di((Z)-non-2-en-1-yl)O'1,O1-(2-((4-oxohexadecyl)oxy)propane-1,3-diyl)di(azelate)

[0556]

[0557] Using the method employed with intermediate 1d, intermediate 22d was synthesized from intermediate 22c and intermediate 1a with a yield of 38%. 1 H NMR(400MHz, CDCl3) δ5.66(dtt,J=11.0,7.3,1.3Hz,2H),5.54(dtt,J=11.0,6.9,1.5Hz,2H),5. 26(tt,J=5.9,4.3Hz,1H),4.64(dd,J=6.9,1.3Hz,4H),4.31(dd,J=11.9,4.3Hz,2H),4.17(dd,J =11.9,5.9Hz,2H),2.74(t,J=6.7Hz,2H),2.68–2.56(m,2H),2.45(t,J=7.5Hz,2H),2.33(td,J= 7.6,6.0Hz,8H),2.19–2.07(m,4H),1.70–1.57(m,12H),1.43–1.23(m,46H),0.98–0.84(m,9H).

[0558] Intermediate 22e: O'1,O1-(2-((4-((((4-nitrophenoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl)9,9'-di((Z)-non-2-en-1-yl)di(azelate)

[0559]

[0560] Using the method employed with intermediate 1e, intermediate 22e was synthesized from intermediate 22d with a yield of 51%. 1H NMR (400MHz, CDCl3) δ8.38–8.31(m,1H),8.31–8.24(m,2H),7.54–7.47(m,1H),7.44–7.36(m,2H),5.64(d tt,J=11.0,7.3,1.3Hz,2H),5.51(dtt,J=11.0,6.8,1.5Hz,2H),5.26(tt,J=5.8,4.3Hz,1H),4.86(ddd,J =11.8,7.5,4.8Hz,1H),4.65–4.57(m,4H),4.36–4.25(m,2H),4.21–4.10(m,2H),2.50–2.44(m,2H),2.30 (td,J=7.5,4.5Hz,8H),2.13–2.05(m,4H),1.62(d,J=37.2Hz,9H),1.46–1.18(m,49H),0.94–0.80(m,9H).

[0561] Compound 22: O'1,O1-(2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl)9,9'-di((Z)-non-2-en-1-yl)di(azelate)

[0562]

[0563] Compound 22 was synthesized from intermediate 22e and 3-(dimethylamino)prop-1-ol using the method employed in Example 1, with a yield of 54%. 1 H NMR (400MHz, CDCl3) δ5.71–5.58(m,2H),5.52(dtt,J=10.9,6.8,1.5Hz,2H),5.25(tt,J=5.7,4 .4Hz,1H),4.72(ddd,J=11.7,7.4,4.5Hz,1H),4.62(dd,J=6.8,1.3Hz,4H),4.34–4.25(m,2H), 4.25–4.08(m,4H),2.39(ddd,J=12.9,8.4,6.4Hz,4H),2.34–2.21(m,13H),2.09(tdd,J=7.4,5 .7,1.5Hz,4H),2.02–1.67(m,7H),1.69–1.51(m,11H),1.42–1.19(m,45H),0.97–0.78(m,8H). MS: 1065.7m / z[M+H].

[0564] Example 23 – Compound 23

[0565] Compound 23: O'1,O1-(2-((4-(((2-(ethyl(methyl(amino)amino)ethyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl)9,9'-di((Z)-non-2-en-1-yl)di(azelate)

[0566]

[0567] Compound 23 was synthesized from intermediate 22e and N1-ethyl-N1-methylethane-1,2-diamine using the method employed in Example 1, with a yield of 59%. 1 H NMR(400MHz, CDCl3)δ5.72–5.59(m,2H),5.52(dtt,J=11.0,6.8,1.5Hz,2H),5.36–5.18(m,2H),4.75(s,1H) ,4.62(dd,J=6.9,1.2Hz,4H),4.28(dd,J=11.9,4.4Hz,2H),4.14(ddd,J=11.9,5.8,3.5Hz,2H),3.35–3.20( m,2H),2.56–2.43(m,4H),2.38(ddd,J=8.7,6.6,4.1Hz,2H),2.34–2.19(m,11H),2.10(qd,J=7.4,1.5Hz,4H ),1.96–1.72(m,3H),1.63(d,J=6.9Hz,11H),1.41–1.18(m,48H),1.07(t,J=7.1Hz,3H),0.93–0.81(m,9H). MS: 1065.2m / z[M+H].

[0568] Example 24 – Compound 24

[0569] Compound 24: O'1,O1-(2-((4-((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl)9,9'-di((Z)-non-2-en-1-yl)di(azelate)

[0570]

[0571] Compound 24 was synthesized from intermediate 22e and (1-methylpyrrolidone-2-yl)methylamine using the method employed in Example 1. 1H NMR (400MHz, CDCl3) δ5.71–5.59(m,2H),5.52(dtt,J=10.9,6.8,1.5Hz,2H),5.24(ddd,J=8.8,5.8,4 .4Hz,2H),4.76(s,1H),4.62(dd,J=6.9,1.3Hz,4H),4.28(dd,J=11.9,4.4Hz,2H),4.14(ddd,J=11.9, 5.8,3.2Hz,2H),3.39(q,J=11.2,10.1Hz,1H),3.10(t,J=14.0Hz,2H),2.50–2.20(m,15H),2.10(qd, J=7.4,1.5Hz,4H),1.97–1.67(m,6H),1.61(d,J=7.3Hz,12H),1.41–1.18(m,47H),0.97–0.79(m,9H). MS: 1076.7m / z[M+H].

[0572] Example 25 – Compound 25

[0573] Intermediate 25a: 5-oxoheptadecanoic acid

[0574]

[0575] Intermediate 25a was synthesized from glutaric anhydride and dodecyl magnesium bromide solution using the method employed in intermediate 1a, with a yield of 64%. 1 H NMR (400MHz, CDCl3) δ2.75(t,J=6.7Hz,1H),2.49(t,J=7.2Hz,2H),2.44–2.35(m,4H),1. 90 (p, J = 7.2 Hz, 2H), 1.56 (t, J = 7.3 Hz, 2H), 1.26 (d, J = 4.0 Hz, 19H), 0.88 (t, J = 6.7 Hz, 3H).

[0576] Intermediate 25b: 2-((5-oxohexadecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0577]

[0578] Using the method employed with intermediate 1d, intermediate 25b was synthesized from intermediate 25a and intermediate 1c with a yield of 55%. 1H NMR (400MHz, CDCl3) δ5.45–5.19(m,9H),4.30(dd,J=11.9,4.3Hz,2H),4.12(dd,J=11.9,5.8Hz,2H),2.76(t,J=6.4Hz,4H),2.46(t,J=7.2Hz,2H) ,2.42–2.24(m,8H),2.04(q,J=6.8Hz,8H),1.88(p,J=7.2Hz,2H),1.57( dp,J=21.3,7.2Hz,6H),1.43–1.18(m,41H),0.87(td,J=6.8,4.1Hz,8H).

[0579] Intermediate 25c: 2-((5-(((4-nitrophenoxy)carbonyl)oxy)heptadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0580]

[0581] Using the method employed with intermediate 1e, intermediate 25c was synthesized from intermediate 25b with a yield of 23%. 1 H NMR (400MHz, CDCl3) δ8.31–8.24(m,2H),7.42–7.35(m,2H),5.44–5.19(m,9H),4.8 3(q,J=5.7Hz,1H),4.31(ddd,J=12.0,4.4,2.9Hz,2H),4.14(ddd,J=11.9,5.8,2.1 Hz,2H),2.76(t,J=6.3Hz,4H),2.34(dt,J=30.7,7.0Hz,6H),2.04(q,J=6.9Hz,8H) ,1.80–1.52(m,14H),1.29(dd,J=19.3,12.3Hz,44H),0.88(td,J=6.8,3.7Hz,9H).

[0582] Compound 25: 2-((5-(((3-(dimethylamino)propoxy)carbonyl)oxy)heptadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0583]

[0584] Compound 25 was synthesized from intermediate 25c and 3-(dimethylamino)prop-1-ol using the method employed in Example 1, with a yield of 94%. 1H NMR (400MHz, CDCl3) δ5.46–5.19(m,9H),4.74–4.65(m,1H),4.29(ddd,J=11.9,4.4,1.7Hz,2H),4.25–4.07(m,4H),2.82–2.72(m,4H),2.39–2.2 8(m,8H),2.22(s,6H),2.05(q,J=6.8Hz,8H),1.84(dq,J=8.2,6.7Hz,2H ),1.73–1.50(m,14H),1.41–1.18(m,50H),0.88(td,J=7.0,4.2Hz,9H). MS: 1016.11m / z[M+H].

[0585] Example 26 – Compound 26

[0586] Compound 26: 2-((5-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)heptadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0587]

[0588] Compound 26 was synthesized from intermediate 25c and 3-((methyl)ethylamino)prop-1-ol using the method employed in Example 1, with a yield of 84%. 1 H NMR (400 MHz, CDCl3) δ5.46–5.20(m,10H),4.69(dq,J=7.5,5.5Hz,1H),4.29(ddd,J=11.9,4.4,1.6 Hz,2H),4.25–4.10(m,4H),2.83–2.72(m,4H),2.45–2.38(m,4H),2.37–2.28(m,6H),2.21(s,3H),2.05(q,J=6.8 Hz, 8H), 1.88–1.79 (m, 2H), 1.74–1.52 (m, 13H), 1.44–1.22 (m, 51H), 1.04 (t, J = 7.2 Hz, 3H), 0.88 (td, J = 6.9, 4.1 Hz, 9H). MS: 1030.1 m / z[M+H].

[0589] Example 27 – Compound 27

[0590] Compound 27: 2-((5-(((3-(pyrrolid-1-yl)propoxy)carbonyl)oxy)heptadecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0591]

[0592] Compound 27 was synthesized from intermediate 25c and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 79%. 1 H NMR (400 MHz, CDCl3) δ5.44–5.21(m,10H),4.69(dt,J=7.1,5.3 Hz,1H),4.29(ddd,J=11.9,4.3,1.6 Hz,2H),4.25–4.10(m,4H),2.81–2.73(m,4H),2.52(dd,J=14.8,7.1Hz,6H),2.33(dt,J=13.4,7.2Hz,6H),2.05(q,J=6.8Hz,8H),1 .89(dq,J=10.8,6.8Hz,2H),1.82–1.74(m,J=3.8,3.3Hz,4H),1.71–1.53(m,13H),1.40–1.21(m,49H),0.88(td,J=6.9,4.2Hz,9H). MS: 1041.8m / z[M+H].

[0593] Example 28 – Compound 28

[0594] Compound 28: 2-((5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)heptadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0595]

[0596] Compound 28 was synthesized from intermediate 25c and N1-ethyl-N1-methylethane-1,2-diamine using the method employed in Example 1, with a yield of 87%. 1H NMR (400MHz, CDCl3) δ5.43–5.21(m,10H),5.12(s,1H),4.80–4.65(m,1H),4.29(dt,J= 11.9,4.5Hz,2H),4.19–4.09(m,2H),3.24(d,J=4.8Hz,2H),2.83–2.72(m,4H),2.44(dt ,J=14.6,6.6Hz,4H),2.38–2.27(m,6H),2.20(s,3H),2.04(dd,J=7.8,5.7Hz,8H),1.74 –1.42(m,15H),1.42–1.19(m,50H),1.04(t,J=7.1Hz,3H),0.88(td,J=7.0,4.4Hz,9H). MS: 1014.1m / z[M+H].

[0597] Example 29 – Compound 29

[0598] Compound 29: 2-((5-((((1-methylpyrrolidone-2-yl)methyl)carbamoyl)oxy)heptadecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0599]

[0600] Compound 29 was synthesized from intermediate 25c and (1-methylpyrrolidone-2-yl)methylamine using the method employed in Example 1, with a yield of 93%. 1 H NMR (400MHz, CDCl3) δ5.43–5.21(m,11H),5.08(d,J=8.8Hz,1H),4.74(s,1H),4.29(dt,J=11. 9,4.6Hz,2H),4.13(ddd,J=11.9,5.8,4.5Hz,2H),3.39(q,J=11.3,10.6Hz,1H),3.08(d,J=25 .9Hz,2H),2.82–2.73(m,4H),2.39–2.18(m,12H),2.05(q,J=6.8Hz,9H),1.91–1.82(m,1H),1 .62(dddd,J=27.4,20.2,13.2,6.7Hz,18H),1.42–1.20(m,52H),0.88(td,J=6.9,4.4Hz,9H). MS: 1026.8m / z[M+H].

[0601] Example 30 – Compound 30

[0602] Intermediate 30a: 3-hydroxypropane-1,2-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0603]

[0604] Linoleic acid (2.05 equivalents), DMAP (0.2 equivalents), DIPEA (2.4 equivalents), EDCI (2.4 equivalents), and 1 mL DMF were added sequentially to a solution of glycerol (1.59 g, 1.0 equivalents) in DCM (0.1 M). The reaction was stirred at room temperature for 65 h. After completion, the mixture was quenched by adding water, and the organic layer was washed once with 1 M HCl and once with 5% NaHCO3. The organic layer was then dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (EtOAc / hexane) to a product as a colorless oil. 1 HNMR (400MHz, CDCl3) δ5.43–5.28(m,8H),5.13–5.04(m,1H),4.32(dd,J=11.9,4.6Hz,1H),4.23(dd,J=11.9,5.6Hz,1H),3.73(ddd,J=6.5,5.0,1. 3Hz, 2H), 2.82–2.73 (m, 4H), 2.33 (dt, J=8.8, 7.5Hz, 4H), 2.03 (dq, J=13. 1,6.6Hz,9H),1.69–1.57(m,4H),1.44–1.23(m,29H),0.96–0.83(m,6H).

[0605] Intermediate 30b: 3-((4-oxohexadecyl)oxy)propane-1,2-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0606]

[0607] Using the method employed in intermediate 1d, intermediate 30b was synthesized from intermediate 30a and intermediate 1a with a yield of 66%. 1H NMR(400MHz, CDCl3)δ5.43–5.26(m,9H),4.29(ddd,J=11.8,4.3,2.1Hz,2H),4.15 (ddd,J=11.9,7.7,6.0Hz,2H),2.82–2.74(m,4H),2.71(td,J=6.3,5.6,2.4Hz,2H ),2.62–2.55(m,2H),2.43(t,J=7.5Hz,2H),2.31(td,J=7.6,5.4Hz,4H),2.05(q, J=6.9Hz,8H),1.66–1.54(m,8H),1.41–1.19(m,47H),0.88(td,J=7.0,4.4Hz,9H).

[0608] Intermediate 30c: 3-((4-(((4-nitrophenoxy)carbonyl)oxy)hexadecyl)oxy)propane-1,2-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0609]

[0610] Using the method employed in intermediate 1e, intermediate 30c was synthesized from intermediate 30b with a yield of 40%.

[0611] Compound 30: 9-dodecyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azahexadecane-15,16-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0612]

[0613] Compound 30 was synthesized from intermediate 30c and 3-((methyl)ethylamino)prop-1-ol using the method employed in Example 1, with a yield of 84%. 1 H NMR (500MHz, CDCl3) δ5.44–5.20(m,8H),4.76–4.67(m,1H),4.29(ddd,J=11.9,4.3,1.7Hz,2H),4.22–4.11(m,4H),2.77(t,J=6.7Hz,4H),2. 45–2.28(m,8H),2.22(s,6H),2.05(q,J=6.9Hz,8H),2.00–1.79(m,4H),1.69–1.51(m,6H),1.40–1.17(m,48H),0.88(td,J=6.9,4.8Hz,8H). MS: 1002.2m / z[M+H].

[0614] Example 31 – Compound 31

[0615] Intermediate 31a: 4-Oxododecanoic acid

[0616]

[0617] Intermediate 31a was synthesized from succinic anhydride and octyl magnesium bromide solution using the method employed in intermediate 1a, with a yield of 9%. 1 H NMR (400MHz, CDCl3) δ2.71(t,J=6.4Hz,2H),2.62(t,J=6.7Hz,2H),2.43(t,J=7 .5Hz, 2H), 1.57 (p, J = 7.1Hz, 2H), 1.26 (d, J = 3.6Hz, 10H), 0.87 (t, J = 6.7Hz, 3H).

[0618] Intermediate 31b: 2-((4-oxododecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0619]

[0620] Using the method employed in intermediate 1d, intermediate 31b was synthesized from intermediate 31a and intermediate 1c with a yield of 38%. 1 H NMR (400MHz, CDCl3) δ5.47–5.19(m,8H),4.28(dd,J=11.9,4.3Hz,2H),4.15(dd,J=11.9,5.9Hz,2H),2.77(t,J=6.4Hz,4H),2.71(t,J=6.5Hz,2H),2.59( t,J=6.5Hz,2H),2.43(t,J=7.5Hz,2H),2.32(t,J=7.6Hz,4H),2.04(q,J=6.9 Hz, 8H), 1.67–1.53 (m, 6H), 1.40–1.20 (m, 37H), 0.88 (td, J = 6.8, 5.0Hz, 9H).

[0621] Intermediate 31c: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0622]

[0623] Intermediate 31c was synthesized using the method employed in intermediate 1e, with a yield of 68%. 1HNMR (400MHz, CDCl3) δ8.26–8.16(m,2H),7.40–7.30(m,2H),5.39–5.05(m,9H),4.80(tdd,J=7.5,5.4,4.0Hz,1H),4.49–4.04(m,4 H),2.77–2.63(m,4H),2.45–2.18(m,6H),2.08–1.85(m,10H),1.74–1.46(m,6H),1.41–1.11(m,40H),0.81(td,J=6.9,2.4Hz,9H).

[0624] Compound 31: 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0625]

[0626] Compound 31 was synthesized from intermediate 31c and 3-(dimethylamino)prop-1-ol using the method employed in Example 1, with a yield of 71%. 1 H NMR(400MHz, CDCl3)δ5.42–5.22(m,9H),4.72(tdd,J=7.4,5.3,4.0Hz,1H),4.3 4–4.26(m,2H),4.23–4.10(m,4H),2.82–2.72(m,4H),2.45–2.36(m,4H),2.31(d dd,J=8.0,7.1,1.2Hz,4H),2.25(s,6H),2.05(q,J=6.6Hz,8H),1.99–1.91(m,1H ),1.91–1.82(m,3H),1.67–1.53(m,6H),1.40–1.21(m,41H),0.94–0.82(m,9H). MS: 945.6 m / z [M+H].

[0627] Example 32 – Compound 32

[0628] Compound 32: 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0629]

[0630] Compound 32 was synthesized from intermediate 31c and 3-(diethylamino)prop-1-ol using the method employed in Example 1, with a yield of 71%. 1 H NMR(400MHz, CDCl3)δ5.43–5.28(m,9H),5.25(ddd,J=5.8,4.4,1.5Hz,1H),4.72(tdd,J=7.6,5.4,4.0Hz,1H), 4.29(dd,J=11.9,4.4Hz,2H),4.25–4.08(m,5H),2.77(td,J=6.1,1.1Hz,4H),2.64–2.53(m,6H),2.40(dt,J=8. 7,6.8Hz,2H),2.31(ddd,J=8.0,7.2,1.1Hz,4H),2.10–2.01(m,10H),1.95(dddd,J=11.1,9.2,6.9,3.5Hz,1H) ,1.91–1.81(m,3H),1.69–1.49(m,7H),1.39–1.20(m,42H),1.05(t,J=7.2Hz,6H),0.88(td,J=7.0,4.9Hz,9H). MS: 974.0m / z[M+H].

[0631] Example 33 – Compound 33

[0632] Compound 33: 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0633]

[0634] Compound 33 was synthesized from intermediate 31c and 3-((methyl)ethylamino)prop-1-ol using the method employed in Example 1, with a yield of 79%. 1H NMR (400MHz, CDCl3) δ5.45–5.21 (m, 10H), 4.72 (tdd, J = 9.5, 6.7, 4.1Hz, 1H), 4.38 –4.25(m,2H),4.25–4.10(m,4H),2.77(td,J=6.2,1.1Hz,4H),2.51–2.38(m,6H),2 .37–2.27(m,5H),2.24(d,J=2.4Hz,3H),2.05(q,J=7.1Hz,8H),1.97–1.83(m,4H) ,1.70–1.50(m,7H),1.39–1.21(m,41H),1.06(t,J=7.2Hz,3H),0.94–0.84(m,9H). 960.1m / z[M+H].

[0635] Example 34 – Compound 34

[0636] Compound 34: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0637]

[0638] Compound 34 was synthesized from intermediate 31c and N1-ethyl-N1-methylethane-1,2-diamine using the method employed in Example 1, with a yield of 59%. 1 H NMR (400MHz, CDCl3) δ5.44–5.13(m,10H),4.75(s,1H),4.28(ddd,J=11.9,4.4,1.4Hz,2H) ,4.15(ddd,J=11.9,5.8,3.3Hz,2H),3.25(q,J=5.7Hz,2H),2.83–2.73(m,4H),2.53–2.35( m,6H),2.35–2.27(m,4H),2.21(d,J=3.3Hz,3H),2.05(q,J=6.8Hz,8H),1.96–1.74(m,3H), 1.66–1.43(m,7H),1.41–1.21(m,41H),1.05(t,J=7.1Hz,3H),0.88(td,J=6.9,5.6Hz,9H). 944.7m / z[M+H].

[0639] Example 35 – Compound 35

[0640] Compound 35: 2-((4-((((1-methylpyrrolidone-2-yl)methyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0641]

[0642] Compound 35 was synthesized from intermediate 31c and (1-methylpyrrolidone-2-yl)methylamine using the method employed in Example 1, with a yield of 71%. 1 H NMR (400MHz, CDCl3) δ5.44–5.21(m,10H),4.86–4.66(m,1H),4.28(dd,J=11 .9,4.4Hz,2H),4.14(ddd,J=11.9,5.8,3.1Hz,2H),3.48–3.35(m,1H),3.22– 3.00(m,2H),2.83–2.71(m,4H),2.47–2.19(m,12H),2.04(q,J=6.9Hz,8H),1 .96–1.67(m,6H),1.67–1.42(m,8H),1.41–1.19(m,41H),0.95–0.84(m,9H). 956.5m / z[M+H].

[0643] Example 36 – Compound 36

[0644] Compound 36: 2-((4-(((3-(pyrrolidone-1-yl)propoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0645]

[0646] Compound 36 was synthesized from intermediate 31c and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 34%. 1H NMR (400MHz, CDCl3) δ5.43–5.22(m,9H),4.72(tdd,J=7.4,5.4,4.0Hz,1H),4.29(dd,J=11.9,4.3Hz,2H),4.25–4.08(m,4H),2.82–2.72(m,4H),2. 67(t,J=7.6Hz,5H),2.47–2.27(m,6H),2.09–1.91(m,11H),1.91–1.80(m ,4H),1.68–1.47(m,7H),1.47–1.19(m,42H),0.88(td,J=7.0,4.8Hz,9H). 971.3m / z[M+H].

[0647] Example 37 – Compound 37

[0648] Compound 37: 2-((4-(((2-(ethyl(methyl)amino)ethyl)(methyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0649]

[0650] Compound 37 was synthesized from intermediate 31c and N1-ethyl-N1,N2-dimethylethane-1,2-diamine using the method employed in Example 1, with a yield of 67%. 1 H NMR (400MHz, CDCl3) δ5.44–5.18(m,8H),4.78(s,1H),4.28(dt,J=11.9,4.3H z,2H),4.14(dd,J=11.9,5.8Hz,2H),3.44–3.24(m,2H),2.91(d,J=8.1Hz,3H ),2.81–2.71(m,4H),2.60–2.21(m,12H),2.05(q,J=6.9Hz,7H),1.98–1.46( m,14H),1.43–1.19(m,37H),1.14–1.00(m,3H),0.88(td,J=7.0,5.3Hz,8H). 958.6 m / z [M+H].

[0651] Example 38 – Compound 38

[0652] Compound 38: 2-((4-(((2-(dimethylamino)ethyl)(methyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0653]

[0654] Compound 38 was synthesized from intermediate 31c and N1,N1,N2-trimethylethane-1,2-diamine using the method employed in Example 1, with a yield of 83%. 1 H NMR (400MHz, CDCl3) δ5.43–5.19(m,8H),4.78(s,1H),4.28(dt,J=11.9,4.1Hz,2H),4.14(dd,J=11.9,5.8Hz,2H),3.37(d,J=23.8Hz,2H),2.91(d, J=6.8Hz,3H),2.82–2.72(m,4H),2.54–2.21(m,13H),2.04(q,J=6.7Hz,7 H),1.95–1.44(m,14H),1.42–1.21(m,37H),0.88(td,J=7.0,5.3Hz,8H). 944.5m / z[M+H].

[0655] Example 39 – Compound 39

[0656] Intermediate 39a: Decylmagnesium bromide

[0657]

[0658] 1-Bromodecane (1.0 equivalent) was added dropwise to a solution of Mg (10.99 g, 2 equivalents) and I₂ (0.0001 equivalents) in THF (250 mL) over 2 hours at 80 °C. After the addition, the mixture was stirred at this temperature for 2 hours. A crude product, decyl magnesium bromide, was obtained as a dark brown solution and could be used immediately for the next step without further purification.

[0659] Intermediate 39b: 4-oxotetradecanoic acid

[0660]

[0661] A solution of succinic anhydride (45 g, 1.0 equivalent) in THF (1.1 M) was cooled to -78 °C, and then intermediate 39a was added. The reaction was maintained at -78 °C for 2 h, and then stirred at 15 °C for 16 h. The reaction was then quenched by adding saturated NH4Cl, diluted with H2O, and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. Purification by column chromatography gave the product as a white solid (7.8 g, 7%). 1H NMR(400MHz, CDCl3)δ2.74(dd,J=6.9,5.2Hz,2H),2.70–2.63(m,2H),2.46(t,J =7.5Hz,2H),1.67–1.54(m,2H),1.29(d,J=5.2Hz,14H),0.90(t,J=6.8Hz,3H).

[0662] Intermediate 39c: 2-((4-oxotetradecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0663]

[0664] Using the method employed in intermediate 1d, intermediate 39c is synthesized from intermediate 39b and intermediate 1c. 1 H NMR (400MHz, CDCl3) δ5.42–5.18(m,9H),4.28(dd,J=11.9,4.3Hz,2H),4.14(dd,J=11.9,5.9Hz,2H),2.74(dt,J=22.1,6.8Hz,6H),2.66–2.56(m,2H) ,2.43(t,J=7.5Hz,2H),2.31(t,J=7.6Hz,4H),2.04(q,J=6.8Hz,8H),1.59 (tq,J=14.2,7.5Hz,7H),1.41–1.18(m,44H),0.88(td,J=6.9,4.6Hz,9H).

[0665] Intermediate 39d: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)tetradecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0666]

[0667] At 5°C, NaBH4 (2.5 equivalents) was slowly added to a solution of intermediate 39c in MeOH (0.13M). The mixture was stirred at this temperature for 2 h. The reaction mixture was quenched by adding H2O and then extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a colorless oil, which was immediately reconstituted in DCM (1M), and then 4-nitrobenzene chloroformate (1.5 equivalents) and pyridine (2 equivalents) were added at 0°C. The mixture was stirred at 20°C for 12 h, after which the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give intermediate 39d in 31% yield. 1 HNMR(400MHz, CDCl3)δ8.33–8.22(m,2H),7.46–7.33(m,2H),5.43–5.23(m,9H),4. 31(dd,J=12.0,4.3Hz,2H),4.15(dd,J=12.0,5.8Hz,2H),2.76(t,J=6.3Hz,4H),2.4 7(t,J=7.5Hz,2H),2.30(td,J=7.6,4.2Hz,4H),2.04(q,J=7.3Hz,10H),1.67(dt,J= 47.7, 6.1Hz, 7H), 1.30 (tq, J=14.3, 4.9, 3.0Hz, 53H), 0.88 (td, J=6.8, 3.5Hz, 10H).

[0668] Compound 39: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tetradecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0669]

[0670] Compound 39 was synthesized from intermediate 39d and N1-ethyl-N1-methylethane-1,2-diamine using the method employed in Example 1, with a yield of 78%. 1H NMR (400MHz, CDCl3) δ5.49–5.17(m,9H),4.75(s,1H),4.28(dd,J=11.9,4.4Hz ,2H),4.15(ddd,J=11.9,5.9,3.3Hz,2H),3.26(q,J=6.0Hz,2H),2.77(t,J=6.4 Hz,4H),2.52–2.27(m,10H),2.23(s,3H),2.05(q,J=6.8Hz,8H),1.95–1.42(m ,14H),1.42–1.18(m,40H),1.05(t,J=7.1Hz,3H),0.88(td,J=6.8,4.5Hz,7H). 972.9 m / z [M+H].

[0671] Example 40 – Compound 40

[0672] Intermediate 40a: 4-Hydroxydecanoic acid

[0673]

[0674] To a solution of 5-hexyltetrahydrofuran-2-one (15 g, 1.0 equivalent) in EtOH (240 mL) and H₂O (80 mL), NaOH (2.0 equivalent) was added. The reaction was stirred at 25 °C for 12 h. The mixture was concentrated to remove EtOH. The pH was then adjusted to 5 with 1 N HCl aqueous solution, and the mixture was extracted three times with EtOAc. The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography (EtOAc / hexane) to give the product (48%) as a white solid.

[0675] Intermediate 40b: 4-Oxodecanoic acid

[0676]

[0677] Intermediate 40a (10 g, 1.0 equivalent) was added to a solution of NaH₂PO₄ (17 equivalents) and Na₂HPO₄ (2.4 equivalents) in H₂O (1000 mL). Then, NaClO (200 mL, 6–8% Cl) was added to the solution. The reaction mixture was stirred at 25 °C for 12 h. Then, H₃PO₄ (200 mL, 85%) and silica gel (300 g) were added to the mixture. The mixture was dried under reduced pressure and washed three times with EtOAc. The organic layer was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel chromatography (EtoAc / hexane) to give a product as a white solid (80%). 1H NMR(400MHz, CDCl3)δ2.70(dd,J=7.0,5.3Hz,2H),2.63–2.58(m,2H),2.43(t ,J=7.5Hz,2H),1.58(q,J=7.4Hz,2H),1.33–1.19(m,6H),0.90–0.83(m,3H).

[0678] Intermediate 40c: 2-((4-oxodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadecano-9,12-dienoate)

[0679]

[0680] Using the method employed in Example 1, intermediate 40c was synthesized from intermediate 40b and intermediate 1c in a yield of 40%. 1 H NMR (400MHz, CDCl3) δ5.42–5.28(m,8H),5.24(tt,J=5.9,4.3Hz,1H),4.28(dd,J =11.9,4.3Hz,2H),4.14(dd,J=11.9,5.9Hz,2H),2.73(dt,J=22.4,6.7Hz,7H),2 .58(t,J=6.5Hz,2H),2.43(t,J=7.5Hz,2H),2.31(t,J=7.6Hz,4H),2.04(q,J=6. 9Hz, 8H), 1.59 (q, J = 6.7Hz, 7H), 1.40–1.22 (m, 35H), 0.88 (td, J = 6.8, 4.0Hz, 9H).

[0681] Intermediate 40d: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0682]

[0683] Using the method employed with intermediate 1e, intermediate 40d was synthesized from intermediate 40c with a yield of 31%. 1H NMR (400MHz, CDCl3) δ8.37–8.27(m,2H),7.43(dd,J=9.1,5.9Hz,2H),5.45–5.24(m,9H),4.34(dd,J=12.0,4.3Hz,2H),4.18(dd,J=11.9,6.0Hz,2H) ,2.79(t,J=6.3Hz,4H),2.49(t,J=7.5Hz,2H),2.41–2.30(m,4H),2.07(q ,J=6.9Hz,9H),1.83–1.58(m,7H),1.48–1.12(m,37H),1.00–0.82(m,9H).

[0684] Compound 40: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0685]

[0686] Compound 40 was synthesized from intermediate 40d and N1-ethyl-N1-methylethane-1,2-diamine using the method employed in Example 1, with a yield of 53%. 1 H NMR (400MHz, CDCl3) δ5.44–5.19(m,9H),4.76(s,1H),4.32–4.22(m,2H),4.15( ddd,J=11.9,5.9,3.4Hz,2H),3.26(s,2H),2.77(t,J=6.5Hz,4H),2.57–2.36(m ,6H),2.36–2.27(m,4H),2.23(s,3H),2.05(q,J=6.6Hz,8H),1.98–1.42(m,15H ),1.41–1.19(m,35H),1.06(t,J=7.2Hz,3H),0.88(td,J=9.7,8.2,4.2Hz,9H). MS: 916.7 m / z [M+H].

[0687] Example 41 – Compound 41

[0688] Intermediate 41a: 4-Cyclohexyl-4-oxobutyric acid

[0689]

[0690] CuI (0.1 equivalent) was added to a solution of succinic anhydride (30 g, 1.0 equivalent) in Et₂O (300 mL). The mixture was cooled to -20 °C. Then, cyclohexylmagnesium bromide (1.0 equivalent) was added to the mixture. The reaction was heated to 25 °C and stirred for 14 h. The reaction was quenched by adding 1 M HCl aqueous solution and stirred for 15 min. The reaction was then filtered, and the filtrate was extracted three times with DCM. The combined organic layers were dried over Na₂SO₄, filtered, and concentrated. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (13%) as a pale pink solid. 1 H NMR (400MHz, CDCl3) δ10.09 (s, 1H), 2.66 (dt, J = 55.4, 6.5Hz, 4H), 2.35 (td, J = 1 0.8, 9.3, 5.2Hz, 1H), 1.75 (ddd, J=43.2, 36.6, 11.9Hz, 5H), 1.46–1.09 (m, 5H).

[0691] Intermediate 41b: 2-((4-cyclohexyl-4-oxobutyryl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0692]

[0693] Using the method employed in Example 1, intermediate 41b was synthesized from intermediate 41a and intermediate 1c with a yield of 34%. 1 H NMR (400MHz, CDCl) 3 )δ5.42–5.28(m,8H),5.24(ddd,J=10.2,5.6,4.2Hz,1H),4.28(dt,J=11.9,4.6Hz,2H),4.14(ddd,J=11.9,5.8,2.3Hz,2H),2.81–2.7 0(m,6H),2.64–2.54(m,3H),2.38–2.27(m,5H),2.04(q,J=6.8Hz,8H),1.89–1.55(m,11H),1.40–1.20(m,34H),0.88(t,J=6.8Hz,6H).

[0694] Intermediate 41c: 2-((4-cyclohexyl-4-(((4-nitrophenoxy)carbonyl)oxy)butyryl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0695]

[0696] Using the method employed in Example 1, intermediate 41c was synthesized from intermediate 41b in a yield of 14%. 1 H NMR (400MHz, CDCl3) δ8.33–8.21(m,2H),7.45–7.34(m,2H),5.44–5.13(m,9H) ,4.70(ddd,J=9.3,6.1,3.4Hz,1H),4.50(dd,J=12.3,3.9Hz,1H),4.35–4.10( m,4H),2.76(t,J=6.4Hz,4H),2.50–2.40(m,2H),2.38–2.22(m,4H),2.04(q,J =6.9Hz,10H),1.85–1.52(m,10H),1.49–1.00(m,31H),0.88(t,J=6.8Hz,6H).

[0697] Compound 41: 2-((4-cyclohexyl-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)butyryl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0698]

[0699] Compound 41 was synthesized from intermediate 41d and N1-ethyl-N1-methylethane-1,2-diamine using the method employed with intermediate 1e, with a yield of 60%. 1 H NMR (400MHz, CDCl3) δ5.45–5.09(m,9H),4.60(s,1H),4.28(ddd,J=11.9,4.5,1.6Hz,2H),4.15(ddd,J=11.9,5.8,2.9Hz,2 H),3.25(s,2H),2.77(t,J=6.5Hz,4H),2.54–2.15(m,12H),2.05(q,J=6.7Hz,8H),1.99–0.95(m,50H),0.93–0.80(m,6H). MS: 914.5m / z[M+H].

[0700] Example 42 – Compound 42

[0701] Intermediate 42a: 4-hydroxyoctanoic acid

[0702]

[0703] Intermediate 42a was synthesized from 5-butyltetrahydrofuran-2-one using the same method as intermediate 39b, with a yield of 53%.

[0704] Intermediate 42b: 4-Octooctanoic acid

[0705]

[0706] Using the same method as intermediate 38b, intermediate 42b was synthesized from intermediate 42a with a yield of 61%. 1 H NMR (400MHz, CDCl3) δ2.65 (ddd, J=7.0, 5.8, 1.1Hz, 2H), 2.56 (ddd, J=7.1, 6.0, 1.2Hz, 2 H), 2.38 (t, J = 7.5Hz, 2H), 1.56–1.44 (m, 2H), 1.30–1.17 (m, 2H), 0.84 (t, J = 7.3Hz, 3H).

[0707] Intermediate 42c: 2-((4-oxooctanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0708]

[0709] Using the method employed in Example 1, intermediate 42c was synthesized from intermediate 42b and intermediate 1c with a yield of 29%. 1 H NMR (400MHz, CDCl3) δ5.35 (tq, J=18.2, 9.4, 7.6Hz, 8H), 5.24 (t, J=5.3Hz, 1H), 4.2 8(dd,J=12.0,3.9Hz,2H),4.14(dd,J=12.1,5.8Hz,2H),2.74(dt,J=20.9,6.7Hz,6 H),2.59(d,J=6.6Hz,2H),2.43(t,J=7.6Hz,2H),2.31(t,J=7.7Hz,4H),2.04(q,J= 7.0Hz, 8H), 1.60 (t, J = 7.5Hz, 8H), 1.31 (d, J = 14.1Hz, 29H), 0.88 (t, J = 6.9Hz, 9H).

[0710] Intermediate 42d: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)octanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0711]

[0712] Using the method employed with intermediate 1e, intermediate 42d was synthesized from intermediate 40c with a yield of 51%. 1H NMR(400MHz, CDCl3)δ8.28(dd,J=7.8,4.7Hz,2H),7.47–7.36(m,2H),5.32(dp,J=19.8,9.2Hz ,8H),5.17(s,1H),4.87(d,J=7.3Hz,1H),4.49(d,J=12.2Hz,1H),4.36–4.09(m,4H),2.75(d,J =6.7Hz,4H),2.49(dt,J=23.8,7.9Hz,2H),2.42–2.14(m,5H),2.03(q,J=7.2Hz,10H),1.73(s, 1H), 1.61 (dd, J=23.8, 11.9Hz, 6H), 1.31 (d, J=14.4Hz, 30H), 0.89 (tt, J=9.5, 7.0, 2.7Hz, 9H).

[0713] Compound 42: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)octanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0714]

[0715] Compound 42 was synthesized from intermediate 42d and N1-ethyl-N1-methylethane-1,2-diamine using the method employed in Example 1, with a yield of 93%. 1 H NMR (400MHz, CDCl3) δ5.43–5.10(m,11H),4.76(s,1H),4.28(ddd,J=11.9,4.5,1.3Hz, 2H),4.15(ddd,J=11.8,5.8,3.3Hz,2H),3.25(s,2H),2.77(t,J=6.5Hz,4H),2.39(ddd ,J=8.7,6.6,4.3Hz,6H),2.35–2.28(m,4H),2.22(s,3H),2.05(q,J=6.7Hz,8H),1.96– 1.74(m,3H),1.61(s,11H),1.42–1.22(m,34H),1.10–0.99(m,3H),0.95–0.83(m,9H).

[0716] Example 43 – Compound 43

[0717] Intermediate 43a: Sodium 4-hydroxybutyrate

[0718]

[0719] To a mixture of tetrahydrofuran-2-one (10 g, 1.0 equivalent) in H₂O (100 mL), NaOH (4.0 equivalent) was added, and the mixture was degassed and purged three times with N₂. The mixture was stirred at 25 °C under a N₂ atmosphere for 24 hours. The reaction mixture was then concentrated under reduced pressure to give a crude residue as a colorless oil, which could be used in the next step without further purification (12 g, crude).

[0720] Intermediate 43b: Benzyl 4-hydroxybutyrate

[0721]

[0722] To a mixture of intermediate 43a (12 g, 1.0 equivalent) in acetone (100 mL), TBAB (0.05 equivalent) and benzyl bromide (1.2 equivalent) were added, and the mixture was degassed and purged three times with N2. The mixture was stirred at 60 °C under N2 atmosphere for 12 hours. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with EtOAc and precipitated with 1N NaH. S The organic layer was washed once each with O4, NaHCO3, and H2O. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude residue was purified by column chromatography (EtOAc / hexane) to obtain a colorless oily product (43%).

[0723] Intermediate 43C: Benzyl 4-(((4-nitrophenoxy)carbonyl)oxy)butyrate

[0724]

[0725] Add 1.0 equivalent of 4-nitrobenzene chloroformate to a mixture of intermediate 43b (5 g, 1.0 equivalent) in DCM (150 mL), and degas the mixture and purge it three times with N2. Cool the reaction to 0 °C, and then add pyridine (2.0 equivalent). Stir the reaction at 25 °C under N2 atmosphere for 5 h. Then concentrate the reaction mixture under reduced pressure, dilute with hexane, and filter. Wash the filtrate 10 times with H2O, and wash the combined organic layers with brine, dry with Na2SO4, filter, and concentrate. Purify the crude material by column chromatography to give a product (32%) as a yellow oil.

[0726] Intermediate 43d: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)benzyl butyrate

[0727]

[0728] To a mixture of intermediate 43c (3 g, 1.0 equivalent) in MeCN (50 mL), N1-ethyl-N1-methylethane-1,2-diamine (2.5 equivalent), pyridine (3.0 equivalent), and DMAP (1.0 equivalent) were added. The mixture was degassed and purged three times with N2, and stirred at 70 °C under N2 for 12 h. The reaction mixture was then concentrated under reduced pressure, and the crude residue was purified by column chromatography to give a product (56%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ7.33–7.23(m,4H),5.24(d,J=12.8Hz,1H),5.05(s,2H),4.04(dt,J=12.9,6.7Hz,2H),3.19(q, J=5.8Hz,2H),3.00(s,1H),2.39(dt,J=10.4,6.6Hz,6H),2.13(s,3H),1.89(p,J=6.9Hz,2H),0.98(t,J=7.1Hz,3H).

[0729] Intermediate 43e: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)butyric acid

[0730]

[0731] The mixture of intermediate 43d (1.5 g, 1.0 equivalent) in MeOH (30 mL) was added to Pd / C (10% by weight, 0.05 equivalent), and the mixture was degassed and purged three times with H2. The reaction mixture was stirred at 25 °C under H2 atmosphere for 1 h, after which the filtrate was concentrated to obtain a crude residue, which could be used for the next step without further purification. 1 H NMR (400MHz, CD3OD) δ4.11(t,J=6.3Hz,2H),3.49(t,J=6.0Hz,2H),3.29–3.21(m,5H ), 2.89 (s, 3H), 2.38 (t, J = 7.2Hz, 2H), 1.92 (p, J = 6.7Hz, 2H), 1.34 (t, J = 7.3Hz, 3H).

[0732] Compound 43: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)butyryl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0733]

[0734] Compound 43 was synthesized from intermediate 43d and intermediate 1c using the method employed with intermediate 1d, with a yield of 45%. 1 H NMR (400MHz, CDCl3) δ5.43–5.22(m,9H),4.29(dd,J=11.9,4.4Hz,2H),4.18–4.06(m ,4H),3.25(q,J=5.8Hz,2H),2.76(t,J=6.4Hz,4H),2.43(dt,J=10.5,7.3Hz,6H),2. 31(t,J=7.6Hz,4H),2.20(s,3H),2.04(q,J=6.9Hz,8H),1.95(q,J=6.9Hz,2H),1.59 (d,J=7.1Hz,7H),1.40–1.22(m,26H),1.04(t,J=7.1Hz,3H),0.88(t,J=6.8Hz,6H).

[0735] Example 44 – Compound 44

[0736] Compound 44: 2-((4-((((1-methylpyrrolidone-2-yl)methyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0737]

[0738] Compound 44 was synthesized from intermediate 40d and (1-methylpyrrolidone-2-yl)methylamine using the method employed with intermediate 1e, with a yield of 35%. 1 H NMR (400MHz, CDCl3) δ5.44–5.20(m,9H),5.16(s,1H),4.76(s,1H),4.28(dd, J=11.9,4.4Hz,2H),4.15(ddd,J=11.9,5.8,3.0Hz,2H),3.39(s,1H),3.10(d, J=27.2Hz,2H),2.81–2.73(m,4H),2.43–2.18(m,11H),2.05(q,J=6.7Hz,8H) ,1.96–1.42(m,17H),1.30(dqt,J=12.8,8.8,5.4Hz,36H),0.92–0.83(m,9H). MS: 928.9 m / z [M+H].

[0739] Example 45 – Compound 45

[0740] Compound 45: 2-((4-(((3-(pyrrolidone-1-yl)propoxy)carbonyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0741]

[0742] Compound 45 was synthesized from intermediate 40d and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 52%. 1 H NMR (400MHz, CDCl3) δ5.44–5.21 (m, 9H), 4.72 (ddd, J = 11.3, 8.8, 5.7Hz, 1H), 4. 29(dd,J=11.9,4.3Hz,2H),4.25–4.10(m,4H),2.81–2.74(m,4H),2.59(s,5H), 2.40(dt,J=8.6,6.4Hz,2H),2.36–2.27(m,4H),2.05(q,J=6.7Hz,8H),1.98–1. 78(m,9H),1.67–1.51(m,7H),1.41–1.21(m,36H),0.88(td,J=7.0,5.0Hz,9H). MS: 943.8 m / z [M+H].

[0743] Example 46 – Compound 46

[0744] Compound 46: 2-((4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0745]

[0746] Compound 46 was synthesized from intermediate 1e and 2-(pyrrolidone-1-yl)ethyl-1-amine using the method employed in Example 1, with a yield of 53%. 1H NMR(400MHz, CDCl3)δ5.55(s,1H),5.46–5.21(m,9H),4.75(s,1H),4.28(dd,J= 11.9,4.4Hz,2H),4.15(ddd,J=11.9,5.8,3.6Hz,2H),3.35(d,J=6.0Hz,2H),2. 81–2.60(m,9H),2.39(ddd,J=8.6,6.7,3.8Hz,2H),2.31(t,J=7.6Hz,4H),2.05 (q,J=6.8Hz,8H),1.84(s,7H),1.66–1.19(m,55H),0.88(td,J=7.0,4.3Hz,9H). MS: 1012.5 m / z [M+H].

[0747] Example 47 – Compound 47

[0748] Compound 47: 2-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0749]

[0750] Pyridine (3.0 equivalents), 1-methylpiperidin-4-ol (1.5 equivalents), and DMAP (1.0 equivalents) were added to a solution of intermediate 1e (250 mg, 1.0 equivalents) in MeCN (0.1 M). The reaction was stirred at room temperature for 18 h. Another portion of 1-methylpiperidin-4-ol (3.0 equivalents) was added, and the mixture was heated to 50 °C. After 2 h, another portion of DMAP (1.0 equivalents) was added, and the reaction was maintained for another 2 h. After completion, heptane (0.1 M) was added to the reaction mixture, and the heptane layer was washed three times with MeCN. The combined MeCN layers were back-extracted three times with heptane, and the combined heptane layers were washed once more with MeCN. The heptane was then removed under vacuum, and the crude material was purified by column chromatography (MeOH / DCM) to give a product (62 mg, 32%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ5.44–5.20(m,9H),4.77–4.60(m,2H),4.29(dd,J=11.9,4.4Hz,2H),4.14(ddd,J=11.9,5.8,3.6Hz,2H),2 .83–2.66(m,6H),2.46–2.26(m,11H),2.08–1.71(m,18H),1.71–1.47(m,8H),1.40–1.21(m,47H),0.88(td,J=7.0,4.2Hz,9H). MS: 1013.7m / z[M+H].

[0751] Example 48 – Compound 48

[0752] Compound 48: 2-((4-(((3-(bis(2-hydroxyethyl)amino)propyl)carbamoyl)oxy)hexadecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0753]

[0754] Pyridine (3.0 equivalents) and 2-[(3-aminopropyl)(2-hydroxyethyl)amino]ethane-1-ol (1.5 equivalents) were added to a solution of intermediate 1e (250 mg, 1.0 equivalents) in MeCN (0.1 M). The reaction was stirred at room temperature for 2 h. After completion, heptane (0.1 M) was added to the reaction mixture, and the heptane layer was washed three times with MeCN. The combined MeCN layers were back-extracted three times with heptane, and the combined heptane layers were washed once more with MeCN. The heptane was then removed under vacuum, and the crude material was purified by column chromatography (MeOH / DCM) to give the product (97 mg, 48%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.43–5.20(m,9H),4.76(s,1H),4.29(ddd,J=11.9,4.6,1 .8Hz,2H),4.16(dd,J=11.9,5.8Hz,2H),3.72(t,J=5.0Hz,4H),3.35–3.22(m,2H ),2.98–2.46(m,14H),2.41–2.28(m,6H),2.09–1.86(m,9H),1.80(dd,J=14.2, 7.2Hz, 3H), 1.67–1.42 (m, 7H), 1.42–1.18 (m, 46H), 0.88 (td, J = 7.0, 4.3Hz, 9H). MS: 1060.6 m / z [M+H].

[0755] Example 49 – Comparison Compound 49

[0756] Compound 49: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoic acid ester

[0757]

[0758] Compound 49.1H was synthesized according to the method described in WO 2015 / 095340 A1 (Example 13). NMR(CDCl3,400MHz)δ5.35(m,4H),4.48(t,J=5.6Hz,1H),4.17(m,8H),3.56(m ,2H),3.40(m,2H),2.77(t,J=6.6Hz,2H),2.55(q,J=7.2Hz,6H),2.40(m,3H), 2.30(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,4H),1.92(m,2H),1.84(m,2H),1.57( m, 6H), 1.30 (m, 34H), 1.03 (t, J = 7.2Hz, 6H), 0.88 (m, 9H) ppm; MS: 853m / z [M+H].

[0759] Example 50 – Compound 50

[0760] Compound 50: 2-((4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0761]

[0762] Compound 50 was synthesized from intermediate 40d and 2-(pyrrolidone-1-yl)ethyl-1-amine using the method employed in Example 1, with a yield of 36%. 1H NMR (400MHz, CDCl3) δ5.49–5.29(m,8H),5.27–5.20(m,1H),4.76(s,1H),4.28(d d,J=11.9,4.4Hz,2H),4.15(ddd,J=11.9,5.8,3.8Hz,2H),3.35(s,2H),2.83–2.4 4(m,10H),2.39(ddd,J=8.4,6.7,3.3Hz,2H),2.36–2.25(m,4H),2.05(q,J=6.8H z,8H),1.98–1.43(m,15H),1.42–1.20(m,33H),0.89(td,J=6.1,5.4,0.9Hz,9H). MS: 928.6 m / z [M+H].

[0763] Example 51 – Compound 51

[0764] Compound 51: 2-((4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0765]

[0766] Compound 51 was synthesized from intermediate 31c and 2-(pyrrolidone-1-yl)ethyl-1-amine using the method employed in Example 1, with a yield of 27%. 1 H NMR(400MHz, CDCl3)δ5.55(s,1H),5.44–5.28(m,8H),5.28–5.22(m,1H),4.76(s ,1H),4.28(dd,J=11.9,4.4Hz,2H),4.15(ddd,J=11.9,5.8,3.9Hz,2H),3.37(s, 2H),2.83–2.51(m,10H),2.39(ddd,J=8.4,6.8,3.1Hz,2H),2.35–2.27(m,4H),2 .05(q,J=6.7Hz,8H),1.97–1.41(m,17H),1.41–1.20(m,40H),0.94–0.84(m,9H). MS: 957.0 m / z [M+H].

[0767] Example 52 – Compound 52

[0768] Intermediate 52a: Benzyl 3-hydroxyundecanoate

[0769]

[0770] Zn (22.99 g, 1.0 equivalent) and nonanal (25 g, 1.0 equivalent) were added dropwise to a THF (150 mL) solution at 66 °C and N2. Next, benzyl 2-bromoacetate (50.00 g, 1.24 equivalent) was slowly and carefully added dropwise to the reaction mixture. After the addition of benzyl 2-bromoacetate was complete, the mixture was cooled and stirred at 20 °C for 2 h. The reaction mixture was then concentrated under reduced pressure to remove THF, and the resulting residue was diluted with water and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure and purified by column chromatography to give a product (25 g, 49%) as a yellow oil. 1 H NMR(400MHz, CDCl3)δ7.33–7.24(m,5H),5.09(s,2H),3.95(tt,J=7.7,3.8Hz,1H),2 .75(d,J=3.9Hz,1H),2.56–2.35(m,2H),1.54–1.11(m,18H),0.81(t,J=6.7Hz,3H).

[0771] Intermediate 52b: Benzyl 3-(((4-nitrophenoxy)carbonyl)oxy)undecanoate

[0772]

[0773] Under a nitrogen atmosphere, (4-nitrophenyl)carbonochloridate (17.23 g, 1.0 equivalent) and pyridine (13.80 mL, 2.0 equivalent) were added to a solution of intermediate 52a (25 g, 1.0 equivalent) in DCM (250 mL). The mixture was stirred at 20 °C for 2 h. After completion, the reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with water and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure and purified by column chromatography to give a product (12 g, 31%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ8.31–8.01(m,4H),7.28(s,5H),5.22–5.14(m,2H),5.13–5.01(m,4H), 2.68(dd,J=14.8,6.4Hz,3H),1.75–1.64(m,2H),1.30–1.19(m,18H),0.80(t,J=6.6Hz,3H).

[0774] Intermediate 52c: 3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)benzyl undecanoate

[0775]

[0776] To a solution of intermediate 52b (14 g, 1.0 equivalent) and N'-ethyl-N'-methyl-ethane-1,2-diamine (6.25 g, 2.0 equivalent) in MeCN (400 mL), pyridine (7.41 mL, 3.0 equivalent) and DMAP (3.74 g, 1.0 equivalent) were added. The mixture was stirred at 20 °C for 12 h. After completion, the reaction mixture was concentrated under reduced pressure to remove MeCN, and the resulting residue was diluted with water and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure and purified by column chromatography to give a product (6 g, 47%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ7.39–7.28(m,5H),5.11(s,4H),3.21(h,J=6.8Hz,2H),2.62(td,J=14.4,13.6,6.4Hz,2H), 2.50–2.39(m,4H),1.59(h,J=8.2Hz,2H),1.25(d,J=16.1Hz,14H),1.03(t,J=7.2Hz,3H),0.86(t,J=6.7Hz,3H).

[0777] Intermediate 52d: 3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoic acid

[0778]

[0779] Pd / C (2 g, 10% w / w) was added to a solution of intermediate 52c (6 g, 1.0 equivalent) in MeOH (60 mL). The mixture was stirred at 20 °C and H2 for 2 h. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give a product that was a brown oil. 1 H NMR (400MHz, CDCl3) δ12.76(s,1H),6.28(t,J=5.8Hz,1H),5.01(p,J=6.1,5.6Hz,1H),3.30(qq,J=13.8,6.0Hz,2H),2.70(hd,J=12.1,11.0 ,5.4Hz,3H),2.37(d,J=14.1Hz,4H),1.51(ddq,J=21.6,14.2,6.7Hz,2H),1.34–1.14(m,11H),1.10(t,J=7.1Hz,3H),0.80(t,J=6.7Hz,3H).

[0780] Compound 52: 2-((3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0781]

[0782] DMAP (0.25 equivalents), DIPEA (3.5 equivalents), and EDCI (1.5 equivalents) were added sequentially to a solution of intermediate 52d (1.0–1.1 equivalents) and intermediate 1c (1.0–1.1 equivalents) in DCM (0.1–0.25 equivalents). The reaction was stirred at 23°C for at least 16 h. Additional DMAP (0.25 equivalents) was added to the reaction, and the reaction was stirred at 23°C for another 6 h. The reaction was then quenched by adding water, washed once with 1M HCl, and once with 5% NaHCO3. The organic layer was dried over Na2SO4, filtered, and concentrated. Purification by column chromatography yielded a colorless oily product (28%). 1 H NMR (400MHz, CDCl3) δ5.53–5.28(m,8H),5.24(s,1H),5.08(s,1H),4.28(ddd,J=15.9,11.9,4.5Hz,2H),4.15(dt,J=13.4,6.8Hz,2H),2.77(t,J=6 .5Hz,4H),2.59(tt,J=15.9,9.0Hz,6H),2.37–2.14(m,6H),2.05(q,J=6. 8Hz, 7H), 1.59 (d, J = 14.2Hz, 12H), 1.46–0.97 (m, 42H), 0.96–0.81 (m, 9H). MS: 930.8m / z[M+H].

[0783] Example 53 – Compound 53

[0784] Intermediate 53a: 2-(hydroxymethyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0785]

[0786] To a solution of linoleic acid (20 g, 1.0 equivalence) and 2-(hydroxymethyl)propane-1,3-diol (7.57 g, 1.0 equivalence) in DCM (200 mL), EDCI (13.67 g, 1.0 equivalence), DMAP (1.39 g, 0.16 equivalence), and DIPEA (37.27 mL, 3.0 equivalence) were added. The mixture was stirred at 20 °C for 2 hours. After completion, the reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with water and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (6.3 g, 11%) as a colorless oil. 1 H NMR (400MHz, CD3OD) δ5.44–5.30(m,8H),4.17(qd,J=11.1,6.0Hz,4H),3.63(d,J=6.0Hz,2H),2.80(t,J=6.1Hz,4H),2.35(t ,J=7.4Hz,4H),2.21(h,J=6.0Hz,1H),2.14–2.03(m,8H),1.64(p,J=7.4Hz,4H),1.46–1.28(m,27H),0.93(t,J=6.7Hz,6H).

[0787] Compound 53: 2-(11-methyl-5-octyl-3,7-dioxo-2,6-dioxa-8,11-diazatetranyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadecyl-9,12-dienolate)

[0788]

[0789] Compound 53 was synthesized from intermediates 52d and 53a using the same method as compound 52, with a yield of 45%. 1 H NMR (400MHz, CDCl3) δ5.48–5.24(m,8H),5.07(t,J=6.5Hz,1H),4.20–4.07(m,6H),2.77(t,J=6.5Hz,4H),2.62–2 .46(m,4H),2.45–2.37(m,2H),2.34–2.15(m,6H),2.05(q,J=6.8Hz,8H),1.43–0.97(m,43H),0.95–0.83(m,9H). MS: 944.8m / z[M+H].

[0790] Example 54 – Compound 54

[0791] Intermediate 54a: 4-hydroxybenzyl dodecanoate

[0792]

[0793] To a solution of 20 g (1.0 equivalent) of 5-octyltetrahydrofuran-2-one in water (200-250 mL), NaOH (1.1 equivalent) was added. The mixture was stirred at 20 °C under a N2 atmosphere for 24 h. Next, the reaction mixture was concentrated under reduced pressure to remove the solvent water. The resulting crude material was reconstituted in acetone (200 mL), followed by the addition of TBAB (0.05 equivalent) and BnBr (1.2-1.5 equivalent). The mixture was heated to 60 °C and stirred under a N2 atmosphere for 12 h. After completion, the mixture was concentrated under reduced pressure, and the resulting residue was poured into a saturated NaHCO3 solution. The product was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (20 g, 39%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ7.45–7.29(m,5H),5.12(s,2H),3.65–3.57(m,1H),2.51(td,J=7.2,2.7Hz,2H),1.86(dtd,J=14.6 ,7.4,3.5Hz,1H),1.71(ddd,J=14.2,8.6,7.1Hz,1H),1.61(d,J=5.0Hz,1H),1.52–1.17(m,17H),0.88(t,J=6.7Hz,3H).

[0794] Intermediate 54b: 4-(((4-nitrophenoxy)carbonyl)oxy)benzyl dodecanoate

[0795]

[0796] (4-Nitrophenyl)chloroformate (1.5 equivalents) was added to a solution of intermediate 54a (10 g, 1.0 equivalents) in DCM (80–100 mL). The reaction mixture was degassed and purged three times with N2, cooled to 0 °C, and then pyridine (2.0 equivalents) was added. The reaction mixture was stirred at 20 °C under N2 atmosphere for 5 h. After completion, the mixture was concentrated under reduced pressure, and the resulting residue was reconstituted in hexane and filtered. The resulting filtrate was washed three times with water, and the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (9 g, 29%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ8.27(dd,J=9.8,7.8Hz,2H),7.50–7.30(m,8H),5.12(d,J=8.3Hz,2H),4.87(ddt,J=12.2,7.3,4.1Hz,1H),2.50(t,J=7.5Hz,2H),2 .16–2.06(m,1H),1.99(dq,J=14.8,7.5Hz,1H),1.73(ddd,J=14.0,12.0,7.0 Hz,1H),1.63(tt,J=8.6,4.5Hz,1H),1.45–1.19(m,12H),0.94–0.84(m,3H).

[0797] Intermediate 54c: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)benzyl dodecanoate

[0798]

[0799] To a solution of intermediate 54b (9 g, 1.0 equivalent) in MeCN (45–90 mL), N'-ethyl-N'-methyl-ethane-1,2-diamine (1.5–2.5 equivalents), pyridine (3–4 equivalents), and DMAP (2.33 g, 1.0 equivalent) were added, and the resulting mixture was degassed and purged three times with N2. The reaction was heated to 70 °C and stirred under N2 atmosphere for 2 h. After completion, the mixture was concentrated to remove the solvent, and the resulting residue was diluted with EtOAc, washed three times with saturated NaHCO3, and then washed three times with water. The organic layer was then dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (5 g, 60%) as a pale yellow oil. 1 H NMR (400MHz, CDCl3) δ7.38–7.22(m,5H),5.04(s,2H),4.69(s,1H),3.16(q,J=6.0Hz,2H),2.45–2.27(m,6H),2.13( s,3H),1.95–1.71(m,2H),1.40(d,J=7.3Hz,2H),1.34–1.09(m,13H),0.96(t,J=7.1Hz,3H),0.80(t,J=6.7Hz,3H).

[0800] Intermediate 54d: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoic acid

[0801]

[0802] Pd / C (1.8 g, 10% w / w) was added to a solution of intermediate 54c (6 g, 1.0 equivalent) in MeOH (60 mL). The mixture was stirred at 20 °C and H2 for 5 h. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a product (3.2 g, 67%) as a brown oil. 1 H NMR (400MHz, CD3OD) δ4.76 (dp, J=8.1, 4.1Hz, 1H), 3.65 (dt, J=14.0, 7.0Hz, 1H), 3 .28–3.10(m,4H),3.07(ddd,J=11.3,7.5,2.1Hz,1H),2.79(s,3H),2.27(ddd,J=1 4.0,8.2,4.4Hz,1H),2.20–1.99(m,2H),1.86–1.74(m,1H),1.71–1.56(m,1H),1. 48(dq,J=13.7,7.6,6.8Hz,1H), 1.31(dd,J=14.1,7.0Hz,14H), 0.94–0.83(m,3H).

[0803] Compound 54: 2-(12-methyl-6-octyl-3,8-dioxo-2,7-dioxa-9,12-diazatetradecyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienolate)

[0804]

[0805] Compound 54 was synthesized from intermediates 54d and 53a using the same method as compound 52, with a yield of 47%. 1 H NMR (400MHz, CDCl3) δ5.44–5.26(m,8H),4.75(s,1H),4.12(d,J=6.0Hz,6H),2.77(t,J=6.5Hz,4H),2.55–2.34(m,6H),2. 34–2.19(m,6H),2.05(q,J=6.8Hz,8H),1.97–1.70(m,4H),1.61(t,J=7.2Hz,10H),1.41–1.00(m,43H),0.94–0.82(m,9H). MS: 959.1m / z[M+H].

[0806] Example 55 – Compound 55

[0807] Intermediate 55a: Benzyl 5-hydroxytridecanoate

[0808]

[0809] Intermediate 55a was prepared from 6-octyltetrahydropyran-2-one using the same method as intermediate 54a, with a yield of 25%. 1 H NMR (400MHz, CDCl3) δ7.34 (qt, J=9.5, 4.5Hz, 5H), 5.11 (s, 2H), 3.57 (s, 1H), 2. 39(t,J=7.3Hz,2H),1.85–1.65(m,2H),1.60–1.11(m,18H),0.98–0.80(m,3H).

[0810] Intermediate 55b: 5-(((4-nitrophenoxy)carbonyl)oxy)benzyl tridecanoate

[0811]

[0812] Intermediate 55b was prepared from intermediate 55a using the same method as intermediate 54b, with a yield of 37%. 1 H NMR (400MHz, CDCl3) δ8.32–8.24(m,3H),7.39(dddd,J=20.3,9.9,5.7,1.9Hz,8H),5.12(s,2H),4.87–4.75(m,1H ), 2.41 (t, J = 6.8 Hz, 2H), 1.69 (dddt, J = 33.5, 23.3, 14.2, 6.0 Hz, 6H), 1.51–1.01 (m, 13H), 0.87 (t, J = 6.6 Hz, 3H).

[0813] Intermediate 55c: 5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)benzyl tridecanoate

[0814]

[0815] Intermediate 55c was prepared from intermediate 55b using the same method as intermediate 54c, with a yield of 53%. 1 H NMR (400MHz, CDCl3) δ7.41–7.18(m,5H),5.04(s,2H),4.65(p,J=6.5Hz,1H),3.19(q,J=6.4,5.9Hz,2H),2.49–2.23(m,6H),2.16(d,J=2 9.0Hz, 3H), 1.69–1.54 (m, J = 6.7Hz, 2H), 1.44 (tt, J = 13.6, 7.1Hz, 4H), 1.37–1.05 (m, 14H), 0.98 (t, J = 7.1Hz, 3H), 0.80 (t, J = 6.6Hz, 3H).

[0816] Intermediate 55d: 5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridecanoic acid

[0817]

[0818] Pd / C (2.5 g, 10% w / w) was added to a solution of intermediate 55c (2.2 g, 1.0 equivalent) in THF (20 mL). The mixture was stirred at 20 °C and H2 for 5 h. The reaction mixture was then filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a product (1 g, 58%) as a brown oil. 1 H NMR (400MHz, CDCl3) δ12.81(s,1H),5.93–5.72(m,1H),4.61(q,J=7.1Hz,1H),3.68–3.47(m,1H),3.29–3.04(m ,1H),2.76(tt,J=17.2,9.1Hz,3H),2.40(s,3H),2.34–2.06(m,2H),1.85–0.87(m,22H),0.80(t,J=6.4Hz,3H).

[0819] Compound 55: 2-(13-methyl-7-octyl-3,9-dioxo-2,8-dioxa-10,13-diazapentadecanyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0820]

[0821] Compound 55 was prepared from intermediates 53a and 55d using the same method as compound 52, with a yield of 58%. 1 H NMR (400MHz, CDCl3) δ5.44–5.27(m,8H),4.73(s,1H),4.12(d,J=6.0Hz,6H),3.32(s,2H),2.81–2.73(m,4H),2.49(d,J=45.9 Hz,4H),2.43–2.22(m,10H),2.05(q,J=6.8Hz,8H),1.75–1.42(m,13H),1.42–1.21(m,40H),1.11(s,3H),0.95–0.82(m,9H). MS: 972.8m / z[M+H].

[0822] Example 56 – Compound 56

[0823] Intermediate 56a: 9-(decoxy)-9-oxononanoic acid

[0824]

[0825] DMF (0.1-0.2 equivalents) and (COCl)₂ (1.0 equivalent) were added dropwise to a mixture of azelaic acid (90 g, 1.0 equivalent) in THF (300-900 mL) at 0 °C. The mixture was degassed and purged three times with N₂, and then stirred at 20 °C under N₂ atmosphere for 2 h. Decyl-1-ol (1.0 equivalent) was added dropwise to the reaction mixture at 0 °C, and stirred at 20 °C under N₂ atmosphere for 2 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, and the resulting solution was poured into water and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (20 g, 6%) as a white solid. 1 H NMR (400MHz, CDCl3) δ4.05(t,J=6.7Hz,2H),2.34(t,J=7.5Hz,2H),2.29(t,J= 7.5Hz, 2H), 1.62 (q, J = 7.3Hz, 6H), 1.41–1.15 (m, 19H), 0.88 (t, J = 6.7Hz, 3H).

[0826] Intermediate 56b: 9,9'-decylO'1,O1-(2-oxopropane-1,3-diyl)bis(azelate)

[0827]

[0828] Intermediate 56a (1.0 equivalent) was added fractionally to a mixture of 1,3-dihydroxyprop-2-one (1.37 g, 0.5 equivalent), EDCI (1.0 equivalent), and DMAP (1.0 equivalent) in DCM (100–200 mL) at 0 °C. The mixture was degassed and purged three times with N2. The mixture was stirred at 20 °C under N2 atmosphere for 5 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent, and the resulting solution was poured into water and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (10 g, 46%) as a white solid. 1H NMR(400MHz, CDCl3) δ4.74(d,J=2.1Hz,4H),4.05(t,J=6.8Hz,4H),2.41(t,J=7.5Hz,4H), 2.28(t,J=7.5Hz,4H), 1.62(d,J=8.8Hz,12H), 1.39–1.19(m,39H), 0.87(t,J=6.7Hz,6H).

[0829] Intermediate 56c: 9,9'-decyl O'1,O1-(2-hydroxypropane-1,3-diyl)di(azelate)

[0830]

[0831] At 0 °C and N2, NaBH4 (1.5 equivalents) was added to a solution of intermediate 56b (13.4 g, 1.0 equivalent) in THF / toluene / water (4:1:2, 0.4 M). The mixture was stirred at 15–20 °C and N2 for 5 h. After completion, the reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give the product (5.1 g, 38%) as a white solid. 1 H NMR (400MHz, CDCl3) δ4.15–4.03(m,5H),3.98(t,J=6.8Hz,4H),2.44(d,J=4.8Hz,1H),2.25(d t, J=24.2, 7.5Hz, 8H), 1.55 (q, J=7.1Hz, 12H), 1.22 (d, J=22.7Hz, 38H), 0.81 (t, J=6.7Hz, 6H).

[0832] Compound 56: 9,9'-DdecylO'1,O1-(2-((3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecyl)oxy)propane-1,3-diyl)di(azelate)

[0833]

[0834] Compound 56 was prepared from intermediates 52d and 56c using the same method as compound 52, with a yield of 36%. 1H NMR (400MHz, CDCl3) δ5.24 (t, J=5.2Hz, 1H), 5.13–5.04 (m, 1H), 4.28 (ddd, J=14. 5,11.9,4.5Hz,2H),4.14(ddd,J=11.9,8.3,5.8Hz,2H),4.05(t,J=6.8Hz,4H),3. 31(s,2H),2.58(qd,J=15.3,6.4Hz,6H),2.30(dt,J=10.3,7.6Hz,10H),1.61(dd, J=10.1, 4.1Hz, 16H), 1.29 (dt, J=17.6, 3.3Hz, 54H), 0.88 (td, J=6.9, 1.9Hz, 9H). MS: 1027.2 m / z [M+H].

[0835] Example 57 – Compound 57

[0836] Compound 57: 9,9'-DdecylO'1,O1-(2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecyl)oxy)propane-1,3-diyl)di(azelate)

[0837]

[0838] Compound 57 was prepared from intermediates 54d and 56c using the same method as compound 52, with a yield of 55%. 1 H NMR (400MHz, CDCl3) δ5.28–5.21(m,1H),4.76(s,1H),4.28(ddd,J=11.8,4.4,1 .2Hz,2H),4.15(ddd,J=11.9,5.9,4.6Hz,2H),4.05(t,J=6.8Hz,4H),3.29(s,2 H),2.38(ddd,J=8.6,6.9,2.7Hz,5H),2.35–2.12(m,10H),1.98–1.73(m,3H),1 .68–1.37(m,17H),1.29(dt,J=19.5,4.1Hz,54H),0.88(td,J=6.9,1.9Hz,9H). MS: 1040.8 m / z [M+H].

[0839] Example 58 – Compound 58

[0840] Compound 58: 9,9'-DdecylO'1,O1-(2-((5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridecyl)oxy)propane-1,3-diyl)di(azelate))

[0841]

[0842] Compound 58 was prepared from intermediates 55d and 56c using the same method as compound 52, with a yield of 60%. 1 H NMR (400MHz, CDCl3) δ5.27–5.20(m,1H),4.73(s,1H),4.28(ddd,J=11.9,4.4,2.8Hz,2H),4.13(ddd,J=11.9,5.9,2.6Hz,2H),4.05(t ,J=6.7Hz,4H),3.31(s,2H),2.39–2.09(m,12H),1.62(dq,J=13.8,7.2,6.5Hz,22H),1.41–0.99(m,54H),0.88(td,J=6.9,2.0Hz,9H). MS: 1055.0m / z[M+H].

[0843] Example 59 – Compound 59

[0844] Intermediate 59a: 9-oxo-9-(undecyloxy)nonanoic acid

[0845]

[0846] Using the same method as intermediate 56a, intermediate 59a was prepared from azelaic acid and undecane-1-ol in a yield of 41%. 1 H NMR (400MHz, CDCl3) δ4.05 (t, J = 6.7Hz, 2H), 2.31 (dt, J = 22.6, 7.5Hz, 4H), 1.62 (q, J = 7.1Hz, 6H), 1.40–1.18 (m, 23H), 0.87 (t, J = 6.7Hz, 3H).

[0847] Intermediate 59b: O'1,O1-(2-oxopropane-1,3-diyl)9,9'-di(undecyl)di(azelate)

[0848]

[0849] Intermediate 59b was prepared from 1,3-dihydroxyprop-2-one and intermediate 59a using the same method as intermediate 56b, with a yield of 23%. 1H NMR (400MHz, CDCl3) δ3.98(t,J=6.7Hz,2H),2.35(t,J=7.5Hz,2H),2.22(t,J=7 .5Hz,2H),1.57(dt,J=18.6,6.9Hz,6H),1.36–1.12(m,23H),0.89–0.76(m,3H).

[0850] Intermediate 59c: O'1,O1-(2-hydroxypropane-1,3-diyl)9,9'-di(undecyl)di(azelate)

[0851]

[0852] Intermediate 59c was prepared from intermediate 59b using the same method as intermediate 56c, with a yield of 47%. 1 H NMR (400MHz, CDCl3) δ4.23–4.00(m,9H),2.52(d,J=4.7Hz,1H),2.31(dt,J=24.2,7 .5Hz, 8H), 1.62 (q, J = 7.0Hz, 13H), 1.29 (d, J = 23.9Hz, 43H), 0.88 (t, J = 6.7Hz, 6H).

[0853] Compound 59: O'1,O1-(2-((3-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoyl)oxy)propane-1,3-diyl)9,9'-di(undecyl)di(azelate)

[0854]

[0855] Compound 59 was prepared from intermediates 52d and 59c using the same method as compound 52, with a yield of 35%. 1 H NMR (400MHz, CDCl3) δ5.23 (s, 1H), 5.08 (d, J = 6.5Hz, 1H), 4.28 (ddd, J = 15.0, 11.9, 4.5Hz, 2H), 4.14 (dt, J = 13.1, 6.7Hz, 2H), 4.05 (t, J = 6.8Hz, 4H),2.58(qd,J=15.4,6.6Hz,4H),2.30(dt,J=10.4,7.5Hz,10H),1.60( q, J=7.7, 6.9Hz, 20H), 1.43–0.97 (m, 58H), 0.88 (td, J=6.9, 1.9Hz, 9H). MS: 1054.9m / z[M+H].

[0856] Example 60 – Compound 60

[0857] Compound 60: O'1,O1-(2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl)9,9'-di(undecyl)di(azelate)

[0858]

[0859] Compound 60 was prepared from intermediates 54d and 59c using the same method as compound 52, with a yield of 79%. 1 H NMR (400MHz, CDCl3) δ5.25(p,J=5.1Hz,1H),4.76(s,1H),4.28(ddd,J=11.9,4.5,2.7Hz,2H),4.15(dt,J=11.7,5.6Hz,2H),4.05( t,J=6.8Hz,4H),2.38(t,J=7.5Hz,5H),2.35–2.11(m,10H),1.98–1.41(m,23H),1.41–0.96(m,57H),0.88(td,J=6.9,1.8Hz,9H). MS: 1068.9m / z[M+H].

[0860] Example 61 – Compound 61

[0861] Compound 61: O'1,O1-(2-((5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridecyl)oxy)propane-1,3-diyl)9,9'-di(undecyl)di(azelate)

[0862]

[0863] Compound 61 was prepared from intermediates 55d and 59c using the same method as compound 52, with a yield of 61%. 1 H NMR (400MHz, CDCl3) δ5.29–5.20(m,1H),4.72(d,J=7.4Hz,1H),4.28(ddd,J=11.9,4.4,2.3Hz,2H),4.14(ddd,J=11.9,5.9, 2.0Hz,2H),4.05(t,J=6.8Hz,4H),2.82–2.13(m,15H),1.82–1.39(m,21H),1.39–0.97(m,56H),0.88(td,J=6.9,1.9Hz,9H). MS: 1083.0m / z[M+H].

[0864] Example 62 – Compound 62

[0865] Intermediate 62a: 1-((tert-butyldimethylsilyl)oxy)-3-hydroxyprop-2-one

[0866]

[0867] At 0°C, TBSCl (83.66 g, 1.0 equivalent) was added to a mixture of 1,3-dihydroxyprop-2-one (50 g, 1.0 equivalent) and imidazole (37.79 g, 1.0 equivalent) in THF (40 mL), and the mixture was stirred at 20°C under a N2 atmosphere for 2 h. After completion, the reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by column chromatography (EtOAc / hex) to give a product (15 g, 13%) as a colorless oil.

[0868] Intermediate 62b: 1-(3-((tert-butyldimethylsilyl)oxy)-2-oxopropyl)9-undecyl azelate

[0869]

[0870] Intermediate 59a (16.76 g, 1.0 equivalent) was added to a mixture of intermediate 62a (10 g, 1.0 equivalent), EDCI (9.38 g, 1.0 equivalent), and DMAP (5.98 g, 1.0 equivalent) in DCM (300 mL) at 0 °C, and the mixture was stirred at 20 °C under a N2 atmosphere for 12 h. After completion, the reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with water. The crude material was extracted three times with EtOAc, and the combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (11 g, 43%) as a colorless oil. 1 HNMR (400MHz, CDCl3) δ4.84(s,2H),4.16(s,2H),3.95(t,J=6.8Hz,2H),2.32(t,J=7.5Hz,2H),2 .18(t,J=7.5Hz,2H),1.61–1.48(m,6H),1.30–1.12(m,22H),0.82(s,9H),0.78(t,J=6.8Hz,3H).

[0871] Intermediate 62c: 1-(3-hydroxy-2-oxopropyl)9-undecyl azelate

[0872]

[0873] HF·pyridine (3.75 mL, 8.0 equivalent) was added to a mixture of intermediate 62b (11 g, 1.0 equivalent) in THF (50 mL). The mixture was stirred at 20 °C under a N2 atmosphere for 5 h. After completion, the reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (6 g, 70%) as a white solid. 1 H NMR (400MHz, CDCl3) δ4.70(s,2H),4.30(s,2H),3.98(t,J=6.8Hz,2H),2.95(s,1H),2.36(t,J=7.5Hz ,2H),2.22(t,J=7.6Hz,2H),1.57(dt,J=20.3,7.1Hz,6H),1.37–1.10(m,22H),0.81(t,J=6.7Hz,3H).

[0874] Intermediate 62d: 1-(3-(((9Z,12Z)-octadec-9,12-dienoyl)oxy)-2-oxopropyl)9-undecyl azelate

[0875]

[0876] EDCI (1.0 equivalent) and DMAP (1.0 equivalent) were added to a solution of intermediate 62c (6 g, 1.0 equivalent) in DCM (100 mL) at 0 °C. The solution was degassed and purged three times with N2, and then linoleic acid (4 mL, 1.0 equivalent) was added. The mixture was stirred at 20 °C under N2 atmosphere for 12 h. After completion, the reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with water. The crude material was extracted three times with EtOAc, and the combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude residue was purified by column chromatography (EtOAc / hexane) to give a product (7.5 g, 55%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ4.70(s,2H),4.30(s,2H),3.98(t,J=6.8Hz,2H),2.95(s,1H),2.36(t,J=7.5Hz ,2H),2.22(t,J=7.6Hz,2H),1.57(dt,J=20.3,7.1Hz,6H),1.37–1.10(m,22H),0.81(t,J=6.7Hz,3H).

[0877] Intermediate 62e: 1-(2-hydroxy-3-(((9Z,12Z)-octadecano-9,12-dienoyl)oxy)propyl)9-undecyl azelate

[0878]

[0879] At 0 °C, NaBH4 (5 equivalents) was added to a solution of intermediate 62d (7 g, 1.0 equivalent) in THF / toluene / water (4:1:2, 0.04 M), and the mixture was stirred at 20 °C under a N2 atmosphere for 5 h. After completion, the mixture was quenched with saturated NH4Cl at 5 °C and extracted three times with EtOAc. The combined organic layers were washed twice with brine, dried over Na2SO4, filtered, and concentrated to obtain the residue. The crude residue was purified by column chromatography (EtOAc / hex) to give a product (6 g, 85%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.42–5.25(m,4H),4.25–3.90(m,8H),2.76(t,J=6.4Hz,2H),2.55(s,1H),2.30(dt,J=24 .5,7.6Hz,6H),2.03(q,J=6.8Hz,4H),1.61(q,J=7.0Hz,8H),1.48–1.06(m,35H),0.87(td,J=6.8,3.8Hz,6H).

[0880] Compound 62: 1-(3-methyl-13-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)-9-octyl-7,11-dioxo-8,12-dioxa-3,6-diazatetradecane-14-yl)9-undecyl azelate

[0881]

[0882] Compound 62 was prepared from intermediates 52d and 62e using the same method as compound 52, with a yield of 36%. 1H NMR(400MHz, CDCl3)δ5.47–5.28(m,4H),5.23(t,J=5.2Hz,1H),5.14–5.04(m,1H),4.28 (ddd,J=15.3,11.9,4.6Hz,2H),4.14(ddd,J=11.8,7.5,5.8Hz,2H),4.05(t,J=6.8Hz,2 H),3.38(s,2H),2.77(t,J=6.5Hz,2H),2.58(qd,J=15.2,6.3Hz,5H),2.37–2.22(m,7H) ,2.05(q,J=6.7Hz,4H),1.62(d,J=6.8Hz,14H),1.48–0.97(m,50H),0.97–0.79(m,9H). MS: 992.3m / z[M+H].

[0883] Example 63 – Compound 63

[0884] Compound 63: 1-(3-methyl-14-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)-9-octyl-7,12-dioxo-8,13-dioxa-3,6-diazapentadecan-15-yl)9-undecyl azelate

[0885]

[0886] Compound 63 was prepared from intermediates 54d and 62e using the same method as compound 52, with a yield of 79%. 1 H NMR(400MHz, CDCl3)δ5.44–5.28(m,4H),5.25(ddd,J=5.8,4.5,1.4Hz,1H),4.76(s,1H) ,4.28(dd,J=11.9,4.4Hz,2H),4.15(ddd,J=11.9,5.8,4.2Hz,2H),4.05(t,J=6.8Hz,2H) ,3.31(s,2H),2.77(t,J=6.7Hz,2H),2.70–2.12(m,15H),2.05(q,J=6.9Hz,4H),1.97–1 .72(m,3H),1.61(h,J=6.5Hz,12H),1.40–1.19(m,47H),1.11(s,3H),0.96–0.81(m,9H). MS: 1006.9m / z[M+H].

[0887] Example 64 – Compound 64

[0888] Compound 64: 1-(3-methyl-15-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)-9-octyl-7,13-dioxo-8,14-dioxa-3,6-diazahexadecane-16-yl)9-undecyl azelate

[0889]

[0890] Compound 64 was prepared from intermediates 55d and 62e using the same method as compound 52, with a yield of 79%. 1 H NMR (400MHz, CDCl3) δ5.48–5.28(m,4H),5.24(h,J=4.9,4.4Hz,1H),4.73(s,1 H),4.29(ddd,J=11.9,4.4,3.3Hz,2H),4.14(ddd,J=11.9,5.9,3.1Hz,2H),4.0 5(t,J=6.8Hz,2H),3.31(s,2H),2.77(t,J=6.5Hz,2H),2.67–2.11(m,14H),2.0 5(q,J=6.8Hz,4H),1.73–1.41(m,17H),1.41–0.95(m,50H),0.94–0.82(m,9H). MS: 1021.0 m / z [M+H].

[0891] Example 65 – Compound 65

[0892] Compound 65: 2-((4-((((1-methylpyrrolidone-2-yl)methyl)carbamoyl)oxy)tetradecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0893]

[0894] Compound 65 was prepared from intermediate 39d and 1-(1-methylpyrrolidone-2-yl)methylamine using the method employed in Example 1, with a yield of 75%. 1H NMR (400MHz, CDCl3) δ5.45–5.28(m,8H),5.25(ddd,J=10.2,5.7,4.4Hz,1H),4.76(s,1H),4.28(dd,J=11.9,4.4Hz,2H),4.20–4.11(m,2H),3.45(s, 1H),3.21(s,2H),2.82–2.74(m,4H),2.46–2.24(m,9H),2.05(q,J=6.8Hz, 8H), 2.01–1.42 (m, 16H), 1.41–1.20 (m, 45H), 0.88 (td, J = 7.0, 4.7Hz, 9H). MS: 984.9m / z[M+H].

[0895] Example 66 – Compound 66

[0896] Compound 66: 2-((4-(((3-(pyrrolidone-1-yl)propoxy)carbonyl)oxy)tetradecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0897]

[0898] Compound 66 was prepared from intermediate 39d and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 65%. 1 H NMR (400MHz, CDCl3) δ5.57–5.21(m,9H),4.72(dt,J=12.2,5.2Hz,1H),4.29(dd,J=11.9,4.4Hz,2H),4.24–4.07(m,4H),2.82–2.74(m,4H),2 .60(d,J=102.7Hz,5H),2.45–2.38(m,2H),2.38–2.26(m,4H),2.13–1. 73(m,16H),1.73–1.43(m,9H),1.41–1.16(m,42H),1.04–0.74(m,9H). MS: 999.4m / z[M+H].

[0899] Example 67 – Compound 67

[0900] Compound 67: 2-((4-((((1-methylpyrrolidone-2-yl)methyl)carbamoyl)oxy)octanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0901]

[0902] Compound 67 was prepared from intermediate 42d and 1-(1-methylpyrrolidone-2-yl)methylamine using the method employed in Example 1, with a yield of 50%. 1 H NMR (400MHz, CDCl3) δ5.42–5.08(m,9H),4.77(s,1H),4.28(ddd,J=11.8,4.5,1.8Hz,2H),4.20–4.12(m,2H),3.48(s,1H),3.33(s,2H ), 2.77(td,J=6.1,1.2Hz,4H),2.63–2.23(m,11H),2.05(q,J=6.8Hz,9H),1.98–1.42(m,14H),1.42–1.22(m,32H),0.96–0.81(m,9H). MS: 900.0m / z[M+H].

[0903] Example 68 – Compound 68

[0904] Compound 68: 2-((4-(((3-(pyrrolidone-1-yl)propoxy)carbonyl)oxy)octanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0905]

[0906] Compound 68 was prepared from intermediate 42d and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 43%. 1 H NMR (400MHz, CDCl3) δ5.43–5.04(m,9H),4.72(tt,J=7.4,5.1Hz,1H),4.40–4.10(m,6H),2.81–2.74(m,4H),2.54(d,J=58.1Hz,5H), 2.40(ddd,J=8.4,6.8,4.4Hz,2H),2.36–2.27(m,4H),2.11–1.73(m,16H),1.73–1.43(m,8H),1.43–1.21(m,31H),0.99–0.77(m,9H). MS: 915.7m / z[M+H].

[0907] Example 69 – Compound 69

[0908] Intermediate 69a: 4-Oxononanoic acid

[0909]

[0910] NaOH (2.0 equivalents) was added in a single batch to a mixture of 50 g (1.0 equivalent) of 5-pentyltetrahydrofuran-2-one in 3:1 EtOH / H2O (500 mL) at 15 °C and N2. The mixture was stirred at 15 °C for 12 h. After completion, the reaction mixture was concentrated under reduced pressure, and the resulting residue was extracted twice with DCM. The combined organic layers were dried over Na2SO4 and concentrated to obtain a crude residue.

[0911] The crude residue was then reconstituted in DCM (1 L), followed by the addition of DMP (3 equivalents) in a single batch at 15 °C under N2. The mixture was stirred at 15 °C for 4 h. After completion, the reaction was concentrated under reduced pressure, and the solution was adjusted to pH 3 with 1 M HCl. The slurry was filtered and washed with H2O. The resulting filter cake was dissolved in EtOAc, dried over Na2SO4, filtered, and concentrated to give a product as a white solid (22 g, 45%). 1 H NMR (400MHz, CDCl3) δ2.72 (dd, J=7.0, 5.4Hz, 2H), 2.63 (ddd, J=7.2, 5.9, 1.2Hz, 2H) ,2.44(t,J=7.4Hz,2H),1.64–1.54(m,2H),1.36–1.20(m,4H),0.89(t,J=6.9Hz,3H).

[0912] Intermediate 69b: 2-((4-oxonanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0913]

[0914] Using the method employed with intermediate 1d, intermediate 69b was synthesized from intermediate 69a and intermediate 1c with a yield of 74%. 1 H NMR(400MHz, CDCl3) δ5.46–5.16(m,9H),4.29(dd,J=11.9,4.3Hz,2H),4.14( dt,J=11.8,6.0Hz,2H),2.74(dt,J=21.5,6.5Hz,6H),2.59(t,J=6.6Hz,2H), 2.43(t,J=7.5Hz,2H),2.32(t,J=7.6Hz,4H),2.04(dd,J=8.6,5.3Hz,8H),1. 60(ddd,J=14.2,8.4,4.8Hz,6H),1.50–1.06(m,32H),0.89(t,J=6.8Hz,9H).

[0915] Intermediate 69c: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)nonanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0916]

[0917] NaBH4 (5.0 equivalents) was added to a solution of intermediate 69b (10 g, 1.0 equivalent) in THF (300 mL), toluene (150 mL), and H2O (75 mL) at 5 °C. The mixture was stirred at 15 °C under a N2 atmosphere for 3 h. After stirring, Na2SO4 was added to the reaction mixture at 0 °C, and the resulting mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give an alcohol intermediate as a colorless oil.

[0918] The intermediate was immediately reconstituted in DCM (80 mL), followed by the addition of pyridine (1.67 equivalents) and (4-nitrophenyl)chloroformate (2 equivalents) at 0 °C. The mixture was stirred at 0 °C under a N2 atmosphere for 5 h. After completion, the reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by column chromatography to give a product (4.05 g, 33%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ8.26–8.19(m,2H),7.40–7.28(m,2H),5.43–5.11(m,9H),4.80(tt,J=7. 5,4.4Hz,1H),4.25(dd,J=11.9,4.3Hz,2H),4.09(dd,J=11.9,5.8Hz,2H),2.70(t,J=6.4Hz,4H ),2.40(t,J=7.4Hz,2H),2.33–2.16(m,4H),1.97(q,J=6.8Hz,10H),1.67(ddt,J=13.4,9.9,4 .7Hz,1H),1.55(dtd,J=15.7,8.4,7.4,4.8Hz,5H),1.43–1.16(m,36H),0.83(q,J=6.8Hz,9H).

[0919] Compound 69: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)nonanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0920]

[0921] Compound 69 was prepared from intermediate 69c and N1-ethyl-N1-methylethane-1,2-diamine using the method employed in Example 1, with a yield of 77%. 1 H NMR(500MHz, CDCl3) δ5.35(pt,J=8.8,4.4Hz,8H),5.25(p,J=5.1Hz,1H),4.76(dt,J=12.3,6.7Hz,1H),4.28(dd ,J=11.9,4.5Hz,2H),4.15(dh,J=11.4,5.9Hz,2H),3.31(s,2H),2.77(t,J=6.8Hz,4H),2.62–2.45(m,4H),2.38( dt,J=9.0,6.2Hz,2H),2.30(q,J=13.0,10.4Hz,8H),2.05(q,J=7.0Hz,9H),1.91(tt,J=9.5,4.5Hz,1H),1.81(d t,J=14.7,7.8Hz,1H),1.65–1.43(m,7H),1.40–1.17(m,36H),1.09(q,J=8.8,8.2Hz,3H),0.88(q,J=6.5Hz,9H). MS: 902.6m / z[M+H].

[0922] Example 70 – Compound 70

[0923] Compound 70: 2-((4-((((1-methylpyrrolidone-2-yl)methyl)carbamoyl)oxy)nonanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0924]

[0925] Compound 70 was prepared from intermediate 69c and 1-(1-methylpyrrolidone-2-yl)methylamine using the method employed in Example 1, with a yield of 62%. 1H NMR (500MHz, CDCl3) δ5.45–5.29 (m, 8H), 5.25 (p, J = 5.2Hz, 1H), 4.76 (s, 1H), 4 .28(dd,J=11.9,4.5Hz,2H),4.15(dq,J=11.7,5.8Hz,2H),3.46(d,J=25.9Hz,1 H),3.19(s,2H),2.77(t,J=6.8Hz,4H),2.34(dt,J=31.1,8.3Hz,12H),2.05(q ,J=7.0Hz,8H),1.99–1.42(m,16H),1.42–1.19(m,35H),0.88(q,J=6.8Hz,9H). MS: 914.4 m / z [M+H].

[0926] Example 71 – Compound 71

[0927] Compound 71: 2-((4-(((3-(pyrrolid-1-yl)propoxy)carbonyl)oxy)nonanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0928]

[0929] Compound 71 was prepared from intermediate 69c and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 78%. 1 H NMR(500MHz,Chloroform-d)δ5.50–5.28(m,8H),5.25(p,J=5.1Hz,1H),4.72( tt,J=7.8,4.5Hz,1H),4.36–4.26(m,2H),4.23–4.11(m,4H),2.77(t,J=6.7Hz, 4H),2.41(dt,J=15.0,9.0Hz,2H),2.31(t,J=7.5Hz,5H),2.05(q,J=6.9Hz,8H) ,2.00–1.75(m,9H),1.72–1.48(m,8H),1.43–1.20(m,35H),1.00–0.75(m,9H). MS: 929.4 m / z [M+H].

[0930] Example 72 – Compound 72

[0931] Compound 72: 2-((4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0932]

[0933] Compound 72 was prepared from intermediate 69c and 2-(pyrrolidone-1-yl)ethyl-1-amine using the method employed in Example 1, with a yield of 72%. 1 H NMR (500MHz, CDCl3) δ5.49 (s, 1H), 5.34 (dtt, J = 18.0, 6.9, 3.9Hz, 8H), 5.25 (p, J = 5.1Hz, 1 H),4.76(s,1H),4.28(dd,J=11.9,4.4Hz,2H),4.15(dt,J=11.6,5.4Hz,2H),3.40–3.27(m ,2H),2.80–2.50(m,11H),2.45–2.35(m,2H),2.31(t,J=7.6Hz,5H),2.05(q,J=7.0Hz,9H) ,1.97–1.67(m,8H),1.60(q,J=7.3Hz,8H),1.33(d,J=26.5Hz,36H),0.88(q,J=6.5Hz,9H). MS: 914.7m / z[M+H].

[0934] Example 73 – Compound 73

[0935] Intermediate 73a: 4-Oxoundecanoic acid

[0936]

[0937] To a solution of 20 g (1.0 equivalent) of 5-heptyltetrahydrofuran-2-one in 2:1 EtOH / H2O (400 mL), NaOH (1.1 equivalent) was added. The mixture was degassed and purged three times with N2, and then stirred at 20 °C under N2 atmosphere for 12 h. After completion, the reaction mixture was concentrated under reduced pressure to remove the solvent, and 1 N HCl was added to the resulting residue until pH = 3. The resulting slurry was filtered, and the filter cake was concentrated under reduced pressure to obtain the residue.

[0938] The resulting residue was reconstituted in DCM (200 mL), followed by the addition of DMP (3 equivalents). The mixture was stirred at 20 °C for 5 h. After completion, the mixture was concentrated under reduced pressure, and the resulting residue was diluted with H2O and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography to give a product as a white solid (10 g, 46%). 1H NMR (400MHz, CDCl3) δ2.65 (dd, J=7.0, 5.3Hz, 2H), 2.56 (t, J=6.8Hz, 2H), 2.37 (t,J=7.5Hz,2H),1.51(q,J=7.2Hz,2H),1.27–1.13(m,8H),0.86–0.76(m,3H).

[0939] Intermediate 73b: 2-((4-oxoundecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0940]

[0941] Using the method employed with intermediate 1d, intermediate 73b was synthesized from intermediate 73a and intermediate 1c with a yield of 42%. 1 H NMR(400MHz, CDCl3)δ5.28(dddd,J=12.3,10.7,8.7,5.5Hz,8H),5.21–5.15(m,1H), 4.22(dd,J=11.9,4.3Hz,2H),4.13–4.05(m,2H),2.70(t,J=6.4Hz,4H),2.64(t,J=6. 5Hz,2H),2.52(t,J=6.5Hz,2H),2.36(t,J=7.5Hz,2H),2.25(t,J=7.6Hz,4H),2.02– 1.95(m,8H),1.53(h,J=7.0Hz,6H),1.33–1.16(m,37H),0.81(td,J=6.8,4.7Hz,9H).

[0942] Intermediate 73c: 2-((4-(((4-nitrophenoxy)carbonyl)oxy)undecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0943]

[0944] NaBH4 (5.0 equivalents) was added to a solution of intermediate 73b (25 g, 1.0 equivalent) in THF (500 mL), toluene (125 mL), and H2O (250 mL) at 0 °C and N2. The mixture was stirred at 5 °C and N2 atmosphere for 1 h. After completion, the residue was poured into a saturated NH4Cl solution and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography to give an intermediate alcohol as a colorless residue.

[0945] The intermediate was immediately reconstituted in DCM (100 mL), followed by the addition of pyridine (2 equivalents) and (4-nitrophenyl)chloroformate (2 equivalents) at 0 °C. The mixture was stirred at 20 °C under a N2 atmosphere for 5 h, after which the reaction mixture was concentrated under reduced pressure. The resulting residue was diluted with hexane and filtered. The filtrate was washed three times with water and once with brine, then dried over Na2SO4, filtered, and concentrated. Purification by column chromatography yielded a colorless oily product (5.2 g, 40%). 1H NMR (400MHz, chloroform-d) δ 8.28 (d, J = 8.8Hz, 2H), 7.41 (dd, J = 8.6, 6.1Hz, 2H), 5.33 (tdd, J = 23.5, 11.3, 6.1Hz, 9H), 4.87 (tt, J = 7.7, 4.6Hz, 1H), 4.32 (dd, J = 12.0, 4.4Hz, 2H), 4.16 ( dd,J=12.0,5.8Hz,2H),2.77(t,J=6.4Hz,4H),2.47(t,J=7.5Hz,2H),2.39–2.25(m,4H) ,2.04(q,J=6.9Hz,10H),1.79–1.55(m,7H),1.48–1.15(m,36H),0.89(t,J=6.7Hz,9H).

[0946] Compound 73: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)undecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0947]

[0948] Compound 73 was prepared from intermediate 73c and (2-aminoethyl)(ethyl)methylamine using the method employed in Example 1, with a yield of 53%. 1H NMR (500MHz, CDCl3) δ5.47–5.27(m,8H),5.24(q,J=5.1Hz,1H),4.76(s,1H),4.28(dd,J=11. 9,4.4Hz,2H),4.15(dt,J=11.6,5.4Hz,2H),3.31(s,2H),2.77(t,J=6.7Hz,4H),2.61–2.35(m ,6H),2.31(t,J=7.6Hz,7H),2.05(q,J=7.0Hz,8H),1.90(q,J=11.5,9.0Hz,1H),1.81(dt,J= 14.5, 7.6Hz, 1H), 1.65–1.41 (m, 9H), 1.39–1.19 (m, 38H), 1.10 (s, 3H), 0.88 (q, J = 7.0Hz, 9H). MS: 930.3m / z[M+H].

[0949] Example 74 – Compound 74

[0950] Compound 74: 2-((4-((((1-methylpyrrolidone-2-yl)methyl)carbamoyl)oxy)undecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0951]

[0952] Compound 74 was prepared from intermediate 73c and 1-(1-methylpyrrolidone-2-yl)methylamine using the method employed in Example 1, with a yield of 55%. 1 H NMR (500MHz, CDCl3) δ5.34 (dtt, J=18.0, 6.9, 3.8Hz, 8H), 5.25 (p, J=5.2Hz, 1H), 4. 81–4.72(m,1H),4.28(dd,J=11.9,4.4Hz,2H),4.15(dt,J=11.7,5.2Hz,2H),3.43( s,1H),3.17(s,2H),2.77(t,J=6.8Hz,4H),2.34(dt,J=31.2,8.3Hz,11H),2.05(q, J=7.0Hz,8H),1.99–1.43(m,16H),1.43–1.13(m,39H),0.88(q,J=7.7,7.2Hz,9H). MS: 942.8m / z[M+H].

[0953] Example 75 – Compound 75

[0954] Compound 75: 2-((4-(((3-(pyrrolidone-1-yl)propoxy)carbonyl)oxy)undecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0955]

[0956] Compound 75 was prepared from intermediate 73c and 3-(pyrrolidone-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 73%. 1 H NMR (500MHz, CDCl3) δ5.46–5.28(m,8H),5.25(p,J=5.1Hz,1H),4.72(tt,J=7.8,4 .5Hz,1H),4.29(dd,J=11.9,4.4Hz,2H),4.22–4.11(m,4H),2.77(t,J=6.8Hz,4H), 2.45–2.37(m,2H),2.31(t,J=7.5Hz,4H),2.05(q,J=7.0Hz,8H),2.01–1.91(m,3H) ,1.91–1.78(m,5H),1.67–1.50(m,9H),1.39–1.22(m,39H),0.88(q,J=6.6Hz,9H). MS: 957.4m / z[M+H].

[0957] Example 76 – Compound 76

[0958] Compound 76: 2-((4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)undecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0959]

[0960] Compound 76 was prepared from intermediate 73c and 2-(pyrrolidone-1-yl)ethyl-1-amine using the method employed in Example 1, with a yield of 81%. 1H NMR (500MHz, CDCl3) δ5.56 (s, 1H), 5.46–5.28 (m, 8H), 5.25 (p, J = 5.2Hz, 1H), 4.76 (s ,1H),4.28(dd,J=11.9,4.4Hz,2H),4.15(dt,J=11.7,5.5Hz,2H),3.43–3.31(m,2H), 2.77(t,J=6.7Hz,11H),2.45–2.35(m,2H),2.31(t,J=7.6Hz,4H),2.05(q,J=7.0Hz,8 H),1.97–1.74(m,7H),1.69–1.43(m,8H),1.43–1.09(m,39H),0.88(q,J=7.0Hz,9H). MS: 943.2m / z[M+H].

[0961] Example 77 – Compound 77

[0962] Compound 77: 2-((4-(((2-(azacyclobutan-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0963]

[0964] Compound 77 was prepared from intermediate 40d and 2-(azacyclobutan-1-yl)ethyl-1-amine using the method employed in Example 1, with a yield of 36%. 1 H NMR (400MHz, CDCl3) δ5.45–5.29(m,8H),5.25(h,J=4.6,4.1Hz,1H),4.75(s,1H),4.28(ddd,J=11 .9,4.5,1.9Hz,2H),4.15(ddt,J=11.9,5.8,3.6Hz,2H),3.74–3.54(m,3H),3.48–3.38(m,1H),3. 31(d,J=6.3Hz,1H),2.86(s,1H),2.81–2.73(m,4H),2.41–2.26(m,7H),2.05(q,J=6.8Hz,8H),1. 97–1.87(m,2H),1.87–1.77(m,2H),1.60(d,J=7.2Hz,8H),1.44–1.15(m,37H),0.96–0.84(m,9H). MS: 914.7m / z[M+H].

[0965] Example 78 – Compound 78

[0966] Compound 78: 2-((4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0967]

[0968] Compound 78 was prepared from intermediate 40d and 2-(piperidin-1-yl)ethyl-1-amine using the method employed in Example 1, with a yield of 60%. 1 H NMR (400MHz, CDCl3) δ5.47–5.28(m,8H),5.25(h,J=4.8Hz,1H),4.76(s,1H),4.28( ddd,J=11.9,4.4,1.3Hz,2H),4.15(dt,J=11.8,5.7Hz,2H),3.42(s,2H),2.80–2.72 (m,4H),2.44(d,J=6.7Hz,2H),2.39(ddd,J=8.6,6.9,2.5Hz,2H),2.36–2.27(m,5H) ,2.05(q,J=6.9Hz,8H),1.95–1.41(m,18H),1.41–1.20(m,37H),0.96–0.81(m,9H). MS: 942.4m / z[M+H].

[0969] Example 79 – Compound 79

[0970] Compound 79: 2-((4-(((2-methyl-1-(pyrrolid-1-yl)prop-2-yl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0971]

[0972] Compound 79 was prepared from intermediate 40d and 2-methyl-1-(pyrrolidone-1-yl)propyl-2-amine using the method employed in Example 1, with a yield of 48%. 1H NMR (400MHz, CDCl3) δ5.42–5.21(m,9H),4.72(s,1H),4.28(ddt,J=11.9,4.4,2.2Hz,2H),4.15(ddd,J=11.9,5.8,4.6Hz,2H),2.86–2.49(m,8H), 2.39(td,J=8.2,7.4,2.2Hz,1H),2.31(td,J=7.6,1.8Hz,4H),2.05(q,J =6.8Hz,8H),2.00–1.45(m,17H),1.45–1.03(m,41H),0.98–0.77(m,9H). MS: 956.5m / z[M+H].

[0973] Example 80 – Compound 80

[0974] Intermediate 80a: (5-methylhexyl)magnesium bromide

[0975]

[0976] 1-Bromo-5-methylhexane (1.0 equivalent) was added to a solution of Mg (1.63 g, 1.2 equivalent) and I2 (0.1 equivalent) in THF (100 mL) at 15 °C. The mixture was stirred at 40 °C under a N2 atmosphere for 2 h. No further work-up was performed. The reaction mixture was used directly for the next step. Magnesium bromo(5-methylhexyl) was obtained as a gray solution in THF.

[0977] Intermediate 80b: Ethyl 9-methyl-4-oxodecanoate

[0978]

[0979] At -78°C, CuI (1.0 equivalent) and intermediate 80a (2.0 equivalent) were added to a solution of ethyl 4-chloro-4-oxobutyrate (16 g, 1.0 equivalent) in THF (150 mL). The mixture was stirred at -78°C under a N2 atmosphere for 2 h. After completion, the reaction mixture was quenched with water and extracted three times with EtOAc. The combined organic layers were washed twice with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The crude residue was purified by column chromatography to give the product (10 g, 45% yield) as a colorless oil. 1H NMR (400MHz, CDCl3) δ4.06(q,J=7.1Hz,2H),2.64(t,J=6.6Hz,2H),2.50(t,J=6.8Hz,2H),2.38( t,J=7.5Hz,2H),1.55–1.45(m,3H),1.28–1.15(m,6H),1.15–1.06(m,2H),0.79(d,J=6.6Hz,6H).

[0980] Intermediate 80c: 9-methyl-4-oxodecanoic acid

[0981]

[0982] At 20°C, KOH (3.0 equivalents) was added to a solution of intermediate 80b (10 g, 1.0 equivalent) in EtOH (100 mL). The mixture was stirred at 50°C under a N2 atmosphere for 12 h. The reaction was then concentrated to remove EtOH, and the pH was adjusted to 5.5 with HCl (1 M). The resulting mixture was diluted with water and extracted twice with EtOAc. The combined organic layers were washed twice with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by column chromatography to give a product (4.4 g, 50%) as a white solid. 1 H NMR (400MHz, CDCl3) δ2.71(t,J=6.8Hz,2H),2.63(ddd,J=7.0,5.8,1.1Hz,2H),2.44(t,J=7.5Hz,2H),1 .53(dq,J=19.8,7.0,6.6Hz,3H),1.31–1.24(m,2H),1.16(dt,J=8.6,6.7Hz,2H),0.85(d,J=6.6Hz,6H).

[0983] Intermediate 80d: 2-((9-methyl-4-oxodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadecano-9,12-dienoate)

[0984]

[0985] The method used with intermediate 1d was used to synthesize intermediate 80d from intermediate 80c and intermediate 1c, with a yield of 40%. 1H NMR (400MHz, CDCl3) δ5.45–5.28(m,8H),5.24(p,J=5.1Hz,1H),4.28(dd,J=11.9,4. 3Hz,2H),4.14(dd,J=11.9,5.9Hz,2H),2.76(t,J=6.4Hz,4H),2.71(t,J=6.5Hz,2H), 2.59(t,J=6.5Hz,2H),2.43(t,J=7.5Hz,2H),2.31(t,J=7.6Hz,4H),2.04(q,J=6.8Hz ,8H),1.67–1.50(m,8H),1.39–1.21(m,29H),1.22–1.12(m,3H),0.93–0.82(m,12H).

[0986] Intermediate 80e: 2-((9-methyl-4-(((4-nitrophenoxy)carbonyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0987]

[0988] NaBH4 (5.0 equivalents) was added to a solution of intermediate 80d (1.5 g, 1.0 equivalent) in THF (30 mL), toluene (15 mL), and H2O (7.5 mL) at 0 °C. The mixture was stirred at 5 °C for 5 h. After complete reduction, the reaction mixture was partitioned between THF and H2O, and the resulting organic phase was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give the intermediate alcohol as a yellow oil.

[0989] The resulting intermediate was immediately reconstituted in DCM (10 mL), and then pyridine (2.0 equivalents) and (4-nitrophenyl)chloroformate (2.0 equivalents) were added at 0 °C. The mixture was stirred at 0 °C for 5 h. The reaction mixture was then filtered and concentrated under reduced pressure to give a crude residue, which was purified by column chromatography to give a product as a colorless oil. 1HNMR (400MHz, CDCl3) δ8.25–8.18(m,2H),7.38–7.28(m,2H),5.39–5.15(m,9H),4.80(tt,J=7.1,4.3 Hz,1H),4.25(dd,J=12.0,4.3Hz,2H),4.13–4.04(m,2H),2.70(t,J=6.4Hz,4H),2.40(dd,J=8.3,6.6 Hz,2H),2.33–2.19(m,4H),2.05–1.85(m,10H),1.67(ddt,J=14.6,9.4,4.8Hz,1H),1.52(dddq,J=32 .6,26.2,13.2,6.6,6.1Hz,8H),1.37–1.17(m,30H),1.11(dt,J=9.0,6.5Hz,2H),0.94–0.66(m,12H).

[0990] Compound 80: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)-9-methyldecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[0991]

[0992] Compound 80 was prepared from intermediate 80e and (2-aminoethyl)(ethyl)methylamine using the method employed in Example 1, with a yield of 68%. 1 H NMR (400MHz, CDCl3) δ5.43–5.22(m,9H),4.76(s,1H),4.28(ddd,J=12.0,4.5,1.5Hz,2H),4 .15(ddd,J=11.9,5.8,4.7Hz,2H),3.37(s,3H),2.77(td,J=6.0,1.3Hz,4H),2.39(ddd,J=8 .4,6.9,2.4Hz,2H),2.35–2.28(m,5H),2.05(q,J=6.8Hz,8H),1.86(ddt,J=37.3,14.4,7.1 Hz, 3H), 1.77–1.38 (m, 12H), 1.39–1.19 (m, 34H), 1.15 (d, J = 6.7Hz, 3H), 0.94–0.79 (m, 12H). MS: 929.9m / z[M+H].

[0993] Example 81 – Compound 81

[0994] Intermediate 81a: 1-Bromo-6-methylheptane

[0995]

[0996] At 0°C, Br2 (1.33 equivalents) was added to a solution of PPh3 (66.97 g, 1.33 equivalents) in DCM (300 mL). Then, 6-methylhepta-1-ol (1.0 equivalent) was added in a single batch. The mixture was stirred at 0°C under a N2 atmosphere for 2 h. After completion, the reaction mixture was diluted with H2O, and the organic phase was separated. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Pentane was added, and the precipitated triphenylphosphine oxide was filtered off. The organic phase was concentrated under reduced pressure to obtain the residue, which was purified by column chromatography to give a product (30 g, 81%) as a yellow oil. 1 H NMR(400MHz, CDCl3)δ3.41(td,J=6.9,1.1Hz,2H),1.91–1.79(m,2H),1.59–1.49(m,1H) ,1.45–1.35(m,2H),1.34–1.25(m,3H),1.21–1.12(m,2H),0.86(dd,J=6.6,1.1Hz,6H).

[0997] Intermediate 81b: (6-methylheptyl)magnesium bromide

[0998]

[0999] Intermediate 81a (1.0 equivalent) was added to a solution of Mg (4.53 g, 1.2 equivalent) and I2 (0.1 equivalent) in THF (300 mL) at 15 °C. The mixture was stirred at 40 °C under a N2 atmosphere for 2 h. No further post-treatment was performed. The reaction mixture was used directly for the next step. Magnesium bromo(6-methylheptyl)bromo was obtained as a gray solution in THF.

[1000] Intermediate 81c: Ethyl 10-methyl-4-oxodecanoate

[1001]

[1002] Intermediate 81b (1.0 equivalent) was added to a solution of ethyl 4-chloro-4-oxobutyrate (25 g, 1.0 equivalent) and CuI (1.0 equivalent) in THF (200 mL) at -78 °C. The mixture was stirred at -78 °C under a N2 atmosphere for 5 h. The reaction mixture was then diluted with water and extracted three times with EtOAc. The combined organic layers were separated, washed twice with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a product (10 g, 27%) as a yellow oil.1 H NMR (400MHz, CDCl3) δ4.06(q,J=7.1Hz,2H),2.64(t,J=6.6Hz,2H),2.50(t,J=6.7Hz,2H),2.37(t,J=7.5Hz,2H), 1.51(q,J=7.5Hz,2H),1.43(dq,J=13.2,6.6Hz,1H),1.26–1.15(m,7H),1.14–1.04(m,2H),0.79(d,J=6.6Hz,6H).

[1003] Intermediate 81d: 10-methyl-4-oxoundecanoic acid

[1004]

[1005] The mixture of intermediate 81c (10 g, 1.0 equivalent) and KOH (3.0 equivalent) in EtOH (50 mL) was degassed and purged three times with N2, and then stirred at 50 °C under N2 atmosphere for 12 h. The mixture was concentrated to remove EtOH, and then adjusted to pH 5.5 with HCl (1 M). The reaction mixture was then diluted with water and extracted three times with EtOAc. The combined organic layers were separated, washed twice with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give the product (4 g, 45%) as a white solid. 1 H NMR (400MHz, CDCl3) δ2.65(t,J=6.1Hz,2H),2.56(ddd,J=7.1,5.9,1.2Hz,2H),2.37(t,J=7.5Hz,2H),1.51(q,J= 7.3Hz, 2H), 1.42 (dt, J = 13.2, 6.6Hz, 1H), 1.20 (tt, J = 4.9, 2.5Hz, 4H), 1.14–1.05 (m, 2H), 0.79 (d, J = 6.6Hz, 6H).

[1006] Intermediate 81e: 2-((10-methyl-4-oxoundecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1007]

[1008] Using the method employed with intermediate 1d, intermediate 81e was synthesized from intermediate 81d and intermediate 1c with a yield of 40%. 1H NMR (400MHz, CDCl3) δ5.39–5.20 (m, 8H), 5.17 (ddd, J=10.1, 5.8, 4.3Hz, 1H), 4.21 (dd, J= 11.9,4.3Hz,2H),4.07(dd,J=11.9,5.9Hz,2H),2.69(t,J=6.4Hz,4H),2.64(t,J=6.5Hz, 2H),2.52(t,J=6.5Hz,2H),2.36(t,J=7.5Hz,2H),2.24(t,J=7.5Hz,4H),1.97(q,J=6.8H z, 8H), 1.66–1.46 (m, 7H), 1.33–1.13 (m, 35H), 1.08 (q, J = 6.9Hz, 2H), 0.84–0.77 (m, 12H).

[1009] Intermediate 81f: 2-((10-methyl-4-(((4-nitrophenoxy)carbonyl)oxy)undecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1010]

[1011] NaBH4 (5.0 equivalents) was added to a solution of intermediate 81e (6 g, 1.0 equivalent) in THF (120 mL), toluene (30 mL), and H2O (60 mL) at 0 °C. The mixture was stirred at 5 °C for 5 h. After complete reduction, the reaction mixture was partitioned between THF and H2O, and the resulting organic phase was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give an intermediate alcohol as a yellow oil.

[1012] The resulting intermediate was immediately reconstituted in DCM (10 mL), and then pyridine (2.0 equivalents) and (4-nitrophenyl)chloroformate (2.0 equivalents) were added at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction mixture was then filtered and concentrated under reduced pressure to give a crude residue, which was purified by column chromatography to give a product (4.3 g, 59%) as a yellow oil. 1H NMR (400MHz, CDCl3) δ8.27–8.16(m,2H),7.37–7.29(m,2H),5.43–5.12(m,9H) ,4.80(s,1H),4.25(dd,J=11.9,4.4Hz,2H),4.14–4.06(m,2H),2.70(t,J=6.4H z,4H),2.40(t,J=7.5Hz,2H),2.24(td,J=7.5,4.1Hz,4H),2.05–1.88(m,9H), 1.71–1.62(m,1H),1.62–1.47(m,6H),1.45–0.97(m,35H),0.89–0.66(m,12H).

[1013] Compound 81: 2-((4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)-10-methylundecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1014]

[1015] Compound 81 was prepared from intermediate 81f and (2-aminoethyl)(ethyl)methylamine using the method employed in Example 1, with a yield of 85%. 1 H NMR (400MHz, CDCl3) δ5.42–5.21(m,9H),4.76(s,1H),4.33–4.23(m,2H),4.15(dt ,J=11.8,5.7Hz,2H),3.38(d,J=40.4Hz,3H),2.77(t,J=6.5Hz,4H),2.38(t,J=7. 7Hz,2H),2.32(td,J=7.6,1.1Hz,5H),2.05(q,J=6.7Hz,8H),1.96–1.78(m,3H),1 .68–1.43(m,12H),1.43–1.20(m,36H),1.14(d,J=6.6Hz,3H),0.93–0.80(m,12H). MS: 944.5m / z[M+H].

[1016] Example 82 – Compound 82

[1017] Compound 82: 2-((9-methyl-4-(((2-(pyrrolid-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1018]

[1019] Compound 82 was synthesized from intermediate 80e and 2-(pyrrolidone-1-yl)ethyl-1-amine using the method employed in Example 1, with a yield of 86%. 1 H NMR(400MHz, CDCl3)δ5.87(s,1H),5.43–5.23(m,9H),4.76(s,1H),4.28(ddd,J=12.0,4.5 ,2.6Hz,2H),4.15(ddd,J=11.9,5.8,4.6Hz,2H),3.52–3.42(m,2H),2.94(s,5H),2.82–2. 75(m,4H),2.39(ddd,J=8.4,6.9,2.0Hz,2H),2.36–2.29(m,4H),2.05(q,J=6.8Hz,8H),2. 01–1.76(m,7H),1.67–1.45(m,8H),1.40–1.21(m,33H),1.15(s,2H),0.93–0.81(m,12H). MS: 942.3m / z[M+H].

[1020] Example 83 – Compound 83

[1021] Compound 83: 2-((10-methyl-4-(((2-(pyrrolid-1-yl)ethyl)carbamoyl)oxy)undecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1022]

[1023] Compound 83 was synthesized from intermediate 81f and 2-(pyrrolidone-1-yl)ethyl-1-amine using the method employed in Example 1, with a yield of 87%. 1H NMR(400MHz, CDCl3)δ5.99(s,1H),5.45–5.19(m,9H),4.76(s,1H),4.28(ddd,J=11.9,4.5,3 .2Hz,2H),4.15(ddd,J=11.9,5.8,4.7Hz,2H),3.50(d,J=6.3Hz,2H),2.99(s,5H),2.81–2.73 (m,4H),2.39(ddd,J=8.2,6.9,1.8Hz,2H),2.35–2.28(m,4H),2.05(q,J=6.8Hz,8H),2.02–1. 77(m,7H),1.66–1.43(m,8H),1.41–1.20(m,35H),1.14(d,J=6.7Hz,2H),0.95–0.81(m,12H). MS: 956.5m / z[M+H].

[1024] Example 84 – Compound 84

[1025] Compound 84: 2-((4-(((1-methylazacyclobutane-3-yl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1026]

[1027] Compound 84 was prepared from intermediate 40d and 1-methylazacyclobutane-3-amine using the method employed in Example 1, with a yield of 87%. 1 H NMR (500MHz, CDCl3) δ5.44–5.28(m,8H),5.23(p,J=5.2Hz,1H),4.77(d,J=7.0Hz,1H),4.46(s,1H ),4.28(dt,J=10.9,5.2Hz,2H),4.16(ddd,J=8.7,5.6,2.8Hz,2H),3.99(s,2H),3.81(s,2H),2.7 7(t,J=6.7Hz,4H),2.65(s,2H),2.40–2.29(m,6H),2.05(q,J=7.0Hz,8H),1.93(dtd,J=15.1,7.6 ,3.8Hz,1H),1.83(q,J=7.4Hz,1H),1.66–1.44(m,7H),1.40–1.17(m,37H),0.88(q,J=7.0Hz,9H). MS: 900.4m / z[M+H].

[1028] Example 85 – Compound 85

[1029] Intermediate 85a: 4-hydroxybenzyl octanoate

[1030]

[1031] Add 5.63 g of NaOH (1.0 equivalent) to a solution of 20 g of 5-butyltetrahydrofuran-2-one (1.0 equivalent) in 50 mL of H₂O. Stir the mixture at 25 °C for 12 h. After completion, concentrate the reaction mixture under reduced pressure to remove H₂O.

[1032] The resulting residue was reconstituted in DMF (100 mL), and then BnBr (14.08 g, 1.0 equivalent) was added. The mixture was stirred at 25 °C for 2 h. The reaction mixture was then concentrated under reduced pressure to remove DMF. The residue was diluted with H2O (150 mL), extracted three times with EtOAc, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a product (15 g, 43%) as a yellow oil.

[1033] Intermediate 85b: 4-(((4-nitrophenoxy)carbonyl)oxy)benzyl octanoate

[1034]

[1035] Under N2 atmosphere, (4-nitrophenyl)chloroformate (32.21 g, 5.0 equivalent) was added to a mixture of intermediate 85a (15 g, 1.0 equivalent) in DCM (150 mL), and then pyridine (5.16 mL, 2.0 equivalent) was added to the mixture. The reaction mixture was stirred at 20 °C under N2 atmosphere for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with hexane and filtered. The filtrate was washed three times with H2O. The organic layer was washed once with brine, dried over Na2SO4 and filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a product (12 g, 48%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ8.31–8.17(m,2H),7.42–7.30(m,6H),5.13(s,2H),4.87(qd,J=7.6,4.8Hz,1H),2.50(t,J=7.4Hz,2H),2.11(dtd,J=15. 3,7.8,4.0Hz,1H),2.00(dt,J=14.7,7.6Hz,1H),1.82–1.69(m,1H),1.63(ddt,J=14.3,8.5,5.0Hz,1H),1.45–1.27(m,4H),1.01–0.83(m,3H).

[1036] Intermediate 85c: 4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)benzyl octanoate

[1037]

[1038] At 0 °C, DMAP (59 mg, 0.1 equivalent) was added to a solution of intermediate 85b (4 g, 2.0 equivalent) and 2-pyrrolidine-1-ylethylamine (550 mg, 1.0 equivalent) in MeCN (20 mL), followed by the addition of pyridine (1.17 mL, 3.0 equivalent). The mixture was stirred at 20 °C under a N2 atmosphere for 12 h. The reaction mixture was concentrated under reduced pressure to remove MeCN (20 mL). The residue was diluted with H2O and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a product (1.8 g, 48%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ7.32–7.23(m,5H),5.03(d,J=10.2Hz,3H),4.69(s,1H),3.23(dq,J=18.7,5.9Hz,3H),2.70(s,2H),2.60–2.40(m,7H),2.34 (td,J=8.3,7.6,2.7Hz,2H),1.87(d,J=10.5Hz,1H),1.70(t,J=3.8Hz,6H),1.44(d,J=24.4Hz,2H),1.22(q,J=11.9Hz,4H),0.80(t,J=4.2Hz,3H).

[1039] Compound 85: 2-((4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1040]

[1041] Under an Ar atmosphere, Pd / C (0.5 g, 10% w / w) was added to a solution of intermediate 85c (1.8 g, 1.0 equivalent) in THF (9 mL). The mixture was purged three times with H2, and then stirred for 5 h at 20 °C under an H2 atmosphere (15 Psi). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to remove THF.

[1042] The obtained crude material was dissolved in DCM (10 mL), and then DMAP (65 mg, 0.16 equivalents), DIPEA (1.74 mL, 3.0 equivalents), and EDCI (638 mg, 1.0 equivalents) were added. Then, intermediate 1c (2.05 g, 1.0 equivalents) was added at 0 °C. The mixture was stirred at 20 °C under a N2 atmosphere for 16 h. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with H2O and extracted twice with EtOAc. The combined organic layers were concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a product (1.1 g, 50%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.39–5.21(m,7H),5.18(p,J=5.0Hz,1H),5.09(s,1H),4.76–4.65(m,1H),4.22(dd,J =12.0,4.4Hz,2H),4.08(ddd,J=11.9,5.9,3.4Hz,2H),3.21(q,J=5.9Hz,2H),2.70(t,J=6.4Hz,4H),2.51(t ,J=6.1Hz,2H),2.43(d,J=5.8Hz,4H),2.32(ddd,J=8.9,6.6,3.9Hz,2H),2.25(t,J=7.6Hz,4H),1.98(q,J= 6.9Hz,7H),1.91–1.66(m,6H),1.63–1.35(m,8H),1.25(tq,J=7.2,4.6,3.6Hz,30H),0.82(t,J=6.7Hz,9H). MS: 900.4m / z[M+H].

[1043] Example 86 – Compound 86

[1044] Intermediate 86a: Ethyl 1-nonanoylcyclopropane-1-carboxylate

[1045]

[1046] A solution of magnesium bromo(octyl)magnesium (2M, 40.28 mL, 1.0 equivalent) was added dropwise to a solution of diethyl cyclopropane-1,1-dicarboxylate (15 g, 1.0 equivalent) in THF (200 mL) at -78 °C and N2. The reaction mixture was stirred for 5 h at -78 °C and N2. The reaction mixture was then poured into an aqueous solution of NH4Cl. The aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography in a separate batch to give a product (20 g, 48%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ4.13 (q, J = 7.1Hz, 2H), 2.75 (t, J = 7.4Hz, 2H), 1.57–1.49 (m, 2H), 1.35 (s, 4H), 1.21 (dd, J = 8.7, 5.5Hz, 16H), 0.81 (t, J = 6.9Hz, 6H).

[1047] Intermediate 86b: 1-Nonanoylcyclopropane-1-carboxylic acid

[1048]

[1049] Under N2 atmosphere, NaOH (2.36 g, 1.5 equivalent) and LiOH (1.41 g, 1.5 equivalent) were added to a solution of intermediate 86a (10 g, 1.0 equivalent) in EtOH (60 mL) and H2O (30 mL). The mixture was then stirred at 20 °C under N2 atmosphere for 12 h. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was adjusted to pH 3 by adding 2N HCl. The mixture was then stirred at 20 °C under N2 atmosphere for 1 h. The mixture was extracted three times with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under vacuum. The product (5 g, 28%) was collected as a colorless oil. 1 HNMR(400MHz, CDCl3) δ2.25(t,J=7.3Hz,2H),1.96(q,J=4.4Hz,2H),1.71(q,J =4.4Hz,2H),1.64–1.55(m,2H),1.26(d,J=3.7Hz,11H),0.87(t,J=6.8Hz,3H).

[1050] Intermediate 86c: 2-((1-nonanoylcyclopropane-1-carbonyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1051]

[1052] At 0 °C, Et3N (5.54 mL, 1.5 equivalent) was added to a solution of intermediate 86b (6 g, 1.0 equivalent), Int.1C (16.36 g, 1.0 equivalent), EDCI (6.10 g, 1.5 equivalent), and DMAP (324 mg, 0.1 equivalent) in DCM (60 mL). The mixture was stirred at 20 °C under a N2 atmosphere for 12 h. It was then poured into water and extracted three times with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under vacuum to obtain the residue. The residue was purified by column chromatography to give the product (8 g, 37%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.28(dtt,J=15.4,10.8,5.8Hz,8H),4.26(dd,J=12.1,4.1Hz,2H),4.08(dd,J=12.0,6.1Hz,2H),2.76(t,J=7.4Hz,2H),2.7 0(t,J=6.4Hz,4H),2.24(t,J=7.6Hz,4H),1.98(q,J=6.9Hz,8H),1.54(p, J=7.0Hz,9H),1.43–1.33(m,4H),1.33–1.15(m,40H),0.85–0.79(m,9H).

[1053] Intermediate 86d: 2-((1-(1-hydroxynonyl)cyclopropane-1-carbonyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1054]

[1055] A solution of intermediate 86c (8 g, 1.0 equivalent) in 4:2:1 THF / water / toluene (0.05–0.1 M) was cooled to 0–5 °C, and then NaBH4 (5.0 equivalent) was added. The reaction was maintained at 0–25 °C for at least 2 h. The reaction mixture was then diluted with water and EtOAc, and the resulting two-phase mixture was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (EtOAc / hexane) to give a product (4 g, 50%) as a colorless oil. 1H NMR(400MHz, CDCl3)δ5.33(tdq,J=20.3,10.0,5.6,4.9Hz,9H),4.28(dt,J=12.0,4.0Hz,2 H),4.15(ddd,J=12.1,8.0,6.0Hz,2H),3.18–3.12(m,1H),2.77(t,J=6.4Hz,4H),2.68(s, 1H),2.31(t,J=7.6Hz,4H),2.05(q,J=6.9Hz,8H),1.68–1.48(m,9H),1.40–1.16(m,43H), 0.98(ddd,J=10.7,6.7,4.1Hz,1H), 0.93–0.84(m,9H), 0.81(ddd,J=9.7,6.8,4.2Hz,1H).

[1056] Intermediate 86e: 2-((1-(1-(((4-nitrophenoxy)carbonyl)oxy)nonyl)cyclopropane-1-carbonyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1057]

[1058] At 0 °C, (4-nitrophenyl)chloroformate (1.95 g, 2.0 equivalent) and pyridine (781 μL, 2.0 equivalent) were added to a solution of intermediate 86d (4 g, 1.0 equivalent) in DCM (40 mL). The reaction mixture was then stirred at 20 °C under a N2 atmosphere for 5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was reconstituted in petroleum ether and filtered. The filtrate was washed with H2O. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a product (2.8 g, 59%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ8.25–8.16(m,2H),7.36–7.26(m,2H),5.27(tdd,J=17.5,8.5,4.0Hz,9H),4.84(dd, J=9.1,4.4Hz,1H),4.24(dt,J=11.9,3.8Hz,2H),4.08(dd,J=11.8,5.8Hz,2H),2.70(t,J=6.3Hz,4H),2.23 (t,J=7.6Hz,4H),2.02–1.93(m,8H),1.82(qd,J=9.4,4.9Hz,1H),1.69(ddt,J=14.4,10.0,5.1Hz,1H),1.5 3(t,J=7.2Hz,5H),1.35–1.16(m,41H),1.07–1.01(m,1H),0.95–0.89(m,1H),0.81(td,J=6.8,3.8Hz,9H).

[1059] Compound 86: 2-((1-(1-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)nonyl)cyclopropane-1-carbonyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1060]

[1061] Compound 86 was synthesized from intermediate 86e and N1-ethyl-N1-methylethane-1,2-diamine using the method employed in Example 1, with a yield of 90%. 1 H NMR (400MHz, CDCl3) δ5.53–5.22(m,9H),4.75(dd,J=8.7,4.5Hz,1H),4.29(ddd ,J=22.5,11.8,4.3Hz,2H),4.14(dd,J=11.9,5.9Hz,2H),3.30(s,2H),2.77(t,J =6.5Hz,4H),2.54(d,J=13.7Hz,4H),2.38–2.21(m,7H),2.05(q,J=6.7Hz,8H),1 .60(t,J=7.4Hz,4H),1.40–1.17(m,40H),1.12–1.00(m,4H),0.91–0.85(m,9H). MS: 956.1 m / z [M+H].

[1062] Example 87 – Compound 87

[1063] Intermediate 87a: 5-hydroxy-1-methyl-5-octylpyrrolidone-2-one

[1064]

[1065] At 15 °C and N2, magnesium bromo(octyl)magnesium (1 M, 265 mL, 1.5 equivalent) was added dropwise to a solution of 20 g, 1.0 equivalent, of 1-methylpyrrolidone-2,5-dione in 150 mL of THF. The mixture was stirred at 15 °C and N2 for 12 h. The reaction mixture was quenched at 0 °C by adding NH4Cl. It was then diluted with H2O and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give the product (20 g, 50%) as a white solid. 1 H NMR (400MHz, CDCl3) δ3.75(s,1H),2.68(s,3H),2.43(ddd,J=16.4,10.0,6.4Hz,1H),2.24(ddd,J=16.7,10.0,4.5Hz,1H),2.13(ddd,J=13.6,1 0.0, 6.4Hz, 1H), 1.93 (ddd, J=14.0, 10.0, 4.7Hz, 1H), 1.73 (td, J=13.4, 3.7Hz, 1H), 1.65–1.53 (m, 1H), 1.35–1.07 (m, 12H), 0.88–0.76 (m, 3H).

[1066] Intermediate 87b: 4-hydroxy-N-methyldodecanoic acid

[1067]

[1068] NaBH4 (4.99 g, 2.0 equivalent) was added fractionally to a solution of intermediate 87a (15 g, 1.0 equivalent) in MeOH (150 mL). The mixture was stirred at 15 °C for 1 h. The reaction mixture was quenched at 0 °C by adding NH4Cl, and then diluted with H2O and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give the product (8 g, 53%) as a white solid. 1 HNMR (400MHz, CDCl3) δ5.93 (s, 1H), 3.60–3.50 (m, 1H), 3.11 (s, 1H), 2.73 (d, J = 4.8Hz, 3H), 2.36–2.20 (m, 2H), 1.7 8(dddd,J=14.3,7.7,6.5,3.1Hz,1H),1.58(ddt,J=13.8,8.9,6.7Hz,1H),1.46–1.09(m,14H),0.87–0.77(m,3H).

[1069] Intermediate 87c: (R)-4-hydroxy-N-methyldodecanoic acid

[1070]

[1071] Novozym 435 (0.89 equivalents) and vinyl acetate (2.0 equivalents) were added to a solution of intermediate 87b (1.0 equivalents) in Et2O (0.2–1 M). The mixture was stirred at 40 °C for 96 h. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by column chromatography. The material collected from the column was subjected to reaction conditions at least three more times to give (R)-88c (24% yield) as a yellow solid. 1 H NMR (400MHz, CDCl3) δ5.57(s,1H),3.54(tt,J=7.7,3.6Hz,1H),2.74(d,J=4.8Hz,3H),2.64(d,J=4.6Hz,1H),2.28(td,J=6.9 ,3.2Hz,2H),1.79(dtd,J=14.3,7.0,3.1Hz,1H),1.60(ddd,J=14.3,8.8,6.8Hz,2H),1.47–1.12(m,14H),0.87–0.74(m,3H).

[1072] Intermediate 87d: (R)-5-octyldihydrofuran-2(3H)-one

[1073]

[1074] To a solution of intermediate 87d (1.0 equivalent) in MeOH (0.1–1.0 M), add NaOH (10 equivalent). Stir the mixture at 80 °C for at least 2 h, then add HCl (1–2 M, 10.0 equivalent) and stir the mixture at 25 °C for 1 h. The reaction mixture is then diluted with H₂O and extracted three times with EtOAc. The combined organic layers are concentrated under reduced pressure to give the residue. The residue is purified by column chromatography to give a product as a yellow oil. 1 H NMR (400MHz, CDCl3) δ4.41(p,J=6.9Hz,1H),2.46(dd,J=9.5,6.8Hz,2H),2.25(dq,J=13.4,6.7Hz,1H),1.7 8(dq,J=12.7,9.3Hz,1H),1.72–1.62(m,1H),1.60–1.47(m,1H),1.45–1.10(m,14H),0.81(t,J=6.6Hz,3H).

[1075] Intermediate 87e: (R)-4-hydroxydodecanoic acid

[1076]

[1077] Add NaOH (1.0–1.5 equivalents) to a solution of intermediate 87d (1.0 equivalent) in H₂O (0.1–0.5 M). Stir the mixture at 20 °C for at least 12 h. Concentrate the reaction mixture under reduced pressure to obtain a residue. The residue can be used directly in the next step without further purification.

[1078] Intermediate 88f: (R)-4-hydroxybenzyl dodecanoate

[1079]

[1080] Add BnBr (1.0–1.5 equivalents) and TBAB (0.05 equivalents) to a solution of intermediate 87e (1.0 equivalent) in acetone (0.3 M). Stir the mixture at 60 °C for at least 12 h. Concentrate the reaction mixture under reduced pressure to obtain a residue. Purify the residue (1) by column chromatography, or (2) dilute with saturated NaHCO3, extract three times with EtOAc, dry to Na2SO4, filter and concentrate, and then purify by column chromatography. Both methods yielded a product as a yellow oil (54% yield).

[1081] Intermediate 87g: (R)-4-(((4-nitrophenoxy)carbonyl)oxy)benzyl dodecanoate

[1082]

[1083] At 0–20 °C, pyridine (3.0 equivalents) was added to a solution of intermediate 87f (1.0 equivalents) and (4-nitrophenyl)chloroformate (3.0 equivalents) in DCM (0.3–1.2 M). The mixture was stirred at 20 °C for at least 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was (1) purified by column chromatography, or (2) used for the next step without further purification. The product (36%) was collected as a pale yellow oil. 1H NMR (400MHz, CDCl3) δ8.22–8.13(m,2H),7.29(d,J=2.0Hz,2H),5.06(s,2H),4.80(tt,J=7.6,4.5Hz,1H),2.43(t,J=7.5Hz,2H),2.04(ddt,J=15.3 ,7.8,3.9Hz,1H),1.92(dq,J=14.8,7.4Hz,1H),1.71–1.61(m,1H),1.55( ddd,J=14.3,9.1,5.2Hz,1H),1.36–1.13(m,11H),0.81(t,J=6.6Hz,3H).

[1084] Intermediate 87h: (R)-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)benzyl dodecanoate

[1085]

[1086] To a solution of 87 g (1.0 equivalent) of intermediate and N'-ethyl-N'-methyl-ethane-1,2-diamine (3.0 equivalent) in MeCN (0.1–0.2 M), pyridine (3.0 equivalent) and DMAP (1.0 equivalent) were added. The mixture was stirred at 20 °C for 12 h. The reaction mixture was then concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a product (88%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ7.60–7.23(m,5H),5.04(s,2H),4.77–4.64(m,1H),3.15(t,J=6.0Hz,2H),2.36(ddt,J=12.3,8.2,4.3H z,5H),2.13(s,3H),1.96–1.70(m,3H),1.55–1.33(m,2H),1.29–1.10(m,13H),0.96(t,J=7.1Hz,3H),0.80(t,J=6.7Hz,4H).

[1087] Compound 87: 2-(((R)-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1088]

[1089] Pd / C (10% w / w, 1.0 equivalent) was added to a solution of intermediate 87h (1.0 equivalent) in THF (0.1–0.3 M). The mixture was stirred at 15–20 °C and H2 (15 Psi) for 5 h. After completion, the reaction mixture was filtered and concentrated under reduced pressure to obtain the residue.

[1090] Add EDCI (1.0 equivalent), DMAP (0.1 equivalent), and Et3N (1.05 equivalent) to a solution of the residue and intermediate 1c (1.0 equivalent) in DCM (10 mL). Stir the mixture at 15–20 °C for at least 12 h. Concentrate the reaction mixture under reduced pressure to remove DCM. Dilute the residue with H2O and extract three times with EtOAc. Dry the combined organic layers over Na2SO4, filter, and concentrate the filtrate under reduced pressure to obtain the residue. Purify the residue by column chromatography to give a product (61%) as a pale yellow oil. 1 H NMR (400MHz, CDCl3) δ5.40–5.14(m,8H),5.08(s,1H),4.69(s,1H),4.21(dd,J=11.9,4.4Hz,2H ),4.08(ddd,J=12.0,5.9,3.3Hz,2H),3.17(q,J=6.0Hz,2H),2.70(t,J=6.4Hz,4H),2.43–2.28( m,5H),2.24(t,J=7.6Hz,4H),2.14(s,3H),1.98(q,J=6.8Hz,7H),1.79(ddd,J=42.6,15.1,7.5H z,3H),1.53(q,J=7.6Hz,8H),1.33–1.06(m,37H),0.97(t,J=7.1Hz,3H),0.81(q,J=6.3Hz,9H). MS: 944.1m / z[M+H].

[1091] Example 88 – Compound 88

[1092] Intermediate 88a: (S)-1-(methylamino)-1-oxododecane-4-ylacetate

[1093]

[1094] Using the method employed with intermediate 87c, intermediate 88a was synthesized from intermediate 87b with a yield of 23%. 1H NMR (400MHz, CDCl3) δ5.81(s,1H),4.79(tq,J=8.5,3.8Hz,1H),2.73(d,J=4.8Hz,3H),2.17–2.03(m,2 H), 1.98 (s, 3H), 1.95–1.63 (m, 3H), 1.56–1.36 (m, 2H), 1.19 (d, J = 8.4Hz, 11H), 0.81 (t, J = 6.8Hz, 3H).

[1095] Intermediate 88b: (S)-5-octyldihydrofuran-2(3H)-one

[1096]

[1097] Intermediate 88b is prepared from intermediate 88a using the same method as intermediate 87d.

[1098] Intermediate 88c: (S)-4-hydroxybenzyl dodecanoate

[1099]

[1100] Intermediate 88c is prepared from intermediate 88b using the same method employed with intermediates 87e and 87f.

[1101] Intermediate 88d: (S)-4-(((4-nitrophenoxy)carbonyl)oxy)benzyl dodecanoate

[1102]

[1103] Intermediate 88d was prepared from intermediate 88c using the method employed with intermediate 88g.

[1104] Intermediate 88e: (S)-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)benzyl dodecanoate

[1105]

[1106] Using the method employed with intermediate 87h, intermediate 88e was synthesized from intermediate 88e with a yield of 47%.

[1107] Compound 88: 2-(((S)-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1108]

[1109] Compound 88 was synthesized from intermediate 88d using the method employed in compound 87, with a yield of 42%.

[1110] Example 89 – Compound 89

[1111] Intermediate 89a: 4-hydroxy-N-methyldecanoamide

[1112]

[1113] AlMe3 (2M, 152.72mL, 2.08 equivalents) was added dropwise to a mixture of methylamine hydrochloride (19.83g, 2.0 equivalents) in DCM (250mL) at 0°C and N2. The mixture was stirred at 0°C and N2 for 2 h. This solution was then added dropwise to a solution of 5-hexyltetrahydrofuran-2-one (25g, 1.0 equivalents) in DCM (100mL) under N2. The mixture was stirred at 20°C and N2 for 12 h. The reaction mixture was quenched at 0°C by adding HCl (1N). The organic layer was separated and the aqueous layer was extracted three times with DCM. The combined organic phases were washed three times with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography to give the product (67%) as a white solid. 1 H NMR (400MHz, CD3OD) δ3.50(td,J=7.7,3.8Hz,1H),2.70(s,3H),2.29(ddt,J=25.9,16.6,6.8Hz,2H),1.77(dddd ,J=13.3,9.2,6.8,3.9Hz,1H),1.66–1.55(m,1H),1.42(h,J=4.0Hz,3H),1.39–1.25(m,8H),0.95–0.87(m,3H).

[1114] Intermediate 89b: (S)-1-(methylamino)-1-oxodecane-4-ylacetate

[1115]

[1116] Intermediate 89b was synthesized using the method employed in intermediate 87c, with a yield of 23%. 1HNMR(400MHz, CDCl3)δ5.70(s,1H),4.79(tt,J=8.4,4.4Hz,1H),2.74(d,J=4.8Hz,3H),2.11(dp,J=21.3,7.7,7.2Hz,2H),1.98(s,3H),1 .86(tdd,J=13.6,6.7,3.2Hz,1H),1.81–1.70(m,1H),1.45(ddp,J=19.0,13.6,6.6,6.1Hz,2H),1.27–1.12(m,8H),0.80(t,J=6.7Hz,3H).

[1117] Intermediate 89c: (S)-5-hexyldihydrofuran-2(3H)-one

[1118]

[1119] Using the method employed in intermediate 87d, intermediate 89b was synthesized from intermediate 89c with a yield of 78%. 1 H NMR(400MHz, CDCl3) δ4.48(ddd,J=13.7,7.5,5.6Hz,1H),2.52(dd,J=9.5,6.9Hz,2H),2.31(dq,J=13.4,6.7Hz,1 H),1.91–1.80(m,1H),1.80–1.68(m,1H),1.58(dq,J=13.8,5.2Hz,1H),1.51–1.21(m,8H),0.87(t,J=6.6Hz,3H).

[1120] Intermediate 89d: (S)-4-hydroxydecanoic acid benzyl ester

[1121]

[1122] Under N2 atmosphere, NaOH (1.1 equivalents) was added to a solution of intermediate 89c (3 g, 1.0 equivalents) in EtOH (25 mL) and H2O (5 mL). The mixture was then stirred at 20 °C under N2 atmosphere for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent.

[1123] The resulting residue was processed according to the method used in intermediate 87f to obtain a product (3g, 50%) that is a colorless oil.

[1124] Intermediate 89e: (S)-4-(((4-nitrophenoxy)carbonyl)oxy)benzyl decanoate

[1125]

[1126] Under N2 atmosphere, (4-nitrophenyl)chloroformate (3.26 g, 1.5 equivalent) was added to a solution of intermediate 89d (3 g, 1.0 equivalent) in DCM (30 mL). Then, pyridine (1.74 mL, 2.0 equivalent) was added to the mixture at 0 °C. The reaction mixture was stirred at 20 °C under N2 atmosphere for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a residue. The residue was diluted with hexane and filtered. The filtrate was washed three times with H2O. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give a product (2 g, 42%) as a colorless oil. 1 H NMR (400MHz, CD3OD) δ8.23–8.13(m,2H),7.37–7.18(m,7H),5.39(s,1H),5.03(s,2H),4.47(s,1H),4.00(q,J=7.1Hz,2H),2.42(t,J=7.1Hz,2H ), 2.00(ddt,J=15.0,7.6,3.8Hz,1H),1.85(dt,J=14.7,7.3Hz,1H),1.57(ddt,J=21.1,14.0,6.8Hz,2H),1.35–1.16(m,8H),0.91–0.75(m,3H).

[1127] Intermediate 89f: (S)-4-(((2-(pyrrolid-1-yl)ethyl)carbamoyl)oxy)benzyl decanoate

[1128]

[1129] Under N2 atmosphere, 2-pyrrolidine-1-ylethylamine (1.16 g, 3.0 equivalent), pyridine (819 μL, 3.0 equivalent), and DMAP (413 mg, 1.0 equivalent) were added to a solution of intermediate 89e (1.5 g, 1.0 equivalent) in MeCN (15 mL). The mixture was stirred at 20 °C under N2 atmosphere for 5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with EtOAc and washed 5 times with 1N NaHCO3 and 3 times with H2O. The organic layer was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a product (0.65 g, 41%) as a pale yellow oil. 1H NMR (400MHz, CD3OD) δ7.41–7.26(m,5H),5.11(d,J=1.1Hz,2H),4.73(dd,J=10.9,5.7Hz,1H),3.26(dd,J=6.7,1.7Hz, 2H),2.79–2.57(m,7H),2.41(t,J=7.4Hz,2H),1.99–1.74(m,7H),1.53(s,3H),1.36–1.24(m,9H),0.92–0.86(m,3H).

[1130] Compound 89: 2-(((S)-4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1131]

[1132] Intermediate 89f (1.0 equivalent) was added to a suspension of Pd / C (10% w / w, 1.0–3.0 equivalents) in THF (0.2–0.5 M). The mixture was stirred at 15–25 °C under a H2 atmosphere for at least 5 h. The reaction mixture was filtered and the filtrate was concentrated to give a residue. The residue was purified by column chromatography to give a product that was a viscous brown oil.

[1133] The brown oily substance was immediately reconstituted in DCM (0.2–0.5 M) at 0 °C and N2, followed by the addition of intermediate 1c (1.0 equivalent), EDCI (1.2 equivalent), DMAP (0.1–0.2 equivalent), and Et3N (1.5 equivalent). The mixture was stirred at 20 °C and N2 atmosphere for at least 12 h. The mixture was diluted with H2O and extracted three times with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography to give a product (23%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ5.49–5.22(m,8H),5.18(t,J=5.0Hz,1H),5.08(s,1H),4.69(s,1 H),4.22(dd,J=11.9,4.4Hz,2H),4.12–4.04(m,2H),3.28–3.15(m,2H),2.70(t,J=6.5 Hz,4H),2.50(d,J=6.4Hz,2H),2.44(s,3H),2.32(s,2H),2.25(t,J=7.6Hz,4H),1.98( q,J=6.9Hz,8H),1.83(s,1H),1.70(s,5H),1.38–0.94(m,37H),0.81(q,J=6.6Hz,9H). MS: 928.4m / z[M+H].

[1134] Example 90 – Compound 90

[1135] Intermediate 90a: (R)-4-hydroxydecanoic acid benzyl ester

[1136]

[1137] Intermediate 90a was synthesized from (5R)-5-hexyltetrahydrofuran-2-one using the method employed in intermediate 89d, with a yield of 27%.

[1138] Intermediate 90b: (R)-4-(((4-nitrophenoxy)carbonyl)oxy)benzyl decanoate

[1139]

[1140] Using the method employed in intermediate 89e, intermediate 90a was synthesized from intermediate 90b with a yield of 57%.

[1141] Intermediate 90c: (R)-4-(((2-(pyrrolid-1-yl)ethyl)carbamoyl)oxy)benzyl decanoate

[1142]

[1143] Intermediate 90c was synthesized from intermediate 90b using the method employed in intermediate 89f, with a yield of 47%.

[1144] Compound 90: 2-(((R)-4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1145]

[1146] Compound 90 was synthesized from intermediate 90c using the method employed in compound 89, with a yield of 27%.

[1147] Example 91 – Compound 91

[1148] Intermediate 91a: 4-Hydroxydodecanoic acid

[1149]

[1150] To a solution of 20 g (1.0 equivalent) of 5-octyltetrahydrofuran-2-one in 200 mL of H₂O, 4.03 g (1.0 equivalent) of NaOH was added. The mixture was stirred at 20 °C under a N₂ atmosphere for 12 h. The reaction mixture was concentrated under reduced pressure to remove H₂O. This yielded a crude product (14 g, 65%) as a white solid. 1 H NMR (400MHz, D2O) δ3.55(ddd,J=9.5,7.5,4.6Hz,1H),2.19(qdd,J=15.0,9.3,6.3Hz,2H),1.69(dddd,J=13.9,9.4,6. 4,4.4Hz,1H),1.59(dddd,J=14.0,9.2,7.7,6.4Hz,1H),1.46–1.30(m,3H),1.23(d,J=6.6Hz,11H),0.86–0.77(m,3H).

[1151] Intermediate 91b: 4-hydroxybenzyl dodecanoate

[1152]

[1153] BnBr (7.91 g, 1.0 equivalent) was added to a solution of intermediate 91a (10 g, 1.0 equivalent) in DMF (100 mL). The mixture was stirred at 20 °C under a N2 atmosphere for 2 h. The mixture was diluted with H2O and extracted twice with EtOAc. The combined organic layers were concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a product (5.7 g, 40%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ7.46–7.28(m,5H),5.12(s,2H),3.61(h,J=4.6Hz,1H),2.51(td,J=7.3,2.9Hz,2H),1.86(dtd,J= 14.6,7.4,3.5Hz,1H),1.76–1.59(m,3H),1.42(q,J=12.1,9.1Hz,4H),1.28(d,J=11.9Hz,12H),0.88(t,J=6.8Hz,3H).

[1154] Intermediate 91c: 4-(((4-nitrophenoxy)carbonyl)oxy)benzyl dodecanoate

[1155]

[1156] At 0 °C, pyridine (3.95 mL, 1.5 equivalent) was added to a solution of intermediate 91b (10 g, 1.0 equivalent) and (4-nitrophenyl)chloroformate (9.87 g, 1.05 equivalent) in DCM (100 mL). The mixture was stirred at 20 °C under a N2 atmosphere for 1 h. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with H2O and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a product (7.5 g, 49%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ8.30–8.21(m,2H),7.48–7.26(m,7H),5.22(d,J=69.6Hz,2H),4.87(tt,J=7 .5,4.5Hz,1H),2.59–2.42(m,2H),1.80–1.57(m,2H),1.52–1.16(m,13H),0.88(t,J=6.6Hz,3H).

[1157] Intermediate 91d: 4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)benzyl dodecanoate

[1158]

[1159] At 0°C, a diamine or amino alcohol (N',N'-dimethylethane-1,2-diamine (2.0-3.0 equivalents)), pyridine (3.0 equivalents), and DMAP (0.1 equivalents) were added to a solution of intermediate 91c (1.0 equivalents) in MeCN (0.1–0.5 M). The mixture was stirred at 15–25°C for at least 2 h. After completion, the reaction mixture was concentrated under reduced pressure, diluted with H2O, and extracted three times with EtOAc. The combined organic layers were concentrated to give a residue, which was purified by column chromatography to give a product (79%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ7.46–7.28(m,5H),5.08(s,1H),4.76(s,1H),3.23(q,J=5.9Hz,2H),2.40(ddt,J=12.0,8.9,4.5Hz,4H ), 2.21 (s, 7H), 1.94 (d, J = 9.9Hz, 1H), 1.82 (p, J = 7.6Hz, 1H), 1.61–1.40 (m, 3H), 1.39–1.16 (m, 14H), 0.87 (t, J = 6.7Hz, 3H).

[1160] Compound 91: 2-((4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1161]

[1162] Pd / C (10% w / w) was added to a solution of intermediate 91d in THF (0.1–0.5 M). The mixture was stirred at 25 °C and 15 Psi under a H2 atmosphere for at least 5 h. The resulting product was diluted in THF and filtered to remove solids. The filtrate was concentrated under vacuum. The residue was purified by column chromatography to give a carboxylic acid intermediate as a brown oil.

[1163] Compound 91 was then synthesized from the resulting oil using the method employed in intermediate 90f, with a yield of 15%. 1HNMR(400MHz, CDCl3)δ5.44–5.26(m,8H),5.27–5.20(m,1H),5.12(s,1H),4.75(s,1H),4.28(ddd,J =11.9,4.5,1.3Hz,2H),4.14(ddd,J=11.9,5.8,3.4Hz,2H),3.24(q,J=5.7Hz,2H),2.77(t,J=6.7Hz, 4H),2.44–2.35(m,4H),2.31(t,J=7.6Hz,4H),2.22(s,6H),2.04(q,J=6.9Hz,8H),1.97–1.84(m,2H ),1.80(dt,J=14.9,7.7Hz,1H),1.67–1.42(m,8H),1.42–1.14(m,39H),0.88(td,J=6.8,5.2Hz,9H). MS: 930.4m / z[M+H].

[1164] Example 92 – Compound 92

[1165] Intermediate 92a: 4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)benzyl dodecanoate

[1166]

[1167] Intermediate 92a was synthesized from intermediate 91c and 1-methylpiperidin-4-ol using the method employed in intermediate 91d, with a yield of 94%. 1 H NMR (400MHz, CDCl3) δ7.39–7.28(m,5H),5.11(s,2H),4.73(dtd,J=11.8,5.4 ,4.6,2.7Hz,1H),4.62(tt,J=8.6,4.2Hz,1H),2.68(s,2H),2.48–2.37(m,2H) ,2.29(s,5H),2.01–1.84(m,4H),1.78(dtt,J=12.5,8.2,3.5Hz,2H),1.67–1. 58(m,1H),1.58–1.46(m,1H),1.27(d,J=15.6Hz,13H),0.87(t,J=6.8Hz,3H).

[1168] Compound 92: 2-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)dodecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1169]

[1170] Compound 92 was synthesized from intermediate 92a using the method employed in Compound 91, with a yield of 28%. 1 H NMR (400MHz, CDCl3) δ5.39–5.21(m,8H),5.21–5.16(m,1H),4.65(d,J=6.9Hz,1H),4.57–4.52(m,1H), 4.22(dd,J=11.9,4.3Hz,2H),4.08(ddd,J=11.9,5.8,4.1Hz,2H),2.70(t,J=6.5Hz,4H),2.61(s,2H), 2.36–2.29(m,2H),2.29–2.18(m,7H),2.14(s,2H),1.98(q,J=6.8Hz,8H),1.93–1.84(m,4H),1.83–1. 77(m,1H),1.77–1.65(m,3H),1.54(t,J=7.3Hz,13H),1.33–1.12(m,41H),0.81(td,J=6.9,5.3Hz,9H). MS: 957.4m / z[M+H].

[1171] Example 93 – Compound 93

[1172] Intermediate 93a: 4-(((2-(azacyclobutane-1-yl)ethyl)carbamoyl)oxy)benzyl dodecanoate

[1173]

[1174] Intermediate 93a was synthesized from intermediate 91c and 2-(azacyclobutane-1-yl)ethylamine using the method employed in intermediate 92d, with a yield of 80%. 1 H NMR (400MHz, CDCl3) δ7.49–7.26(m,5H),5.11(s,2H),5.04(s,1H),4.74(s,1H ),3.20(t,J=7.0Hz,4H),3.11(q,J=5.8Hz,2H),2.50(t,J=5.9Hz,2H),2.41(d dd,J=8.6,6.7,3.2Hz,2H),2.10–2.00(m,2H),1.96–1.88(m,1H),1.82(d,J=1 5.4Hz, 4H), 1.46 (d, J = 6.9Hz, 3H), 1.34–1.18 (m, 14H), 0.87 (t, J = 6.8Hz, 3H).

[1175] Compound 93: 2-((4-(((2-(azacyclobutan-1-yl)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1176]

[1177] Compound 93 was synthesized from intermediate 93a using the method employed in Compound 91, with a yield of 18%. 1 H NMR (400MHz, CDCl3) δ5.36–5.22(m,8H),5.18(p,J=5.0Hz,1H),4.98(t,J=5.6Hz,1H),4.68(s,1H),4.22(d dd,J=11.9,4.5,1.6Hz,2H),4.08(ddq,J=11.7,5.8,2.8Hz,2H),3.12(t,J=7.0Hz,4H),3.05(q,J=5.9Hz,2H ),2.70(t,J=6.4Hz,4H),2.43(t,J=5.9Hz,2H),2.31(ddd,J=8.8,6.6,4.2Hz,2H),2.25(t,J=7.6Hz,4H),1 .99(dq,J=10.0,6.9Hz,10H),1.88–1.68(m,3H),1.59–1.36(m,7H),1.34–1.11(m,42H),0.86–0.77(m,9H). MS: 942.1m / z[M+H].

[1178] Example 94 – Compound 94

[1179] Intermediate 94a: 4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)benzyl dodecanoate

[1180]

[1181] Intermediate 94a was synthesized from intermediate 91c and 2-(piperidin-1-yl)ethyl-1-amine using the method employed in intermediate 91d, with a yield of 68%. 1H NMR (400MHz, CDCl3) δ7.38–7.28(m,5H),5.10(s,3H),4.75(s,1H),3.22(t,J=6.0Hz,2H),2.64(s,1H),2.45–2.29(m,8H), 1.99–1.88(m,1H),1.82(dq,J=15.0,8.0Hz,1H),1.48(dt,J=53.9,5.8Hz,9H),1.34–1.17(m,12H),0.86(t,J=6.7Hz,3H).

[1182] Compound 94: 2-((4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1183]

[1184] Compound 94 was synthesized from intermediate 94a using the method employed in Compound 91, with a yield of 22%. 1 H NMR (400MHz, CDCl3) δ5.29 (qq, J=11.7, 6.7Hz, 8H), 5.18 (p, J=5.1Hz, 1H), 5.08 (s, 1H), 4. 69(s,1H),4.22(ddd,J=11.9,4.5,2.2Hz,2H),4.08(ddd,J=11.9,5.9,3.6Hz,2H),3.18(q, J=5.9Hz,2H),2.70(t,J=6.5Hz,4H),2.39–2.18(m,12H),1.98(q,J=6.9Hz,8H),1.91–1.79 (m,1H),1.79–1.68(m,1H),1.61–1.33(m,15H),1.33–1.10(m,42H),0.81(q,J=6.4Hz,9H). MS: 970.2m / z[M+H].

[1185] Example 95 – Compound 95

[1186] Intermediate 95a: 4-(((4-(diethylamino)butoxy)carbonyl)oxy)benzyl dodecanoate

[1187]

[1188] Intermediate 95a was synthesized from intermediate 91c and 4-(diethylamino)but-1-ol using the method employed in intermediate 92d, with a yield of 83%. 1H NMR (400MHz, CDCl3) δ7.44–7.28(m,5H),5.11(s,2H),4.72(tdd,J=7.6,5.4,4.0Hz,1H),4.11( td,J=6.7,3.8Hz,2H),2.49(q,J=7.1Hz,4H),2.42(dtd,J=7.7,5.1,4.4,2.5Hz,4H),1.99(ddd d,J=14.4,8.9,7.0,4.1Hz,1H),1.89(dtd,J=14.7,8.4,6.5Hz,1H),1.64(ddt,J=17.4,10.5,6 .2Hz, 3H), 1.56–1.46 (m, 3H), 1.34–1.21 (m, 13H), 1.00 (t, J = 7.1Hz, 6H), 0.87 (t, J = 6.8Hz, 3H).

[1189] Compound 95: 2-((4-(((4-(diethylamino)butoxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1190]

[1191] Compound 95 was prepared from intermediate 95a using the method employed in compound 91, with a yield of 27%. 1 H NMR(400MHz, CDCl3)δ5.35(qdd,J=10.6,4.4,1.7Hz,8H),5.27–5.22(m,1H),4.71(tt,J=7.6,2.7Hz,1 H),4.28(dd,J=11.9,4.3Hz,2H),4.13(dtt,J=10.4,6.6,3.7Hz,4H),2.76(t,J=6.6Hz,4H),2.50(q,J= 7.2Hz, 4H), 2.41 (ddd, J=15.2, 8.0, 6.4Hz, 4H), 2.31 (t, J=7.6Hz, 4H), 2.04 (q, J=6.8Hz, 8H), 1.98–1. 82(m,3H),1.72–1.47(m,12H),1.39–1.21(m,42H),1.00(t,J=7.1Hz,6H),0.88(td,J=6.8,4.8Hz,9H). MS: 987.5m / z[M+H].

[1192] Example 96 – Compound 96

[1193] Intermediate 96a: 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)benzyl dodecanoate

[1194]

[1195] Using the method employed in intermediate 91d, intermediate 96a was synthesized from intermediate 91c and N',N'-diethylethane-1,2-diamine in 58% yield. 1 H NMR (400MHz, CDCl3) δ7.39–7.28(m,5H),5.19(s,1H),5.11(s,2H),4.76(s,1 H),3.51(s,2H),3.27–3.15(m,2H),2.54(dd,J=8.7,5.9Hz,6H),2.41(ddd,J =9.0,6.9,2.6Hz,2H),1.94(d,J=7.5Hz,1H),1.83(p,J=7.3Hz,1H),1.63–1. 40(m,3H),1.35–1.17(m,13H),1.01(t,J=7.1Hz,6H),0.87(t,J=6.8Hz,3H).

[1196] Compound 96: 2-((4-(((2-(diethylamino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1197]

[1198] Compound 96 was prepared from intermediate 96a using the method employed in compound 91, with a yield of 42%. 1H NMR (400MHz, CDCl3) δ5.29 (qq, J=11.4, 6.5Hz, 8H), 5.18 (p, J=5.2Hz, 1H), 5.08 (s, 1H), 4.69 (s, 1H) ,4.21(ddd,J=11.8,4.5,2.0Hz,2H),4.08(ddd,J=12.0,5.9,2.7Hz,2H),3.12(dt,J=13.0,6.3Hz,2 H),2.70(t,J=6.5Hz,4H),2.45(q,J=7.3Hz,6H),2.28(dt,J=27.2,8.0Hz,6H),1.98(q,J=6.9Hz,8H ),1.83(s,1H),1.78–1.68(m,1H),1.54(t,J=7.2Hz,9H),1.35–1.11(m,42H),0.81(q,J=6.3Hz,9H). MS: 958.4m / z[M+H].

[1199] Example 97 – Compound 97

[1200] Compound 97: 2-((5-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)tridecyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1201]

[1202] Compound 96 was prepared from intermediates 55d and 1c using the same method as compound 52, with a yield of 75%. 1 H NMR (400MHz, CDCl3) δ5.42–5.28(m,8H),5.27–5.22(m,1H),5.12(d,J=5.7Hz,1H),4.76–4.67(m ,1H),4.28(dt,J=11.9,4.5Hz,2H),4.13(ddd,J=12.0,5.9,4.5Hz,2H),3.23(h,J=7.2,6.2Hz,2H ),2.76(t,J=6.6Hz,4H),2.43(dt,J=14.5,6.6Hz,4H),2.35–2.27(m,6H),2.19(s,3H),2.04(q, J=6.8Hz,8H),1.71–1.46(m,13H),1.40–1.19(m,42H),1.03(t,J=7.1Hz,3H),0.92–0.83(m,9H). MS: 958.5m / z[M+H].

[1203] Example 98

[1204] Intermediate 98a: Ethyl 2,2-dimethyl-3-oxodecanoate

[1205]

[1206] At -78°C and N2, n-BuLi (2.5M, 56.60mL, 1.0 equivalent) was added to a solution of N-isopropylpropyl-2-amine (20.0mL, 1.0 equivalent) in THF (1-3M). The mixture was stirred at -78°C for 20 min. Then, ethyl 2-methylpropionate (18.96mL, 1.0 equivalent) was added at -78°C. The mixture was stirred again at -78°C for 20 min. Nonanoyl chloride (26.60mL, 1.0 equivalent) in THF (200mL) was added dropwise to the above solution. The mixture was warmed to 15°C and stirred at 15°C under N2 atmosphere for 12 h. The reaction mixture was quenched by adding ice water, filtered, and concentrated under reduced pressure to obtain the residue. The residue was diluted with H2O and extracted three times with EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to obtain a product (15 g, 41%) that was a yellow oil. 1 H NMR (400MHz, CDCl3) δ4.11 (q, J = 7.2Hz, 2H), 2.36 (t, J = 7.3Hz, 2H), 1.56–1.46 (m, 2H), 1.28 (s, 6H), 1.25–1.13 (m, 13H), 0.80 (t, J = 6.7Hz, 3H).

[1207] Intermediate 98b: 2,2-Dimethyl-3-oxoundecanoic acid

[1208]

[1209] The mixture of intermediate 98a (15 g, 1.0 equivalent), LiOH (2.10 g, 1.5 equivalent), and KOH (4.92 g, 1.5 equivalent) in EtOH (120 mL) and H₂O (40 mL) was degassed and purged three times with N₂, and then stirred at 40 °C under N₂ atmosphere for 3 h. The mixture was concentrated to remove EtOH. The pH was then adjusted to 5-6 with HCl (1 M). The mixture was partitioned between H₂O and EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a product (5 g, 37%) as a yellow oil. ¹H NMR (400 MHz, chloroform-d) δ 2.46 (t, J = 7.3 Hz, 2H), 1.58–1.47 (m, 2H), 1.33 (s, 6H), 1.20 (s, 10H), 0.81 (t, J = 6.6 Hz, 3H).

[1210] Example 99 – Compound 99

[1211] Intermediate 99a: 2-(((2,2-dimethyl-3-oxoundecanoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1212]

[1213] Intermediate 98b (4 g, 1.0 equivalent), DBAD (8.07 g, 2.0 equivalent), and PPh3 (9.19 g, 2.0 equivalent) were added to a solution of intermediate 53a (12.16 g, 1.1 equivalent) in THF (20 mL) at 0 °C. The mixture was stirred at 0 °C for 15 min. Then the mixture was stirred at 15 °C under a N2 atmosphere for 16 h. The reaction mixture was diluted with H2O and extracted three times with EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a product (3 g, 20%) as a yellow oil. 1H NMR (400MHz, CDCl3) δ5.27(dtt,J=10.8,8.7,6.1Hz,8H),4.10(d,J=6.0Hz,2H),4.03(d,J=6.0Hz,4H),2.70(t,J=6.4Hz,4H),2.37( t,J=7.3Hz,2H),2.23(t,J=7.6Hz,4H),1.98(q,J=6.8Hz,8H),1.52(tt,J=14.2,7.4Hz,8H),1.31–1.17(m,46H),0.85–0.77(m,12H).

[1214] Intermediate 99b: 2-(((3-hydroxy-2,2-dimethylundecyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1215]

[1216] At 0 °C, NaBH4 (5.0 equivalents) was added to a solution of intermediate 99a (1.0 equivalent) in a 4:2:1 THF:H2O:toluene (0.025–0.1). The mixture was stirred at 15 °C under a N2 atmosphere for 5 h. The reaction mixture was partitioned between THF and H2O, and the organic phase was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a product (33%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.41–5.25(m,8H),4.20–4.07(m,7H),3.63(dd,J=10.2,6.7Hz,1H),2.76(t,J=6.4Hz,4H),2.40(t,J=6.0Hz,2H),2. 30(t,J=7.6Hz,4H),2.04(q,J=6.8Hz,8H),1.59(q,J=7.3Hz,6H),1.40–1.22(m,40H),1.16(d,J=4.5Hz,6H),0.88(td,J=6.8,4.0Hz,9H).

[1217] Intermediate 99c: 2-(((2,2-dimethyl-3-(((4-nitrophenoxy)carbonyl)oxy)undecanoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1218]

[1219] Pyridine (191 μL, 2.0 equivalent) was added to a solution of intermediate 99b (1 g, 1.0 equivalent) and (4-nitrophenyl)chloroformate (478 mg, 2.0 equivalent) in DCM (10 mL) at 0 °C and N2. The mixture was stirred for 5 h at 15–25 °C under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a product (0.8 g, 67%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ8.20(d,J=8.8Hz,2H),7.30(d,J=8.9Hz,2H),5.29(dq,J=1 3.1,7.1,6.1Hz,8H),5.03(dd,J=10.4,2.3Hz,1H),4.08(dd,J=6.0,2.5Hz,6H),2 .69(t,J=6.4Hz,4H),2.35(p,J=6.0Hz,1H),2.23(q,J=8.4,7.7Hz,4H),1.97(q,J =6.9Hz,8H),1.53(h,J=7.1,5.9Hz,8H),1.36–1.12(m,56H),0.85–0.77(m,12H).

[1220] Compound 99: 2-(4,4,11-trimethyl-5-octyl-3,7-dioxo-2,6-dioxa-8,11-diazatetranyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadecyl-9,12-dienolate)

[1221]

[1222] At 0 °C, diamine (N'-ethyl-N'-methyl-ethane-1,2-diamine (3.0 equivalents)), DMAP (1.0 equivalents), and pyridine (3.0 equivalents) were added to a solution of intermediate 99c (1.0 equivalents) in MeCN (0.1 M). The mixture was stirred at 15 °C under a N2 atmosphere for at least 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O and extracted three times with EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a product (66%) as a yellow oil. 1H NMR (400MHz, CDCl3) δ5.28 (pt, J=11.5, 5.8Hz, 8H), 4.90 (t, J=6.4Hz, 1H), 4.11– 4.02(m,5H),3.17(dt,J=13.4,7.3Hz,2H),2.70(t,J=6.4Hz,4H),2.37(dq,J=12. 3,6.2Hz,5H),2.23(t,J=7.6Hz,4H),2.14(s,3H),1.98(q,J=6.8Hz,7H),1.54(p ,J=6.8Hz,4H),1.37–1.06(m,49H),0.97(t,J=7.1Hz,3H),0.81(q,J=6.9Hz,9H). MS: 973.5 m / z [M+H].

[1223] Example 100 – Compound 100

[1224] Intermediate 100a: Ethyl 2,2-dimethyl-3-oxononanoate

[1225]

[1226] Intermediate 100a was synthesized from N-isopropylpropyl-2-amine, ethyl 2-methylpropionate, and heptanyl chloride using the method employed in intermediate 99a, with a yield of 26%. 1 H NMR (400MHz, CDCl3) δ4.14 (q, J = 7.1Hz, 2H), 2.40 (t, J = 7.3Hz, 2H), 1.60–1.47 (m, 2H), 1.36–1.14 (m, 13H), 0.84 (t, J = 6.8Hz, 3H).

[1227] Intermediate 100b: 2,2-Dimethyl-3-oxononanoic acid

[1228]

[1229] Under a nitrogen atmosphere, a mixture of intermediate 100a (20 g, 1.0 equivalent), LiOH (3.15 g, 1.5 equivalent), and KOH (7.37 g, 1.5 equivalent) in EtOH (6 mL) and H2O (2 mL) was stirred for 4 h at 40 °C. The mixture was concentrated to remove EtOH. The pH was then adjusted to 5-6 with HCl (1 M). The mixture was partitioned between H2O and EtOAc and extracted twice with EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a product (6 g, 34%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ2.53 (t, J = 7.3Hz, 2H), 1.58 (p, J = 7.2Hz, 2H), 1.40 (s, 6H), 1.34–1.23 (m, 6H), 0.92–0.81 (m, 3H).

[1230] Intermediate 100c: 2-(((2,2-dimethyl-3-oxonanoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1231]

[1232] At 0 °C, PPh3 (14.41 g, 2.0 equivalents) and DBAD (12.65 g, 2.0 equivalents) and intermediate 100b (5.5 g, 1.0 equivalents) were added to a solution of intermediate 1c (19.06 g, 1.1 equivalents) in THF (50 mL). The mixture was stirred at 0 °C for 20 min, then warmed to 15 °C and stirred at 15 °C under a N2 atmosphere for 16 h. The reaction mixture was diluted with H2O and extracted three times with EtOAc. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give the product (4 g, 18%) as a yellow oil. 1 HNMR (400MHz, CDCl3) δ5.28(tt,J=11.1,5.4Hz,8H),4.10(d,J=6.0Hz,2H),4.05(dd,J=10.4,6.0Hz,4H),2.70(t,J=6.4Hz,4H),2.34(dt,J=17 .5,6.6Hz,3H),2.23(t,J=7.6Hz,4H),1.98(q,J=6.8Hz,8H),1.61–1.44 (m,8H),1.39(d,J=14.2Hz,3H),1.35–1.14(m,41H),0.85–0.76(m,9H).

[1233] Intermediate 100d: 2-(((3-hydroxy-2,2-dimethylnonanoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1234]

[1235] At 0 °C, NaBH4 (5.0 equivalents) was added to a solution of intermediate 100c (1.0 equivalent) in a 4:2:1 THF:H2O:toluene (0.025–0.1). The mixture was stirred at 5 °C under a N2 atmosphere for 5 h. The reaction mixture was partitioned between THF and H2O, and the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a product (20%) as a colorless oil.

[1236] Intermediate 100e: 2-(((2,2-dimethyl-3-(((4-nitrophenoxy)carbonyl)oxy)nonanoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1237]

[1238] At 0 °C, pyridine (119 μL, 3.0 equivalent) was added to a solution of intermediate 100d (0.4 g, 1.0 equivalent) and (4-nitrophenyl)chloroformate (890 mg, 9.0 equivalent) in DCM (5 mL). The mixture was stirred at 15 °C under N2 atmosphere for 3 h. Then the mixture was stirred at 25 °C under N2 atmosphere for 2 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a product (0.2 g, 42%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ8.21(dd,J=9.3,2.5Hz,2H),7.31(dd,J=9.4,2.8Hz,2H),5.43–5.16(m,8H),5.03(dd,J=10.3,2.3Hz,1H),4.12–3.99(m,6H),2. 70(t,J=6.4Hz,4H),2.35(h,J=6.0Hz,1H),2.22(t,J=7.6Hz,4H),1.97(t,J =6.9Hz,7H),1.63–1.47(m,6H),1.40–1.12(m,41H),0.82(t,J=6.7Hz,9H).

[1239] Compound 100: 2-(((2,2-dimethyl-3-(((2-(pyrrolid-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1240]

[1241] Compound 100 was synthesized from intermediate 100e and 2-pyrrolidine-1-ylethylamine using the method employed in compound 99, with a yield of 90%. 1 H NMR (400MHz, CDCl3) δ5.28 (tdq, J=17.2, 12.4, 5.8, 5.3Hz, 8H), 4.90 (t, J=6.4Hz, 1H), 4.13–4. 00(m,6H),3.22(ddq,J=19.5,12.8,6.3,5.8Hz,2H),2.70(t,J=6.4Hz,4H),2.51(t,J=6.3Hz,2 H),2.44(d,J=6.0Hz,4H),2.34(p,J=6.0Hz,1H),2.23(t,J=7.6Hz,4H),1.98(q,J=6.8Hz,8H), 1.70(h,J=7.5Hz,4H), 1.54(p,J=7.0Hz,4H), 1.41–1.06(m,44H), 0.81(dt,J=9.6,6.7Hz,9H). MS: 956.4m / z[M+H].

[1242] Example 101 – Compound 101

[1243] Compound 101: 2-((4-(((3-(azacyclobutane-1-yl)propoxy)carbonyl)oxy)decyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1244]

[1245] Compound 101 was synthesized from intermediate 40d and 3-(azacyclobutan-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 65%. 1H NMR (400MHz, CDCl3) δ5.45–5.28(m,8H),5.25(tt,J=5.8,4.3Hz,1H),4.71(ddd,J=11.5,7.6,4.7Hz,1 H),4.29(dd,J=11.9,4.3Hz,2H),4.15(dddd,J=13.3,7.2,5.5,3.2Hz,4H),3.18(t,J=7.0Hz,4H),2.8 2–2.72(m,4H),2.48(t,J=7.3Hz,2H),2.40(dt,J=8.8,6.8Hz,2H),2.31(td,J=7.6,1.2Hz,4H),2.10– 1.99(m,10H),1.99–1.84(m,2H),1.77–1.50(m,15H),1.42–1.21(m,37H),0.88(td,J=6.9,5.4Hz,9H). MS: 929.3m / z[M+H].

[1246] Example 102 – Compound 102

[1247] Compound 102: 2-((4-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)decanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1248]

[1249] Compound 102 was synthesized from intermediate 40d and 3-(piperidin-1-yl)prop-1-ol using the method employed in Example 1, with a yield of 72%. 1 H NMR(400MHz, CDCl3)δ5.44–5.28(m,8H),5.25(ddd,J=5.8,4.3,1.5Hz,1H) ,4.76–4.69(m,1H),4.37–4.24(m,2H),4.24–4.10(m,4H),2.82–2.71(m,4 H),2.48–2.26(m,12H),2.05(q,J=6.8Hz,8H),2.01–1.81(m,4H),1.71–1. 49(m,17H),1.43(s,2H),1.40–1.24(m,37H),0.88(td,J=6.9,5.0Hz,9H). MS: 957.1m / z[M+H].

[1250] Example 103 – Compound 103

[1251] Intermediate 103a: (2-Ethylbutyl)magnesium bromide

[1252]

[1253] 3-(bromomethyl)pentane (1.0 equivalent) was added dropwise to a solution of Mg (1.1 equivalent) and I₂ (0.1 equivalent) in THF (0.4–0.5 M). The mixture was stirred at 40 °C for at least 1 h. The reaction mixture could be used directly for the next step without further purification.

[1254] Intermediate 103b: Ethyl 6-ethyl-4-oxooctanoate

[1255]

[1256] At -78°C, CuI (1-2 equivalents) and intermediate 103a (1-2 equivalents) were added dropwise to a solution of ethyl 4-chloro-4-oxobutyrate (1.0 equivalent) in THF (0.3 M). The mixture was stirred at -78°C for at least 1.5 h. After completion, saturated NH4Cl was added, and the reaction was extracted three times with EtOAc. The organic layer was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give the product (82%) as a brown oil. 1 H NMR (400MHz, CDCl3) δ4.05(q,J=7.1Hz,2H),2.65(t,J=6.6Hz,2H),2.49(d,J=13.1Hz,2H),2 .30(d,J=6.8Hz,2H),1.82–1.71(m,J=6.5Hz,1H),1.33–1.13(m,7H),0.78(t,J=7.4Hz,6H).

[1257] Intermediate 103c: 6-Ethyl-4-oxooctanoic acid

[1258]

[1259] To a solution of intermediate 103c (1.0 equivalent) in 3:1 EtOH / H2O (0.2 M), add NaOH (1.5 equivalent) and LiOH (1.5 equivalent). Stir the mixture at 40 °C for at least 1 h. After completion, adjust the pH of the reaction to 3 using 1 M HCl, dilute the reaction mixture with H2O, and extract three times with EtOAc. Dry the organic layer with Na2SO4, filter, and concentrate under reduced pressure to obtain the residue. Purify the residue by column chromatography to give a product (46%) as a yellow oil. 1H NMR (400MHz, CDCl3) δ2.69–2.60(m,2H),2.56(t,J=6.6Hz,2H),2.30(d,J=6.7Hz,2 H), 1.75 (hept, J = 6.4Hz, 1H), 1.22 (qp, J = 13.8, 6.6Hz, 4H), 0.78 (t, J = 7.4Hz, 6H).

[1260] Intermediate 103d: 2-((6-ethyl-4-oxooctanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1261]

[1262] At 0 °C and N2, intermediate 1c (1.0 equivalent), EDCI (1.2 equivalent), DMAP (0.1–0.2 equivalent), and Et3N (1.5 equivalent) were added to a solution of intermediate 103c (1.0 equivalent) in DCM (0.2–0.5 M). The mixture was stirred at 20–25 °C under N2 atmosphere for at least 12 h. The mixture was diluted with H2O and extracted three times with EtOAc. The combined organic phases were dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography to give a product (40%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ5.40–5.21(m,8H),5.18(p,J=5.0Hz,1H),4.23(dt,J=11.9 ,5.0Hz,2H),4.08(dd,J=11.9,5.9Hz,2H),2.67(dt,J=20.4,6.5Hz,6H),2.51(t d,J=6.5,2.9Hz,2H),2.32–2.22(m,5H),1.98(q,J=6.8Hz,8H),1.75(p,J=6.4Hz ,1H),1.53(q,J=7.2Hz,4H),1.35–1.09(m,31H),0.80(dt,J=16.7,6.9Hz,12H).

[1263] Intermediate 103e: 2-((6-ethyl-4-(((4-nitrophenoxy)carbonyl)oxy)octanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1264]

[1265] Intermediate 103e was synthesized from intermediate 103d using the method employed in intermediate 1e, with a yield of 46%. 1 H NMR (400MHz, CDCl3) δ8.21(d,J=8.1Hz,2H),7.32(d,J=8.4Hz,2H),5.27(tt,J=14.1,7.8Hz,8H),4.89(p,J=7.4Hz,1H),4.32–4.02(m,4H),2.70( t,J=6.5Hz,4H),2.41(t,J=7.5Hz,2H),2.32–2.19(m,4H),1.98(q,J=7.2 Hz, 10H), 1.69–1.40 (m, 7H), 1.39–1.16 (m, 36H), 0.82 (t, J = 6.9Hz, 12H).

[1266] Compound 103: 2-((6-ethyl-4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)octanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

[1267]

[1268] Compound 103 was synthesized from intermediate 103e and N'-ethyl-N'-methyl-ethane-1,2-diamine using the method employed in intermediate 91d, with a yield of 98%. 1 H NMR (400MHz, CDCl3) δ5.42–5.21(m,8H),5.18(q,J=5.0Hz,1H),5.07(s,1H),4.79(s,1H),4.21(ddd,J=11.8,4 .4,1.8Hz,2H),4.08(ddd,J=11.9,5.8,3.4Hz,2H),3.17(d,J=6.2Hz,2H),2.70(t,J=6.4Hz,4H),2.44–2.27(m, 6H),2.24(t,J=7.6Hz,4H),2.13(s,3H),1.98(q,J=6.8Hz,8H),1.84(dq,J=8.4,4.4Hz,1H),1.71(dd,J=14.8, 7.5Hz, 1H), 1.58–1.43 (m, 8H), 1.29–1.16 (m, 38H), 0.96 (d, J = 7.2Hz, 3H), 0.80 (ddd, J = 13.4, 6.7, 3.7Hz, 13H). MS: 916.3m / z[M+H].

[1269] Example 104

[1270] Intermediate 104a: (3,7-dimethyloctyl)magnesium bromide

[1271]

[1272] Intermediate 104a was synthesized from 1-bromo-3,7-dimethyl-octane using the method employed in intermediate 104a.

[1273] Intermediate 104b: Ethyl 7,11-dimethyl-4-oxododecanoate

[1274]

[1275] Intermediate 104b was synthesized from intermediate 104a using the method employed in intermediate 103b, with a yield of 66%. 1 HNMR (400MHz, CDCl3) δ4.11(qd,J=7.1,2.5Hz,2H),2.71(t,J=6.6Hz,2H),2.56(t,J=6.6Hz,2H),2.49–2.35(m,2H),1.68–1.54(m,1H),1.49( dq,J=13.2,6.6Hz,1H),1.43–1.31(m,2H),1.24(td,J=8.0,7.1,3.6Hz,6H),1.10(dddd,J=16.9,9.9,4.7,2.0Hz,3H),0.84(d,J=6.5Hz,9H).

[1276] Intermediate 104c: 7,11-dimethyl-4-oxododecanoic acid

[1277]

[1278] Intermediate 104c was synthesized from intermediate 104b using the method employed in intermediate 103c, with a yield of 51%. 1 HNMR (400MHz, CDCl3) δ2.72(t,J=6.1Hz,2H),2.63(t,J=6.5Hz,2H),2.50–2.36(m,2H),1.61(ddt,J=11.8,9.0,4.3Hz,1H),1.51(h ept,J=6.6Hz,1H),1.45–1.34(m,2H),1.24(ddt,J=16.2,13.5,4.2Hz...

Claims

1. A compound of formula IA; in, Independently at each occurrence, X 1 C 1-3 Alkylene or X 2 is selected from O, NH, NMe and a bond, X 3 C 2-4 Alkylene, X 4 is a C1 alkylene group or a bond, X 5 is a C1 alkylene group or a bond, R 1 C 1-3 alkyl, R 2 C 1-3 Alkyl, or R 2 With nitrogen atoms and R 1 or X 3 The carbon atoms of Y 1 is selected from a bond, -CH=CH-, -(C=O)O- and -O(C=O)-, Y 2 Selected from -CH2-CH=CH- and C3-C4 alkylene, R 3 Selected from H, C 5-7 Cycloalkyl, C8-C 10 Alkenyl and C 3-18 alkyl, R 4 C 4-8 alkyl, or a salt thereof, The condition is that when R 2 For Me, R 3 For straight chain C 12 Alkyl, X 1 is C2 alkylene, X 2 O, X 3 is C3 alkylene, X 4 is the key, X 5 is C1 alkylene, Y 1 is cis-CH=CH-, and -Y 2 –R 4 When it is cis-CH2-CH=CH-(C5-alkyl), then R 1 C 2-3 alkyl.

2. The compound of claim 1, wherein the compound is a compound of formula I: in, Independently at each occurrence, X 1 C 2-3 Alkylene, X 2 is selected from O, NH, NMe and a bond, X 3 C 2-4 Alkylene, X 4 is a C1 alkylene group or a bond, X 5 is a C1 alkylene group or a bond, R 1 C 1-2 alkyl, R 2 C 1-2 Alkyl, or R 2 With nitrogen atoms and R 1 or X 3 The carbon atoms of the Y 1 is selected from -CH=CH-, -(C=O)O- and -O(C=O)-, R 3 Selected from H, C 5-7 Cycloalkyl and C 3-18 alkyl, R 4 C 4-7 alkyl, or a salt thereof, The condition is that when R 2 For Me, R 3 For straight chain C 12 Alkyl, X 1 is C2 alkylene, X 2 O, X 3 is C3 alkylene, X 4 is the key, X 5 is C1 alkylene, Y 1 is cis-CH=CH-, and R 4 When it is a C5 alkyl group, R 1 It is a C2 alkyl group.

3. The compound of claim 1 or 2, wherein the compound is enantiomerically enriched or diastereomerically enriched.

4. A compound as claimed in any one of the preceding claims, wherein X 1 It is a C2 alkylene group.

5. The compound as claimed in claim 1, wherein X 1 It is a C1 alkylene group.

6. The compound of any one of claims 1 to 3, wherein X 1 It is a straight chain C3 alkylene group.

7. A compound as described in any one of claims 1 to 3, wherein X 1 It is a branched C3 alkylene group.

8. A compound as claimed in any one of the preceding claims, wherein X 2 For NH.

9. The compound of any one of claims 1 to 7, wherein X 2 is O.

10. The compound of any one of claims 1 to 7, wherein X 2 For NMe.

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