Ionizable amine lipids and lipid nanoparticles

Abstract

To provide ionizable amine lipids and salts thereof useful for the delivery of biologically active agents, e.g., delivering biologically active agents to cells to prepare engineered cells.SOLUTION: An ionizable amine lipid is represented by the general formula in the figure.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 838,551, filed April 25, 2019, and U.S. Provisional Patent Application No. 62 / 843,854, filed May 6, 2019, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Lipid nanoparticles formulated with ionizable amine-containing lipids can serve as cargo vehicles for delivering biologically active 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 gene editing components and compositions into living cells. Biologically active agents that are particularly difficult to deliver into cells include proteins, nucleic acid-based drugs, and their derivatives, particularly drugs containing relatively large oligonucleotides such as mRNA. Of particular interest are compositions for delivering promising gene editing technologies into cells, such as CRISPR / Cas9 system components (e.g., mRNAs encoding nucleases and associated guide RNAs (gRNAs)).

[0003] There is a need for compositions for delivering CRISPR / Cas protein and nucleic acid components to cells, such as patient cells. In particular, compositions for delivering mRNA encoding CRISPR protein components and for delivering CRISPR gRNA are of particular interest. Compositions that can stabilize and deliver RNA components and have useful properties for in vitro and in vivo delivery are also of particular interest. Summary of the Invention

[0004] The present disclosure provides amine-containing lipids useful for formulating lipid nanoparticle (LNP) compositions that may have advantageous properties for delivering nucleic acid cargo, such as CRISPR / Cas gene editing components, into cells.

[0005] In some embodiments, the lipid is a compound having the structure of Formula II, [ka] During the ceremony, X 1 But, O, NR 1 , or a direct bond, X 2 But C 2-5 is alkylene, X 3 is C(=O) or a direct bond, R 1 is H or Me, R 3 But C 1-3 is alkyl, R 2 But C 1-3 is alkyl, or R 2 The nitrogen atom to which it is bonded, and X 2 together with 1 to 3 carbon atoms of the formula (I) to form a 4-, 5-, or 6-membered ring; or X 1 But NR 1 , R 1 , and R 2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R 2 But R 3 and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y 1 But C 2-12 is alkylene, Y 2 but, [ka] is selected from n is 0 to 3, R 4 But C 1-15 is alkyl, Z 1 But C 1-6 is an alkylene or a direct bond, Z 2 but, [ka] (in either direction) or absent, except that Z 1 If is a direct bond, Z 2 is assumed not to exist, R 5 But C 5-9 Alkyl or C 6-10 is an alkoxy, R 6 But C 5-9 Alkyl or C 6-10 is an alkoxy, W is methylene or a direct bond; R 7 is H or Me, However, R 3 and R 2 is a C2 alkyl, and X 1 is O and X 2 is a linear C3 alkylene, and X 3 is C(=O) and Y 1 is a linear C6 alkylene, (Y 2 ) n -R 4 but, [ka] and R 4 is a linear C5 alkyl, and Z 1 is a C2 alkylene, and Z 2 is absent, W is methylene, and R 7 If H, then R 5 and R 6 is not C8 alkoxy, or a salt thereof.

[0006] In certain embodiments, the lipid is a compound having the structure of formula (I): [ka] X 1 But, O, NR 1 , or a direct bond, X 2 But C 2-5 is alkylene, X 3 is C(=O) or a direct bond, R 1 is H or Me, R 3 But C 1-3 is alkyl, R 2 But C 1-3 is alkyl, or R 2 The nitrogen atom to which it is bonded, and X 2 together with 1 to 3 carbon atoms of the formula (I) to form a 4-, 5-, or 6-membered ring; or X 1 But NR 1 , R 1 , and R 2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R 2 But R 3 and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y 1 But C 2-12 is alkylene, Y 2 but, [ka] is selected from R 4 But C 3-15 is alkyl, Z 1 But C 1-6 is an alkylene or a direct bond, Z2 but, [ka] (in either direction) or absent, except that Z 1 If is a direct bond, Z 2 is assumed not to exist, R 5 But C 5-9 Alkyl or C 6-10 is an alkoxy, R 6 But C 5-9 Alkyl or C 6-10 is an alkoxy, W is methylene or a direct bond; R 7 is H or Me, However, R 3 and R 2 is a C2 alkyl, and X 1 is O and X 2 is a linear C3 alkylene, and X 3 is C(=O) and Y 1 is a linear C6 alkylene, and Y 2 but, [ka] and R 4 is a straight chain C4 alkyl, and Z 1 is a C2 alkylene, and Z 2 is absent, W is methylene, and R 7 If H, then R 5 and R 6 is not C8 alkoxy, or a salt thereof.

[0007] In certain embodiments, the present invention relates to any compound described herein, wherein the pKa of the protonated form of the compound is from about 5.1 to about 9.0, e.g., from about 5.7 to about 7.6, or from about 6 to about 7.5.

[0008] In certain embodiments, the present invention relates to a composition comprising any compound described herein and a lipid component, e.g., about 50% (e.g., about 50% of the lipid component) of a compound described herein and a lipid component, e.g., an amine lipid, preferably a compound of Formula (I) or Formula (II).

[0009] In certain embodiments, the present invention relates to any composition described herein, wherein the composition is an LNP composition. For example, the present invention relates to an LNP composition comprising any compound described herein and a lipid component. In certain embodiments, the present invention relates to any LNP composition described herein, wherein the lipid component comprises a helper lipid and a PEG-lipid. In certain embodiments, the present invention relates to any LNP composition described herein, wherein the lipid component comprises a helper lipid, a PEG-lipid, and a neutral lipid. In certain embodiments, the present invention relates to any LNP composition described herein, further comprising a cryoprotectant. In certain embodiments, the present invention relates to any LNP composition described herein, further comprising a buffer.

[0010] In certain embodiments, the present invention relates to any LNP composition described herein, further comprising a nucleic acid component. In certain embodiments, the present invention relates to any LNP composition described herein, further comprising an RNA or DNA component. In certain embodiments, the present invention relates to any LNP composition described herein, wherein the LNP composition has an N / P ratio of about 3 to 10, e.g., an N / P ratio of about 6±1, or an N / P ratio of about 6±0.5. In certain embodiments, the present invention relates to any LNP composition described herein, wherein the LNP composition has an N / P ratio of about 6.

[0011] In certain embodiments, the invention relates to any of the LNP compositions described herein, wherein the RNA component comprises an mRNA. In certain embodiments, the invention relates to any of the LNP compositions described herein, wherein the RNA component comprises an RNA-guided DNA-binding agent, e.g., a Cas nuclease mRNA, such as a class 2 Cas nuclease mRNA or a Cas9 nuclease mRNA.

[0012] In certain embodiments, the present invention relates to any LNP composition described herein, wherein the mRNA is a modified mRNA. In certain embodiments, the present invention relates to any LNP composition described herein, wherein the RNA component comprises a gRNA nucleic acid. In certain embodiments, the present invention relates to any LNP composition described herein, wherein the gRNA nucleic acid is a gRNA.

[0013] In certain embodiments, the invention relates to any LNP composition described herein, wherein the RNA component comprises a Class 2 Cas nuclease mRNA and a gRNA. In certain embodiments, the invention relates to any LNP composition described herein, wherein the gRNA nucleic acid is or encodes a dual guide RNA (dgRNA). In certain embodiments, the invention relates to any LNP composition described herein, wherein the gRNA nucleic acid is or encodes a single guide RNA (sgRNA).

[0014] In certain embodiments, the present invention relates to any LNP composition described herein, wherein the gRNA is a modified gRNA. In certain embodiments, the present invention relates to any LNP composition described herein, wherein the modified gRNA comprises a modification in one or more of the first five nucleotides at the 5' end. In certain embodiments, the present invention relates to any LNP composition described herein, wherein the modified gRNA comprises a modification in one or more of the last five nucleotides at the 3' end.

[0015] In certain embodiments, the present invention relates to any of the LNP compositions described herein, further comprising at least one template nucleic acid.

[0016] In certain embodiments, the present invention relates to methods of gene editing comprising contacting a cell with an LNP, hi certain embodiments, the present invention relates to any method of gene editing described herein comprising cleaving DNA.

[0017] In certain embodiments, the present invention relates to a method of cleaving DNA comprising contacting a cell with an LNP composition. In certain embodiments, the present invention relates to any method of cleaving DNA described herein, wherein the cleaving step comprises introducing a single-stranded DNA nick. In certain embodiments, the present invention relates to any method of cleaving DNA described herein, wherein the cleaving step comprises introducing a double-stranded DNA break. In certain embodiments, the present invention relates to any method of cleaving DNA described herein, wherein the LNP composition comprises a Class 2 Cas mRNA and a gRNA nucleic acid. In certain embodiments, the present invention relates to any method of cleaving DNA described herein, further comprising introducing at least one template nucleic acid into the cell. In certain embodiments, the present invention relates to any method of cleaving DNA described herein, comprising contacting a cell with an LNP composition comprising a template nucleic acid.

[0018] In certain embodiments, the invention relates to any of the methods of gene editing described herein, comprising administering an LNP composition to an animal, e.g., a human. In certain embodiments, the invention relates to any of the methods of gene editing described herein, comprising administering an LNP composition to a cell, such as a eukaryotic cell.

[0019] In certain embodiments, the invention relates to any method of gene editing described herein, comprising administering mRNA formulated in a first LNP composition and a second LNP composition comprising one or more of mRNA, gRNA, gRNA nucleic acid, and template nucleic acid. In certain embodiments, the invention relates to any method of gene editing described herein, wherein the first and second LNP compositions are administered simultaneously. In certain embodiments, the invention relates to any method of gene editing described herein, wherein the first and second LNP compositions are administered sequentially. In certain embodiments, the invention relates to any method of gene editing described herein, comprising administering mRNA and gRNA nucleic acid formulated in a single LNP composition.

[0020] In certain embodiments, the invention relates to any of the methods of gene editing described herein, wherein the gene editing results in a gene knockout.

[0021] In certain embodiments, the invention relates to any of the methods of gene editing described herein, wherein the gene editing results in a gene correction. [Brief explanation of the drawings]

[0022] [Figure 1A] 1 is a graph showing the percentage of TTR editing in mouse liver cells after delivery using LNPs containing Compound 1, Compound 29, Compound 31, or Compound 32. Dose-response data is also shown. See Example 122. [Figure 1B] 1 is a graph showing the percentage of TTR editing in mouse liver cells after delivery using LNPs containing Compound 1, Compound 40, Compound 53, or Compound 54. Dose-response data is also shown. See Example 122. [Figure 1C] 1 is a graph showing the percentage of B2M and TTR editing in mouse liver cells after delivery using LNPs containing Compound 1 or Compound 59. Dose-response data is also shown. See Example 122. [Figure 1D] 1 is a graph showing the percentage of TTR editing in mouse liver cells after delivery using LNPs containing Compound 1, Compound 24, Compound 59, Compound 61, or Compound 94. Dose-response data is also shown. See Example 122. [Figure 2] 1 is a graph showing the percentage of TTR editing in mouse liver cells after delivery using LNPs containing Compound 1, Compound 100, Compound 101, Compound 102, or Compound 103. See Example 123. [Figure 3] 12 is a graph showing the percentage of TTR editing in mouse liver cells after delivery using LNPs containing Compound 1, Compound 114, Compound 115, or Compound 116. See Example 123. [Figure 4] 1 is a graph showing the percentage of TTR editing in rat liver cells after delivery using LNPs containing Compound 1, Compound 12, Compound 59, or Compound 94. Dose-response data is shown. See Example 125. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure provides lipids, particularly ionizable lipids, useful for delivering biologically active agents, including nucleic acids such as CRISPR / Cas component RNA ("cargo"), to cells, as well as methods for preparing and using such compositions. The lipids and pharmaceutically acceptable salts thereof are optionally provided as lipid-containing compositions, including LNP compositions. In certain embodiments, the LNP compositions may comprise a lipid component comprising a biologically active agent, e.g., an RNA component, and a compound of Formula (II) or (I), as defined herein. In certain embodiments, the RNA component comprises RNA. In some embodiments, the lipids are used to deliver biologically active agents, e.g., mRNA, to cells, such as liver cells. In certain embodiments, the RNA component comprises a gRNA and, optionally, an mRNA encoding a class 2 Cas nuclease. Methods of gene editing and generating engineered cells using these compositions are also provided.

[0024] Lipid Nanoparticle Composition Disclosed herein are various LNP compositions for delivering biologically active agents, such as nucleic acids, e.g., mRNA and gRNA, including CRISPR / Cas cargo. Such LNP compositions include "ionizable amine lipids" along with neutral lipids, PEG lipids, and helper lipids. "Lipid nanoparticle" or "LNP" refers, without limitation, to a particle comprising multiple (i.e., two or more) LNP components physically bound to each other by intermolecular forces.

[0025] lipids The present disclosure provides lipids that can be used in LNP compositions. In some embodiments, the lipid is a compound having the structure of Formula II: [ka] During the ceremony, X 1 But, O, NR 1 , or a direct bond, X 2 But C 2-5 is alkylene, X 3 is C(=O) or a direct bond, R 1 is H or Me, R 3 But C 1-3 is alkyl, R 2 But C 1-3 is alkyl, or R 2 The nitrogen atom to which it is bonded, and X 2 together with 1 to 3 carbon atoms of the formula (I) to form a 4-, 5-, or 6-membered ring; or X 1 But NR 1 , R 1 , and R 2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R 2 But R 3 and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y 1 But C 2-12 is alkylene, Y 2 but, [ka] is selected from n is 0 to 3, R 4 But C 1-15 is alkyl, Z 1 But C1-6 is an alkylene or a direct bond, Z 2 but, [ka] (in either direction) or absent, except that Z 1 If is a direct bond, Z 2 is assumed not to exist, R 5 But C 5-9 Alkyl or C 6-10 is an alkoxy, R 6 But C 5-9 Alkyl or C 6-10 is an alkoxy, W is methylene or a direct bond; R 7 is H or Me, However, R 3 and R 2 is a C2 alkyl, and X 1 is O and X 2 is a linear C3 alkylene, and X 3 is C(=O) and Y 1 is a linear C6 alkylene, (Y 2 ) n -R 4 but, [ka] and R 4 is a linear C5 alkyl, and Z 1 is a C2 alkylene, and Z 2 is absent, W is methylene, and R 7 If H, then R 5 and R 6 is not C8 alkoxy, or a salt thereof.

[0026] In some embodiments, n is 1 to 3, for example, n is 1. In certain embodiments, n is 2. In some embodiments, n is 3.

[0027] In certain embodiments, the lipid is a compound having the structure of formula (I): [ka] X 1 But, O, NR 1 , or a direct bond, X 2 But C 2-5 is alkylene, X 3 is C(=O) or a direct bond, R 1 is H or Me, R 3 But C 1-3 is alkyl, R 2 But C 1-3 is alkyl, or R 2 The nitrogen atom to which it is bonded, and X 2 together with 1 to 3 carbon atoms of the formula (I) to form a 4-, 5-, or 6-membered ring; or X 1 But NR 1 , R 1 , and R 2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R 2 But R 3 and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y 1 But C 2-12 is alkylene, Y 2 but, [ka] is selected from R 4 But C 3-15 is alkyl, Z 1 But C 1-6 is an alkylene or a direct bond, Z2 but, [ka] (in either direction) or absent, except that Z 1 If is a direct bond, Z 2 is assumed not to exist, R 5 But C 5-9 Alkyl or C 6-10 is an alkoxy, R 6 But C 5-9 Alkyl or C 6-10 is an alkoxy, W is methylene or a direct bond; R 7 is H or Me, However, R 3 and R 2 is a C2 alkyl, and X 1 is O and X 2 is a linear C3 alkylene, and X 3 is C(=O) and Y 1 is a linear C6 alkylene, and Y 2 but, [ka] and R 4 is a straight chain C4 alkyl, and Z 1 is a C2 alkylene, and Z 2 is absent, W is methylene, and R 7 If H, then R 5 and R 6 is not C8 alkoxy (e.g., the compound is not Compound 1), or a salt thereof.

[0028] Preferably, the compound is a compound having the structure of formula (I), except that R 3 and R 2 is a C2 alkyl, and X 1 is O and X 2 is a linear C3 alkylene, and X 3is C(=O) and Y 1 is a linear C6 alkylene, and Y 2 but, [ka] and R 4 is a straight chain C4 alkyl, and Z 1 is a C2 alkylene, and Z 2 is absent, W is methylene, and R 7 If H, then R 5 and R 6 But C 6-10 It is not alkoxy.

[0029] In some embodiments, the compound is a compound of Formula (Ia). [ka]

[0030] In some embodiments, [ka] teeth, [ka] Selected from JPEG2025066792000021.jpg19143.

[0031] In some embodiments, X 2 is a straight chain C2 alkylene, or a straight chain C3 alkylene, or a straight chain C4 alkylene.

[0032] In other embodiments, R 3 is a C1 alkyl or a C2 alkyl.

[0033] In certain embodiments, R 2 is a C alkyl or a C alkyl. In some other embodiments, R 2 is a nitrogen atom, and X 2together with one or two carbon atoms of R to form a five-membered ring. 2 is a nitrogen atom, and X 2 may be taken together with 1 to 3 carbon atoms thereof to form a 6-membered ring.

[0034] In yet other embodiments, R 2 and R 3 together with the nitrogen atom form a 5-membered ring. 1 is NH or a direct bond.

[0035] In some embodiments, Y 1 is a linear C 3-10 Alkylene, e.g., linear C 4-8 Alkylene, e.g., linear C 5-7 It is alkylene.

[0036] In certain embodiments, R 4 is a linear C 4-14 alkyl, preferably linear C 6-12 It is alkyl.

[0037] In some embodiments, Z 1 is a linear C 2-4 It is alkylene.

[0038] In certain embodiments, R 5 and R 6 are each independently a linear C 6-8 Linear C such as alkyl 5-9 It is alkyl.

[0039] In some embodiments, R 5 and R 6 are each independently a linear C 7-9 It is an alkoxy.

[0040] In certain embodiments, R 5 and R 6 are identical. Alternatively, R 5 and R 6 is different.

[0041] In some embodiments, Y 2 teeth, [ka] In another embodiment, Y 2 teeth, [ka] In some embodiments, Y 1 is a straight chain C7 alkylene, and Y 2 teeth, [ka] where n is 1 and R 4 is a linear C 10 It is alkyl.

[0042] In certain embodiments, Z 2 teeth, [ka] is.

[0043] In some embodiments, Z 1 , Z 2 , and R 5 is selected to form a linear chain of 6 to 18 atoms including the carbon and oxygen atoms of the ester and acetal.

[0044] In some embodiments, Y 1 , Y 2 , and R 4 is selected to form a linear chain of 14 to 24 atoms including the carbon and oxygen atoms of the ester.

[0045] Representative compounds of formula (II) include: [ka] JPEG2025066792000027.jpg250163JPEG2025066792000028.jpg251164JPEG202506 6792000029.jpg251164JPEG2025066792000030.jpg251163JPEG2025066792000031. jpg252164JPEG2025066792000032.jpg252164JPEG2025066792000033.jpg216163JP EG2025066792000034.jpg252164JPEG2025066792000035.jpg252164JPEG202506679 2000036.jpg252164JPEG2025066792000037.jpg226164JPEG2025066792000038.jpg253165JPEG2025066792000039.jpg236165JPEG2025066792000040.jpg232165JPEG2025066792000041.jpg238166JPEG2025066792000042.jpg247167JPEG2025066792000043.jpg247167JPEG2025066792000044.jpg154167 or a salt thereof such as a pharmaceutically acceptable salt thereof. In certain embodiments, at least 75% of the compound of Formula (II) or (I) in the lipid composition formulated as disclosed herein is cleared from the plasma of the subject within 8, 10, 12, 24, or 48 hours, or within 3, 4, 5, 6, 7, or 10 days after administration. In certain embodiments, at least 50% of the lipid composition comprising a compound of Formula (II) or (I) disclosed herein is cleared from the plasma of the subject within 8, 10, 12, 24, or 48 hours, or within 3, 4, 5, 6, 7, or 10 days after administration. This can be determined, for example, by measuring lipids (e.g., compounds of Formula (II) or (I)), RNA (e.g., mRNA), or other components in the plasma. In certain embodiments, the lipid-encapsulated versus free lipid, RNA, or nucleic acid components of the lipid composition are measured.

[0046] Lipid clearance may be measured as described in the literature. See Maier, MA, et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78 ("Maier"). For example, in Maier, an LNP-siRNA system containing siRNA targeting luciferase was administered to 6-8 week-old male C57Bl / 6 mice at 0.3 mg / kg via intravenous bolus injection via the lateral tail vein. Blood, liver, and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 hours post-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. Furthermore, Maier describes a procedure for evaluating the toxicity of LNP-siRNA compositions after administration. For example, siRNA targeting luciferase was administered to male Sprague-Dawley rats at 0, 1, 3, 5, and 10 mg / kg (5 animals / group) via a single intravenous bolus injection at a dose of 5 mL / kg. After 24 hours, approximately 1 mL of blood was obtained from the jugular vein of conscious animals, and serum was isolated. Seventy-two hours after administration, all animals were euthanized for necropsy. Clinical signs, body weight, serum chemistry, organ weight, and histopathology were evaluated. While Maier describes methods for evaluating siRNA-LNP compositions, these methods can be applied to assessing the clearance, pharmacokinetics, and toxicity of lipid compositions, such as LNP compositions, of the present disclosure.

[0047] In certain embodiments, lipid compositions using compounds of Formula (II) or (I) disclosed herein exhibit increased clearance rates compared to alternative ionizable amine lipids. In some such embodiments, the clearance rate is the lipid clearance rate, e.g., the rate at which a compound of Formula (II) or (I) is removed from blood, serum, or plasma. In some embodiments, the clearance rate is the clearance rate of a cargo (e.g., a biologically active agent), e.g., the rate at which a cargo moiety is removed from blood, serum, or plasma. In some embodiments, the clearance rate is the RNA clearance rate, e.g., the rate at which mRNA or gRNA is removed from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNPs are removed from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNPs are removed from tissues such as liver or spleen tissue. Desirably, a high clearance rate can result in a safety profile without substantial adverse effects and / or reduced LNP accumulation in the circulation and / or tissues.

[0048] The compounds of formula (II) or (I) of the present disclosure may form salts depending on the pH of the medium in which they are contained. For example, in a slightly acidic medium, the compounds of formula (II) or (I) may be protonated and therefore positively charged. Conversely, in a slightly basic medium, such as blood, which has a pH of approximately 7.35, the compounds of formula (II) or (I) may not be protonated and therefore uncharged. In some embodiments, the compounds of formula (II) or (I) of the present disclosure may be mostly protonated at a pH of at least about 9. In some embodiments, the compounds of formula (II) or (I) of the present disclosure may be mostly protonated at a pH of at least about 10.

[0049] The pH at which a compound of Formula (II) or (I) is largely protonated is related to its inherent pKa. In preferred embodiments, salts of compounds of Formula (II) or (I) of the present disclosure have a pKa in the range of about 5.1 to about 8.0, even more preferably about 5.5 to about 7.6. In other embodiments, salts of compounds of Formula (II) or (I) of the present disclosure have a pKa in the range of about 5.7 to about 7.6, e.g., about 6 to about 7.5. Alternatively, salts of compounds of Formula (II) or (I) of the present disclosure have a pKa in the range of about 6 to about 8. The pKa of a salt of a compound of Formula (II) or (I) can be an important consideration when formulating LNPs, as LNPs formulated with certain lipids having a pKa in the range of about 5.5 to about 7.0 have been found to be effective for in vivo cargo delivery, for example, to the liver. Furthermore, LNPs formulated with certain lipids having a pKa ranging from about 5.3 to about 6.4 have been found to be effective for in vivo delivery, for example, to tumors. See, e.g., WO2014 / 136086.

[0050] Additional fats "Neutral lipids" suitable for use in the lipid compositions of the present disclosure include, for example, a variety of neutral, uncharged, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2- These include, but are not limited to, diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In certain embodiments, the neutral phospholipid may be selected from distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE), preferably distearoylphosphatidylcholine (DSPC).

[0051] "Helper lipids" include steroids, sterols, and alkylresorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In certain embodiments, the helper lipid may be cholesterol or a derivative thereof, such as cholesterol hemisuccinate.

[0052] PEG lipids can affect the length of time nanoparticles can persist in vivo (e.g., in blood). PEG lipids can be useful in the formulation process, for example, by reducing particle aggregation and controlling particle size. PEG lipids used herein can modulate the pharmacokinetic properties of LNPs. Typically, PEG lipids comprise a lipid moiety and a PEG-based polymer moiety (sometimes referred to as poly(ethylene oxide)) (PEG moiety). PEG lipids suitable for use in lipid compositions comprising compounds of formula (II) or (I) of the present disclosure, and information regarding the biochemistry of 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 WO2015 / 095340 (page 31, line 14 to page 37, line 6), WO2006 / 007712, and WO2011 / 076807 ("stealth lipids").

[0053] In some embodiments, the lipid moiety may be derived from a diacylglycerol or diacylglycamide, including those containing a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently containing from about C4 to about C40 saturated or unsaturated carbon atoms, where the chain may contain one or more functional groups, such as, for example, an amide or ester. In some embodiments, the alkyl chain length includes from about C10 to C20. The dialkylglycerol or dialkylglycamide group may further contain one or more substituted alkyl groups. The chain length may be symmetric or asymmetric.

[0054] Unless otherwise specified, as used herein, the term "PEG" refers to any polyethylene glycol or other polyalkylene ether polymer, e.g., an optionally substituted, linear or branched, polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety is unsubstituted. Alternatively, the PEG moiety may be substituted, e.g., with one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. For example, the PEG moiety may comprise a PEG copolymer, such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)). Alternatively, the PEG moiety may be a PEG homopolymer. In certain embodiments, the PEG moiety has a molecular weight of about 130 to about 50,000, e.g., about 150 to about 30,000, or even about 150 to about 20,000. Similarly, the PEG moiety can 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 certain 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.

[0055] In certain preferred embodiments, the PEG moiety is "PEG-2K," also known as "PEG2000," which has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (II), where n is 45, meaning that it contains a number-average degree of polymerization of about 45 subunits. [ka] However, other embodiments of PEG known in the art may be used, for example, having a number average degree of polymerization of about 23 subunits (n=23) and / or 68 subunits (n=68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl, such as methyl.

[0056] In any of the embodiments described herein, the PEG lipid may be PEG-dilaurylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (Cat. No. GM-020, manufactured by NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (Cat. No. DSPE-020CN, manufactured by NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene)-2-yl]-1,3-dimethyl-2,4-diphenyl ... -3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (Cat. No. 880120C The PEG lipid may be selected from Avanti Polar Lipids (Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In certain 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 still other embodiments, the PEG lipid may be PEG2k-C-DMA.In certain embodiments, the PEG lipid may be compound S027, which is disclosed in WO2016 / 010840 (paragraphs

[0240] to

[0244] ). 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 embodiments, the PEG lipid may be PEG2k-C18.

[0057] Cationic lipids suitable for use in the lipid compositions of the present invention include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)prolyl)-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-dimethylammonium propane (DODAP ... N-(1-(2,3-dioleoyloxy)prolyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)prolyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)prolyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)prolyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)prolyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-di (oxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-dilineoyl-3-dimethylammonium-propane (DLINDAP), dilauryl (C12:0) trimethylammonium-propane (DLTAP), dioctadecylamidoglycylspermine (DOGS), DC-Choi, dioleoyloxy-N-[2-(sperm aminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), N,N-dimethyl-2,3-dioleyloxy) Examples of cationic lipids include, but are not limited to, N,N-dimethyl-3,4-dioleyloxybenzylamine (DODMA), 2-[5'-(cholest-5-ene-3[beta]-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP). In one embodiment, the cationic lipid is DOTAP or DLTAP.

[0058] Anionic lipids suitable for use in the present invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine cholesterol hemisuccinate (CHEMS), and lysylphosphatidylglycerol.

[0059] lipid composition The present invention provides a lipid composition comprising at least one compound of formula (II) or (I) or a salt thereof (e.g., a pharmaceutically acceptable salt thereof) and at least one other lipid component. Such compositions can also contain a biologically active 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 biologically active agent.

[0060] In one embodiment, the lipid composition comprises a compound of formula (II) or (I) or a pharmaceutically acceptable salt thereof and at least one other lipid component. In another embodiment, the lipid composition further comprises a biologically active agent, optionally in combination with one or more other lipid components. In another embodiment, the lipid composition is in the form of a liposome. In another embodiment, the lipid composition is in the form of a lipid nanoparticle (LNP). In another embodiment, the lipid composition is suitable for delivery to the liver.

[0061] In one embodiment, the lipid composition comprises a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof and another lipid component. Such other lipid components include, but are not limited to, a neutral lipid, a helper lipid, a PEG lipid, a cationic lipid, and an anionic lipid. In certain embodiments, the lipid composition comprises a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof and a neutral lipid, such as DSPC, optionally with one or more additional lipid components. In another embodiment, the lipid composition comprises a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof and a helper lipid, such as cholesterol, optionally with one or more additional lipid components. In a further embodiment, the lipid composition comprises a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof and a PEG lipid, optionally with one or more additional lipid components. In a further embodiment, the lipid composition comprises a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof and a cationic lipid, optionally with one or more additional lipid components. In a further embodiment, the lipid composition comprises a compound of formula (II) or (I) or a pharmaceutically acceptable salt thereof, and an anionic lipid, optionally together with one or more additional lipid components. In a subembodiment, the lipid composition comprises a compound of formula (II) or (I) or a pharmaceutically acceptable salt thereof, a helper lipid, and a PEG lipid, optionally together with a neutral lipid. In a further subembodiment, the lipid composition comprises a compound of formula (II) or (I) or a pharmaceutically acceptable salt thereof, a helper lipid, a PEG lipid, and a neutral lipid.

[0062] Compositions containing lipids of formula (II) or (I), or pharmaceutically acceptable salts thereof, or lipid compositions thereof, may be in various forms, including, but not limited to, particle-forming delivery agents, including microparticles, nanoparticles, and transfection agents, useful for delivering various molecules to cells. Certain compositions are effective for transfecting or delivering biologically active agents. Preferred biologically active agents are RNA and DNA. In further embodiments, the biologically active agent is selected from mRNA, gRNA, and DNA. The gRNA may be a dgRNA or sgRNA. In certain embodiments, the cargo includes an mRNA encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease, a class 2 Cas nuclease, or Cas9), a gRNA, or a nucleic acid encoding a gRNA, or a combination of mRNA and gRNA.

[0063] Exemplary compounds of Formula (I) or (II) for use in the lipid compositions described above are shown in Examples 2-99, 100-103, and 113-118. In certain embodiments, the compound of Formula (I) is Compound 2. In certain embodiments, the compound of Formula (I) is Compound 3. In certain embodiments, the compound of Formula (I) is Compound 4. In certain embodiments, the compound of Formula (I) is Compound 5. In certain embodiments, the compound of Formula (I) is Compound 6. In certain embodiments, the compound of Formula (I) is Compound 7. In certain embodiments, the compound of Formula (I) is Compound 8. In certain embodiments, the compound of Formula (I) is Compound 9. In certain embodiments, the compound of Formula (I) is Compound 10. In certain embodiments, the compound of Formula (I) is Compound 11. In certain embodiments, the compound of Formula (I) is Compound 12. In certain embodiments, the compound of Formula (I) is Compound 13. In certain embodiments, the compound of Formula (I) is Compound 14. In certain embodiments, the compound of Formula (I) is Compound 15. In certain embodiments, the compound of Formula (I) is Compound 16. In certain embodiments, the compound of Formula (I) is Compound 17. In certain embodiments, the compound of Formula (I) is Compound 18. In certain embodiments, the compound of Formula (I) is Compound 19. In certain embodiments, the compound of Formula (I) is Compound 20. In certain embodiments, the compound of Formula (I) is Compound 21. In certain embodiments, the compound of Formula (I) is Compound 22. In certain embodiments, the compound of Formula (I) is Compound 23. In certain embodiments, the compound of Formula (I) is Compound 24. In certain embodiments, the compound of Formula (I) is Compound 25. In certain embodiments, the compound of Formula (I) is Compound 26. In certain embodiments, the compound of Formula (I) is Compound 27. In certain embodiments, the compound of Formula (I) is Compound 28. In certain embodiments, the compound of Formula (I) is Compound 29. In certain embodiments, the compound of formula (I) is compound 30. In certain embodiments, the compound of formula (I) is compound 31.In certain embodiments, the compound of Formula (I) is Compound 32. In certain embodiments, the compound of Formula (I) is Compound 33. In certain embodiments, the compound of Formula (I) is Compound 34. In certain embodiments, the compound of Formula (I) is Compound 35. In certain embodiments, the compound of Formula (I) is Compound 36. In certain embodiments, the compound of Formula (I) is Compound 37. In certain embodiments, the compound of Formula (I) is Compound 38. In certain embodiments, the compound of Formula (I) is Compound 39. In certain embodiments, the compound of Formula (I) is Compound 40. In certain embodiments, the compound of Formula (I) is Compound 41. In certain embodiments, the compound of Formula (I) is Compound 42. In certain embodiments, the compound of Formula (I) is Compound 43. In certain embodiments, the compound of Formula (I) is Compound 44. In certain embodiments, the compound of Formula (I) is Compound 45. In certain embodiments, the compound of Formula (I) is Compound 46. In certain embodiments, the compound of Formula (I) is Compound 47. In certain embodiments, the compound of Formula (I) is Compound 48. In certain embodiments, the compound of Formula (I) is Compound 49. In certain embodiments, the compound of Formula (I) is Compound 50. In certain embodiments, the compound of Formula (I) is Compound 51. In certain embodiments, the compound of Formula (I) is Compound 52. In certain embodiments, the compound of Formula (I) is Compound 53. In certain embodiments, the compound of Formula (I) is Compound 54. In certain embodiments, the compound of Formula (I) is Compound 55. In certain embodiments, the compound of Formula (I) is Compound 56. In certain embodiments, the compound of Formula (I) is Compound 57. In certain embodiments, the compound of Formula (I) is Compound 58. In certain embodiments, the compound of Formula (I) is Compound 59. In certain embodiments, the compound of Formula (I) is Compound 60. In certain embodiments, the compound of Formula (I) is Compound 61. In certain embodiments, the compound of Formula (I) is Compound 62. In certain embodiments, the compound of Formula (I) is Compound 63.In certain embodiments, the compound of Formula (I) is Compound 64. In certain embodiments, the compound of Formula (I) is Compound 65. In certain embodiments, the compound of Formula (I) is Compound 66. In certain embodiments, the compound of Formula (I) is Compound 67. In certain embodiments, the compound of Formula (I) is Compound 68. In certain embodiments, the compound of Formula (I) is Compound 69. In certain embodiments, the compound of Formula (I) is Compound 70. In certain embodiments, the compound of Formula (I) is Compound 71. In certain embodiments, the compound of Formula (I) is Compound 72. In certain embodiments, the compound of Formula (I) is Compound 73. In certain embodiments, the compound of Formula (I) is Compound 74. In certain embodiments, the compound of Formula (I) is Compound 75. In certain embodiments, the compound of Formula (I) is Compound 76. In certain embodiments, the compound of Formula (I) is Compound 77. In certain embodiments, the compound of Formula (I) is Compound 78. In certain embodiments, the compound of Formula (I) is Compound 79. In certain embodiments, the compound of Formula (I) is Compound 80. In certain embodiments, the compound of Formula (I) is Compound 81. In certain embodiments, the compound of Formula (I) is Compound 82. In certain embodiments, the compound of Formula (I) is Compound 83. In certain embodiments, the compound of Formula (I) is Compound 84. In certain embodiments, the compound of Formula (I) is Compound 85. In certain embodiments, the compound of Formula (I) is Compound 86. In certain embodiments, the compound of Formula (I) is Compound 87. In certain embodiments, the compound of Formula (I) is Compound 88. In certain embodiments, the compound of Formula (I) is Compound 89. In certain embodiments, the compound of Formula (I) is Compound 90. In certain embodiments, the compound of Formula (I) is Compound 91. In certain embodiments, the compound of Formula (I) is Compound 92. In certain embodiments, the compound of Formula (I) is Compound 93. In certain embodiments, the compound of formula (I) is compound 94. In certain embodiments, the compound of formula (I) is compound 95.In certain embodiments, the compound of Formula (I) is Compound 96. In certain embodiments, the compound of Formula (I) is Compound 97. In certain embodiments, the compound of Formula (I) is Compound 98. In certain embodiments, the compound of Formula (I) is Compound 99. In certain embodiments, the compound is Compound 100. In certain embodiments, the compound is Compound 101. In certain embodiments, the compound is Compound 102. In certain embodiments, the compound is Compound 103. In certain embodiments, the compound is Compound 113. In certain embodiments, the compound is Compound 114. In certain embodiments, the compound is Compound 115. In certain embodiments, the compound is Compound 116. In certain embodiments, the compound is Compound 117. In certain embodiments, the compound is Compound 118.

[0064] LNP composition The lipid composition may be provided as an LNP composition. The lipid nanoparticle may be, for example, a microsphere (unilamellar and multilamellar vesicle, e.g., "liposome," which in some embodiments is substantially spherical and, in more specific embodiments, comprises a lamellar phase lipid bilayer that may include an aqueous core, e.g., containing a substantial portion of RNA molecules), the dispersed phase in an emulsion, a micelle, or the internal phase in a suspension.

[0065] LNPs range in size from about 1 to about 1,000 nm, about 10 to about 500 nm, about 20 to about 500 nm, in subembodiments about 50 to about 400 nm, in subembodiments about 50 to about 300 nm, in subembodiments about 50 to about 200 nm, and in subembodiments about 50 to about 150 nm, and in other subembodiments about 60 to about 120 nm. Preferably, LNPs have a size of about 60 nm to about 100 nm. The average size (diameter) of fully formed LNPs can be measured by dynamic light scattering on a Malvern Zetasizer or Wyatt NanoStar. LNP samples are diluted with phosphate-buffered saline (PBS) to achieve a count rate of approximately 200 to 400 kcps. Data are presented as a weighted average of intensity measurements.

[0066] Embodiments of the present disclosure provide lipid compositions described according to the respective molar ratios of the lipid components in the composition. All mole percentages are expressed as the lipid composition, or more specifically, as the lipid component fraction of the LNP composition. In certain embodiments, the mole percentage of the compound of Formula (II) or (I) may be from about 30 mole percent to about 70 mole percent. In certain embodiments, the mole percentage of the compound of Formula (II) or (I) may be at least 30 mole percent, at least 40 mole percent, at least 50 mole percent, or at least 60 mole percent.

[0067] In certain embodiments, the mole % of neutral lipids can be about 0 mole % to about 30 mole %. In certain embodiments, the mole % of neutral lipids can be about 0 mole % to about 20 mole %. In certain embodiments, the mole % of neutral lipids can be about 10 mole %. In certain embodiments, the mole % of neutral lipids can be about 9 mole %.

[0068] In certain embodiments, the mole percent of helper lipid may be about 0 mole percent to about 80 mole percent. In certain embodiments, the mole percent of helper lipid may be about 20 mole percent to about 60 mole percent. In certain embodiments, the mole percent of helper lipid may be about 30 mole percent to about 50 mole percent. In certain embodiments, the mole percent of helper lipid may be 30 mole percent to about 40 mole percent, or about 35 mole percent to about 45 mole percent. In certain embodiments, the mole percent of helper lipid is adjusted based on the compound of Formula (II) or (I), neutral lipid, and / or PEG lipid concentration to bring the lipid component to 100 mole percent.

[0069] In certain embodiments, the mole % of PEG lipid may be about 1 mole % to about 10 mole %. In certain embodiments, the mole % of PEG lipid may be about 1 mole % to about 4 mole %. In certain embodiments, the mole % of PEG lipid may be about 1 mole % to about 2 mole %. In certain embodiments, the mole % of PEG lipid may be about 1.5 mole %.

[0070] In various embodiments, the LNP composition comprises a compound of Formula (II) or (I) or a salt thereof (e.g., a pharmaceutically acceptable salt thereof (e.g., as disclosed herein)), a neutral lipid (e.g., DSPC), a helper lipid (e.g., cholesterol), and a PEG-lipid (e.g., PEG2k-DMG). In some embodiments, the LNP composition comprises a compound of Formula (II) or (I) 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 certain preferred embodiments, the LNP composition comprises a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof, cholesterol, DSPC, and PEG2k-DMG.

[0071] In certain embodiments, lipid compositions, such as LNP compositions, include a lipid component and a nucleic acid component, e.g., an RNA component, and the molar ratio of a compound of Formula (II) or (I) to nucleic acid can be determined. Embodiments of the present disclosure also provide lipid compositions having a defined molar ratio between the positively charged amine groups (N) of a pharmaceutically acceptable salt of a compound of Formula (II) or (I) and the negatively charged phosphate groups (P) of the encapsulated nucleic acid. This can be mathematically represented by the equation N / P. In some embodiments, lipid compositions, such as LNP compositions, can include a lipid component comprising a compound of Formula (II) or (I), or a pharmaceutically acceptable salt thereof, and a nucleic acid component, and the N / P ratio is about 3-10. In some embodiments, LNP compositions can include a lipid component comprising a compound of Formula (II) or (I), or a pharmaceutically acceptable salt thereof, and an RNA component, and the N / P ratio is about 3-10. For example, the N / P ratio can be about 4-7. Alternatively, the N / P ratio may be about 6, for example, 6±1, or 6±0.5.

[0072] In some embodiments, the aqueous component comprises a biologically active agent. In some embodiments, the aqueous component comprises a polypeptide, optionally in combination 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, which may be referred to as a DNA component. In some embodiments, the nucleic acid component comprises RNA. In some embodiments, an aqueous component, such as an RNA component, may comprise an mRNA, such as an mRNA encoding an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is a Cas nuclease. In certain embodiments, the aqueous component may comprise an mRNA encoding Cas9. In certain embodiments, the aqueous component may comprise a gRNA. In some compositions comprising an mRNA encoding an RNA-guided DNA-binding agent, the composition further comprises a gRNA nucleic acid, such as a gRNA. In some embodiments, the aqueous component comprises an RNA-guided DNA-binding agent and a gRNA. In some embodiments, the aqueous component comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the aqueous component comprises a Class 2 Cas nuclease mRNA and a gRNA.

[0073] In certain embodiments, a lipid composition, such as an LNP composition, may comprise an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof, a helper lipid, optionally a neutral lipid, and a PEG-lipid. In certain compositions comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the helper lipid is cholesterol. In other compositions comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, for example, Cas9, the neutral lipid is DSPC. In additional embodiments comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, for example, Cas9, the PEG-lipid is PEG2k-DMG. In certain compositions, the composition comprises an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, and a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof. In certain compositions, the composition further comprises a gRNA, such as a dgRNA or sgRNA.

[0074] In some embodiments, a lipid composition, such as an LNP composition, may include a gRNA. In certain embodiments, a composition may include a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof, a gRNA, a helper lipid, optionally a neutral lipid, and a PEG-lipid. In certain LNP compositions including a gRNA, the helper lipid is cholesterol. In some compositions including a gRNA, the neutral lipid is DSPC. In additional embodiments including a gRNA, the PEG-lipid is PEG2k-DMG. In certain compositions, the gRNA is selected from a dgRNA and an sgRNA.

[0075] In certain embodiments, a lipid composition, such as an LNP composition, comprises an mRNA and a gRNA (which may be an sgRNA) encoding an RNA-guided DNA-binding agent in an aqueous component, and a compound of Formula (II) or (I) in a lipid component. For example, an LNP composition may comprise a compound of Formula (II) or (I) or a pharmaceutically acceptable salt thereof, an mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG-lipid. In certain compositions comprising an mRNA and a gRNA encoding a Cas nuclease, the helper lipid is cholesterol. In some compositions comprising an mRNA and a gRNA encoding a Cas nuclease, the neutral lipid is DSPC. In additional embodiments comprising an mRNA and a gRNA encoding a Cas nuclease, the PEG-lipid is PEG2k-DMG.

[0076] In certain embodiments, a lipid composition, such as an LNP composition, comprises an RNA-guided DNA-binding agent, such as a Class 2 Cas mRNA, and at least one gRNA. In certain embodiments, the LNP composition comprises a ratio of gRNA to RNA-guided DNA-binding agent mRNA, such as a Class 2 Cas nuclease mRNA, of about 1:1 or about 1:2. In some embodiments, this 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.

[0077] The lipid compositions disclosed herein, such as LNP compositions, may include a template nucleic acid, e.g., a DNA template. The template nucleic acid may be delivered together with or separately from a lipid composition containing a compound of formula (II) or (I) or a pharmaceutically acceptable salt thereof, including as an 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, for example, have a region of homology with the target DNA within the target DNA sequence and / or a sequence adjacent to the target DNA.

[0078] In some embodiments, LNPs are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution. Suitable solutions or solvents may include or contain water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, or isopropanol. For example, the organic solvent may be 100% ethanol. For example, a pharmaceutically acceptable buffer for in vivo administration of LNPs may be used. In certain embodiments, a buffer is used to maintain the pH of a composition comprising LNPs at pH 6.5 or above. In certain embodiments, a buffer is used to maintain the pH of a composition comprising LNPs at pH 7.0 or above. In certain embodiments, the composition has a pH in the range of about 7.2 to about 7.7. In additional embodiments, the composition has a pH in the range of about 7.3 to about 7.7 or about 7.4 to about 7.6. In further embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of the composition may be measured with a micro pH probe. In certain 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 certain embodiments, the composition may include tris saline sucrose (TSS). In certain embodiments, the LNP composition may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In certain 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 include a buffer solution. In some embodiments, the buffer solution may include phosphate buffer solution (PBS), Tris buffer solution, citrate buffer solution, and mixtures thereof. In certain exemplary embodiments, the buffer solution includes NaCl. In certain embodiments, the buffer solution lacks NaCl. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM.An exemplary amount of NaCl may 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. An exemplary amount of Tris may range from about 20 mM to about 60 mM. An exemplary amount of Tris may 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 LNP compositions contain 5% sucrose and 45 mM NaCl in a Tris buffer. In other exemplary embodiments, the composition contains about 5% w / v sucrose, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The amounts of salt, buffer, and cryoprotectant may be varied to maintain the osmolality of the overall composition. For example, the final osmolality may be maintained at less than 450 mOsm / L. In further embodiments, the osmolality is between 350 and 250 mOsm / L. Certain embodiments have a final osmolality of 300±20 mOsm / L or 310±40 mOsm / L.

[0079] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing of aqueous RNA and lipid solutions in organic solvents is used. In certain aspects, the flow rate, junction size, junction geometry, junction shape, tubing diameter, solution, and / or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be concentrated or purified, for example, via dialysis, centrifugal filtration, tangential flow filtration, or chromatography. LNPs may be stored, for example, as a suspension, emulsion, or lyophilized powder. In some embodiments, LNP compositions are stored at 2-8°C; in certain aspects, LNP compositions are stored at room temperature. In additional embodiments, LNP compositions are stored 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, for example, on ice, at room temperature, or at 25°C, prior to use.

[0080] LNPs may be, for example, microspheres (unilamellar and multilamellar vesicles, e.g., "liposomes," lamellar phase lipid bilayers, which in some embodiments are substantially spherical and, in more specific embodiments, may contain an aqueous core containing, for example, the majority of RNA molecules), the dispersed phase in an emulsion, a micelle, or the internal phase in a suspension.

[0081] Preferred lipid compositions, such as LNP compositions, are biodegradable in that they do not accumulate to cytotoxic levels in vivo at therapeutically effective doses.In some embodiments, the compositions do not induce the innate immune response that causes substantial adverse effects at therapeutic dose levels.In some embodiments, the compositions provided herein do not cause toxicity at therapeutic dose levels.

[0082] In some embodiments, the LNPs disclosed herein have a polydispersity index (PDI) that may range from about 0.005 to about 0.75. In some embodiments, the LNPs have a PDI that may range from about 0.01 to about 0.5. In some embodiments, the LNPs have a PDI that may range from about 0 to about 0.4. In some embodiments, the LNPs have a PDI that may range from about 0 to about 0.35. In some embodiments, the LNPs have a PDI that may range from about 0 to about 0.3. In some embodiments, the LNPs have a PDI that may range from about 0 to about 0.25. In some embodiments, the LNPs have a PDI that may range from about 0 to about 0.2. In some embodiments, the LNPs have a PDI that may be less than about 0.08, 0.1, 0.15, 0.2, or 0.4.

[0083] The LNPs disclosed herein have a size (e.g., Z-average diameter) of about 1 to about 250 nm. In some embodiments, the LNPs have a size of about 10 to about 200 nm. In further embodiments, the LNPs have a size of about 20 to about 150 nm. In some embodiments, the LNPs have a size of about 50 to about 150 nm. In some embodiments, the LNPs have a size of about 50 to about 100 nm. In some embodiments, the LNPs have a size of about 50 to about 120 nm. In some embodiments, the LNPs have a size of about 60 to about 100 nm. In some embodiments, the LNPs have a size of about 75 to about 150 nm. In some embodiments, the LNPs have a size of about 75 to about 120 nm. In some embodiments, the LNPs have a size of about 75 to about 100 nm. Unless otherwise specified, all sizes referred to herein are the average size (diameter) of fully formed nanoparticles as measured by dynamic light scattering on a Malvern Zetasizer or Wyatt NanoStar. Nanoparticle samples are diluted in phosphate-buffered saline (PBS) to achieve a count rate of approximately 200–400 kcps. Data are presented as a weighted average of intensity measurements (Z-average diameter).

[0084] In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 95%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 95%.

[0085] cargo The cargo delivered via the LNP composition may be a biologically active agent. In certain embodiments, the cargo is one or more biologically active agents, such as mRNA, gRNA, expression vectors, template nucleic acids, RNA-guided DNA binding agents, antibodies (e.g., monoclonal, chimeric, humanized, nanobodies, and fragments thereof), cholesterol, hormones, peptides, proteins, chemotherapeutic agents and other types of anti-tumor drugs, low molecular weight drugs, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex-forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozymes, decoys and analogs thereof, plasmids, and the like. and other types of vectors, as well as small nucleic acid molecules, RNAi agents, small interfering nucleic acids (siNAs), small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and "self-replicating RNAs" (which encode replicase enzyme activity and can induce its own replication or amplification in vivo), peptide nucleic acids (PNAs), locked nucleic acid ribonucleotides (LNAs), morpholino nucleotides, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), sisiRNAs (small internal segmented interfering RNAs), and iRNAs (asymmetric interfering RNAs). The above list of biologically active agents is merely exemplary and is not intended to be limiting. Such compounds may be purified or partially purified, naturally occurring or synthetic, or chemically modified.

[0086] The cargo delivered via the LNP composition may be RNA, such as an mRNA molecule encoding a protein of interest. Examples include mRNA for expressing a protein such as green fluorescent protein (GFP), an RNA-guided DNA binder, or a Cas nuclease. LNP compositions are provided that include a Cas nuclease mRNA, e.g., a Class 2 Cas nuclease mRNA that enables cellular expression of a Class 2 Cas nuclease, such as a Cas9 or Cpf1 protein. Additionally, the cargo may contain one or more gRNAs or nucleic acids encoding the gRNAs. For example, a template nucleic acid for repair or recombination may also be included in the composition, or the template nucleic acid may be used in the methods described herein. In a subembodiment, the cargo includes an mRNA encoding Streptococcus pyogenes Cas9 and, optionally, an S. pyogenes gRNA. In a further subembodiment, the cargo includes an mRNA encoding Neisseria meningitidis Cas9 and, optionally, an Nme (Neisseria meningitidis) gRNA.

[0087] "mRNA" refers to a polynucleotide that contains an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylated tRNAs). mRNA can include a phosphate-sugar backbone that includes a ribose residue or an analog thereof (e.g., 2'-methoxyribose residues). In some embodiments, the sugars of the mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxyribose residues, or a combination thereof. Generally, mRNA does not contain significant amounts of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues, or a thymidine content of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). mRNA can contain modified uridines at some or all of its uridine positions.

[0088] CRISPR / Cas cargo In certain embodiments, the disclosed compositions comprise mRNA encoding an RNA-guided DNA-binding agent, such as a Cas nuclease, hi certain embodiments, the disclosed compositions comprise mRNA encoding a class 2 Cas nuclease, such as S. pyogenes Cas9.

[0089] As used herein, "RNA-guided DNA binding agent" refers to a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, where the DNA-binding activity is sequence-specific and dependent on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas cleavage enzymes / nickases and their inactivated forms ("dCas DNA binders"). As used herein, "Cas nuclease" encompasses Cas cleavage enzymes, Cas nickases, and dCas DNA binders. Cas cleavage enzymes / nickases and dCas DNA binders 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, a "class 2 Cas nuclease" is a single-chain polypeptide with RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavage enzymes / nickases that additionally have RNA-guided DNA cleavage enzyme or nickase activity (e.g., H840A, D10A, or N863A variants), and Class 2 dCas DNA binders in which the cleavage enzyme / nickase activity has been 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 modifications thereof. The Cpf1 protein, Zetsche et al., Cell, 163:1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpf1 sequence of Zetsche is incorporated by reference in its entirety. See, e.g., Tables 2 and 4 of Zetsche.See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0090] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a gRNA combined with an RNA-guided DNA-binding agent, such as a Cas nuclease, e.g., a Cas cleavage enzyme, a Cas nickase, or a dCas DNA-binding agent (e.g., Cas9). In some embodiments, the gRNA guides the RNA-guided DNA-binding agent, such as Cas9, to a target sequence, where the gRNA hybridizes to the target sequence and the binding agent binds to the target sequence. If the binding agent is a cleavage enzyme or nickase, binding can be followed by cleavage or nicking.

[0091] In some embodiments of the present disclosure, the cargo of the LNP composition comprises at least one gRNA comprising a guide sequence that directs an RNA-guided DNA-binding agent, which may be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA. The gRNA may guide the 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 the Class 2 Cas nuclease, thereby providing specificity for cleavage. In some embodiments, the gRNA and Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex, such as a CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type V CRISPR / Cas complex, such as a Cpf1 / gRNA complex. The Cas nuclease and cognate gRNA may be paired. The gRNA scaffold structure paired with each Class 2 Cas nuclease varies depending on the specific CRISPR / Cas system.

[0092] "Guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to the cognate guide nucleic acid for an RNA-guided DNA-binding agent. Guide RNAs may include modified RNAs described herein. gRNAs may be either crRNAs (also known as CRISPR RNAs) or a combination of crRNA and trRNA (also known as tracrRNAs). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA may be a naturally occurring sequence or a trRNA sequence with modifications or changes compared to the naturally occurring sequence.

[0093] As used herein, "guide sequence" refers to a sequence within a gRNA that is complementary to a target sequence and functions to guide the gRNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA-binding agent. A "guide sequence" may also be referred to as a "targeting sequence" or a "spacer sequence." A guide sequence can be 20 base pairs in length, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences, e.g., 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length, can also be used as guides. In some embodiments, the target sequence is, for example, in a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and target region may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides. In other embodiments, the guide sequence and target region may contain at least one mismatch. For example, the guide sequence and target sequence may contain one, two, three, or four mismatches, where 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 target region may contain one to four mismatches, where the guide sequence comprises at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches, and the guide sequence comprises 20 nucleotides.

[0094] Because the nucleic acid substrate of a Cas protein is a double-stranded nucleic acid, the target sequence of an RNA-guided DNA-binding protein, such as a Cas protein, includes both the plus and minus strands of genomic DNA (i.e., the given sequence and its reverse complementary strand). Thus, when a guide sequence is said to be "complementary to a target sequence," it should be understood that the guide sequence can guide a gRNA to bind to the reverse complementary strand of the target sequence. Thus, in some embodiments, when the guide sequence binds to the reverse complementary strand of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence without the PAM) except for the substitution of T for U in the guide sequence.

[0095] The length of the targeting sequence can depend on the CRISPR / Cas system and components used. For example, various Class 2 Cas nucleases derived from various bacterial species have different optimal targeting sequence lengths. Thus, the targeting sequence may comprise 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 length. In some embodiments, the length of the targeting sequence is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of a naturally occurring CRISPR / Cas system. In certain embodiments, the Cas nuclease and gRNA scaffold will be derived from the same CRISPR / Cas system. In some embodiments, the targeting sequence may comprise or consist of 18 to 24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19 to 21 nucleotides, hi some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

[0096] In some embodiments, the sgRNA is a "Cas9 sgRNA" capable of mediating RNA-guided DNA cleavage by the Cas9 protein. In some embodiments, the sgRNA is a "Cpf1 sgRNA" capable of mediating RNA-guided DNA cleavage by the Cpf1 protein. In certain embodiments, the gRNA comprises sufficient crRNA and tracrRNA to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises sufficient crRNA to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0097] Certain embodiments of the present invention also provide nucleic acids, e.g., expression cassettes, encoding the gRNAs described herein. "Guide RNA nucleic acid" is used herein to refer to gRNAs (e.g., sgRNAs or dgRNAs) and gRNA expression cassettes, which are nucleic acids that encode one or more gRNAs.

[0098] Modified RNA In certain embodiments, a lipid composition, such as an LNP composition, comprises a modified nucleic acid, including a modified RNA.

[0099] Modified nucleosides or nucleotides can be present in an RNA, e.g., a gRNA or mRNA. For example, a gRNA or mRNA that includes one or more modified nucleosides or nucleotides is referred to as a "modified" RNA to account for the presence of one or more non-natural and / or naturally occurring components or arrangements used in place of or in addition to the standard A, G, C, and U residues. In some embodiments, the modified RNA is synthesized with non-standard nucleosides or nucleotides and is referred to herein as "modified."

[0100] Modified nucleosides and modified nucleotides include (i) modifications in the phosphodiester backbone linkage, e.g., replacement of one or both of the non-linked phosphate oxygens and / or one or more of the linkage phosphate oxygens (exemplary backbone modifications); (ii) modifications, e.g., replacement of the ribose sugar components, e.g., the 2' hydroxyl on the ribose sugar (exemplary sugar modifications); (iii) wholesale replacement of the phosphate moiety with a "dephosphorylated" linker (exemplary backbone modifications); and (iv) non-canonical nucleobases. (v) a modification or substitution of a naturally occurring nucleic acid base (exemplary base modification), including (vi) a substitution or modification of a ribose phosphate backbone (exemplary backbone modification), (vi) a modification of the 3' or 5' end of the polynucleotide, e.g., removal, modification, or substitution of a terminal phosphate group, or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications may include sugar and / or backbone modifications), and (vii) a modification or substitution of a sugar (exemplary sugar modification). Certain embodiments include 5' end modifications to an mRNA, gRNA, or nucleic acid. Certain embodiments include modifications to an mRNA, gRNA, or nucleic acid. Certain embodiments include 3' end modifications to an mRNA, gRNA, or nucleic acid. Modified RNAs may contain 5' and 3' end modifications. Modified RNAs may contain one or more modified residues at non-terminal positions. In certain embodiments, a gRNA includes at least one modified residue. In certain embodiments, the mRNA comprises at least one modified residue.

[0101] Unmodified nucleic acids may be susceptible to degradation, for example, by intracellular nucleases or nucleases found in serum. For example, nucleases may hydrolyze phosphodiester bonds of nucleic acids. Thus, in one aspect, the RNAs (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 RNA molecules described herein may exhibit a reduced innate immune response when introduced into a cell population both in vivo and ex vivo. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single-stranded nucleic acids, involving the induction of cytokine expression and release (particularly interferon) and cell death.

[0102] Thus, in some embodiments, an RNA or nucleic acid comprises at least one modification that confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms "modified" and "modified," when referring to the nucleic acids provided herein, preferably include at least one alteration that enhances stability and renders the RNA or nucleic acid more stable (e.g., more resistant to nuclease digestion) than a wild-type or naturally occurring version of the RNA or nucleic acid. As used herein, the terms "stable" and "stability," when referring to the nucleic acids of the invention, particularly with respect to RNA, refer to, for example, increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that are normally capable of degrading such RNA. Increased stability can include, for example, reduced susceptibility to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within a target cell or tissue, thereby increasing or enhancing the retention of such RNA or nucleic acid in the target cell, tissue, subject, and / or cytoplasm. The stabilized RNA or nucleic acid molecules provided herein exhibit a longer half-life compared to their naturally occurring, unmodified counterparts (e.g., wild-type versions of the molecules). Also contemplated by the terms "modification" and "modified" as they relate to the mRNAs of the LNP compositions disclosed herein are modifications that improve or enhance translation of the mRNA nucleic acid, including, for example, the inclusion of a sequence that functions in the initiation of protein translation (e.g., a Kozak consensus sequence). (Kozak, M., Nucleic Acids Res 15(20):8125-48(1987)).

[0103] In some embodiments, RNA or nucleic acid is chemically or biologically modified to make it more stable.Exemplary modifications to RNA or nucleic acid include base depletion (for example, by deletion or by replacing one nucleotide with another nucleotide) or base modification, for example, base chemical modification.As used herein, the phrase "chemical modification" includes the modification that introduces chemical properties different from those found in naturally occurring RNA or nucleic acid, for example, covalent modification such as the introduction of modified nucleotide (for example, by including nucleotide analogues or pendant groups that are not naturally found in such RNA or nucleic acid molecules).

[0104] In some embodiments of backbone modifications, the phosphate group of the modified residue may be modified by replacing one or more of the oxygens with different substituents. Furthermore, modified residues, such as those present in modified nucleic acids, may contain large-scale replacement of unmodified phosphate moieties with modified phosphate groups, as described herein. In some embodiments, backbone modifications of the phosphate backbone may include modifications that result in either uncharged linkers or charged linkers with asymmetric charge distribution.

[0105] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphate atom in an unmodified phosphate group is achiral. However, the phosphorus atom can be made chiral by replacing one of the non-bridging oxygens with one of the atoms or groups of atoms described above. The stereogenic phosphorus atom can possess either the "R" configuration (referred to herein as Rp) or the "S" configuration (referred to herein as Sp). The backbone can also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate to the nucleoside) with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), or carbon (bridged methylene phosphonates). Substitutions can occur at either or both linking oxygens. The phosphate group can be replaced with a non-phosphorus-containing linking group in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties that can replace the phosphate group can include, but are not limited to, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0106] mRNA In some embodiments, the compositions or formulations disclosed herein comprise an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or Class 2 Cas nuclease, described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or Class 2 Cas nuclease, is provided, used, or administered. The mRNA may comprise one or more of a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenine tail. The mRNA may comprise an open reading frame that has been modified, for example, to encode a nuclear localization sequence or to encode a protein using alternative codons.

[0107] The mRNA in the disclosed LNP compositions may encode, for example, a secreted hormone, enzyme, receptor, polypeptide, peptide, or other normally secreted protein of interest. In one embodiment of the present invention, 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 facilitate protein production.

[0108] Further, suitable modifications include alterations in one or more nucleotides of a codon that encode the same amino acid but are more stable than the codon found in the wild-type version of the mRNA. For example, an inverse correlation between RNA stability and the number of cytidine (C) and / or uridine (U) residues has been demonstrated, and RNA lacking C and U residues has been found to be stable against 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 substituting one codon encoding a particular amino acid with another codon encoding the same or a related amino acid. Contemplated modifications of the mRNA nucleic acids of the present invention also include the incorporation of pseudouridine. The incorporation of pseudouridine into the mRNA nucleic acids of the present invention may enhance stability and translation ability and reduce immunogenicity in vivo. See, for example, Kariko, K., et al., Molecular Therapy 16(11):1833-1840(2008). Substitutions and modifications to the mRNA of the present invention can be carried out by methods well known to those skilled in the art.

[0109] Constraints on reducing the number of C and U residues in a sequence may be greater in the coding regions of an mRNA compared to untranslated regions (i.e., it is likely not possible to eliminate all C and U residues present in a message while retaining the function of the message to encode a desired amino acid sequence). However, the degeneracy of the genetic code provides opportunities that allow for the number of C and / or U residues present in a sequence to be reduced while maintaining the same coding capacity (i.e., several different possibilities for modification of an RNA sequence may be possible, depending on which amino acids are encoded by the codons).

[0110] The term modified also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the invention (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include the addition of bases to the mRNA sequence (e.g., inclusion of a polyA tail or a longer polyA tail), alteration of the 3' or 5' UTR, complexation of the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and the inclusion of elements that alter the structure of the mRNA molecule (e.g., forming a secondary structure).

[0111] Poly-A tails are believed to stabilize natural messengers. Thus, in one embodiment, adding a long poly-A tail to an mRNA molecule can make the mRNA more stable. Poly-A tails can be added using various art-recognized techniques. For example, long poly-A tails can be added to synthetic or in vitro transcribed mRNA using poly-A polymerase (Yokoe, et al. Nature Biotechnology. 1996;14:1252-1256). Transcription vectors can also encode long poly-A tails. Additionally, poly-A tails can be added by direct transcription from PCR products. In one embodiment, the length of the poly-A tail is at least about 90, 200, 300, 400, or at least 500 nucleotides. In one embodiment, the length of the poly-A tail is adjusted to control the stability of the modified mRNA molecules of the present invention and, therefore, protein transcription. For example, because the length of the poly-A tail can affect the half-life of an mRNA molecule, the length of the poly-A tail can be adjusted to change the level of resistance of the mRNA to nucleases, thereby controlling the time course of protein expression in cells. In one embodiment, the stabilized mRNA molecule is sufficiently resistant to in vivo degradation (e.g., by nucleases) so that it can be delivered to a target cell without a transfer vehicle.

[0112] In one embodiment, mRNA can be modified by incorporating 3' and / or 5' untranslated (UTR) sequences not naturally found in wild-type mRNA. In one embodiment, 3' and / or 5' flanking sequences naturally adjacent to the mRNA and encoding a second, unrelated protein can be incorporated into the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein in order to modify it. For example, 3' or 5' sequences from a stable mRNA molecule (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzyme) can be incorporated into the 3' and / or 5' regions of a sense mRNA nucleic acid molecule to increase the stability of the sense mRNA molecule. See, e.g., US2003 / 0083272.

[0113] A more detailed description of mRNA modifications is provided on pages 57-68 of US2017 / 0210698A1, the contents of which are incorporated herein.

[0114] template nucleic acid The compositions and methods disclosed herein may include a template nucleic acid. The template may be used to modify or insert a nucleic acid sequence at or near a target site for an RNA-guided DNA-binding protein, such as a Cas nuclease, e.g., 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 for editing a single gene in a cell or two different genes in a cell.

[0115] In some embodiments, the template may be used in homologous recombination. In some embodiments, the template sequence or a portion of the template sequence may be incorporated into the target nucleic acid molecule by homologous recombination. In other embodiments, the template may be used in homology-directed repair, which involves invasion of a DNA strand at the cleavage site of the nucleic acid. In some embodiments, homology-directed repair may result in the inclusion of the template sequence in the edited target nucleic acid molecule. In still other embodiments, the template may be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence is dissimilar to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is integrated. In some embodiments, the template includes adjacent inverted terminal repeat (ITR) sequences.

[0116] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. The template sequence 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 that is native to the cell. The term "exogenous sequence" refers to a sequence that is not native to the cell or a sequence that is in a location different from its natural location in the genome of the cell. 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.

[0117] In some embodiments, the template contains ssDNA or dsDNA containing flanking inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, a plasmid, a minicircle, a nanocircle, or a PCR product.

[0118] In some embodiments, nucleic acids are purified. In some embodiments, nucleic acids are purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent method, e.g., those described herein). In some embodiments, nucleic acids are purified using a chromatography-based method, such as an HPLC-based method or equivalent method (e.g., those described herein). In some embodiments, nucleic acids are purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method. In some embodiments, nucleic acids are purified by tangential flow filtration (TFF).

[0119] The present compounds or compositions generally, but not necessarily, contain one or more pharmaceutically acceptable excipients. The term "excipient" includes any ingredient other than the compound(s) of the present disclosure, other lipid component(s), and biologically active agent(s). An excipient may impart either functional (e.g., controlling the drug release rate) and / or non-functional (e.g., processing aid or diluent) properties to the present composition. The choice of excipient will largely depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0120] Parenteral formulations are typically aqueous or oily solutions or suspensions. When the formulation is aqueous, the excipients may be sugars (including but not limited to glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffers (preferably pH 3-9), but for some applications, they may be more suitably formulated using sterile non-aqueous solutions or as a dry form used in combination with a suitable vehicle such as sterile pyrogen-free water (WFI).

[0121] While the invention will be described in conjunction with illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, including equivalents of specific features, which may be included within the scope of the invention as defined by the appended claims.

[0122] Both the foregoing general and detailed descriptions, as well as the following examples, are exemplary and explanatory only and are not intended to limit the present teachings. The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. In the event that a document incorporated by reference conflicts with a term defined herein, the present specification shall control. Unless otherwise specified, all ranges specified in this application include the endpoints.

[0123] definition It should be noted that, as used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. Thus, for example, a reference to "a composition" includes a plurality of compositions, a reference to "a cell" includes a plurality of cells, etc. The use of "or" is inclusive and means "and / or" unless otherwise stated.

[0124] Unless otherwise stated in the specification above, embodiments of the specification that describe various components as "comprising" are considered to "consist" or "consist essentially of" the listed components. Embodiments of the specification that describe various components as "consisting" are also contemplated as "comprising" or "consisting essentially of" the listed components. Embodiments of the specification that describe various components as "about" are also contemplated as "at" the listed components. Also, embodiments of the specification that describe various components as "consisting essentially of" are contemplated as "consisting" or "comprising" the listed components (this interchangeability does not apply to the use of these terms in the claims).

[0125] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values ​​are understood to be approximate, taking into account significant digits and the error associated with measurement. As used herein, the terms "about" and "approximately" have the meaning understood in the art, and the use of one rather than the other does not necessarily imply a different range. Unless otherwise specified, numbers used herein, with or without modifiers such as "about" or "approximately," should be understood to encompass normal deviations and / or variations as understood by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 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) of the stated reference value, unless otherwise stated or clear from the context (except where such number exceeds 100% of possible values).

[0126] As used herein, the term "contacting" 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 nanoparticle share a physical connection. Methods for contacting cells with external entities, both in vivo and ex vivo, are well known in the field of biology. For example, contacting a nanoparticle composition with mammalian cells placed within a mammal may be performed by various routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve various amounts of the nanoparticle composition. Furthermore, more than one mammalian cell may be contacted by the nanoparticle composition.

[0127] As used herein, the term "delivering" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject can involve administering to the subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes) a nanoparticle composition comprising the therapeutic and / or prophylactic agent. Administering a nanoparticle composition to a mammal or mammalian cells can involve contacting one or more cells with the nanoparticle composition.

[0128] As used herein, "encapsulation efficiency" refers to the amount of a therapeutic and / or prophylactic agent that becomes part of a nanoparticle composition relative to the initial total amount of the therapeutic and / or prophylactic agent used to prepare the nanoparticle composition. For example, if 97 mg of a therapeutic and / or prophylactic agent is encapsulated in the nanoparticle composition out of a total of 100 mg of the therapeutic and / or prophylactic agent initially provided in the composition, an encapsulation efficiency of 97% may be given. As used herein, "encapsulation" may refer to complete, substantial, or partial enclosing, confining, surrounding, or packaging.

[0129] As used herein, the term "biodegradable" is used to refer to a material that, when introduced into a cell, is broken down by cellular mechanisms (e.g., enzymatic degradation) or by hydrolysis into components that the cell can reuse or discard without significant toxic effect(s) to the cell. In certain embodiments, the components produced by degradation of the biodegradable material do not induce inflammation and / or other adverse effects in vivo. In some embodiments, the biodegradable material is enzymatically degraded. Alternatively, or in addition, in some embodiments, the biodegradable material is hydrolytically degraded.

[0130] As used herein, "N / P ratio" refers to the molar ratio of ionizable nitrogen atom-containing lipids (e.g., compounds of Formula I) to phosphate groups in a nanoparticle composition comprising a lipid component and RNA, e.g., a lipid component and RNA.

[0131] The composition may also include one or more salts of the compounds. The salt may be a pharmaceutically acceptable salt. As used herein, "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 (for example, by reacting a free base group 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 or organic salts of acidic residues such as carboxylic acids, etc. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, Examples of suitable salts include lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two (generally, 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, 17. th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. 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.

[0132] As used herein, "polydispersity index" is a ratio that represents 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 embodiments, the polydispersity index may be less than 0.1.

[0133] As used herein, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0134] As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-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. For example, alkyl groups can be substituted with one or more groups, including, but not limited to, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, carboxylate, or thiol, as described herein. A "lower alkyl" group is an alkyl group containing 1 to 6 (e.g., 1 to 4) carbon atoms.

[0135] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one carbon-carbon double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter of which refers to an alkenyl moiety having substituents replacing a hydrogen on one or more carbon atoms of the alkenyl group. Such substituents may be on one or more carbons included or not included in one or more double bonds. Furthermore, such substituents include all of those contemplated for alkyl groups, as described below, except where prohibited by stability. For example, alkenyl groups that may be substituted with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups are contemplated. Exemplary alkenyl groups include, but are not limited to, vinyl (-CH=CH), allyl (-CHCH=CH), cyclopentenyl (-CH), and 5-hexenyl (-CHCHCHCHCH=CH).

[0136] An "alkylene" group refers to a divalent alkyl radical, which may be branched or unbranched (i.e., straight-chain). Any of the monovalent alkyl groups described above may be converted to an alkylene by abstraction of a second hydrogen atom from the alkyl. Representative alkylenes include C 2-4 Alkylene and C 2-3 Examples of alkylene groups include alkylene. Typical alkylene groups include, but are not limited to, -CH(CH)-, -C(CH)-, -CHCH-, -CHCH(CH)-, -CHC(CH)-, -CHCHCH-, -CHCHCHCH-, and the like. Alkylene groups can also be substituted or unsubstituted. For example, alkylene groups can be substituted with one or more groups, including, but not limited to, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, carboxylate, or thiol, as described herein.

[0137] The term "alkenylene" includes divalent, straight or branched, unsaturated, acyclic hydrocarbyl groups having at least one carbon-carbon double bond and, in one embodiment, no carbon-carbon triple bonds. Any of the monovalent alkenyl groups described above can be converted to an alkenylene by abstraction of a second hydrogen atom from the alkenyl. Representative alkenylenes include C 2-6 Examples include alkenylene.

[0138] "C x-y The term "alkyl" when used with a chemical moiety such as alkyl or alkylene is meant to include groups containing x to y carbons in the chain. For example, "C x-y The term "alkyl" refers to a substituted or unsubstituted saturated hydrocarbon group, including straight-chain and branched-chain alkyl and alkylene groups, containing x to y carbons in the chain.

[0139] Incorporation by Reference The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicant reserves the right to physically incorporate into this application all material and information from such articles, patents, patent applications, or other physical and electronic documents. [Example]

[0140] General information All reagents and solvents were purchased from commercial suppliers and used as received or synthesized according to the cited procedures. All intermediates and final compounds were purified using flash column chromatography on silica gel. NMR spectra were recorded on Bruker or Varian 400 MHz spectrometers, and NMR data were collected in CDCl3 at ambient temperature. Chemical shifts are reported in parts per million (ppm) relative to CDCl3 (7.26). 1 H NMR data are reported as chemical shifts, multiplicities (br = broadband, s = singlet, d = doublet, t = triplet, dd = doublet doublet, dt = triplet doublet, q = quartet, m = multiplet, ddd = doublet doublet doublet, td = doublet triplet, tt = triplet triplet, tdd = doublet doublet triplet, dddd = doublet doublet doublet doublet doublet), 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 chromatography instrument equipped with an SQD2 mass spectrometer equipped with a photodiode array (PDA) and evaporative light scattering (ELS) detector.

[0141] [ka] JPEG2025066792000047.jpg251161JPEG2025066792000048.jpg251161JPEG202 5066792000049.jpg251161JPEG2025066792000050.jpg252161JPEG20250667920 00051.jpg251161JPEG2025066792000052.jpg251161JPEG2025066792000053.j pg215161JPEG2025066792000054.jpg250161JPEG2025066792000055.jpg250161 JPEG2025066792000056.jpg246160JPEG2025066792000057.jpg231161JPEG202 5066792000058.jpg246161JPEG2025066792000059.jpg231161JPEG20250667920 00060.jpg231161JPEG2025066792000061.jpg220161JPEG2025066792000062.j pg240161JPEG2025066792000063.jpg230161JPEG2025066792000064.jpg216161

[0142] Synthesis of Examples Synthesis of Example 1 Intermediate 1a: 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of linoleic acid (13.2 g, 47.1 mmol), DMAP (1.15 g, 9.42 mmol), DIPEA (12.3 mL, 70.6 mmol), and 2-(hydroxymethyl)propane-1,3-diol (5 g, 47.1 mmol) in DCM (100 mL) was added EDC·HCl (13.5 g, 70.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 24 h, concentrated in vacuo, and directly purified using silica gel chromatography (120 g HCl Si, 0–50% EtOAc in hexanes) to give 6.7 g (18.1 mmol, 39% yield) of the desired product as an oil. 1 H NMR(400MHz,CDCl3)δ5.44-5.25(m,4H),4.25(d,J=6.3Hz,2H),3.76(m,4H),2.77(t,J=6.5Hz,2H) ,2.43-2.27(m,4H),2.11-1.96(m,5H),1.62(m,2H),1.43-1.22(m,14H),0.89(t,J=6.8Hz,3H)ppm.

[0143] Intermediate 1b: 4,4-bis(octyloxy)butanenitrile [ka] To a mixture of 4,4-diethoxybutanenitrile (9.4 g, 60 mmol, 1 equiv.) and octan-1-ol (3 equiv.) was added pyridinium p-toluenesulfonate (0.05 equiv.) at room temperature. The reaction mixture was warmed to 105 °C and stirred for at least 18 h with the reaction vessel open to air and no reflux condenser attached. The reaction mixture was then cooled to room temperature and directly purified using silica gel chromatography (a gradient of EtOAc in hexanes) to give 10.1 g (31.0 mmol, 52% yield) of the desired product as a clear oil. 1H NMR(400MHz,CDCl3)δ4.55(t,J=5.3Hz,1H),3.60(dt,J=9.2,6.6Hz,2H),3.43(dt,J=9.2,6.6Hz,2H),2.42(t,J=7.4H MS:348m / z[M+Na].

[0144] Intermediate 1c: 4,4-bis(octyloxy)butanoic acid [ka] To a solution of intermediate 1b (8.42 g, 31 mmol, 1 equiv.) in ethanol (1 M) was added aqueous potassium hydroxide (2.5 M, 2.5 equiv.) at room temperature. The reaction vessel was fitted with a reflux condenser, and the reaction mixture was warmed to 110 °C and stirred for 20–24 h. The reaction mixture was then cooled to room temperature, acidified to pH 5 with 1 N aqueous HCl, and extracted into hexane. The combined organic extracts were washed with water and brine, dried over anhydrous sodium sulfate or magnesium sulfate, filtered, and concentrated in vacuo to afford 8.15 g (23.6 mmol, 76% yield) of the desired product as a clear oil, which was used crude without further purification. 1 H NMR(400MHz,CDCl3)δ4.50(t,J=5.5Hz,1H),3.57(dt,J=9.4,6.7Hz,2H),3.41(dt,J=9.3,6.7Hz,2H),2.40(t,J= 7.4Hz,2H),1.92(td,J=7.4,5.3Hz,2H),1.56(m,4H),1.37-1.21(m,20H),0.92-0.83(m,6H)ppm;MS:343m / z[MH].

[0145] Intermediate 1d: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 1a (0.8–1.2 equiv.) and intermediate 1c (1.1 g, 3.2 mmol, 1 equiv.) in DCM (0.08–0.4 M) was added DMAP (0.1–0.2 equiv.), DIPEA (1.4–3 equiv.), and EDC·HCl (1.4–1.6 equiv.) at room temperature. The reaction mixture was stirred at room temperature for at least 5 h, concentrated in vacuo, and directly purified using silica gel chromatography (gradient of EtOAc in hexanes) to afford 1.08 g (1.55 mmol, 48% yield) of the desired product as a clear oil. 1 H NMR(400MHz,CDCl3)δ5.36(m,4H),4.49(t,J=5.4Hz,1H),4.17(m,4H),3.66-3.53(m,4H),3.40(m,2H),2.77(t,J=6.4Hz,2H),2 .41(t,J=7.6Hz,2H),2.32(t,J=7.6Hz,2H),2.19(m,2H),2.05(m,4H),1.93(m,2H),1.58(m,7H),1.31(m,32H),0.88(m,9H)ppm.

[0146] Example 1: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 1d (1.08 g, 1.55 mmol, 1 equiv.) in acetonitrile (0.04–0.1 M), pyridine (2 equiv.), DMAP (0.2 equiv.), and 4-nitrophenyl chloroformate (1.5 equiv.) were added sequentially at room temperature. After stirring for at least 2 h, 3-(diethylamino)propan-1-ol (3 equiv.) was added, and the resulting reaction mixture was stirred at room temperature for an additional 2–24 h. The reaction mixture was extracted into hexane (20 mL) and washed with water. The resulting aqueous layer was re-extracted with hexane. The combined hexane layers were dried over anhydrous MgSO4 or Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (gradient of EtOAc in hexane or methanol in DCM) to afford 711 mg (0.834 mmol, 54% yield) of the desired product as a clear oil. 1 H 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].

[0147] Synthesis of Examples 2 to 24 The following examples were synthesized from intermediate 1d and an amino alcohol or diamine reagent using the methodology used in Example 1.

[0148] Example 2: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 42%; 1H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.23-4.08(m ,8H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5 Hz,2H),2.47-2.34(m,5H),2.30(t,J=7.6Hz,2H),2.24(s,6H),2.05(q,J=6.9 Hz,4H),1.97-1.79(m,4H),1.58(m,6H),1.41-1.24(m,34H),0.88(m,9H)ppm. MS: 825 m / z [M+H].

[0149] Example 3: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 38%; 1 H NMR(400MHz,,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.24-4.08(m,8H),3. 56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H),2.56(m,6 H),2.47-2.35(m,3H),2.30(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,4H),1.97-1.85(m,4H), 1.86-1.73(m,4H),1.58(m,6H),1.41-1.24(m,34H),0.88(m,9H)ppm;MS:851m / z[M+H].

[0150] Example 4: 13-(((4,4-bis(octyloxy)butanoyl)oxy)methyl)-2,5-dimethyl-10-oxo-9,11-dioxa-2,5-diazatetradecan-14-yl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 25%; 1 H NMR(400MHz, CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.22-4.11(m,8H ),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H) ,2.58-2.38(m,9H),2.33-2.28(m,8H),2.25(s,3H),2.05(q,J=6.8Hz,4H),1.97- 1.79(m,4H),1.58(m,6H),1.41-1.23(m,34H),0.88(m,9H)ppm;MS:882m / z[M+H].

[0151] Example 5: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 19%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.6Hz,1H),4.13-3.95(m,8H),3 .56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.96(m,2H),2.77(t,J=6.5 Hz,2H),2.54-2.35(m,5H),2.30(t,J=7.6Hz,2H),2.05(q,J=6.9Hz,5H),2.02-1.54 (m,15H),1.42-1.24(m,31H),1.16-1.00(m,5H),0.88(m,9H)ppm;MS:865m / z[M+H].

[0152] Example 6: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(dimethylamino)propyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 50%; 1 H NMR(400MHz,CDCl3)δ5.70(br m,1H),5.44-5.27(m,4H),4.48(t,J=5.6Hz,1H),4.13(br m,6H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.26(q,J=6.2Hz,2H),2.77(t,J=6.5Hz,2H),2.55(t,J=6.8Hz,2H),2.40(m, 7H),2.30(t,J=7.6Hz,2H),2.21-2.02(m,11H),1.92(m,2H),1.77(m,2 H),1.57(m,4H),1.42-1.25(m,31H),0.88(m,9H)ppm;MS:824m / z[M+H].

[0153] Example 7: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 37%; 1 H NMR(400MHz,CDCl3)δ6.14(s,1H),5.44-5.28(m,4H),4.48(t,J=5.6Hz,1H),4.18-4.06(m,6H), 3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.27(q,J=5.8Hz,2H),2.74(m,7H),2.3 9(m,3H),2.30(t,J=7.6Hz,2H),2.13-2.00(m,4H),1.92(td,J=7.6,5.5Hz,2H),1.78(t,J=6.5Hz ,2H),1.57(m,6H),1.40-1.22(m,35H),1.14(t,J=7.2Hz,6H),0.88(m,9H)ppm;MS:852m / z[M+H].

[0154] Example 8: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(pyrrolidin-1-yl)propyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 44%; 1 H NMR(400MHz,CDCl3)δ5.87(br m,1H),5.43-5.27(m,4H),4.48(t,J=5.6Hz,1H),4.11(m,6H),3.56(dt,J=9.2,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.29(q,J=6.1Hz,2H ),2.95-2.68(m,8H),2.44-2.26(m,5H),2.05(m,4H),1.98-1.81(m,8H),1.58(m,7H),1.41-1.18(m,33H),0.88(m,9H)ppm;MS:850m / z[M+H].

[0155] Example 9: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(diethylamino)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 84%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.28-4.06(m,8H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.79-2 .60(m,8H),2.46-2.35(m,3H),2.30(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,4 H),1.92(td,J=7.6,5.4Hz,2H),1.58(m,9H),1.42-1.17(m,31H),1.05(br m,6H),0.88(m,9H)ppm;MS:839m / z[M+H].

[0156] Example 10: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(pyrrolidin-1-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 74%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.27(t,J=5.9Hz,2H),4.23- 4.08(m,6H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(m,4H),2.59(br s,4H),2.47-2.35(m,3H),2.30(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,4H),1.92(td,J=7.6,5.4Hz,2H),1.86-1.74(br m,4H),1.58(m,7H),1.41-1.16(m,33H),0.88(m,9H)ppm;MS:837m / z[M+H].

[0157] Example 11: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((4-(diethylamino)butoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 74%; 1H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.6Hz,1H),4.22-4.08(m,8H),3.56( dt,J=9.2,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H),2.65-2.35(m, 8H),2.30(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,4H),1.92(td,J=7.6,5.4Hz,2H),1.74-1.5 2(m,12H),1.42-1.18(m,33H),1.03(t,J=7.2Hz,6H),0.88(m,9H)ppm;MS:867m / z[M+H].

[0158] Example 12: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(diethylamino)ethyl)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 72%; 1 H NMR(400MHz,CDCl3)δ5.43-5.27(m,4H),4.48(t,J=5.6Hz,1H),4.12(m,6H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.25(br m,2H),2.77(t,J=6.5Hz,2H),2.57(s,5H),2.44-2.26(m,5H),2.05(q,J=6.8H z,4H),1.92(td,J=7.6,5.5Hz,2H),1.58(m,9H),1.41-1.18(m,32H),1.05(br m,6H),0.88(m,9H)ppm;MS:838m / z[M+H].

[0159] Example 13: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((4-(pyrrolidin-1-yl)butoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 58%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.22-4.11(m,8H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz, 2H),2.77(t,J=6.5Hz,2H),2.60-2.38(m,8H),2.30(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,4H),1.92(td,J=7.6,5.4Hz,2H),1.86-1.77(br m,4H),1.77-1.52(m,13H),1.41-1.24(m,32H),0.88(m,9H)ppm;MS:865m / z[M+H].

[0160] Example 14: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-methylpyrrolidin-2-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 60%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.21-4.05(m,8H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.11(br m,1H),2.77(t,J=6.5Hz,2H),2.50-2.35(m,6H),2.30(t,J=7.6Hz,3H),2.07-1.52(m,19H),1.30(m,32H),0.88(m,9H)ppm;MS:837m / z[M+H].

[0161] Example 15: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(dipropylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 79%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.23-4.07(m,8H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt ,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H),2.55-2.28(m,11H),2.10-2.00(m,4H),1.92(td,J=7.6,5.4Hz,2H),1.81(br m,2H),1.62-1.24(m,44H),0.88(m,15H)ppm;MS:881m / z[M+H].

[0162] Example 16: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-phenyl-3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 58%; 1 H NMR(400MHz,CDCl3)δ7.36-7.17(m,5H),5.44-5.27(m,4H),4.53-4.43(m,2H),4.33(dd,J=10 .7,7.6Hz,1H),4.11(m,6H),3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),3.24(br m,1H),2.92(br m,1H),2.77(t,J=6.5Hz,2H),2.61-2.33(m,7H),2.28(t,J=7.6Hz,2H),2.05(q,J=6.9Hz,4H),1.91(td,J=7.6,5.4Hz,2H),1.74(br m,4H),1.66-1.51(m,8H),1.41-1.24(m,33H),0.88(m,9H)ppm;MS:927m / z[M+H].

[0163] Example 17: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-4-yl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 40%; 1 H NMR(400MHz,CDCl3)δ5.43-5.28(m,4H),4.66(br s,1H),4.48(t,J=5.5Hz,1H),4.22-4.11(m,6H),3.56(dt,J=9.3,6.7Hz, 2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(m,4H),2.50-2.24(m,8H),2.05(m ,6H),1.92(td,J=7.6,5.4Hz,2H),1.87-1.74(m,2H),1.58(m,10H),1.41 -1.24(m,31H),1.11(t,J=7.2Hz,3H),0.88(m,9H)ppm;MS:851m / z[M+H].

[0164] Example 18: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 52%; 1H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.6Hz,1H),4.29-4.18(m,4H),4.18-4.08( m,4H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H),2.64(br t,J=5.8Hz,2H),2.40(m,3H),2.31(m,7H),2.04(dd,J=8.8,5.0Hz,4H),1.92(td, J=7.6,5.4Hz,2H),1.56(m,10H),1.30(m,31H),0.88(m,9H)ppm;MS:811m / z[M+H].

[0165] Example 19: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-(dimethylamino)propan-2-yl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 63%; 1 H NMR(400MHz,CDCl3)δ5.44-5.26(m,4H),4.88(m,1H),4.48(t,J=5.6Hz,1H),4.16(m,7H) ),3.56(dt,J=9.2,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H),2.57 (dd,J=13.0,7.7Hz,1H),2.49-2.35(m,3H),2.29(m,8H),2.09-1.99(m,4H),1.92(td,J =7.6,5.5Hz,2H),1.69-1.53(m,10H),1.30(m,33H),0.88(m,9H)ppm;MS:825m / z[M+H].

[0166] Example 20: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 30%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.24-4.09(m,8H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2. 77(t,J=6.5Hz,2H),2.57-2.38(m,6H),2.32-2.20(m,5H),2.05(q,J=6 .8Hz,4H),1.95-1.82(m,4H),1.58(m,7H),1.42-1.25(m,34H),1.09(br m,3H),0.88(m,9H)ppm;MS:839m / z[M+H].

[0167] Example 21: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diisopropylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 31%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.22-4.08(m,8H),3.56(dt,J=9.3,6.7 Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.98(m,J=6.7Hz,2H),2.77(t,J=6.4Hz,2H),2.50(t,J=6.9Hz,2H ),2.47-2.35(m,3H),2.30(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,4H),1.92(td,J=7.6,5.4Hz,2H),1.74(m ,2H),1.63-1.51(m,10H),1.42-1.24(m,36H),0.98(d,J=6.6Hz,9H),0.88(m,9H)ppm;MS:881m / z[M+H].

[0168] Example 22: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(dimethylamino)butoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 35%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.29-4.21(m ,1H),4.27-4.10(m,8H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz ,2H),2.77(t,J=6.4Hz,3H),2.46-2.35(m,3H),2.29(m,7H),2.05(q,J=6.8H z,4H),1.92(td,J=7.6,5.4Hz,3H),1.60(m,8H),1.42-1.25(m,33H),1.02(br s,3H),0.88(m,9H)ppm;MS:839m / z[M+H].

[0169] Example 23: 3-(((3-(azepan-1-yl)propoxy)carbonyl)oxy)-2-(((4,4-bis(octyloxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 59%; 1 H NMR(400MHz,CDCl3)δ5.35(m,4H),4.48(t,J=5.5Hz,1H),4.24-4.09(m,8H),3.56( dt,J=9.4,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H),2.64(br m,6H),2.47-2.35(m,3H),2.30(t,J=7.6Hz,2H),2.04(dd,J=7.9,5.9Hz,4H),1.95-1. 81(m,4H),1.70-1.52(m,14H),1.41-1.23(m,34H),0.88(m,9H)ppm;MS:879m / z[M+H].

[0170] Example 24: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(1-methylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 56%; 1 H NMR(400MHz,CDCl3)δ5.35(m,4H),4.48(t,J=5.5Hz,1H),4.29-4.07(m,8H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.13(br MS:851m / z[M+H]

[0171] Synthesis of Example 25 Example 25: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(1-methylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 1d (300 mg, 0.43 mmol, 1 equiv.), 5-(dimethylamino)pentanoic acid (1–3 equiv.), DIPEA (1.4–3 equiv.), and DMAP (0.1–0.2 equiv.) in DCM (0.10–0.15 M) was added EDC·HCl (1.4–1.5 equiv.) at room temperature. After stirring at room temperature for at least 2 h, the reaction mixture was diluted with water, and the organic layer was separated and concentrated in vacuo. The crude residue was purified using silica gel chromatography (gradient of EtOAc in hexanes or MeOH in DCM) to afford 159 mg (0.19 mmol, 44% yield) of the desired product. 1H NMR(400MHz,CDCl3)δ5.35(m,4H),4.48(t,J=5.5Hz,1H),4.12(m,6H),3.56(dt ,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H),2.70(br m,2H),2.56(br s,6H),2.39(m,5H),2.30(t,J=7.6Hz,2H),2.04(m,4H),1.92(td,J=7.6,5.4Hz,2 H),1.81-1.52(m,10H),1.41-1.24(m,34H),0.88(m,9H)ppm;MS:823.42m / z[M+H].

[0172] Synthesis of Examples 26 to 32 The following example was synthesized from intermediate 1d and a carboxylic acid reagent using the method used in Example 25.

[0173] Example 26: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 39%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.13(m,J=6.0,2.2Hz, 6H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H),2 .44-2.28(m,9H),2.21(s,6H),2.10-2.00(m,4H),1.92(td,J=7.6,5.5Hz,2H),1.78(m ,J=7.4Hz,2H),1.58(m,6H),1.42-1.22(m,34H),0.88(m,9H)ppm;MS:809.47m / z[M+H].

[0174] Example 27: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 78%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.13(m,6H),3.56( dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(m,4H),2.56-2.34(m,9H) ,2.30(t,J=7.6Hz,2H),2.10-2.00(m,4H),1.92(td,J=7.6,5.5Hz,2H),1.58(m,6H) ),1.42-1.22(m,34H),1.01(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:823.42m / z[M+H].

[0175] Example 28: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((5-(diethylamino)pentanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 77%; 1 H NMR(400MHz,CDCl3)δ5.43-5.28(m,4H),4.48(t,J=5.6Hz,1H),4.13(m,6H),3.56( dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.7Hz,2H),2.50(q, J=7.2Hz,4H),2.45-2.27(m,9H),2.04(dd,J=7.8,6.2Hz,4H),1.92(td,J=7.6,5.5 Hz,2H),1.64-1.44(m,11H),1.31(m,33H),1.01(t,J=7.1Hz,6H),0.88(m,9H)ppm. MS:851.86m / z[M+H].

[0176] Example 29: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(diethylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 53%; 1 H NMR(500MHz,CDCl3)δ5.43-5.28(m,4H),4.48(t,J=5.5Hz,1H),4.13(m,6H),3.56(dt, J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.7Hz,2H),2.50(m,5H),2. 45-2.29(m,10H),2.05(q,J=6.9Hz,4H),1.92(td,J=7.6,5.5Hz,2H),1.75(m,2H),1.6 6-1.52(m,7H),1.40-1.23(m,30H),1.00(m,6H),0.88(m,9H)ppm;MS:837.01m / z[M+H].

[0177] Example 30: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1,3-dimethylpyrrolidine-3-carboxylate [ka] Yield 45%; 1H NMR(500MHz,CDCl3)δ5.43-5.28(m,4H),4.48(t,J=5.5Hz,1H)4.18-4.11(m,6H),3.56( dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.93(d,J=9.4Hz,1H),2.77(t,J=6.7 Hz,2H),2.64-2.52(m,2H),2.47-2.27(m,11H),2.05(q,J=7.0Hz,4H),1.92(td,J=7.6,5 .5Hz,2H),1.71-1.52(m,9H),1.40-1.24(m,34H),0.88(m,9H)ppm;MS:821.30m / z[M+H].

[0178] Example 31: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-methylpiperidine-4-carboxylate [ka] Yield 87%; 1 H NMR(500MHz, CDCl3)δ5.43-5.28(m,4H),4.48(t,J=5.5Hz,1H),4.16-4.09(m,6H ),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.78(m,4H),2.43-2. 34(m,3H),2.34-2.22(m,6H),2.05(q,J=6.9Hz,4H),2.00-1.87(m,6H),1.76(m,2 H),1.65-1.52(m,7H),1.42-1.19(m,33H),0.88(m,9H)ppm;MS:821.80m / z[M+H].

[0179] Example 32: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1,4-dimethylpiperidine-4-carboxylate [ka] Yield 39%; 1 H NMR(500MHz,CDCl3)δ5.43-5.28(m,4H),4.48(t,J=5.5Hz,1H),4.17-4.09(m,6H),3.56(dt ,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.7Hz,2H),2.58(m,2H),2.41( m,3H),2.31(t,J=7.6Hz,2H),2.24(s,3H),2.15-2.01(m,8H),1.92(td,J=7.6,5.3Hz,2H), 1.63-1.48(m,9H),1.42-1.24(m,33H),1.19(s,3H),0.88(m,9H)ppm;MS:835.79m / z[M+H].

[0180] Synthesis of Examples 33 to 36 The following examples were synthesized from intermediate 1d and an amino alcohol or diamine reagent using the methodology used in Example 1.

[0181] Example 33: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(diethylamino)ethyl)(methyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 62%; 1H NMR(500MHz,CDCl3)δ5.43-5.28(m,4H),4.48(t,J=5.6Hz,1H),4.13(m,6H),3.56(dt,J=9.3,6. 7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.30(m,2H),2.94(s,1.5H),2.91(s,1.5H),2.77(t,J=6. 7Hz,2H),2.54(m,6H),2.39(m,3H),2.30(t,J=7.6Hz,2H),2.04(m,4H),1.92(td,J=7.6,5.5Hz, 2H),1.64-1.54(m,7H),1.39-1.22(m,33H),1.01(m,6H),0.88(m,9H)ppm;MS:852.80m / z[M+H].

[0182] Example 34: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(dimethylamino)ethyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 44%; 1 H NMR(500MHz,CDCl3)δ5.43-5.28(m,4H),5.20(br t,J=5.1Hz,1H),4.48(t,J=5.5Hz,1H),4.13(m,6H),3.56(dt,J=9.3,6.7Hz,2H),3 .40(dt,J=9.3,6.7Hz,2H),3.24(m,2H),2.77(t,J=6.7Hz,2H),2.43-2.34(m,5H), 2.30(t,J=7.6Hz,2H),2.22(s,6H),2.05(q,J=6.9Hz,5H),1.92(td,J=7.6,5.4Hz, 2H),1.63-1.52(m,7H),1.40-1.24(m,32H),0.88(m,9H)ppm;MS:810.73m / z[M+H].

[0183] Example 35: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 53%; 1 H NMR(500MHz,CDCl3)δ5.43-5.28(m,4H),5.20(t,J=5.1Hz,1H),4.48(t,J=5.6Hz,1H),4.12(m,6H), 3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.24(m,2H),2.77(t,J=6.7Hz,2H),2.48-2 .33(m,7H),2.30(t,J=7.6Hz,2H),2.20(s,3H),2.05(q,J=6.9Hz,4H),1.92(td,J=7.7,5.5Hz,2H), 1.62-1.52(m,7H),1.35-1.23(m,33H),1.04(t,J=7.2Hz,3H),0.88(m,9H)ppm;MS:824.77m / z[M+H].

[0184] Example 36: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 46%; 1H NMR(400MHz,CDCl3)δ5.43-5.27(m,4H),5.20(br s,1H),4.48(t,J=5.5Hz,1H),4.12(m,6H),3.78(br s,1H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.4Hz,2H),2.50-2.19(m,8H),2.05(q,J=6.8Hz,4H), 1.92(td,J=7.6,5.5Hz,2H),1.73-1.52(m,14H),1.41-1.23(m,33H),1.04(t,J=7.2Hz,3H),0.88(m,9H)ppm;MS:850.48m / z[M+H].

[0185] Synthesis of Example 37 Example 37: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((1-ethylpiperidin-4-yl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 1d (400 mg, 0.57 mmol) in acetonitrile (6 mL) were added pyridine (93 μL, 1.15 mmol), DMAP (14 mg, 0.08 mmol), and 4-nitrophenyl chloroformate (173 mg, 0.86 mmol) sequentially at room temperature. After stirring for 2 h, 1-ethylpiperidin-4-amine dihydrochloride (344 mg, 1.72 mmol) and DIPEA (800 μL, 4.60 mmol) were added, and the resulting reaction mixture was stirred at room temperature for an additional 4 h. The reaction mixture was extracted into hexane and washed with water. If necessary, the resulting aqueous layer was re-extracted with hexane. The combined hexane layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (gradient of EtOAc in hexane or methanol in DCM) to afford 157 mg (0.18 mmol, 32% yield) of the desired product as a clear oil. 1H NMR(400MHz,CDCl3)δ5.43-5.27(m,4H),4.59(br d,J=8.1Hz,1H),4.48(t,J=5.6Hz,1H),4.12(m,6H),3.59-3.49(m,3H),3.40(dt,J=9.3,6.7Hz,2H),2.85(br d,J=11.1Hz,2H),2.77(t,J=6.7Hz,2H),2.45-2.35(m,5H),2.30(t,J=7.6Hz,2H),2.05(q,J=7.2Hz,6H ),1.99-1.87(m,4H),1.65-1.21(m,43H),1.07(t,J=7.2Hz,3H),0.88(m,9H)ppm;MS:850.34m / z[M+H].

[0186] Synthesis of Examples 38 to 49 The following example was synthesized from intermediate 1d and a diamine reagent using the method used in Example 1.

[0187] Example 38: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-2-yl)methyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 30%; 1 H NMR(400MHz,CDCl3)δ5.41-5.27(m,4H),5.14(br s,1H),4.48(t,J=5.6Hz,1H),4.16-4.09(m,6H),3.56(dt,J=9.3,6.7Hz, 2H),3.45-3.23(m,4H),2.89(m,1H),2.77(m,3H),2.49-2.26(m,7H),2.19 (m,1H),2.05(q,J=6.8Hz,4H),1.92(td,J=7.5,5.5Hz,2H),1.73-1.25(m ,46H),1.02(t,J=7.0Hz,3H),0.93-0.84(m,9H)ppm;MS:864.39m / z[M+H].

[0188] Example 39: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-methylpyrrolidin-2-yl)methyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 18%; 1 H NMR(400MHz,CDCl3)δ5.43-5.27(m,4H),5.13(s,1H),4.48(t,J=5.6Hz,1H),4 .13(m,6H),3.56(dt,J=9.3,6.7Hz,2H),3.40(m,3H),3.20-2.98(m,2H),2.77( t,J=6.4Hz,2H),2.41-2.17(m,10H),2.05(q,J=6.8Hz,4H),1.98-1.78(m,3H), 1.72-1.52(m,11H),1.41-1.24(m,32H),0.88(m,9H)ppm;MS:836.27m / z[M+H].

[0189] Example 40: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-ethylpyrrolidin-2-yl)methyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 13%; 1H NMR(400MHz,CDCl3)δ5.42-5.27(m,4H),5.15(br d,J=7.7Hz,1H),4.48(t,J=5.6Hz,1H),4.13(m,6H),3.56(dt,J=9.3,6.7Hz,2H),3.43-3.31(m,3H),3.19-3.05(m,2H),2.78(m,3H),2.50(br m,1H),2.39(m,3H),2.30(t,J=7.6Hz,2H),2.26-2.08(m,2H),2.05(q,J=6.8Hz,4H),1.97-1.79(m,3H) ,1.76-1.51(m,11H),1.40-1.22(m,32H),1.09(t,J=7.2Hz,3H),0.88(m,9H)ppm;MS:850.20m / z[M+H].

[0190] Example 41: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((1-ethylpyrrolidin-3-yl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 34%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),5.01(d,J=8.3Hz,1H),4.48(t,J=5.6Hz,1H),4. 20-4.02(m,7H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.86-2.73(m, 3H),2.62-2.19(m,11H),2.05(q,J=6.9Hz,4H),1.92(td,J=7.6,5.5Hz,2H),1.66-1.51( m,9H),1.44-1.21(m,32H),1.10(t,J=7.2Hz,3H),0.88(m,9H)ppm;MS:836.33m / z[M+H].

[0191] Example 42: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((1-methylpiperidin-3-yl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 57%; 1 H NMR(400MHz,CDCl3)δ5.43-5.27(m,4H),5.18(br m,1H),4.48(t,J=5.6Hz,1H),4.13(m,6H),3.78(br m,1H),3.56(dt,J=9.2,6.7Hz,2H),3.40(dt,J=9.2,6.7Hz,2H),2.77(t,J=6.4Hz,2H),2.47-2.13(m,11H),2.05(q,J= 6.9Hz,4H),1.92(td,J=7.6,5.5Hz,3H),1.71-1.52(m,11H),1.41-1.24(m,33H),0.88(m,9H)ppm;MS:836.51m / z[M+H].

[0192] Example 43: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-methylpyrrolidin-3-yl)methyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 30%; 1H NMR(400MHz,CDCl3)δ5.44-5.30(m,4H),5.19(t,J=5.1Hz,1H),4.51(t,J=5.5Hz,1H),4.18 -4.11(m,6H),3.58(dt,J=9.2,6.7Hz,2H),3.42(dt,J=9.3,6.7Hz,2H),3.19(t,J=5.9Hz,2H) ),2.84-2.79(t,J=6.4Hz,2H),2.64(m,1H),2.56(t,J=8.0Hz,1H),2.44-2.31(m,11H),2.10 -1.92(m,7H),1.71-1.46(m,9H),1.44-1.19(m,33H),0.91(m,9H)ppm;MS:836.31m / z[M+H].

[0193] Example 44: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((1-(diethylamino)propan-2-yl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 78%; 1 H NMR(400MHz,CDCl3)δ5.42-5.27(m,4H),5.07(d,J=5.6Hz,1H),4.48(t,J=5.6Hz,1H),4.12( m,6H),3.67-3.51(m,3H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.4Hz,2H),2.61-2.26(m,1 0H),2.04(dd,J=7.8,5.9Hz,4H),1.92(td,J=7.6,5.5Hz,2H),1.68-1.50(m,7H),1.41-1.24 (m,34H),1.17(d,J=6.4Hz,3H),0.99(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:852.59m / z[M+H].

[0194] Example 45: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((1-(dimethylamino)propan-2-yl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 68%; 1 H NMR(400MHz,CDCl3)δ5.42-5.27(m,4H),5.02(br m,1H),4.48(t,J=5.6Hz,1H),4.18-4.07(m,6H),3.66(m,1H),3.56(dt,J=9.3,6.7Hz,2H ),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.5Hz,2H),2.42-2.27(m,6H),2.22(s,6H),2.1 4(dd,J=12.4,5.5Hz,1H),2.05(q,J=6.9Hz,4H),1.92(td,J=7.6,5.5Hz,2H),1.65-1.50 (m,8H),1.41-1.25(m,32H),1.18(d,J=6.4Hz,3H),0.88(m,9H)ppm;MS:824.28m / z[M+H].

[0195] Example 46: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(4-hydroxypiperidin-1-yl)propyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 26%; 1H NMR(400MHz,CDCl3)δ6.72(br s,1H),5.42-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.23-4.05(m,6H),3.62(br s,1H)3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.27(q,J=5.7Hz,2H),2.83(d,J=11.2Hz,2H),2.77(m,2H),2.4 9-2.26(m,7H),2.09-2.02(m,6H),1.93(m,4H),1.68-1.52(m,11H),1.41-1.24(m,34H),0.88(m,9H)ppm;MS:880.46m / z[M+H].

[0196] Example 47: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-methylpiperazine-1-carboxylate [ka] Yield 61%; 1 H NMR(400MHz,CDCl3)δ5.42-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.18-4.09(m,6H),3.56(dt,J=9.3,6.7Hz,2H),3.47(br s,4H),3.40(dt,J=9.3,6.7Hz,2H),2.81-2.73(m,2H),2.45-2.26(m,11H),2.05(q,J=6.8Hz,4H),1.9 2(td,J=7.6,5.5Hz,2H),1.65-1.49(m,7H),1.41-1.21(m,34H),0.88(m,9H)ppm;MS:822.25m / z[M+H].

[0197] Example 48: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-ethylpiperazine-1-carboxylate [ka] Yield 66%; 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.6Hz,1H),4.19-4.09(m,6H),3.56(dt,J=9.3,6.7Hz,2H),3.48(br s,4H),3.40(dt,J=9.3,6.7Hz,2H),2.81-2.73(m,2H),2.47-2.35(m,8H),2.30(t,J=7.6Hz,2H),2.05(q,J=6.9Hz,4H),1.92 (td,J=7.6,5.5Hz,2H),1.65-1.52(m,7H),1.41-1.24(m,34H),1.09(t,J=7.2Hz,3H),0.88(m,9H)ppm;MS:836.30m / z[M+H].

[0198] Example 49: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(4-hydroxypiperidin-1-yl)ethyl)carbamoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 58%; 1 H NMR(400MHz,CDCl3)δ5.43-5.27(m,3H),5.18(s,1H),4.48(t,1H),4.17-4.09(m,6H ),3.76-3.65(m,1H),3.56(d,2H),3.40(dt,2H),3.30-3.21(m,2H),2.81-2.66(m,4 H),2.53-2.34(m,6H),2.30(t,2H),2.21-2.10(m,2H),2.05(q,4H),1.97-1.85(m,5 H),1.67-1.51(m,10H),1.40-1.25(m,35H),0.94-0.84(m,9H);MS:866.41m / z[M+H].

[0199] Synthesis of Example 50 Example 50: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-ethylazetidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 1d (300 mg, 0.43 mmol) in acetonitrile (6 mL) were added pyridine (69 μL, 86 mmol), DMAP (11 mg, 0.086 mmol), and 4-nitrophenyl chloroformate (134 mg, 0.65 mmol) sequentially at room temperature. After stirring for 2 h, (1-ethylazetidin-3-yl)methanol hydrochloride (194 mg, 1.29 mmol) and EtN (359 μL, 2.58 mmol) were added, and the resulting reaction mixture was stirred at room temperature for an additional 4 h. The reaction mixture was extracted into hexane and washed with water. If necessary, the resulting aqueous layer was re-extracted with hexane. The combined hexane layers were dried over anhydrous MgSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (gradient of EtOAc in hexane) to give 181 mg (0.22 mmol, 50% yield) of the desired product as a clear oil. 1 H NMR(400MHz,CDCl3)δ5.35(m,4H),4.48(t,J=5.5Hz,1H),4.27-4.14(m,8H),3.66-3.36(m,4H),2.94(br s,1H),2.78(m,3H),2.42(m,5H),2.30(t,J=7.6Hz,2H),2.05(q,J=7.0Hz,4H),1.92(m,2H),1 .62-1.52(m,8H),1.31(m,35H),0.95(t,J=7.2Hz,3H),0.89(m,9H)ppm;MS:837.30m / z[M+H].

[0200] Synthesis of Examples 51 to 53 The following example was synthesized from intermediate 1d and an amino alcohol reagent using the methodology used in Example 1.

[0201] Example 51: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-ethylpyrrolidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 57%; 1 H NMR(500MHz,CDCl3)δ5.42-5.29(m,4H),4.48(t,J=5.5Hz,1H),4.22-4.09(m,8H) ,3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.6Hz,2H),2.96-2.49(m,7H),2.4 5-2.36(m,4H),2.30(t,J=7.6Hz,2H),2.20-2.29(m,5H),1.91(td,J=7.6,5.3Hz, 2H),1.75-1.52(m,8H),1.40-1.16(m,37H),0.88(m,9H)ppm;MS:851.48m / z[M+H].

[0202] Example 52: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-ethyl-3-methylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 66%; 1 H NMR(500MHz,CDCl3)5.43-5.28(m,4H),4.48(t,J=5.5Hz,1H),4.22-4.12(m,8H),3.56(dt,J=9.3,6.7Hz, 2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.7Hz,2H),2.46-2.37(m,3H),2.31(t,J=7.6Hz,2H),2.21(br s,6H),2.05(q,J=7.0Hz,4H),1.92(td,J=7.5,5.4Hz,2H),1.57(m,10H),1.45-1.17(m,40H),0.88(m,9H)ppm;MS:865.58m / z[M+H].

[0203] Example 53: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-methylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 42%; 1 H NMR(400MHz,CDCl3)δ5.45-5.24(m,4H),4.48(t,J=5.5Hz,1H),4.24-3.93(m,8H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.2,6.7Hz,2H),3.09(br m,2H),2.77(t,J=6.5Hz,1H),2.70-2.16(m,7H),2.05(q,J=6.9Hz,4H),1.92(td,J=7.6,5.5Hz, 2H),1.87-1.71(m,3H),1.57(m,10H),1.42-1.00(m,36H),0.88(m,9H)ppm;MS:850.77m / z[M+H].

[0204] Synthesis of Example 54 Intermediate 54a: (Z)-9-(non-2-en-1-yloxy)-9-oxononanoic acid [ka] To a solution of nonanedioic acid (25 g, 132 mmol, 1 equiv.) in THF (0.4–0.5 M) was added oxalyl chloride (1.1–1.5 equiv.) at 15–25 °C. After stirring for at least 10 min, DMF (0.01–0.1 equiv.) was added, followed by an additional stirring period of at least 10 min. (Z)-non-2-en-1-ol (24.3 g, 171 mmol, 1.3 equiv.) was then added dropwise, and the resulting mixture was stirred for at least 30 min at 15–25 °C. The reaction mixture was concentrated and directly purified using silica gel chromatography to afford 17.3 g (55.4 mmol, 42% yield) of the desired product as an oil. 1H NMR(400MHz,CDCl3)δ5.65(m,1H),5.53(m,1H),4.62(d,J=6.8Hz,2H),2.33(m,4H),2. 10(m,2H),1.63(m,4H),1.28(m,14H),0.88(t,J=6.8Hz,3H)ppm;MS:335.27m / z[M+Na].

[0205] Intermediate 54b: 3-Hydroxy-2-(hydroxymethyl)propyl 4,4-bis(octyloxy)butanoate [ka] To a solution of intermediate 1c (16.0 g, 46.4 mmol), DMAP (1.1 g, 9.28 mmol), DIPEA (24.1 mL, 139 mmol), and 2-(hydroxymethyl)propane-1,3-diol (6.39 g, 60.3 mmol) in DCM (231 mL) was added EDC·HCl (10.6 g, 55.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 18 h, diluted with water, and the organic layer was concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–100% EtOAc in hexanes) to afford 6.7 g (15.4 mmol, 33% yield) of the desired product. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.24(d,J=6.3Hz,2H),3.75(m,4H),3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.54(br ppm.

[0206] Intermediate 54c: (Z)-1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)9-(non-2-en-1-yl)nonanedioate [ka] To a solution of intermediate 54a (1-1.2 equiv.), DMAP (0.15 equiv.), DIPEA (1.6-3.0 equiv.), and intermediate 54b (900 mg, 2.08 mmol, 1 equiv.) in DCM (0.2 M) was added EDCI·HCl (1.6 equiv.) at room temperature. The reaction mixture was stirred at room temperature for at least 2 h, concentrated in vacuo, and directly purified using silica gel chromatography (gradient of EtOAc in hexanes) to give 503 mg (0.69 mmol, 33% yield) of the desired product as a clear oil. 1 H NMR(400MHz,CDCl3)δ5.64(m,1H),5.51(m,1H),4.61(d,J=6.7Hz,2H),4.48(t,J=5.5 Hz,1H),4.17(m,4H),3.65-3.51(m,4H),3.39(dt,J=9.4,6.7Hz,2H),2.40(t,J=7.5Hz ,2H),2.29(m,5H),2.19(m,1H),2.09(q,J=7.2Hz,2H),1.93(td,J=7.5,5.4Hz,2H),1. 67-1.50(m,8H),1.40-1.19(m,32H),0.87(t,J=6.7Hz,9H)ppm;MS:749.69m / z[M+Na].

[0207] Example 54: (Z)-1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-(non-2-en-1-yl)nonanedioate [ka] Example 54 was synthesized in 48% yield from intermediate 54c and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1H NMR(400MHz,CDCl3)δ5.68-5.58(m,1H),5.55-5.46(m,1H),4.61(dd,J=6.8,1.2Hz,2H),4.47(t ,J=5.5Hz,1H),4.21-4.06(m,8H),3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.5 0(m,6H),2.45-2.35(m,3H),2.29(t,J=7.6Hz,4H),2.09(m,2H),1.91(m,2H),1.80(m,2H),1.67 -1.49(m,8H),1.40-1.21(m,34H),1.00(t,J=7.1Hz,6H),0.87(m,9H)ppm;MS:885.65m / z[M+H].

[0208] Synthesis of Example 55 Intermediate 55a: (Z)-7-(non-2-en-1-yloxy)-7-oxoheptanoic acid [ka] Intermediate 54a was synthesized from heptanedioic acid and (Z)-non-2-en-1-ol using the method used for intermediate 54a. 1 H NMR (400MHz, CDCl3) δ5.58(m,1H),5.44(m,1H),4.55(d,J=6.8Hz,2H),2.26(m,4H),2.03(m,2H),1.59(m,4H),1.30(m,10H),0.81(t,J=6.8Hz,3H)ppm.

[0209] Intermediate 55b: (Z)-1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) 7-(non-2-en-1-yl)heptanedioate [ka] Intermediate 55b was synthesized in 30% yield from intermediate 55a and intermediate 54b using the method used for intermediate 54c. 1H NMR(400MHz,CDCl3)δ5.64(m,1H),5.51(m,1H),4.62(dd,J=6.8,1.2Hz,2H),4.48(t,J= 5.5Hz,1H),4.22-4.07(m,4H),3.67-3.51(m,4H),3.40(dt,J=9.3,6.7Hz,2H),2.41(t,J =7.5Hz,2H),2.32(td,J=7.5,5.9Hz,4H),2.19(m,1H),2.09(m,2H),1.93(td,J=7.5,5. 4Hz, 2H), 1.68-1.50 (m, 8H), 1.39-1.23 (m, 30H), 0.88 (m, 9H) ppm; MS: 721.67m / z [M+Na].

[0210] Example 55: (Z)-1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 7-(non-2-en-1-yl)heptanedioate [ka] Example 55 was synthesized in 46% yield from intermediate 55b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR (400 MHz, CDCl3) δ 1 H NMR(400MHz,CDCl3)δ5.69-5.59(m,1H),5.55-5.47(m,1H),4.62(dd,J=6.9,1.3Hz,2H),4.48(t,J=5.6H z,1H),4.20(m,4H),4.13(m,4H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.94-2.57(br m,6H),2.47-2.36(m,3H),2.31(td,J=7.5,2.0Hz,4H),2.19-1.89(m,6H) 1.70-1.49(m,9H),1.41-1.08(m,35H),0.88(m,9H);MS:857.57m / z[M+H].

[0211] Synthesis of Example 56 Intermediate 56a: (Z)-5-(non-2-en-1-yloxy)-5-oxopentanoic acid [ka] Intermediate 56a was synthesized from pentanedioic acid and (Z)-non-2-en-1-ol using the method used for intermediate 54a. 1 H NMR (400MHz, CDCl3) δ5.66(m,1H),5.52(m,1H),4.64(d,J=6.8Hz,2H),2.43(m,4H),2.15(m,2H),1.97(m,2H),1.28(m,8H),0.89(t,J=6.8Hz,3H)ppm.

[0212] Intermediate 56b: (Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propylnon-2-en-1-yl glutarate [ka] Intermediate 56b was synthesized in 32% yield from intermediate 56a and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ5.65(m,1H),5.51(m,1H),4.63(dd,J=6.9,1.3Hz,2H) ,4.49(t,J=5.5Hz,1H),4.18(m,4H),3.64(m,2H),3.56(dt,J=9.3,6.7Hz,2H ),3.40(dt,J=9.3,6.7Hz,2H),2.40(m,6H),2.19(m,1H),2.20(m,2H),1.94( m,4H),1.56(m,4H),1.39-1.19(m,28H),0.88(m,9H)ppm;MS:693.64[M+Na]

[0213] Example 56: (Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propylnon-2-en-1-yl glutarate [ka] Example 56 was synthesized in 66% yield from intermediate 56b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ5.74-5.58(m,1H),5.58-5.45(m,1H),4.63(dd,J=6.8,1.3Hz,2H),4.48(t,J=5.5Hz,1H),4 .25-4.17(m,4H),4.14(dd,J=6.2,3.1Hz,4H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.71(br m,6H),2.48-2.35(m,7H),2.09(qd,J=7.3,1.5Hz,3H),2.04-1.87(m,5H),1.66-1.52(m,5H),1.42-1.30(m,30H),1.15(br m,3H),0.88(m,9H);MS:829.67m / z[M+H].

[0214] Synthesis of Example 57 Intermediate 57a: 9-(hexyloxy)-9-oxononanoic acid [ka] Intermediate 57a was synthesized from nonanedioic acid and hexan-1-ol using the method used for intermediate 54a. 1 H NMR (400MHz, CDCl3) δ4.05(t,J=6.8Hz,2H),2.36-2.26(m,4H),1.60(m,6H),1.32(m,12H),0.88(t,J=6.8Hz,3H)ppm.

[0215] Intermediate 57b: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)9-hexylnonanedioate [ka] Intermediate 57b was synthesized in 30% yield from intermediate 57a and intermediate 54b using the method used for intermediate 54c. 1H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.21-4.12(m,4H),4.04(t,J=6.7Hz ,2H),3.61(t,J=5.9Hz,2H),3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz, 2H),2.40(t,J=7.5Hz,2H),2.29(m,5H),2.19(m,1H),1.92(td,J=7.5,5.4Hz,2H ),1.66-1.49(m,9H),1.38-1.24(m,32H),0.87(m,9H)ppm;MS:709.64m / z[M+Na].

[0216] Example 57: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-hexylnonanedioate [ka] Example 57 was synthesized in 65% yield from intermediate 57b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ4.47(t,J=5.5Hz,1H),4.23-4.08(m,8H),4.05(t,J=6.8Hz, 2H),3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.50(m,6H),2.40( m,3H),2.29(td,J=7.5,5.9Hz,4H),1.91(m,2H),1.80(m,2H),1.66-1.49(m,10H) ,1.39-1.21(m,32H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:845.63m / z[M+H].

[0217] Synthesis of Example 58 Intermediate 58a: 9-(octyloxy)-9-oxononanoic acid [ka] Intermediate 58a was synthesized from nonanedioic acid and octan-1-ol using the method used for intermediate 54a. 1 H NMR (400MHz, MeOD) δ4.05(t,J=6.6Hz,2H),2.28(m,4H),1.61(m,6H),1.32(m,16H),0.90(t,J=6.6Hz,3H)ppm.

[0218] Intermediate 58b: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) 9-octylnonanedioate [ka] Intermediate 58b was synthesized in 26% yield from intermediate 58a and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.22-4.13(m,4H),4.05(t,J=6. 7Hz,2H),3.62(t,J=5.9Hz,2H),3.56(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.4 ,6.7Hz,2H),2.40(t,J=7.5Hz,2H),2.34-2.24(m,5H),2.19(m,1H),1.93(td ,J=7.5,5.4Hz,2H),1.66-1.49(m,10H),1.39-1.19(m,36H),0.87(m,9H)ppm.

[0219] Example 58: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-octylnonanedioate [ka] Example 58 was synthesized from intermediate 58b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1H NMR(400MHz,CDCl3)δ4.47(t,J=5.5Hz,1H),4.22-4.08(m,8H),4.05(t,J=6.8Hz,2H) ,3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.50(m,6H),2.40(m,3H),2 .29(td,J=7.6,6.2Hz,4H),1.91(td,J=7.6,5.5Hz,2H),1.80(m,2H),1.66-1.51(m,1 0H),1.38-1.21(m,36H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:873.67m / z[M+H].

[0220] Synthesis of Example 59 Intermediate 59a: 9-(decyloxy)-9-oxononanoic acid [ka] Intermediate 59a was synthesized in 39% yield from nonanedioic acid and decan-1-ol using the method used for intermediate 54a. 1 H NMR (400MHz, MeOD) δ4.08(t,J=6.6Hz,2H),2.31(m,4H),1.63(m,6H),1.33(m,20H),0.92(t,J=6.8Hz,3H)ppm.

[0221] Intermediate 59b: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)9-decylnonanedioate [ka] Intermediate 59b was synthesized in 43% yield from intermediate 59a and intermediate 54b using the method used for intermediate 54c. 1H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.22-4.12(m,4H),4.04(t,J=6.8Hz,2 H),3.61(t,J=5.9Hz,2H),3.56(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H) ,2.40(t,J=7.5Hz,2H),2.34-2.24(m,5H),2.19(m,1H),1.93(td,J=7.5,5.4Hz,2H ),1.66-1.50(m,10H),1.37-1.21(m,40H),0.87(m,9H)ppm;MS:765.68m / z[M+Na].

[0222] Example 59: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-decylnonanedioate [ka] Example 59 was synthesized in 65% yield from intermediate 59b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ4.47(t,J=5.5Hz,1H),4.21-4.09(m,8H),4.04(t,J=6.7Hz, 2H),3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.50(m,6H),2.39( m,3H),2.28(m,4H),1.91(td,J=7.6,5.5Hz,2H),1.80(m,2H),1.66-1.49(m,10H) ,1.37-1.21(m,40H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:901.76m / z[M+H].

[0223] Synthesis of Example 60 Intermediate 60a: 12-oxo-12-(pentyloxy)dodecanoic acid [ka] Intermediate 60a was synthesized from dodecanedioic acid and pentan-1-ol using the method used for intermediate 54a. 1 H NMR (400MHz, CDCl3) δ4.07(t,J=6.8Hz,2H),2.30(m,4H),1.63(m,6H),1.33(m,16H),0.93(t,J=6.8Hz,3H)ppm.

[0224] Intermediate 60b: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)12-pentyldodecanedioate [ka] Intermediate 60b was synthesized in 21% yield from intermediate 60a and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.22-4.11(m,4H),4.05(t,J=6.7Hz ,2H),3.61(t,J=5.9Hz,2H),3.56(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz, 2H),2.40(t,J=7.5Hz,2H),2.34-2.24(m,5H),2.19(m,1H),1.93(td,J=7.5,5.4H MS:737.68m / z[M+Na].

[0225] Example 60: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 12-pentyldodecanedioate [ka] Example 60 was synthesized in 65% yield from intermediate 60b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1H NMR(400MHz,CDCl3)δ4.47(t,J=5.5Hz,1H),4.24-4.09(m,8H),4.05(t,J=6.7Hz,2H),3. 55(dt,J=9.2,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.50(m,6H),2.40(m,3H),2.29(td ,J=7.6,5.8Hz,4H),1.91(td,J=7.6,5.5Hz,2H),1.80(dq,J=8.7,6.7Hz,2H),1.68-1.49( m,10H),1.38-1.24(m,36H),1.00(t,J=7.1Hz,6H),0.89(m,9H)ppm;MS:873.67m / z[M+H].

[0226] Synthesis of Example 61 Intermediate 61a: 12-(heptyloxy)-12-oxododecanoic acid [ka] Intermediate 61a was synthesized from dodecanedioic acid and heptan-1-ol using the method used for intermediate 54a. 1 H NMR (400MHz, CDCl3) δ4.05(t,J=6.8Hz,2H),2.31(m,4H),1.61(m,6H),1.30(m,20H),0.88(t,J=6.8Hz,3H)ppm.

[0227] Intermediate 61b: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) 12-heptyldodecanedioate [ka] Intermediate 61b was synthesized in 33% yield from intermediate 61a and intermediate 54b using the method used for intermediate 54c. 1H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.22-4.12(m,4H),4.05(t,J=6.7Hz, 2H),3.64-3.59(m,2H),3.56(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H), 2.40(t,J=7.5Hz,2H),2.35-2.23(m,5H),2.19(m,1H),1.93(td,J=7.5,5.5Hz,2H ),1.66-1.50(m,10H),1.37-1.23(m,40H),0.87(m,9H)ppm;MS:765.68m / z[M+Na].

[0228] Example 61: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 12-heptyldodecanedioate [ka] Example 61 was synthesized in 69% yield from intermediate 61b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ4.47(t,J=5.5Hz,1H),4.22-4.09(m,8H),4.05(t,J=6.7Hz,2H) ,3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.6Hz,2H),2.50(m,6H),2.40(m,3H),2 .29(td,J=7.6,6.3Hz,4H),1.91(td,J=7.6,5.5Hz,2H),1.80(m,2H),1.66-1.49(m,1 0H),1.37-1.24(m,40H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:901.72m / z[M+H].

[0229] Synthesis of Example 62 Intermediate 62a: Methyl 6-hydroxyhexanoate [ka] A mixture of oxepan-2-one (100 g, 876 mmol) in HCl in MeOH (1000 mL) was stirred at 70° C. for 12 h. The reaction mixture was adjusted to pH 8 by the addition of aqueous NaHCO, and then it was extracted into EtOAc (3×1000 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to give 100 g (685 mmol, 78% yield) of crude product as a colorless oil, which required no further purification. 1 H NMR (400MHz, CDCl3) δ3.65(m,5H),2.33(t,J=7.6Hz,2H),1.69-1.57(m,4H),1.41(m,3H)ppm.

[0230] Intermediate 62b: Methyl 6-oxohexanoate [ka] To a solution of intermediate 62a (93 g, 636 mmol) in DCM (1200 mL) was added EtN (266 mL, 1.91 mol) at 0 °C. Then, sulfur trioxide pyridine (203 g, 1.27 mol) in DMSO (497 mL) was added dropwise at 0 °C. The resulting reaction mixture was stirred at 15 °C for 2 h, diluted with water (1000 mL), and extracted into DCM (2 × 1000 mL). The combined organic layers were washed with brine (1000 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–5% EtOAc in petroleum ether) to afford 63 g (437 mmol, 69% yield) of the desired product as a colorless oil. 1 H NMR (400MHz, CDCl3) δ 9.77 (m, 1H), 3.67 (s, 3H), 2.47 (m, 2H), 2.33 (m, 2H), 1.67 (m, 4H) ppm.

[0231] Intermediate 62c: Methyl 6,6-dimethoxyhexanoate [ka] To a solution of intermediate 62b (60 g, 416 mmol) in MeOH (300 mL) was added H2SO4 (2.22 mL, 4.08 g, 41.6 mmol). The reaction mixture was stirred at 80 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was diluted with saturated NaHCO3 until pH 7 and extracted into EtOAc (3 × 500 mL). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–5% EtOAc in petroleum ether) to give 30 g (158 mmol, 38% yield) of the desired product as a colorless oil. 1 H NMR (400MHz, CDCl3) δ4.36(t,J=5.6Hz,1H),3.67(s,3H),3.32(s,6H),2.32(t,J=7.6Hz,2H),1.67-1.60(m,4H),1.35(m,2H)ppm.

[0232] Intermediate 62d: Methyl 6,6-bis(octyloxy)hexanoate [ka] To a solution of intermediate 62c (30 g, 158 mmol) in octan-1-ol (80 mL) was added KHSO (10.7 g, 78.9 mmol). The reaction mixture was stirred at 80 °C for 12 h, diluted with petroleum ether (150 mL), and directly purified using silica gel chromatography (petroleum ether) to give 35 g (90.5 mmol, 57% yield) of the desired product as a colorless oil. 1 H NMR(400MHz,CDCl3)δ4.46(t,J=5.6Hz,1H),3.67(s,3H),3.57(m,2H),3.40(m,2H),2. 32(t,J=7.6Hz,2H),1.67-1.55(m,8H),1.36-1.28(m,22H),0.80(t,J=6.8Hz,6H)ppm.

[0233] Intermediate 62e: 6,6-bis(octyloxy)hexanoic acid [ka] To a solution of intermediate 62d (35 g, 90.5 mmol) in MeOH (150 mL) was added a solution of NaOH (10.9 g, 272 mmol) in HO (50 mL). After stirring at 15 °C for 5 h, the reaction mixture was concentrated under reduced pressure, diluted with water (150 mL), and extracted into petroleum ether (200 mL). The organic layer was washed with brine (200 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–5% EtOAc in petroleum ether) to afford 9.5 g (25.5 mmol, 28% yield) of the desired product as a yellow oil. 1 H NMR(400MHz,CDCl3)δ4.47(t,J=5.6Hz,1H),3.56(m,2H),3.40(m,2H),2.37(t,J=7. 6Hz,2H),1.69-1.55(m,8H),1.33(m,22H),0.89(t,J=6.8Hz,6H)ppm;MS:371.3[MH].

[0234] Intermediate 62f: 3-((6,6-bis(octyloxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate [ka] Intermediate 62f was synthesized in 34% yield from intermediate 1a and intermediate 62e using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ5.45-5.29(m,4H),4.47(t,J=5.7Hz,1H),4.26-4.11(m,4H),3 .63(t,J=5.9Hz,2H),3.57(dt,J=9.3,6.7Hz,2H),3.41(dt,J=9.3,6.7Hz,2H),2.78( m,2H),2.35(td,J=7.6,6.4Hz,4H),2.28(t,J=6.3Hz,1H),2.21(m,1H),2.07(q,J=6. MS: 745.74 [M+Na].

[0235] Example 62: 3-((6,6-bis(octyloxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Example 62 was synthesized in 47% yield from intermediate 62f and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ5.43-5.27(m,4H),4.44(t,J=5.7Hz,1H),4.22-4.07(m,8H),3.55(d t,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.4Hz,2H),2.51(q,J=6.9Hz ,6H),2.41(m,1H),2.31(td,J=7.6,5.4Hz,4H),2.04(q,J=6.8Hz,4H),1.81(m,2H),1.60( m,10H),1.44-1.20(m,36H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:881.76m / z[M+H].

[0236] Synthesis of Example 63 Intermediate 63a: Heptadecan-9-ol [ka] To a solution of nonanal (40 g, 281.22 mmol) in THF (400 mL) was added bromo(octyl)magnesium (1 M, 309.34 mL) at -40 °C. The mixture was stirred at 16 °C for 2 h. The residue was poured into saturated NH4Cl (500 mL) and extracted into ethyl acetate (3 × 700 mL). The combined organic layers were washed with brine (800 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–1.5% EtOAc in petroleum ether) to afford 32 g (124.8 mmol, 44% yield) of the desired product as a white solid. 1 H NMR (400MHz, CDCl3) δ3.52 (m, 1H), 1.42-1.21 (m, 28H), 0.81 (t, J = 6.6Hz, 6H) ppm.

[0237] Intermediate 63b: 5-(heptadecan-9-yloxy)-5-oxopentanoic acid [ka] Intermediate 63b was synthesized from pentanedioic acid and intermediate 63a using the method used for intermediate 54a. 1 H NMR (400MHz, MeOD) δ4.90(m,1H),2.36(m,4H),1.91(m,2H),1.54(m,4H),1.29(m,24H)0.90(t,J=6.6Hz,6H)ppm.

[0238] Intermediate 63c: Heptadecan-9-yl(3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl)glutarate [ka] Intermediate 63c was synthesized in 41% yield from intermediate 1a and intermediate 63b using the method used for intermediate 1d. 1H NMR(400MHz,CDCl3)δ5.43-5.26(m,4H),4.86(m,1H),4.22-4.08(m,4H),3.61(t,J=5.9Hz,2H),2.77(m,2H),2.43-2.28(m,6H),2.26-2. 16(m,2H),2.04(q,J=6.8Hz,4H),1.95(m,2H),1.65-1.58(m,2H),1.50(m,4H),1.40-1.18(m,37H),0.88(m,9H)ppm;MS:721.84m / z[M+H].

[0239] Example 63: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propylheptadecan-9-yl glutarate [ka] Example 63 was synthesized in 59% yield from intermediate 63c and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(500MHz,CDCl3)δ5.42-5.28(m,4H),4.86(m,1H),4.21-4.08(m,8H),2.76(t,J=6.7H z,2H),2.50(q,J=7.0Hz,6H),2.46-2.27(m,7H),2.04(d,J=6.2Hz,4H),1.94(m,J=7.5Hz ,2H),1.80(m,J=6.8Hz,2H),1.60(t,J=7.3Hz,2H),1.50(q,J=6.2Hz,4H),1.35(t,J=7.1 Hz,4H),1.33-1.22(m,34H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:879.78m / z[M+H].

[0240] Synthesis of Example 64 Intermediate 64a: 7-(heptadecan-9-yloxy)-7-oxoheptanoic acid [ka] Intermediate 64 was synthesized from heptanedioic acid and intermediate 63a using the method used for intermediate 54a. 1 H NMR (400MHz, MeOD) δ4.90(m,1H),2.30(m,4H),1.62(m,4H),1.53(m,4H),1.29(m,26H),0.90(t,J=6.8Hz,6H)ppm.

[0241] Intermediate 64b: 1-(heptadecan-9-yl) 7-(3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl)heptanedioate [ka] Intermediate 64b was synthesized in 43% yield from intermediate 1a and intermediate 64a using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ5.42-5.27(m,4H),4.85(m,1H),4.22-4.12(m,4H),3.61(t,J=5.9Hz,2H),2.76(dd,J=7.2,5.9Hz,2H),2.36-2.24(m,6 H),2.19(m,1H),2.04(t,J=3.5Hz,4H),1.69-1.58(m,6H),1.49(t,J=6.2Hz,4H),1.41-1.19(m,40H),0.88(m,9H)ppm;MS:749.83m / z[M+H].

[0242] Example 64: 1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl) 7-(heptadecan-9-yl)heptanedioate [ka] Example 64 was synthesized in 63% yield from intermediate 64b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1H NMR(400MHz,CDCl3)δ5.42-5.26(m,4H),4.85(m,1H),4.22-4.07(m,8H),2.76(m,2H),2.50(m,6H),2.41(m,1H),2.35-2.24(m,7H),2.04(q,J= 6.2Hz,4H),1.80(m,2H),1.63(m,7H),1.49(q,J=6.1Hz,4H),1.38-1.24(m,38H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:907.61m / z[M+H].

[0243] Synthesis of Example 65 Intermediate 65a: 3-((2-hexyldecanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Intermediate 65a was synthesized in 40% yield from intermediate 1a and 2-hexyldecanoic acid using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ5.45-5.25(m,4H),4.25-4.10(m,4H),3.61(t,J=6.0Hz,2H),2.77(t,J=6.4Hz,2H),2.39-2.29(m ,3H),2.27-2.16(m,2H),2.04(q,J=6.8Hz,4H),1.60(m,4H),1.50-1.16(m,36H),0.88(m,9H)ppm;MS:607.77m / z[M+H].

[0244] Example 65: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((2-hexyldecanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Example 65 was synthesized in 63% yield from intermediate 65a and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1H NMR(400MHz,CDCl3)δ5.43-5.27(m,4H),4.22-4.07(m,8H),2.77(t,J=6.5Hz,2H),2.50(q,J=7.1Hz,6H),2.42(m,1H),2.36-2.26(m,3H),2.0 9-2.00(m,4H),1.80(m,2H),1.63-1.51(m,4H),1.43(m,2H),1.39-1.1 9(m,34H),1.00(t,J=7.1Hz,6H),0.87(m,9H)ppm;MS:765.68m / z[M+H].

[0245] Synthesis of Example 66 Intermediate 66a: Heptadecan-9-one [ka] To a solution of intermediate 63a (32.8 g, 127 mmol) in DCM (300 mL) was added Dess-Martin periodinane (81.4 g, 192 mmol). The reaction mixture was stirred at 15° C. for 5 h, concentrated in vacuo, and directly purified using silica gel chromatography (petroleum ether) to give 20 g (70.5 mmol, 56% yield) of the desired product as a white solid. 1 H NMR (400MHz, CDCl3) δ2.39(t,J=7.4Hz,4H),1.56(m,4H),1.30(m,20H),0.88(t,J=6.8Hz,6H)ppm.

[0246] Intermediate 66b: Methyl 3-octylundec-2-enoate [ka] To a solution of methyl 2-dimethoxyphosphoryl acetate (2 equiv.) in DMF (75 mL) was added NaH (2 equiv.) at 15 °C. After stirring for 1 h, intermediate 66a (7.5 g, 29.48 mmol, 1 equiv.) was added, and the reaction mixture was stirred for an additional 1 h. The reaction mixture was then warmed to 90-110 °C and stirred for 10-48 h. The reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography to give 12 g (38.65 mmol, 66% yield) of the desired product as a yellow oil.

[0247] Intermediate 66c: Methyl 3-octylundecanoate [ka] To a solution of intermediate 66b (7.5 g, 24 mmol) in EtOH (75 mL) under N was added Pd / C (1 g). The suspension was degassed under vacuum and purged with H three times. The mixture was stirred under H (15 psi) at 15 °C for 12 h. The reaction mixture was filtered and washed with MeOH (1 L). The filtrate was concentrated under vacuum and directly purified using silica gel chromatography (petroleum ether) to give 10 g (32.00 mmol, 66% yield) of the desired product as a colorless oil. 1 H NMR (400MHz, CDCl3) δ3.59(s,3H),2.16(d,J=7.2Hz,2H),1.77(br m,1H),1.19(m,28H),0.81(t,J=6.8Hz,6H)ppm.

[0248] Intermediate 66d: 3-octylundecanoic acid [ka] To a solution of intermediate 66c (10 g, 32.0 mmol) in EtOH (50 mL) and HO (50 mL) was added LiOH·HO (2.69 g, 64.0 mmol) and NaOH (2.56 g, 64.0 mmol). The reaction mixture was warmed to 60 °C and stirred for 12 h. The reaction mixture was concentrated to remove EtOH and then poured into water (30 mL). The resulting mixture was acidified to pH 6 with 1 M HCl (aq) and then extracted into ethyl acetate (3 × 60 mL). The combined organic layers were washed with brine (2 × 40 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–50% EtOAc in petroleum ether) to afford 3.6 g (12.4 mmol, 38% yield) of the desired product as a colorless oil. 1 H NMR (400MHz, CDCl3) δ2.20 (br m, 2H), 1.82 (br m, 1H), 1.31 (m, 28H), 0.89 (t, J = 6.8Hz, 6H) ppm.

[0249] Intermediate 66e: 3-hydroxy-2-(((3-octylundecanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Intermediate 66e was synthesized in 39% yield from intermediate 1a and intermediate 66d using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ5.45-5.26(m,4H),4.24-4.10(m,4H),3.61(t,J=6.0Hz,2H),2.77(t,J=6.4Hz,2H),2.32(t,J=7.6Hz,2 H),2.28-2.14(m,4H),2.05(q,J=6.8Hz,4H),1.83(m,1H),1.61(m,2H),1.28(m,42H),0.88(m,9H)ppm;MS:649.67m / z[M+H].

[0250] Example 66: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((3-octylundecanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Example 66 was synthesized in 53% yield from intermediate 66e and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(500MHz,CDCl3)δ5.43-5.28(m,4H),4.19-4.13(m,8H),2.77(t,J=6.7Hz,2H),2.50(q,J=7.0Hz,6H),2.42(m,1H),2.30(t,J=7.6Hz,2H),2 .24(d,J=6.8Hz,2H),2.05(m,4H),1.80(m,3H),1.61(m,2H),1.42-1.15(m,42H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:807.58m / z[M+H].

[0251] Synthesis of Example 67 Intermediate 67a: Methyl 3-heptyldec-2-enoate [ka] To a solution of NaH (14.13 g, 353.36 mmol, 2 equiv.) in THF (0.4 M) was added methyl 2-dimethoxyphosphoryl acetate (64.35 g, 353.36 mmol, 2 equiv.) slowly at 0 °C. After stirring for 1 h, pentadecan-8-one (40 g, 176.68 mmol) was added slowly, and the reaction mixture was warmed to 15 °C. After stirring for an additional 1 h, the reaction mixture was heated to 70 °C and stirred for 48 h. The reaction mixture was cooled to 0 °C, diluted with water, and extracted into petroleum ether. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate or magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography to give 20 g (56.65 mmol, 32% yield) of the desired product as a colorless oil.

[0252] Intermediate 67b: 3-heptyldec-2-en-1-ol [ka] To a solution of intermediate 67a (21.5 g, 76.1 mmol) in THF (200 mL) was added DIBAL (1 M, 228.4 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min and then at 15 °C for 12 h. The reaction mixture was diluted with water (20 mL) at 0 °C, followed by the addition of another 200 mL, followed by extraction into EtOAc (2 × 200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–2% EtOAc in petroleum ether) to afford 17 g (40.1 mmol, 53% yield) of the desired product as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.39(t,J=7.0Hz,1H),4.16(d,J=7.2Hz,2H),3.65(t,J=6.6Hz,1H),2.03(m,4H),1.35-1.28(m,20H),0.89(t,J=6.8Hz,6H)ppm.

[0253] Intermediate 67c: 3-heptyldec-2-enal [ka] To a stirred suspension of IBX (1.5-3.5 M) in DMSO (1.5-3.5 M) was added intermediate 67b (17 g, 66.8 mmol, 1 equiv.) in THF (0.5-1 M) at 30 °C. After stirring at 30 °C for at least 2 h, the reaction mixture was diluted with petroleum ether, washed with water and brine, dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography to afford 12 g (47.5 mmol, 71% yield) of the desired product as a colorless oil. 1H NMR (400MHz, CDCl3) δ9.99(d,J=8.4Hz,1H),5.86(d,J=8.4Hz,1H),2.55(m,2H),2.21(m,2H)1.52(m,4H),1.30(m,16H),0.89(t,J=6.8Hz,6H)ppm.

[0254] Intermediate 67d: Methyl (E)-5-heptyldodeca-2,4-dienoate [ka] Intermediate 67d was synthesized in 44% yield from intermediate 67c and methyl 2-dimethoxyphosphorylacetate using the method used for 67a. 1 H NMR(400MHz,CDCl3)δ7.60(dd,J=15.0Hz,11.8Hz,1H),5.97(d,J=11.6Hz,1H),5.79(d,J=15.2Hz,1H), 3.75(s,3H),2.27(t,J=7.6Hz,2H),2.13(t,J=7.6Hz,2H)1.48-1.29(m,20H),0.89(t,J=6.8Hz,6H)ppm.

[0255] Intermediate 67e: Methyl 5-heptyldodecanoate [ka] To a solution of intermediate 67d (8 g, 20.75 mmol) in MeOH (100 mL) was added Pd / C (10 g, 339.59 μmol, 33.96 μL, 10% purity). The suspension was degassed under vacuum and purged with H several times. The mixture was stirred under H (15 psi) at 15 °C for 12 h. The reaction mixture was filtered and concentrated in vacuo to give 6 g (15.36 mmol, 74% yield) of the desired product as a colorless oil, which did not require further purification. 1 H NMR (400MHz, CDCl3) δ3.68(s,3H),2.29(t,J=7.6Hz,2H),1.59(m,2H),1.28(m,27H),0.88(t,J=6.8Hz,6H)ppm.

[0256] Intermediate 67f: 5-heptyldodecanoic acid [ka] To a solution of intermediate 67e (6 g, 15.36 mmol) in THF (120 mL) was added a solution of NaOH (3.07 g, 76.79 mmol) in water (60 mL). The reaction mixture was stirred at 60 °C for 5 h, diluted with water (20 mL), neutralized to pH 4 with 1 M HCl, and extracted into EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–2% EtOAc in petroleum ether) to afford 3.51 g (11.68 mmol, 76.03% yield) of the desired product as a colorless oil. 1 H NMR(400MHz,MeOD)δ2.27(t,J=7.4Hz,2H),1.59(m,2H),1.29(m,27H),0.91(t,J=6.8Hz,6H)ppm;MS:297.2m / z[MH].

[0257] Intermediate 67g: 3-((5-heptyldodecanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Intermediate 67g was synthesized in 42% yield from intermediate 1a and intermediate 67f using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ5.44-5.26(m,4H),4.17(m,4H),3.61(t,J=5.9Hz,2H),2.76(m,2H),2.31(q,J=7 .3Hz,4H),2.26-2.13(m,2H),2.04(q,J=6.8Hz,4H),1.59(m,5H),1.41-1.16(m,39H),0.88(m,9H)ppm. MS:649.67m / z[M+H].

[0258] Example 67: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((5-heptyldodecanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Example 67 was synthesized in 51% yield from Intermediate 67g and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(500MHz,CDCl3)δ5.42-5.28(m,4H),4.22-4.08(m,8H),2.77(t,J=6.7Hz,2H),2.50(q,J=7.0Hz,6H),2.42(m,1H),2.29(m ,4H),2.04(m,4H),1.80(m,2H),1.59(m,4H),1.40-1.20(m,42H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:807.53m / z[M+H].

[0259] Synthesis of Example 68 Intermediate 68a: Methyl 3-hexylnon-2-enoate [ka] Intermediate 68a was synthesized in 55% yield from tridecan-7-one and methyl 2-dimethoxyphosphorylacetate using the method used for 66b. 1 H NMR (400MHz, CDCl3) δ5.63(s,1H),3.68(s,3H),2.60(t,J=7.8Hz,2H),2.14(t,J=7.2Hz,2H),1.45-1.27(m,16H),0.89(t,J=6.4Hz,6H)ppm.

[0260] Intermediate 68b: 3-heptyldec-2-en-1-ol [ka] Intermediate 68b was synthesized in 49% yield from intermediate 68a using the method used for intermediate 67b. 1H NMR (400MHz, CDCl3) δ5.38(t,J=7.0Hz,1H),4.14(d,J=7.2Hz,2H),2.01(m,4H),1.39-1.27(m,16H),0.88(t,J=6.6Hz,6H)ppm.

[0261] Intermediate 68c: 3-heptyldec-2-enal [ka] Intermediate 68c was synthesized in 73% yield from intermediate 68b using the method used for intermediate 67c. 1 H NMR(400MHz,CDCl3)δ9.99(d,J=8.4Hz,1H),5.85(d,J=7.6Hz,1H),2.55(t,J=7.8H z,2H),2.21(t,J=7.2Hz,2H)1.49(m,4H),1.31(m,12H),0.89(t,J=6.8Hz,6H)ppm.

[0262] Intermediate 68d: (E)-7-Hexyltrideca-4,6-dienoic acid [ka] To a solution of intermediate 68c (9 g, 40.1 mmol) in HMPA (8 mL) and THF (104 mL) was added NaHMDS (1 M, 160.4 mL) at 0 °C. 3-Carboxypropyl(triphenyl)phosphonium (22.42 g, 64.18 mmol) in THF (28 mL) was added to the reaction mixture and further stirred at 15 °C for 12 h. The reaction mixture was poured into water (200 mL), acidified with 2 N HCl (aq), and extracted with EtOAc (4 × 150 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (1–100% EtOAc in petroleum ether) to give 8 g (19.0 mmol, 24% yield) of the desired product as a yellow oil. 1H NMR(400MHz,CDCl3)δ6.25(m,1H),6.05(d,J=7.6Hz,1H),5.30(m,1H),2.59-2.45(m,4 H),2.13-2.07(m,4H),1.40-1.29(m,16H),0.89(t,J=6.6Hz,6H)ppm;MS:295.2[M+H].

[0263] Intermediate 68e: 7-Hexyltridecanoic acid [ka] To a solution of intermediate 68d (4 g, 13.6 mmol) in MeOH (50 mL) under N was added Pd / C (0.4 g, 13.58 mmol). The suspension was degassed under vacuum and purged with H several times. The reaction mixture was stirred under H (15 psi) at 35 °C for 12 h, filtered, washed with MeOH (300 mL), and concentrated in vacuo. The crude residue was purified using silica gel chromatography (petroleum ether) to give 5.3 g (16.0 mmol, 59% yield) of the desired product as a yellow oil. 1 H NMR (400MHz, CDCl3) δ2.35(t,J=7.4Hz,2H),1.65(m,2H),1.25(m,27H),0.89(t,J=6.6Hz,6H)ppm;MS:297.2m / z[MH].

[0264] Intermediate 68f: 3-((7-hexyltridecanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Intermediate 68f was synthesized in 60% yield from intermediate 1a and intermediate 68e using the method used for intermediate 1d. 1H NMR(400MHz,CDCl3)δ5.45-5.27(m,4H),4.17(m,4H),3.61(t,J=5.2Hz,2H),2.77(t,J=6.4Hz,2H),2.32(t,J=7.6Hz,4H) ,2.25-2.14(m,2H),2.04(q,J=6.8Hz,4H),1.68-1.55(m,5H),1.40-1.15(m,39H),0.88(m,9H)ppm;MS:649.72m / z[M+H].

[0265] Example 68: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((7-hexyltridecanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Example 68 was synthesized in 43% yield from intermediate 68f and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ5.43-5.27(m,4H),4.22-4.07(m,8H),2.77(t,J=6.5Hz,2H),2.55-2.46(m,6H),2.46-2.37(m,1H),2.30(t,J=7.6Hz,4 H),2.04(m,4H),1.86-1.75(m,2H),1.61(t,J=7.3Hz,4H),1.42-1.11(m,41H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:807.72m / z[M+H].

[0266] Synthesis of Example 69 Intermediate 69a: (Z)-11-(non-2-en-1-yloxy)-11-oxoundecanoic acid [ka] Intermediate 69a was synthesized in 36% yield from undecanedioic acid and (Z)-non-2-en-1-ol using the method used for intermediate 54a. 1H NMR(400MHz,CDCl3)δ 5.65(m,1H),5.52(m,1H),4.61(dd,J=6.9,1.2Hz,2H),2.32(m,4H),2.09(m,2H),1.61(m,4H),1.41-1.20(m,18H),0.88(t,J=6.8Hz,3H)ppm.

[0267] Intermediate 69b: (Z)-1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)11-(non-2-en-1-yl)undecandioate [ka] Intermediate 69b was synthesized in 35% yield from intermediate 69a and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ5.64(m,1H),5.52(m,1H),4.62(d,J=6.7Hz,2H),4.49(t,J=5.5 Hz,1H),4.24-4.10(m,4H),3.67-3.52(m,4H),3.40(dt,J=9.3,6.7Hz,2H),2.41(t,J =7.5Hz,2H),2.30(q,J=7.1Hz,4H),2.21(m,2H),2.14-2.05(m,2H),1.93(td,J=7.5, 5.4Hz,2H),1.57(m,9H),1.40-1.21(m,36H),0.88(m,9H)ppm;MS:777.78m / z[M+Na].

[0268] Example 69: (Z)-1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 11-(non-2-en-1-yl)undecandioate [ka] Example 69 was synthesized in 19% yield from intermediate 69b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1H NMR(400MHz,CDCl3)δ5.62(m,1H),5.53(m,1H),4.61(d,J=7.0,1.3Hz,2H),4.48(t,J =5.5Hz,1H),4.22-4.09(m,8H),3.55(m,2H),3.39(m,2H),2.56(q,J=7.2Hz,6H),2.4 7-2.35(m,3H),2.29(t,J=7.6Hz,4H),2.09(m,2H),1.97-1.78(m,4H),1.65-1.49(m, 8H),1.40-1.24(m,35H),1.03(t,J=7.2Hz,6H),0.88(m,9H)ppm;MS:913.37m / z[M+H].

[0269] Synthesis of Example 70 Intermediate 70a: ((Z)-13-(non-2-en-1-yloxy)-13-oxotridecanoic acid [ka] Intermediate 70a was synthesized in 40% yield from tridecanedioic acid and (Z)-non-2-en-1-ol using the method used for intermediate 54a. 1 H NMR(400MHz,CDCl3)δ5.62(m,1H),5.50(m,1H),4.59(dd,J=6.8,1.2Hz,2H),2.29 (m,4H),2.07(m,2H),1.59(m,4H),1.39-1.18(m,22H),0.85(t,J=6.8Hz,3H)ppm.

[0270] Intermediate 70b: (Z)-1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)13-(non-2-en-1-yl)tridecanedioate [ka] Intermediate 70b was synthesized in 34% yield from intermediate 70a and intermediate 54b using the method used for intermediate 54c. 1H NMR(500MHz,CDCl3)δ5.64(m,1H),5.52(m,1H),4.62(d,J=6.8Hz,2H),4.49(t,J=5.5Hz,1H), 4.17(m,4H),3.62(t,J=5.7Hz,2H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2 .41(t,J=7.5Hz,2H),2.31(q,J=7.9Hz,4H),2.25-2.15(m,2H),2.09(q,J=7.3Hz,2H),1.93(t d,J=7.5,5.4Hz,2H),1.58(m,8H),1.39-1.21(m,41H),0.88(m,9H)ppm;MS:805.63m / z[M+Na].

[0271] Example 70: (Z)-1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 13-(non-2-en-1-yl)tridecanedioate [ka] Example 70 was synthesized in 15% yield from intermediate 70b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ5.64(m,1H),5.51(m,1H),4.61(d,J=6.9,1.2Hz,2H),4.48(t,J=5.6 Hz,1H),4.22-4.09(m,8H),3.55(m,2H),3.40(m,2H),2.51(q,J=7.0Hz,6H),2.46-2.35(m, 3H),2.34-2.26(m,4H),2.15-2.04(m,2H),1.97-1.87(m,2H),1.86-1.75(m,4H),1.67-1.4 9(m,8H),1.40-1.23(m,40H),1.01(t,J=7.2Hz,6H),0.88(m,9H)ppm;MS:942.04m / z[M+H].

[0272] Synthesis of Examples 71 to 74 The following examples were synthesized from intermediate 59b and an amino alcohol or diamine reagent using the methodology used in Example 1.

[0273] Example 71: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-methylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl) 9-decylnonanedioate [ka] Yield 19%; 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.19(d,J=6.0Hz,2H),4.14(dt,J=6.0,1.4Hz ,4H),4.05(t,J=6.7Hz,3H),3.96(dd,J=10.6,7.3Hz,1H),3.56(dt,J=9.3,6.7Hz,2H),3.4 0(dt,J=9.3,6.7Hz,2H),2.78(m,2H),2.47-2.36(m,3H),2.33-2.23(m,7H),2.07-1.86(m, 4H),1.79-1.50(m,16H),1.29(m,38H),1.00(m,1H),0.88(m,9H)ppm;MS:899.87m / z[M+H].

[0274] Example 72: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl) 9-decylnonanedioate [ka] Yield 19%; 1H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.19(d,J=6.0Hz,2H),4.14(dt,J=6.0,1.4Hz,4H),4.05(td,J=6. 9,6.4,3.7Hz,3H),3.96(dd,J=10.7,7.2Hz,1H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.87 m,2H),2.48-2.36(m,5H),2.34-2.24(m,4H),2.05-1.85(m,3H),1.79-1.50(m,16 H),1.39-1.20(m,40H),1.12-0.97(m,3H),0.87(m,9H)ppm;MS:913.46m / z[M+H].

[0275] Example 73: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propyl) 9-decylnonanedioate [ka] Yield 14%; 1 H NMR(400MHz,CDCl3)δ5.22(br s,1H),4.48(t,J=5.6Hz,1H),4.20-4.08(m,6H),4.05(t,J=6.8Hz,2H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3.21(br m,2H),2.52(q,J=7.1,6.2Hz,6H),2.40(t,J=7.6Hz,2H),2.29(td,J=7.6,5.9Hz,4H),1.92(td,J=7.6,5.5H MS: 886.69m / z[M+H].

[0276] Example 74: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(1-methylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl) 9-decylnonanedioate [ka] Yield 26%; 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.6Hz,1H),4.28-4.09(m,8H),4.05(t,J=6.8Hz,2H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),3. 05(m,1H),2.46-2.36(m,3H),2.34-2.24(m,7H),2.21-1.87(m,4H),1.84 -1.43(m,12H),1.40-1.18(m,40H),0.88(m,9H)ppm;MS:899.38m / z[M+H].

[0277] Synthesis of Example 75 Example 75: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(diethylamino)butanoyl)oxy)methyl)propyl) 9-decylnonanedioate [ka] Example 75 was synthesized in 61% yield from intermediate 59b and 4-(diethylamino)butanoic acid using the method used for Example 25. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.12(dd,J=6.1,1.6Hz,6H),4.05(t ,J=6.8Hz,2H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.50(q, J=7.1Hz,4H),2.45-2.24(m,10H),1.92(m,2H),1.75(m,2H),1.65-1.50(m,12H), 1.38-1.20(m,40H),1.00(t,J=7.1Hz,6H),0.88(m,8H)ppm;MS:885.43m / z[M+H].

[0278] Synthesis of Example 76 Intermediate 76a: 1-Decyl 9-(3-hydroxy-2-(hydroxymethyl)propyl)nonanedioate [ka] To a solution of intermediate 59a (15 g, 45.6 mmol, 1 equiv.), (2,2-dimethyl-1,3-dioxan-5-yl)methanol (1-1.2 equiv.), DMAP (0.2 equiv.), and DIPEA (1.5-3 equiv.) in DCM (0.2 M) was added EDC·HCl (1.5 equiv.) at room temperature. The reaction mixture was stirred for at least 16 h, diluted with water, washed successively with 1 M HCl and 5% sodium bicarbonate, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude acetonide-protected intermediate was resuspended in MeOH and Dowex® 50W X8 resin was added. The resulting mixture was stirred for at least 12 h at room temperature, filtered, washed with MeOH, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (gradient of EtOAc in hexanes) to give 12.2 g (29.3 mmol, 65% yield) of the desired product as a white solid. 1 H NMR(400MHz,CDCl3)δ4.24(d,J=6.3Hz,2H),4.04(t,J=6.7Hz,2H),3.76(m,4H),2.52(br s,2H),2.30(dt,J=16.1,7.5Hz,4H),2.03(m,1H),1.61(m,6H),1.38-1.20(m,20H),0.87(t,J=6.8Hz,3H)ppm;MS:439.56m / z[M+Na].

[0279] Intermediate 76b: 1-decyl 9-(3-((7-(heptadecan-9-yloxy)-7-oxoheptanoyl)oxy)-2-(hydroxymethyl)propyl)nonanedioate [ka] Intermediate 76b was synthesized in 37% yield from intermediate 76a and intermediate 64a using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ4.85(m,1H),4.21-4.12(m,4H),4.04(t,J=6.8Hz,2H),3.61(d,J=5.6Hz,2H),2.37-2.2 4(m,8H),2.18(m,1H),1.63(m,11H),1.49(m,7H),1.39-1.19(m,42H),0.87(m,9H)ppm;MS:797.96m / z[M+H].

[0280] Example 76: 1-Decyl 9-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((7-(heptadecan-9-yloxy)-7-oxoheptanoyl)oxy)methyl)propyl)nonanedioate [ka] Example 76 was synthesized in 22% yield from intermediate 76b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ4.85(m,1H),4.22-4.10(m,8H),4.05(t,J=6.8Hz,2H),2.55-2.50(m,6H),2.41(m,1H),2.35-2.24(m,8H), 1.81(m,2H),1.70-1.55(m,12H),1.50(m,4H),1.41-1.23(m,44H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:956.17m / z[M+H].

[0281] Synthesis of Example 77 Intermediate 77a: 1-decyl 9-(3-((5-(heptadecan-9-yloxy)-5-oxopentanoyl)oxy)-2-(hydroxymethyl)propyl)nonanedioate [ka] Intermediate 77a was synthesized in 26% yield from intermediate 76a and intermediate 63b using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ4.86(m,1H),4.23-4.13(m,4H),4.05(t,J=6.8Hz,2H),3.62(t,J=5.5Hz,2H),2.33(m,8H) ,2.19(m,1H),1.95(m,2H),1.61(m,8H),1.50(m,4H),1.38-1.19(m,42H),0.87(m,9H)ppm;MS:769.91m / z[M+H].

[0282] Example 77: 1-Decyl 9-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((5-(heptadecan-9-yloxy)-5-oxopentanoyl)oxy)methyl)propyl)nonanedioate [ka] Example 77 was synthesized from intermediate 77a and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ4.87(m,1H),4.16(m,8H),4.05(t,J=6.8Hz,2H),2.51(m,6H),2.46-2.24(m,9H),1.94(m,2H),1.87-1. 76(m,2H),1.69-1.57(m,6H),1.49(m,4H),1.40-1.17(m,44H),1.00(t,J=7.1Hz,6H),0.89(m,9H)ppm;MS:928.07m / z[M+H].

[0283] Synthesis of Example 78 Intermediate 78a: 4,4-bis(heptyloxy)butanenitrile [ka] Intermediate 78a was synthesized in 99% yield from 4,4-diethoxybutanenitrile and heptan-1-ol using the method used for intermediate 1b. 1H NMR(400MHz,CDCl3)δ4.55(t,J=5.3Hz,1H),3.60(dt,J=9.3,6.6Hz,2H),3.43(dt,J=9.3,6.6Hz,2H), 2.42(t,J=7.4Hz,2H),1.94(td,J=7.4,5.3Hz,2H),1.57(m,4H),1.40-1.23(m,16H),0.88(m,6H)ppm.

[0284] Intermediate 78b: 4,4-bis(heptyloxy)butanoic acid [ka] Intermediate 78b was synthesized in 92% yield from intermediate 78a using the method used for intermediate 1c. 1 H NMR(400MHz,CDCl3)δ8.85(br s,1H),4.46(t,J=5.6Hz,1H),3.52(dt,J=9.4,6.8Hz,2H),3.39(dt,J=9.3,6.8Hz,2H),2.26(t,J=7 MS: 315 m / z [MH].

[0285] Intermediate 78c: 1-(3-((4,4-bis(heptyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)9-decylnonanedioate [ka] Intermediate 78c was synthesized in 46% yield from intermediate 76a and intermediate 78b using the method used for intermediate 1d. 1H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.23-4.11(m,4H),4.04(t,J=6.7Hz,2H),3.65-3.50(m,4H),3.39(dt,J=9.3,6.7Hz,2H),2.40(t,J=7 .5Hz,2H),2.29(m,5H),2.19(m,1H),1.93(td,J=7.5,5.4Hz,2H),1.68-1 .49(m,11H),1.38-1.19(m,34H),0.88(m,9H)ppm;MS:737.82m / z[M+Na].

[0286] Example 78: 1-(3-((4,4-bis(heptyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-decylnonanedioate [ka] Example 78 was synthesized in 35% yield from intermediate 78c and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.6Hz,1H),4.22-4.09(m,8H),4.05(t,J=6.8Hz,2H),3.56(m,2H),3.40(m,2H),2.51(m,6H),2.46-2.35(m,3H) ),2.29(m,4H),1.92(m,2H),1.81(m,2H),1.66-1.49(m,12H),1.39-1.2 3(m,34H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:873.52m / z[M+H].

[0287] Synthesis of Example 79 Intermediate 79a: 4,4-bis(nonyloxy)butanenitrile [ka] Intermediate 79a was synthesized in 16% yield from 4,4-diethoxybutanenitrile and nonan-1-ol using the method used for intermediate 1b. 1H NMR(400MHz,CDCl3)δ4.55(t,J=5.3Hz,1H),3.60(dt,J=9.2,6.6Hz,2H),3.43(dt,J=9.3,6.6Hz,2H),2.42 (t,J=7.4Hz,2H),1.94(td,J=7.4,5.3Hz,2H),1.57(m,4H),1.38-1.24(m,24H),0.88(t,J=6.7Hz,6H)ppm.

[0288] Intermediate 79b: 4,4-bis(nonyloxy)butanoic acid [ka] Intermediate 79b was synthesized in 100% yield from intermediate 79a using the method used for intermediate 1c. 1 H NMR(400MHz,CDCl3)δ5.32(br s,1H),4.44(t,J=5.6Hz,1H),3.49(dt,J=9.3,6.9Hz,2H),3.38(dt,J=9.4,6.9Hz,2H),2.10(t,J=7.6 Hz,2H),1.78(q,J=7.0Hz,2H),1.53(m,4H),1.27(m,24H),0.88(t,J=6.6Hz,6H)ppm;MS:371m / z[MH].

[0289] Intermediate 79c: 1-(3-((4,4-bis(nonyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) 9-decylnonanedioate [ka] Intermediate 79c was synthesized in 43% yield from intermediate 76a and intermediate 79b using the method used for intermediate 1d. 1H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.23-4.12(m,4H),4.05(t,J=6. 8Hz,2H),3.67-3.51(m,4H),3.40(dt,J=9.3,6.7Hz,2H),2.40(t,J=7.5Hz,2H ),2.30(dt,J=11.9,7.6Hz,5H),2.19(m,1H),1.93(td,J=7.6,5.4Hz,2H),1. 66-1.48(m,11H),1.37-1.20(m,42H),0.88(m,9H)ppm;MS:793.91m / z[M+Na].

[0290] Example 79: 1-(3-((4,4-bis(nonyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-decylnonanedioate [ka] Example 79 was synthesized in 35% yield from intermediate 79c and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.22-4.10(m,8H),4.05(t,J=6.8Hz,2H),3.55(m,2H),3.39(m,2H),2.50(m,6H),2.45-2.35(m,3H),2 .29(m,4H),1.92(m,2H),1.86-1.74(m,3H),1.66-1.49(m,10H),1.35-1. 23(m,43H),1.00(t,J=7.1Hz,6H),0.87(m,9H)ppm;MS:929.60m / z[M+H].

[0291] Synthesis of Examples 80-81 The following examples were synthesized from intermediate 76b and an amino alcohol or diamine reagent using the methodology used in Example 1.

[0292] Example 80: 1-Decyl 9-(3-((7-(heptadecan-9-yloxy)-7-oxoheptanoyl)oxy)-2-((((2-(1-methylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl)nonanedioate [ka] Yield 48%; 1 H NMR(400MHz,CDCl3)δ4.85(m,1H),4.26-4.09(m,8H),4.04(t,J=6.7Hz,2H),3.05(m,1H),2.41(m,1H),2.34-2 .26(m,11H),2.19-1.89(m,4H),1.83-1.43(m,18H),1.40-1.17(m,46H),0.87(m,9H)ppm;MS:954.23m / z[M+H].

[0293] Example 81: 1-Decyl 9-(3-(((2-(diethylamino)ethyl)carbamoyl)oxy)-2-(((7-(heptadecan-9-yloxy)-7-oxoheptanoyl)oxy)methyl)propyl)nonanedioate [ka] Yield 43%; 1 H NMR(400MHz,CDCl3)δ5.21(br t,J=5.2Hz,1H),4.85(m,1H),4.12(d,J=6.0Hz,6H),4.05(t,J=6.8Hz,2H),3.21(br q,J=5.8Hz,2H),2.51(q,J=7.1,6.3Hz,6H),2.43-2.23(m,8H),1.62(m,11H),1.50(q,J=6. 1Hz,4H),1.40-1.20(m,46H),0.99(t,J=7.1Hz,6H),0.87(m,9H)ppm;MS:940.46m / z[M+H].

[0294] Synthesis of Example 82 Example 82: 1-Decyl 9-(3-((4-(dimethylamino)butanoyl)oxy)-2-(((7-(heptadecan-9-yloxy)-7-oxoheptanoyl)oxy)methyl)propyl)nonanedioate [ka] Example 82 was synthesized in 55% yield from intermediate 76b and 4-(dimethylamino)butanoic acid using the method used in Example 25. 1 H NMR(400MHz,CDCl3)δ4.85(m,1H),4.12(dd,J=6.0,2.3Hz,6H),4.04(t,J=6.8Hz,2H),2.42-2.23(m,13H),2.20(s ,6H),1.77(m,2H),1.62(m,11H),1.49(q,J=6.0Hz,4H),1.40-1.19(m,45H),0.87(m,9H)ppm;MS:911.44m / z[M+H].

[0295] Synthesis of Example 83 Intermediate 83a: (Z)-1-(3-hydroxy-2-(hydroxymethyl)propyl)9-(non-2-en-1-yl)nonanedioate [ka] Intermediate 83a was synthesized in 84% yield from intermediate 54a and 2-(hydroxymethyl)propane-1,3-diol using the method used for 76a. 1 H NMR(400MHz,CDCl3)δ5.65(m,1H),5.51(m,1H),4.61(dd,J=6.9,1.2Hz,2H),4.24(d,J=6.3Hz,2H),3.76(m,4H),2.52(br s,2H),2.31(m,4H),2.12-1.98(m,3H),1.61(m,4H),1.40-1.21(m,14H),0.87(t,J=6.8Hz,3H)ppm.

[0296] Intermediate 83b: (Z)-1-(3-((4,4-bis(nonyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) 9-(non-2-en-1-yl)nonanedioate [ka] Intermediate 83b was synthesized in 33% yield from intermediate 83a and intermediate 79b using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ5.64(m,1H),5.51(m,1H),4.61(dd,J=6.9,1.2Hz,2H),4.48(t,J=5.5Hz,1H),4.17(m,4H),3.62(br m,2H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.40(t,J=7.5Hz,2H),2.30(m,5H),2.19(m,1H),2.09 (m,2H),1.93(td,J=7.5,5.4Hz,2H),1.66-1.50(m,10H),1.39-1.21(m,36H),0.0.87(m,9H)ppm;MS:777.87m / z[M+Na].

[0297] Example 83: (Z)-1-(3-((4,4-bis(nonyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-(non-2-en-1-yl)nonanedioate [ka] Example 83 was synthesized in 60% yield from intermediate 83b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1H NMR(400MHz,CDCl3)δ5.62(m,1H),5.51(m,1H),4.61(dd,J=6.8,1.2Hz,2H),4.48(t,J=5.5Hz,1 H),4.21-4.09(m,8H),3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.51(m,6H),2.4 5-2.35(m,3H),2.29(t,J=7.6Hz,4H),2.09(m,2H),1.92(td,J=7.6,5.5Hz,2H),1.81(m,2H),1.6 7-1.48(m,8H),1.42-1.20(m,38H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:913.71m / z[M+H].

[0298] Synthesis of Example 84 Intermediate 84a: (Z)-1-(3-((4,4-bis(heptyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)9-(non-2-en-1-yl)nonanedioate [ka] Intermediate 84a was synthesized in 38% yield from intermediate 83a and intermediate 78b using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ5.64(m,1H),5.51(m,1H),4.61(dd,J=6.9,1.2Hz,2H),4.48(t,J=5.5Hz,1H),4.23-4.12(m,4H),3.62(br t,J=5.2Hz,2H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.40(t,J=7.5Hz,2H),2.30(td,J=7.5,5.8Hz,5H),2. 19(m,1H),2.09(m,2H),1.93(td,J=7.5,5.4Hz,2H),1.66-1.50(m,8H),1.39-1.25(m,30H),0.87(m,9H)ppm;MS:721.65[M+Na].

[0299] Example 84: (Z)-1-(3-((4,4-bis(heptyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-(non-2-en-1-yl)nonanedioate [ka] Example 84 was synthesized from intermediate 84a and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ5.64(m,1H),5.51(m,1H),4.61(dd,J=6.8,1.2Hz,2H),4.48(t,J=5.5Hz, 1H),4.22-4.10(m,8H),3.56(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.50(m,6H),2 .46-2.35(m,3H),2.30(t,J=7.5Hz,4H),2.09(m,2H),1.92(m,2H),1.80(dq,J=8.2,6.6Hz,2H) ,1.58(m,8H),1.40-1.21(m,30H),1.00(t,J=7.1Hz,6H),0.89(m,9H)ppm;MS:857.54m / z[M+H].

[0300] Synthesis of Example 85 Intermediate 85a: (Z)-1-(3-((7-(heptadecan-9-yloxy)-7-oxoheptanoyl)oxy)-2-(hydroxymethyl)propyl) 9-(non-2-en-1-yl)nonanedioate [ka] Intermediate 85a was synthesized in 37% yield from intermediate 83a and intermediate 64a using the method used for intermediate 1d. 1H NMR(400MHz,CDCl3)δ5.64(m,1H),5.51(m,1H),4.85(m,1H),4.61(dd,J=6.9,1.2Hz,2H),4.22-4.12(m,4H),3.61(m,2H),2 .30(m,9H),2.19(m,1H),2.09(m,2H),1.64(m,8H),1.49(m,4H),1.41-1.18(m,40H),0.87(m,9H)ppm;MS:781.73m / z[M+H].

[0301] Example 85: (Z)-1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((7-(heptadecan-9-yloxy)-7-oxoheptanoyl)oxy)methyl)propyl) 9-(non-2-en-1-yl)nonanedioate [ka] Example 85 was synthesized in 48% yield from intermediate 85a and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ5.64(m,1H),5.51(m,1H),4.85(m,1H),4.61(dd,J=6. 8,1.2Hz,2H),4.24-4.09(m,8H),2.51(m,6H),2.41(m,1H),2.35-2.21(m,8H ),2.09(m,2H),1.80(m,2H),1.69-1.55(m,10H),1.50(q,J=6.2Hz,4H),1.40 -1.18(m,38H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:939.19m / z[M+H].

[0302] Synthesis of Examples 86 to 88 Intermediate 86a: (Z)-1-(3-((5-(heptadecan-9-yloxy)-5-oxopentanoyl)oxy)-2-(hydroxymethyl)propyl) 9-(non-2-en-1-yl)nonanedioate [ka] Intermediate 86a was synthesized in 39% yield from intermediate 83a and intermediate 63b using the method used for intermediate 1d. 1 H NMR(400MHz,CDCl3)δ5.64(m,1H),5.51(m,1H),4.86(m,1H),4.61(dd,J=7.0,1.2Hz,2H),4.23-4.12(m,4H),3.62(t,J=4.4Hz,2H),2.44- 2.24(m,9H),2.19(m,1H),2.09(m,2H),1.95(m,2H),1.61(m,5H),1.50(m,4H),1.38-1.20(m,37H),0.87(m,9H)ppm;MS:753.74m / z[M+H].

[0303] The following examples were synthesized from intermediate 86a and an amino alcohol or diamine reagent using the methodology used in Example 1.

[0304] Example 86: (Z)-1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((5-(heptadecan-9-yloxy)-5-oxopentanoyl)oxy)methyl)propyl) 9-(non-2-en-1-yl)nonanedioate [ka] Yield 48%; 1 H NMR(400MHz,CDCl3)δ5.65(m,1H),5.51(m,1H),4.86(m,1H),4.61(dd,J=6. 9,1.3Hz,2H),4.22-4.09(m,8H),2.50(q,J=7.1Hz,6H),2.46-2.25(m,9H),2 .09(m,2H),1.94(m,2H),1.86-1.74(m,3H),1.61(m,4H),1.50(m,4H),1.40 -1.23(m,37H),1.00(t,J=7.1Hz,6H),0.87(m,9H)ppm;MS:912.23m / z[M+H].

[0305] Example 87: 1-Decyl 9-(3-((5-(heptadecan-9-yloxy)-5-oxopentanoyl)oxy)-2-((((2-(1-methylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl)nonanedioate [ka] Yield 50%; 1 H NMR(400MHz,CDCl3)δ4.86(m,1H),4.27-4.08(m,8H),4.04(t,J=6.7Hz,2H),3.05(m,1H),2.48-2.24(m, MS:925.43m / z[M+H]

[0306] Example 88: 1-Decyl 9-(3-(((2-(diethylamino)ethyl)carbamoyl)oxy)-2-(((5-(heptadecan-9-yloxy)-5-oxopentanoyl)oxy)methyl)propyl)nonanedioate [ka] Yield 49%; 1 H NMR(500MHz,CDCl3)δ5.23(br t,J=5.3Hz,1H),4.86(m,1H),4.12(m,6H),4.04(t,J=6.8Hz,2H),3.20(br q,J=5.9Hz,2H),2.51(q,J=7.0Hz,6H),2.41-2.25(m,9H),1.93(m,2H),1.61(m,6H),1.50 (m,4H),1.33-1.23(m,44H),0.99(t,J=7.1Hz,6H),0.87(m,9H)ppm;MS:912.43m / z[M+H].

[0307] Synthesis of Example 89 Example 89: 1-Decyl 9-(3-((4-(dimethylamino)butanoyl)oxy)-2-(((5-(heptadecan-9-yloxy)-5-oxopentanoyl)oxy)methyl)propyl)nonanedioate [ka] Example 89 was synthesized in 43% yield from intermediate 86a and 4-(dimethylamino)butanoic acid using the method used in Example 25. 1 H NMR(500MHz,CDCl3)δ4.91-4.82(m,1H),4.12(m,6H),4.05(t,J=6.8Hz,2H),2.42-2.23(m,13H),2.20(s,6H), 1.94(m,2H),1.75(m,2H),1.61(m,6H),1.49(m,4H),1.37-1.16(m,44H),0.87(m,9H)ppm;MS:883.85m / z[M+H].

[0308] Synthesis of Example 90 Intermediate 90a: 11-chloro-11-oxoundecanoic acid [ka] To a solution of undecanedioic acid (15 g, 69.36 mmol, 1 equiv.) in THF (0.6 M) was added oxalyl chloride (1.1–1.3 equiv.). After the addition, the mixture was stirred, and DMF (0.01–0.05 equiv.) was added dropwise. The resulting mixture was stirred at 25 °C for 2 h and then concentrated to give 16 g (47.0 mmol, 99% yield) of the desired crude product as a yellow oil, which required no further purification.

[0309] Intermediate 90b: 11-(decyloxy)-11-oxoundecanoic acid [ka] To a solution of intermediate 90a (16.28 g, 69.36 mmol, 1 equiv.) in THF (0.45 M) was added decan-1-ol (1.1 equiv.). The mixture was stirred at 25° C. for 2 h and then concentrated in vacuo. The crude residue was purified by column chromatography (gradient of EtOAc in petroleum ether) and, if necessary, recrystallized in petroleum ether to give 5.5 g (15.5 mmol, 22% yield) of the desired product as a white solid.1 H NMR (400MHz, CDCl3) δ4.06(t,J=6.8Hz,2H),2.37-2.28(m,4H),1.62(m,6H),1.28(m,24H),0.89(t,J=6.8Hz,3H)ppm;MS:355.2m / z[MH].

[0310] Intermediate 90c: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)11-decylundecandioate [ka] Intermediate 90c was synthesized in 39% yield from intermediate 90b and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.18(m,4H),4.05(t,J=6.8Hz,2H),3.64-3.51(m,4H),3.40(dt,J=9.3,6.7Hz,2H),2.41(t,J=7.5Hz, 2H),2.34-2.23(m,5H),2.19(m,1H),1.93(td,J=7.6,5.5Hz,2H),1.65-1 .50(m,10H),1.37-1.21(m,44H),0.88(m,9H)ppm;MS:793.73m / z[M+Na].

[0311] Example 90: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 11-decylundecandioate [ka] Example 90 was synthesized in 59% yield from intermediate 90c and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1H NMR(500MHz,CDCl3)δ4.47(t,J=5.6Hz,1H),4.21-4.08(m,8H),4.05(t,J=6.7 Hz,2H),3.55(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.50(q,J=7. 0Hz,6H),2.46-2.36(m,3H),2.29(q,J=7.7Hz,4H),1.92(m,2H),1.80(m,2H),1 .65-1.51(m,10H),1.36-1.24(m,44H),1.00(t,J=7.1Hz,6H),0.87(m,9H)ppm. MS: 929.93 m / z [M+H].

[0312] Synthesis of Example 91 Intermediate 91a: 13-chloro-13-oxotridecanoic acid [ka] Intermediate 91a was synthesized in quantitative yield from tridecanedioic acid using the method used for 90a.

[0313] Intermediate 91b: 13-(decyloxy)-13-oxotridecanoic acid [ka] Intermediate 91b was synthesized in 17% yield from intermediate 91a and decan-1-ol using the method used for intermediate 90b. 1 H NMR (400MHz, CDCl3) δ4.06(t,J=6.8Hz,2H),2.37-2.27(m,4H),1.64(m,6H),1.27(m,28H),0.89(t,J=6.8Hz,3H)ppm;MS:383.3m / z[MH].

[0314] Intermediate 91c: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)13-decyltridecanedioate [ka] Intermediate 91c was synthesized in 40% yield from intermediate 91b and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.23-4.12(m,4H),4.05(t,J=6.7Hz,2H),3.65-3.51(m,4H),3.40(dt,J=9.3,6.7Hz,2H),2.41(t,J=7 .5Hz,2H),2.34-2.22(m,5H),2.19(m,1H),1.93(td,J=7.5,5.4Hz,2H),1 .58(m,10H),1.38-1.20(m,48H),0.87(m,9H)ppm;MS:821.77m / z[M+Na].

[0315] Example 91: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 13-decyltridecanedioate [ka] Example 91 was synthesized in 56% yield from intermediate 91c and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(500MHz,CDCl3)δ4.48(t,J=5.6Hz,1H),4.21-4.08(m,8H),4.05(t,J=6.7Hz,2H ),3.56(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.50(q,J=7.1Hz,6H),2 .46-2.36(m,3H),2.29(q,J=7.8Hz,4H),1.92(m,2H),1.81(m,2H),1.65-1.52(m,10 H),1.36-1.22(m,48H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:958.01m / z[M+H].

[0316] Synthesis of Example 92 Intermediate 92a: 9-chloro-9-oxononanoic acid [ka] Intermediate 92a was synthesized in quantitative yield from nonanedioic acid using the method used for 90a.

[0317] Intermediate 92b: 9-(dodecyloxy)-9-oxononanoic acid [ka] Intermediate 92b was synthesized in 31% yield from intermediate 92a and dodecan-1-ol using the method used for intermediate 90b. 1 H NMR (400MHz, CDCl3) δ4.06(t,J=6.8Hz,2H),2.37-2.28(m,4H),1.63(m,6H),1.37-1.27(m,24H),0.89(t,J=6.8Hz,3H)ppm;MS:355.2m / z[MH].

[0318] Intermediate 92c: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)9-dodecylnonanedioate [ka] Intermediate 92c was synthesized in 43% yield from intermediate 92b and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.23-4.12(m,4H),4.05(t,J=6. 8Hz,2H),3.65-3.51(m,4H),3.40(dt,J=9.3,6.7Hz,2H),2.41(t,J=7.5Hz,2H ),2.30(dt,J=12.0,7.6Hz,5H),2.19(m,1H),1.93(td,J=7.5,5.5Hz,2H),1. 66-1.48(m,11H),1.37-1.22(m,43H),0.87(m,9H)ppm;MS:793.77m / z[M+Na].

[0319] Example 92: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-dodecylnonanedioate [ka] Example 92 was synthesized in 53% yield from intermediate 92c and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz, CDCl3)δ4.48(t,J=5.5Hz,1H),4.22-4.09(m,8H),4.05(t,J=6.8Hz ,2H),3.56(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.50(m,6H),2.45 -2.35(m,3H),2.34-2.24(m,4H),1.92(m,2H),1.80(m,2H),1.71-1.51(m,10H), 1.37-1.21(m,44H),1.00(t,J=7.1Hz,6H),0.87(m,9H)ppm;MS:929.53m / z[M+H].

[0320] Synthesis of Example 93 Intermediate 93a: 9-oxo-9-(tetradecyloxy)nonanoic acid [ka] Intermediate 93a was synthesized in 11% yield from intermediate 92a and tetradecan-1-ol using the method used for intermediate 90b. 1 H NMR (400MHz, CDCl3) δ4.06(t,J=6.8Hz,2H),2.37-2.28(m,4H),1.62(m,6H),1.33-1.26(m,30H),0.88(t,J=6.8Hz,3H)ppm;MS:383.3m / z[MH].

[0321] Intermediate 93b: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)9-tetradecylnonanedioate [ka] Intermediate 93b was synthesized in 45% yield from intermediate 93a and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.23-4.12(m,4H),4.05(t,J=6.8Hz,2H),3.65-3.50(m,4H),3.40(dt,J=9.3,6.7Hz,2H),2.41(t,J=7.5 Hz,2H),2.35-2.24(m,5H),2.19(m,1H),1.93(td,J=7.5,5.5Hz,2H),1.66 -1.49(m,11H),1.38-1.21(m,46H),0.87(m,9H)ppm;MS:822.00m / z[M+Na].

[0322] Example 93: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-tetradecylnonanedioate [ka] Example 93 was synthesized in 47% yield from intermediate 93b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ4.48(t,J=5.5Hz,1H),4.22-4.08(m,8H),4.05(t,J=6.8Hz, 2H),3.56(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.50(q,J=7.1Hz,6 H),2.45-2.35(m,3H),2.29(m,4H),1.92(m,2H),1.81(m,2H),1.67-1.49(m,12H) ,1.37-1.21(m,47H),1.00(t,J=7.1Hz,5H),0.88(m,9H)ppm;MS:943.03m / z[M+H].

[0323] Synthesis of Example 94 Intermediate 94a: 9-oxo-9-(undecan-2-yloxy)nonanoic acid [ka] Intermediate 94a was synthesized in 30% yield from intermediate 92a and undecan-2-ol using the method used for intermediate 90b. 1 H NMR(400MHz,CDCl3)δ4.90(m,1H),2.35(t,J=7.6Hz,2H),2.27(t,J=7.4Hz,2H),1.6 6-1.30(m,26H),1.20(d,J=6.0Hz,3H),0.89(t,J=7.0Hz,3H)ppm;MS:341.2m / z[MH].

[0324] Intermediate 94b: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)9-(undecan-2-yl)nonanedioate [ka] Intermediate 94b was synthesized in 39% yield from intermediate 94a and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ4.88(m,1H),4.48(t,J=5.5Hz,1H),4.23-4.11(m,4H),3.66-3.52(m,4H),3.40(dt,J=9.3,6.7Hz,2H),2.41(t,J=7.5Hz,2H ),2.35-2.23(m,5H),2.19(m,1H),1.93(td,J=7.5,5.4Hz,2H),1.66-1.2 2(m,50H),1.19(d,J=6.2Hz,3H),0.87(m,9H)ppm;MS:779.78m / z[M+Na].

[0325] Example 94: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-(undecan-2-yl)nonanedioate [ka] Example 94 was synthesized in 63% yield from intermediate 94b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ4.89(m,1H),4.48(t,J=5.5Hz,1H),4.22-4.07(m,8H),3.5 6(dt,J=9.3,6.7Hz,2H),3.39(dt,J=9.3,6.7Hz,2H),2.55-2.45(m,6H),2.45-2 .35(m,3H),2.34-2.22(m,4H),1.92(m,2H),1.81(m,2H),1.64-1.26(m,50H),1. 19(d,J=6.3Hz,3H),1.00(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:915.44m / z[M+H].

[0326] Synthesis of Example 95 Intermediate 95a: Dodecan-3-ol [ka] To a solution of decanal (12.8 mL, 64 mmol) in THF (100 mL) was added bromo(ethyl)magnesium (3 M, 19.20 mL) dropwise at -78 °C. The reaction mixture was stirred at 25 °C for 2 h, diluted with water (200 mL), and extracted into EtOAc (2 × 200 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (2–2.5% EtOAc in petroleum ether) to afford 6.9 g (37 mmol, 64% yield) of the desired product as a colorless oil. 1 H NMR (400MHz, CDCl3) δ3.53 (m, 1H), 1.49-1.27 (m, 18H), 0.95 (t, J = 7.4 Hz, 3 H), 0.89 (t, J = 6.8 Hz, 3 H) ppm.

[0327] Intermediate 95b: 9-(dodecan-3-yloxy)-9-oxononanoic acid [ka] Intermediate 95b was synthesized in 38% yield from intermediate 92a and dodecan-3-ol using the method used for intermediate 90b. 1 H NMR (400MHz, CDCl3) δ4.82(m,1H),2.37-2.27(m,4H),1.63(m,8H),1.34-1.26(m,20H),0.88(m,6H)ppm;MS:355.2m / z[MH].

[0328] Intermediate 95c: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)9-(dodecan-3-yl)nonanedioate [ka] Intermediate 95c was synthesized in 42% yield from intermediate 95b and intermediate 54b using the method used for intermediate 54c. 1 H NMR(400MHz,CDCl3)δ4.80(m,1H),4.48(t,J=5.5Hz,1H),4.23-4.10(m,4H),3.66-3.50(m,4H),3.39(dt,J=9.3,6.7Hz,2H),2.40(t,J=7.5Hz,2H ),2.36-2.24(m,5H),2.19(m,1H),1.93(td,J=7.6,5.4Hz,2H),1.67-1. 46(m,11H),1.38-1.18(m,40H),0.87(m,12H)ppm;MS:793.77m / z[M+Na].

[0329] Example 95: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl) 9-(dodecan-3-yl)nonanedioate [ka] Example 95 was synthesized in 63% yield from intermediate 95c and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1H NMR(400MHz,CDCl3)δ4.82(m,1H),4.48(t,J=5.5Hz,1H),4.22-4.07(m,8H),3.56 (dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.50(q,J=6.9Hz,6H),2.45-2 .35(m,3H),2.29(q,J=7.5Hz,4H),1.92(m,2H),1.81(m,2H),1.68-1.47(m,11H), 1.37-1.23(m,41H),1.00(t,J=7.1Hz,6H),0.87(m,12H)ppm;MS:929.03m / z[M+H].

[0330] Synthesis of Example 96 Intermediate 96a: (2,2,5-trimethyl-1,3-dioxan-5-yl)methyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of (2,2,5-trimethyl-1,3-dioxan-5-yl)methanol (2 g, 12.4 mmol), linoleic acid (4.23 mL, 13.6 mmol), DMAP (302 mg, 2.48 mmol), and DIPEA (4.32 mL, 24.8 mmol) in DCM (50 mL) was added EDC·HCl (3.56 g, 18.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 48 h and then diluted with water (25 mL). The organic layer was collected, washed with water (25 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–100% EtOAc in hexanes) to afford 2.16 g (5.11 mmol, 41% yield) of the desired product as a clear oil. MS: 867.22 m / z [M+Na].

[0331] Intermediate 96b: 3-Hydroxy-2-(hydroxymethyl)-2-methylpropyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Intermediate 96a (2.16 g, 5.11 mmol) was dissolved in methanol (50 mL) and Dowex 50x8 resin was added. The resulting reaction mixture was stirred at room temperature for 16 h, filtered, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0 to 60% EtOAc in hexanes) to afford 1.43 g (3.73 mmol, 73% yield) of the desired product as a clear oil. 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.20(s,2H),3.58(dd,J=11.3,4.2Hz,2H),3.51(dd,J=11.3,4.9Hz,2H),2.81-2.74(m,2H), 2.71(m,2H),2.36(t,J=7.5Hz,2H),2.05(dt,J=8.5,6.7Hz,4H),1.69-1.56(m,2H),1.40-1.25(m,14H),0.89(m,3H),0.84(s,3H)ppm.

[0332] Intermediate 96c: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)-2-methylpropyl(9Z,12Z)-octadeca-9,12-dienoate [ka] Intermediate 96c was synthesized in 67% yield from intermediate 96b and intermediate 1c using the method used for intermediate 1d. 1 H NMR(400MHz, CDCl3)δ5.44-5.27(m,4H),4.49(t,J=5.5Hz,1H),4.02(d,J=2.7H z,4H),3.56(dt,J=9.3,6.7Hz,2H),3.44-3.36(m,4H),2.80-2.74(m,2H),2.42( t,J=7.5Hz,3H),2.33(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,4H),1.94(td,J=7.5,5 .4Hz,2H),1.69-1.50(m,7H),1.40-1.20(m,34H),0.95(s,3H),0.88(m,9H)ppm.

[0333] Example 96: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-methylpropyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Example 96 was synthesized in 32% yield from intermediate 96c and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.48(t,J=5.5Hz,1H),4.18(t,J=6.6Hz,2H),4.06(s,2H),4.01( m,4H),3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.4Hz,2H),2.51(q,J=7.1Hz,6 H),2.40(t,J=7.6Hz,2H),2.31(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,5H),1.92(td,J=7.6,5.5Hz,2H),1.81 (m,2H),1.67-1.49(m,12H),1.42-1.19(m,38H),1.05-0.96(m,9H),0.88(m,9H)ppm;MS:867.22m / z[M+H].

[0334] Synthesis of Example 97 Intermediate 97a: 2,3-dihydroxypropyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of glycerol (1.59 g, 17.3 mmol) in DCM (172 mL) was added linoleic acid (35.6 mmol), DMAP (423 mg, 3.47 mmol), and DIPEA (7.23 mL, 41.6 mmol) at room temperature. EDC·HCl (8.05 g, 41.6 mmol) was added in three portions over 15 min, followed by DMF (1 mL). The resulting reaction mixture was stirred for 16 h, washed with water, 1 M HCl, and 5% NaHCO3, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (a gradient of EtOAc in hexanes) to give 2.10 g (5.9 mmol, 34% yield) of the desired product. 1 H NMR(400MHz,CDCl3)δ5.44-5.27(m,4H),4.24-4.06(m,2H),3.92(m,1H),3.69(m,1H),3.59(dt,J=11.0,5.1Hz,1H),2.76(m,2H),2 .67(d,J=4.9Hz,1H),2.34(t,J=7.6Hz,2H),2.25(m,1H),2.04(m,4H),1.63(m,2H),1.41-1.22(m,14H),0.89(t,J=6.8Hz,3H)ppm.

[0335] Intermediate 97b: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-hydroxypropyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Intermediate 97b was synthesized in 11% yield from intermediate 97a and intermediate 1c using the method used for intermediate 1d. 1H NMR(400MHz,CDCl3)δ5.35(m,4H),4.50(t,J=5.5Hz,1H),4.23-4.05(m,5H),3.5 6(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(m,2H),2.55(d,J=4.6 Hz,1H),2.44(t,J=7.4Hz,2H),2.35(t,J=7.6Hz,2H),2.05(q,J=6.8Hz,4H),1.9 5(td,J=7.4,5.5Hz,2H),1.70-1.51(m,8H),1.39-1.21(m,34H),0.88(m,9H)ppm.

[0336] Example 97: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 96b (170 mg, 0.25 mmol) in acetonitrile (5 mL) was added pyridine (40 μL, 0.5 mmol) and 4-nitrophenyl chloroformate (70 mg, 0.35 mmol) at room temperature. After stirring for 4 h, 3-diethylamino-1-propanol (111 μL, 0.75 mmol) was added, and the resulting reaction mixture was stirred for an additional 2 h. The reaction mixture was extracted into hexane (10 mL), washed with water, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified using silica gel chromatography (0–100% EtOAc in hexane) to give 52 mg (0.062 mmol, 25% yield) of the desired product as a clear oil. 1H NMR(400MHz,CDCl3)δ5.36(m,4H),5.08(m,1H),4.48(t,J=5.6Hz,1H),4.34(m,2H),4.25-4.13(m,4H) ,3.56(dt,J=9.3,6.7Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.77(t,J=6.6Hz,2H),2.56-2.46(m,6H),2 .41(t,J=7.6Hz,2H),2.36-2.28(m,2H),2.05(q,J=6.9Hz,4H),1.92(td,J=7.6,5.5Hz,2H),1.82(m,2 H),1.67-1.52(10H),1.40-1.23(m,34H),1.01(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:840.03m / z[M+H].

[0337] Synthesis of Example 98 Intermediate 98a: 2-((2,2-dimethyl-1,3-dioxan-5-yl)methoxy)-N,N-diethylethan-1-amine [ka] To a mixture of 95% sodium hydride (2.5 equiv.) in anhydrous DCM (1.7-4.3 M) was added a solution of (2,2-dimethyl-1,3-dioxan-5-yl)methanol (1 equiv.) in anhydrous DCM (0.45-1.1 M) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min, followed by the addition of (2-bromoethyl)diethylamine hydrobromide (1.23 g, 4.78 mmol, 1.4 equiv.). The reaction was warmed to room temperature and stirred for 2 h before being recooled to 0 °C, diluted with water, and extracted into EtOAc. The organic layer was collected, dried over anhydrous magnesium sulfate, and concentrated in vacuo to give 370 mg (1.50 mmol, 44% yield) of the desired product as a clear oil, which was not further purified. 1H NMR(500MHz,CDCl3)δ3.95(m,2H),3.75(m,2H),3.51(t,J=6.2Hz,2H),3.47(d,J=6.7Hz,2H),2.64(t,J=6.2Hz ,2H),2.57(q,J=7.1Hz,4H),1.98(m,1H),1.42(s,3H),1.40(s,3H)1.02(t,J=7.1Hz,6H)ppm;MS:246.40[M+H].

[0338] Intermediate 98b: 3-(2-(diethylamino)ethoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] 4N HCl in 1,4-dioxane (10 mL, 40 equiv.) was added neat to intermediate 98a (1 equiv.). The resulting reaction mixture was stirred at room temperature for 2 h and then concentrated in vacuo to afford the acetonide-deprotected intermediate. The crude residue, linoleic acid (0.9–1.5 equiv.), DIPEA (2–3.2 equiv.), and DMAP (0.15–0.26 equiv.) were dissolved in DCM (0.2–0.3 M). EDC-HCl (1.2–1.9 equiv.) was added to the solution, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated in vacuo and then purified using silica gel chromatography (0–100% EtOAc in hexanes, followed by 0–10% MeOH in DCM) to afford 550 mg (78% yield for two steps) of the desired product as a white viscous material. MS: 469.08 [M+H].

[0339] Example 98: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((2-(diethylamino)ethoxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 98b (1 equiv.), DIPEA (1.5 equiv.), DMAP (0.2 equiv.), and intermediate 1c (1 equiv.) in DCM (0.1-0.5 M) was added EDC-HCl (1.5 equiv.) at room temperature. The reaction mixture was stirred at room temperature for 16 h, concentrated in vacuo, and purified using silica gel chromatography (0-10% MeOH in DCM with 0.1% ammonium hydroxide). Product fractions were pooled, concentrated in vacuo, and azeotroped with 50 mL of toluene (3 times) to remove residual ammonium hydroxide, affording 174 mg (0.22 mmol, 19% yield) of the desired product as a pale yellow oil. 1 H NMR(400MHz,CDCl3)δ5.43-5.28(m,4H),4.48(t,J=5.6Hz,1H),4.18-4.07(m,4H),3.56(dt, J=9.3,6.7Hz,2H),3.52-3.32(m,6H),2.77(t,J=6.4Hz,2H),2.63(t,J=6.2Hz,2H),2.55(q, J=7.2Hz,4H),2.39(t,J=7.6Hz,2H),2.29(m,3H),2.05(q,J=6.8Hz,4H),1.92(td,J=7.6,5. 5Hz,2H),1.57(m,7H),1.31(m,33H),1.02(t,J=7.1Hz,6H),0.93-0.84(m,9H)ppm;MS:795.56 M+H(ESI+).

[0340] Synthesis of Example 99 Intermediate 99a: 3-((2,2-dimethyl-1,3-dioxan-5-yl)methoxy)-N,N-diethylpropan-1-amine [ka] Intermediate 99a was synthesized in 77% yield from (3-bromopropyl)diethylamine hydrobromide and (2,2-dimethyl-1,3-dioxan-5-yl)methanol using the method used for intermediate 98a. MS: 260.58 [M+H].

[0341] Intermediate 99b: 3-(3-(diethylamino)propoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Intermediate 99b was synthesized in 50% yield (2 steps) from intermediate 99a and linoleic acid using the method used for intermediate 98b. MS: 483.12 [M+H].

[0342] Example 99: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((3-(diethylamino)propoxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Example 99 was synthesized in 20% yield from intermediate 99b and intermediate 1c using the method used for example 98. 1 H NMR(400MHz,CDCl3)δ5.42-5.27(m,4H),4.48(t,J=5.6Hz,1H),4.13(m,4H),3.56 (dt,J=9.3,6.7Hz,2H),3.46-3.34(m,6H),2.77(t,J=6.4Hz,2H),2.56-2.43(m,6H ),2.39(t,J=7.6Hz,2H),2.29(m,3H),2.05(q,J=6.8Hz,4H),1.93(m,2H),1.64(m, 9H),1.36-1.26(m,33H),1.01(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:809.74[M+H].

[0343] Synthesis of Example 100 Intermediate 100a: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 1d (3 g, 1.0 equiv.) and (4-nitrophenyl)carbonochloridate (1.74 g, 2.0 equiv.) in DCM (30 mL) was added pyridine (1.05 mL, 3.0 equiv.) at 0° C. The mixture was stirred at 20° C. under a N atmosphere for 2 hours. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with HO and extracted twice 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 (2.5 g, 67%) as a colorless oil.

[0344] Example 100: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 100a (500 mg, 1.0 equiv.) in MeCN (9 mL) was added 2-pyrrolidin-1-ylethanamine (133 mg, 2.0 equiv.), pyridine (94 uL, 2.0 equiv.), and DMAP (71 mg, 1.0 equiv.). The mixture was stirred under a N atmosphere at 20 °C for 5 hours. The mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with EtOAc and washed five times with 1N NaHCO and three times with HO. The organic layer was dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give the product (150 mg, 31%) as a colorless oil. 1H NMR(400MHz,CDCl3)δ5.36-5.22(m,4H),5.20(d,J=15.9Hz,1H),4.42(t,J=5.6Hz,1H),4.05(t,J=5.4Hz,6H),3. 49(dt,J=9.4,6.7Hz,2H),3.38-3.30(m,2H),3.21(q,J=5.8Hz,2H),2.70(t,J=6.4Hz,2H),2.51(t,J=6.1Hz,2H), 2.43(d,J=6.2Hz,4H),2.33(dd,J=9.7,5.5Hz,3H),2.23(t,J=7.6Hz,3H),1.98(q,J=6.9Hz,4H),1.85(td,J=7.6 ,5.5Hz,2H),1.70(q,J=3.3Hz,4H),1.57-1.44(m,12H),1.23(ddt,J=18.4,10.5,6.3Hz,37H),0.84-0.78(m,9H). MS:836.3m / z[M+H].

[0345] Synthesis of Examples 101 to 103 The following examples were synthesized from intermediate 100a and an amino alcohol or diamine reagent using the methodology used in Example 100.

[0346] Example 101 Example 101: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 32%; 1H NMR(400MHz,CDCl3)δ5.36-5.17(m,4H),4.41(t,J=5.5Hz,1H),4.18-4.02(m,8H),3.49(dt,J= 9.3,6.7Hz,2H),3.33(dt,J=9.3,6.6Hz,2H),2.68(dt,J=19.3,6.5Hz,4H),2.50(q,J=7.2Hz,4 H),2.40-2.28(m,3H),2.23(t,J=7.6Hz,2H),1.98(q,J=6.9Hz,4H),1.85(td,J=7.6,5.5Hz,2H ),1.61-1.44(m,12H),1.32-1.14(m,35H),0.96(t,J=7.1Hz,6H),0.82(td,J=6.9,4.0Hz,9H). MS:850.3m / z[M+H].

[0347] Example 102 Example 102: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(piperidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 91%; 1 H NMR(400MHz,CDCl3)δ5.36-5.21(m,4H),4.41(t,J=5.5Hz,1H),4.16-4.00(m,8H),3.49(dt,J=9.3,6.7Hz,2H),3.33(dt,J=9.3,6.7Hz,2H),2.70(t ,J=6.5Hz,2H),2.40-2.19(m,11H),1.98(q,J=6.8Hz,4H),1.89-1.74(m, 4H), 1.60-1.43(m, 14H), 1.41-1.08(m, 36H), 0.82(td, J=6.9, 4.0Hz, 9H). MS:865.3m / z[M+H].

[0348] Example 103 Example 103: 3-(((2-(azepan-1-yl)ethyl)carbamoyl)oxy)-2-(((4,4-bis(octyloxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] Yield 47%; 1 H NMR(400MHz,CDCl3)δ5.67(s,1H),5.42-5.29(m,4H),4.48(t,J=5.6Hz,1H),4.12(qt,J=6.2,4. 1,3.7Hz,7H),3.56(dt,J=9.3,6.7Hz,2H),3.44-3.38(m,2H),3.27(q,J=5.6Hz,2H),2.71(ddt,J =30.8,24.9,12.7Hz,8H),2.35(dt,J=37.4,7.7Hz,6H),2.27-1.99(m,13H),1.92(td,J=7.6,5. 5Hz, 2H), 1.60 (ddt, J=24.3, 21.1, 6.7Hz, 14H), 1.40-1.19 (m, 35H), 0.88 (td, J=6.8, 4.0Hz, 9H). MS:864.3m / z[M+H].

[0349] Synthesis of Example 107 Intermediate 107a: 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 1a (4 g, 1.0 equiv.) in DCM (40 mL) was added EDCI (2.5 g, 1.2 equiv.), DMAP (132 mg, 0.1 equiv.), and DIPEA (3.78 mL, 2.0 equiv.) were added to the above mixture at 0° C. The mixture was stirred at 20° C. under N atmosphere for 2 h. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with HO and extracted twice 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 (4 g, 68%) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ5.37-5.20(m,4H),4.19-4.01(m,4H),3.56(d,J=5.6 Hz,2H),2.70(t,J=6.5Hz,2H),2.25(t,J=7.5Hz,2H),2.13(dt,J=11.6,5.8 Hz,2H),2.04-1.95(m,6H),1.90(q,J=3.2Hz,3H),1.64(dt,J=12.3,3.0Hz, 3H), 1.55 (dd, J=15.4, 2.5Hz, 11H), 1.33-1.15 (m, 15H), 0.87-0.77 (m, 3H).

[0350] Intermediate 107b: 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 107a (3 g, 1.0 equiv.), (4-nitrophenyl)carbonochloridate (3.3 g, 3.0 equiv.) in DCM (30 mL) was added pyridine (1.3 mL, 3.0 equiv.) at 0 °C. The mixture was stirred at 20 °C under a N atmosphere for 2-5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with hexane, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by silica gel chromatography to give the product (3 g, 76% yield) as a colorless oil. 1H NMR(400MHz,CDCl3)δ8.31(dd,J=9.3,3.1Hz,2H),7.41(dd,J=9.3,3.0Hz,2H),5.37(d tp,J=11.1,7.1,3.9Hz,4H),4.39(dd,J=6.0,3.0Hz,2H),4.23(dt,J=9.5,4.1Hz,4H),2 .80(d,J=6.5Hz,2H),2.54(ddt,J=11.6,8.5,4.3Hz,1H),2.35(td,J=7.6,3.0Hz,2H),2 .17-1.95(m,9H),1.78-1.58(m,15H),1.42-1.26(m,15H),0.91(td,J=6.8,3.0Hz,3H).

[0351] Example 107: 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 107b (1 g, 1.0 equiv.) in MeCN (10 mL) was added 3-(diethylamino)propan-1-ol (555 mg, 3.0 equiv.), pyridine (342 uL, 2.0 equiv.), and DMAP (17 mg, 0.1 equiv.). The mixture was stirred at 20 °C under a N atmosphere for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with HO and extracted three times with EtOAc. The organic layer was dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give the product (432 mg, 44%) as a pale yellow oil. 1H NMR(400MHz,CDCl3)δ5.28(tt,J=11.2,5.5Hz,4H),4.21-3.97(m,8H),2.70(t ,J=6.5Hz,2H),2.40(dq,J=29.5,6.5,6.0Hz,7H),2.24(t,J=7.6Hz,2H),1.98 (dd,J=14.6,7.7Hz,6H),1.94-1.85(m,3H),1.74(p,J=6.8Hz,2H),1.68-1.46 (m,16H),1.24(d,J=6.6Hz,14H),0.94(t,J=7.1Hz,6H),0.82(t,J=6.7Hz,3H). MS: 703.3 m / z [M+H].

[0352] Synthesis of Example 113 Intermediate 113a: 1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2-one [ka] To a mixture of 1,3-dihydroxypropan-2-one (20 g, 1.0 equiv.) in THF (150 mL) was added imidazole (15.12 g, 1.0 equiv.). Then, a mixture of TBSCl (1.0 equiv.) in THF (150 mL) was added dropwise to the above mixture at 0 °C. The mixture was stirred at 15 °C under a N atmosphere for 2 hours. After completion, the reaction mixture was poured into water and extracted three times with EtOAc. The combined organic phase was washed twice with brine, dried over anhydrous NaSO, filtered, and concentrated in vacuo. The reaction mixture was purified by column chromatography to give the product (5.5 g, 12%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ4.47(s,2H),4.28(s,2H),0.91-0.87(m,12H),0.14-0.01(m,9H).

[0353] Intermediate 113b: 3-((tert-butyldimethylsilyl)oxy)-2-oxopropyl(9Z,12Z)-octadeca-9,12-dienoate [ka] To a mixture of intermediate 113a (5.5 g, 1.0 equiv.), EDCI (6.19 g, 1.2 equiv.), DMAP (658 mg, 0.2 equiv.), and DIPEA (14.06 mL, 3.0 equiv.) in DCM (55 mL) was added (9Z,12Z)-octadeca-9,12-dienoic acid (7.6 mL, 1.0 equiv.). The reaction was stirred under N at 15 °C for 12 h. After completion, the reaction mixture was poured into water and extracted three times with DCM. The combined organic phases were dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by column chromatography to give the product (7 g, 56%) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ5.33-5.18(m,4H),4.85(s,2H),4.16(s,2H),2.67(t,J=6.5Hz,2H),2.32(t,J =7.5Hz,2H),1.94(q,J=6.9Hz,4H),1.57(q,J=7.3Hz,2H),1.32-1.13(m,14H),0.87-0.74(m,12H).

[0354] Intermediate 113c: 3-hydroxy-2-oxopropyl(9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 113b (7.0 g, 1.0 equiv.) in THF (70 mL) was added HF-pyridine (6.76 mL, 5.0 equiv.) dropwise at 0-15°C. The reaction mixture was stirred at 15°C for 1 h. The mixture was then poured into water and extracted three times with EtOAc. The combined organic phases were washed with brine, dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by column chromatography to give the product (3 g, 57%) as a colorless oil. H NMR (400 MHz, CDCl 3)δ5.35(tp,J=11.2,3.6,3.2Hz,4H),4.76(s,1H),4.37(s,1H),2.93(s,1H),2.77(t,J=6.4Hz,2H),2.43(t,J=7.5Hz,2H),2 .39-2.31(m,1H),2.05(p,J=7.1,6.4Hz,4H),1.65(dp,J=11.8,6.4,5.4Hz,2H),1.41-1.22(m,15H),0.88(t,J=6.7Hz,3H).

[0355] Intermediate 113d: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-oxopropyl(9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 113c (3 g, 1.0 equiv.), EDCI (1.96 g, 1.2 equiv.), DMAP (208 mg, 0.2 equiv.), and DIPEA (4.45 mL, 3.0 equiv.) in DCM (30 mL) was added intermediate 1c (2.93 g, 1.0 equiv.). The reaction mixture was then stirred under N at 15 °C for 12 h. The residue was poured into water and extracted three times with DCM. The combined organic phases were dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by column chromatography to give the product (4 g, 69%) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ5.43-5.26(m,4H),4.74(d,J=1.9Hz,4H),4.50(t,J=5.5Hz,1H),4.11(q, J=7.1Hz,2H),3.56(dt,J=9.3,6.6Hz,2H),3.40(dt,J=9.3,6.7Hz,2H),2.76(t,J=6.4Hz,2H),2 .50(t,J=7.5Hz,2H),2.41(t,J=7.5Hz,2H),2.04(d,J=5.9Hz,6H),1.96(dd,J=7.5,5.5Hz,2H) ,1.65(p,J=7.2Hz,2H),1.55(q,J=6.9Hz,4H),1.40-1.19(m,33H),0.87(td,J=6.8,3.9Hz,8H).

[0356] Intermediate 113e: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-hydroxypropyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 113d (4 g, 1.0 equiv.) in THF (80 mL), HO (40 mL), and toluene (20 mL) was added NaBH (1.11 g, 5.0 equiv.) at 5 °C. The mixture was stirred at 5 °C for 5 h. The reaction mixture was then poured into saturated NH Cl and extracted twice with ethyl acetate. The combined organic phases were washed with brine, dried over anhydrous Na SO , filtered, and concentrated in vacuo. The residue was purified by column chromatography to give the product (2.5 g, 62%) as a colorless oil.

[0357] Intermediate 113f: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-nitrophenoxy)carbonyl)oxy)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 113e (2.5 g, 1.0 equiv.) and (4-nitrophenyl)carbonochloridate (1.48 g, 2.0 equiv.) in DCM (25 mL) was added DMAP (4 mg, 0.01 equiv.) and pyridine (5.93 mL, 20 equiv.). The reaction mixture was stirred at 15° C. for 5 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product (colorless oil) was further purified and used directly in the next step (2.4 g, 79%).

[0358] Example 113: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((2-(diethylamino)ethyl)carbamoyl)oxy)propyl (9Z,12Z)-octadeca-9,12-dienoate [ka] To a solution of intermediate 113f (800 mg, 1.0 equiv.) and N',N'-diethylethane-1,2-diamine (664 uL, 5.0 equiv.) in MeCN (10 mL) was added pyridine (1.53 mL, 20 equiv.) and DMAP (12 mg, 0.1 equiv.). The reaction mixture was stirred at 15 °C for 12 h. The mixture was poured into water and extracted three times with EtOAc. The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography to give the product (320 mg, 41%) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ5.33(ddt,J=19.7,13.2,7.4Hz,4H),5.19-5.09(m,1H),4.47(t,J=5.6Hz,1H),4.25(dt,J=11.5,3.8H z,2H),4.17(dd,J=11.9,5.7Hz,2H),3.55(dt,J=9.4,6.7Hz,2H),3.38(dt,J=9.2,6.7Hz,2H),3.21(q,J=5.7Hz,2H),2.76( t,J=6.5Hz,2H),2.51(q,J=7.3Hz,5H),2.39(t,J=7.6Hz,2H),2.30(t,J=7.6Hz,2H),2.03(q,J=6.9Hz,3H),1.91(td,J=7.6 ,5.5Hz,2H),1.57(dt,J=21.9,7.1Hz,5H),1.29(q,J=10.3,5.5Hz,25H),0.99(t,J=7.1Hz,4H),0.87(td,J=6.7,3.9Hz,6H). MS:825.4m / z[M+H].

[0359] Synthesis of Example 114 Intermediate 114a: 3-Hydroxy-2-(hydroxymethyl)propyl stearate [ka] To a solution of (2,2-dimethyl-1,3-dioxan-5-yl)methanol (1.53 g, 10.5 mmol), stearic acid (3 g, 10.5 mmol), DMAP (256 mg, 2.1 mmol), and DIPEA (4.39 mL, 25.2 mmol) in DCM (25 mL) was added EDC-HCl (2.41 g, 12.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 24 hours and then diluted with water (25 mL). The organic layer was collected and washed with HCl (10 mL), saturated NaHCO solution (10 mL), and then water (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and then concentrated in vacuo. The crude residue was dissolved in methanol, followed by hexane, until the sample was completely solubilized, and Dowex® 50x8 resin was added. The resulting reaction mixture was stirred at room temperature for 24 hours, filtered, and concentrated in vacuo to give 2.74 g (7.35 mmol, 70% yield) of the desired product as a white solid. 1 H NMR(CDCl3,400MHz)δ4.27(d,J=6.3Hz,2H),3.77(m,4H),2.33(m,2H),2.10(s,2H), 2.03(m,1H),1.62(m,2H),1.25(m,29H),0.87(t,J=6.7Hz,3H);MS:373.32m / z[M+H].

[0360] Intermediate 114b: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl stearate [ka] Intermediate 114b was synthesized in 43% yield from intermediate 114a and intermediate 1c using the method used for intermediate 1d. 1H NMR(CDCl3,400MHz)δ4.49(t,J=5.5Hz,1H),4.17(m,4H),3.62(t,J=5.5Hz,2H),3.56(m,2H),3.40(m,2H),2.41(t,J=7.5Hz,2 H),2.32(t,J=7.6Hz,2H),2.20(m,2H),1.62(m,2H),1.55(m,6H),1.27(m,49H),0.88(t,J=6.8Hz,9H);MS:721.78m / z[M+Na].

[0361] Example 114: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl stearate [ka] Example 114 was synthesized in 30% yield from intermediate 114b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(CDCl3,400MHz)δ4.48(t,J=5.5Hz,1H),4.17(m,8H),3.56(m,2H),3.40(m,2H),2.52(s,5H),2.40(m,3H),2.30(t,J=7. 6Hz,2H),1.92(m,2H),1.82(s,2H),1.57(m,7H),1.26(m,48H),1.02(t,J=6.8Hz,6H),0.88(m,9H)ppm;MS:857.14m / z[M+H].

[0362] Synthesis of Example 115 Intermediate 115a: 3-Hydroxy-2-(hydroxymethyl)propyl oleate [ka] To a solution of (2,2-dimethyl-1,3-dioxan-5-yl)methanol (1.53 g, 10.5 mmol), oleic acid (3.37 mL, 10.5 mmol), DMAP (256 mg, 2.1 mmol), and DIPEA (4.39 mL, 25.2 mmol) in DCM (25 mL) was added EDC-HCl (2.41 g, 12.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 24 hours and then diluted with water (25 mL). The organic layer was collected and washed with HCl (10 mL), saturated NaHCO solution (10 mL), and then water (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and then concentrated in vacuo. The crude residue was dissolved in methanol and Dowex® 50x8 resin was added. The resulting reaction mixture was stirred at room temperature for 24 hours, filtered, and concentrated in vacuo to give 2.76 g (7.44 mmol, 70% yield) of the desired product as a clear oil. 1 H NMR(CDCl3,400MHz)δ5.35(m,2H),4.27(d,J=6.3Hz,2H),3.77(m,4H),2.33(t,7.5Hz,2 H),2.02(m,6H),1.62(m,2H),1.29(m,20H),0.88(t,J=6.8Hz,3H);MS:371.29m / z[M+H].

[0363] Intermediate 115b: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl oleate [ka] Intermediate 115b was synthesized in 45% yield from intermediate 115a and intermediate 1c using the method used for intermediate 1d. 1H NMR(CDCl3,400MHz)δ5.34(m,2H),4.49(t,J=5.5Hz,1H)4.18(m,4H),3.62(t,J=5.6Hz,2H),3.56(m,2H),3.40(m,2H),2.41(t,J=7.5Hz,2H),2.3 2(m,2H),2.20(m,2H),2.01(q,J=6.2Hz,4H),1.94(m,2H),1.61(q,J=7. 3Hz, 2H), 1.56 (m, 6H), 1.29 (m, 42H), 0.87 (m, 9H); MS: 720.52m / z [M+Na].

[0364] Example 115: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl oleate [ka] Example 115 was synthesized in 40% yield from intermediate 115b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(CDCl3,400MHz)δ5.34(m,2H),4.48(t,J=5.5Hz,1H),4.17(m,8H),3.56(m,2H),3.40(m,2H),2.52(s,5H),2.40(m,3H),2.30(t,J=7. 6Hz,2H),2.00(m,4H),1.92(m,2H),1.82(m,2H),1.57(m,7H),1.26(m,40H),1.02(t,J=7.1Hz,6H),0.88(m,9H)ppm;MS:855.37m / z[M+H].

[0365] Synthesis of Example 116 Intermediate 116a: 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate [ka] To a solution of (2,2-dimethyl-1,3-dioxan-5-yl)methanol (1.53 g, 10.5 mmol), linolenic acid (3.28 mL, 10.5 mmol), DMAP (256 mg, 2.1 mmol), and DIPEA (4.39 mL, 25.2 mmol) in DCM (25 mL) was added EDC-HCl (2.41 g, 12.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 24 hours and then diluted with water (25 mL). The organic layer was collected and washed with HCl (10 mL), saturated NaHCO solution (10 mL), and then water (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and then concentrated in vacuo. The crude residue was dissolved in methanol and Dowex® 50x8 resin was added. The resulting reaction mixture was stirred at room temperature for 24 hours, filtered, and concentrated in vacuo to give 2.36 g (6.43 mmol, 60% yield) of the desired product as a clear oil. 1 H NMR(CDCl3,400MHz)δ5.36(m,6H),4.27(d,J=6.3Hz,2H),3.77(m,4H),2.81(m,4H),2.33(t,J= 7.5Hz,2H),2.06(m,6H),1.62(m,2H),1.31(m,9H),0.98(t,J=7.5Hz,3H);MS:367.29m / z[M+H].

[0366] Intermediate 116b: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z,15Z)-octadeca-9,12,15-trienoate [ka] Intermediate 116b was synthesized in 45% yield from intermediate 116a and intermediate 1c using the method used for intermediate 1d. 1H NMR(CDCl3,400MHz)δ5.36(m,6H),4.49(t,J=5.5Hz,1H),4.18(m,4H),3.62(t,J= 5.6Hz,2H),3.56(m,2H),3.40(m,2H),2.81(m,4H),2.41(t,J=7.5Hz,2H),2.32(t ,J=7.6Hz,2H),2.20(m,2H),2.07(m,4H),1.93(m,2H),1.62(m,2H),1.56(m,6H), 1.31(m,30H),0.98(t,J=7.5Hz,3H),0.88(t,J=6.9Hz,6H);MS:715.93m / z[M+Na].

[0367] Example 116: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate [ka] Example 116 was synthesized in 31% yield from intermediate 116b and 3-(diethylamino)propan-1-ol using the method used in Example 1. 1 H NMR(CDCl3,400MHz)δ5.34(m,6H),4.48(t,J=5.6Hz,1H),4.17(m,8H),3.56(m,2H),3.40(m,2H),2.81(m,4H)2.52(s,5H),2.40(m,3H),2. 30(t,J=7.6Hz,2H),2.08(m,4H),1.92(m,2H),1.82(m,2H),1.57(m,8H),1.29(m,28H),0.99(m,8H),0.88(m,6H)ppm;MS:851.32m / z[M+H].

[0368] Synthesis of Example 117 Intermediate 117a: Heptadecan-9-yl(3-(((4-nitrophenoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl)glutarate [ka] To a mixture of Intermediate 63a (1.0 equiv.) and (4-nitrophenyl)carbonochloridate (2.0 equiv.) in DCM (0.05-0.2 M) was added pyridine (2.0 equiv.). The mixture was stirred at 20 °C under an inert atmosphere for 5 h. After completion, the mixture was concentrated in vacuo, and the resulting residue was diluted with hexane and filtered. The filtrate was concentrated to give a color residue, which was used directly in the next step without further purification (47%).

[0369] Example 117: 3-(((2-(diethylamino)ethyl)carbamoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propylheptadecan-9-yl glutarate [ka] To a mixture of Intermediate 117a (1.0 equiv.) in MeCN (0.1 M) was added N',N'-diethylethane-1,2-diamine (2.0 equiv.), pyridine (2.0 equiv.), and DMAP (1.0 equiv.) under an inert atmosphere. The mixture was stirred at 20 °C under an inert atmosphere for 12 h, after which time the mixture was concentrated in vacuo. The resulting residue was diluted with EtOAc and washed five times with 1 N NaHCO and three times with HO. The organic layer was dried over NaSO, filtered, concentrated in vacuo, and purified by column chromatography to give the product (36%) as a pale yellow oil. 1 H NMR(400MHz, CDCl3)δ5.36-5.21(m,5H),4.79(p,J=6.2Hz,1H),4.06(t,J=5.9Hz ,6H),3.20(s,2H),2.70(t,J=6.4Hz,2H),2.49(d,J=25.8Hz,6H),2.36-2.20(m,8 H),1.97(dd,J=7.8,5.9Hz,5H),1.87(p,J=7.5Hz,3H),1.54(t,J=7.2Hz,2H),1.4 3(d,J=6.5Hz,4H),1.35-1.06(m,42H),0.98(s,6H),0.81(td,J=6.8,4.9Hz,9H). MS: 863.7 m / z [M+H].

[0370] Synthesis of Example 118 Intermediate 118a: 1-(heptadecan-9-yl) 7-(3-(((4-nitrophenoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl)heptanedioate [ka] Intermediate 118a was synthesized in 61% yield from intermediate 64b using the method used for intermediate 117a.

[0371] Example 118: 1-(3-(((2-(diethylamino)ethyl)carbamoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl) 7-(heptadecan-9-yl)heptanedioate [ka] Example 118 was synthesized in 96% yield from Example 118A using the method used for Example 117. 1 H NMR(400MHz,CDCl3)δ5.28(dtt,J=17.4,10.8,6.0Hz,4H),5.15(s,1H),4.79(p,J=6. 2Hz,1H),4.05(d,J=6.1Hz,6H),3.14(q,J=5.9Hz,2H),2.70(t,J=6.5Hz,2H),2.45(q, J=6.8Hz,6H),2.37-2.18(m,7H),1.98(q,J=7.0Hz,4H),1.56(q,J=7.9Hz,6H),1.43(q ,J=6.3Hz,4H),1.21(d,J=18.3Hz,40H),0.93(t,J=7.1Hz,6H),0.81(q,J=6.4Hz,9H). MS:891.8m / z[M+H].

[0372] Example 119: pKa Measurement The pKa of each amine lipid was determined according to the method of Jayaraman et al. (Angewandte Chemie, 2012), adapted as follows. pKa was determined for unformulated amine lipids in ethanol at a concentration of 2.94 mM. Lipids were diluted to 100 μM in 0.1 M phosphate buffer (Boston Bioproducts), whose pH ranged from 2.0 to 12.0. Fluorescence intensity was measured using excitation and emission wavelengths of 321 nm and 448 nm. Table 1 shows the pKa measurements for the listed compounds.

[0373] [Table 1] JPEG2025066792000287.jpg253160JPEG2025066792000288.jpg253160JPEG2025066792000289.jpg52161

[0374] Example 120 - Materials and Methods LNP Compositions for In Vivo Editing LNPs were prepared using various amine lipids in a four-component lipid system consisting of an ionizable lipid (e.g., amine lipid), DSPC, cholesterol, and PEG-2k-DMG. The molar concentrations of lipids in the lipid components of the LNPs were used at a molar percentage of amine lipid / DSPC / cholesterol / PEG-2k-DMG of 50 / 9 / 38 / 3. For assays for percent liver editing in mice, Cas9 mRNA and chemically modified sgRNA targeting a mouse sequence were formulated in LNPs at either a 1:1 w / w ratio, a 1:2 w / w ratio, or a 1:1.3 w / w ratio.

[0375] LNP formulation The lipid components were dissolved in 100% ethanol at the lipid component molar ratios listed below. The chemically modified sgRNA and Cas9 mRNA were combined and dissolved in 25 mM citric acid, 100 mM NaCl, pH 5.0, to a total RNA cargo concentration of approximately 0.45 mg / mL. LNPs were formulated with an N / P ratio of approximately 6 and a chemically modified sgRNA:Cas9 mRNA ratio of either 1:1 w / w, 1:2 w / w, or 1:1.3 w / w, as described below.

[0376] LNPs were formed by impinging jet mixing of lipids in ethanol with two volumes of RNA solution and one volume of water. Lipids in ethanol were mixed with two volumes of RNA solution through a mixing cross. A fourth water stream was mixed with the cross's outlet stream via an in-line tee (see, e.g., WO2016 / 010840, Figure 2). Alternatively, LNPs were formed by microfluidic mixing of lipid and RNA solutions using a Precision Nanosystems NanoAssemblr™ Benchtop Instrument according to the manufacturer's protocol. A 2:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates. LNPs were kept at room temperature for 1 hour and further diluted with water (approximately 1:1 v / v). The diluted LNP was concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, 100 kD MWCO) and then buffer exchanged by diafiltration into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS). Alternatively, the final buffer exchange into TSS was completed using a PD-10 desalting column (GE). If necessary, the composition was concentrated by centrifugation using an Amicon 100 kDa centrifugal filter (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at 4°C or -80°C until further use.

[0377] LNP composition analysis Dynamic light scattering ("DLS") is used to characterize the polydispersity index ("PDI") and size of the LNPs of the present disclosure. DLS measures the scattering of light that occurs as a result of subjecting a sample to a light source. The PDI determined from DLS measurements represents the distribution of particle sizes (around the mean particle size) within a population, with a completely homogeneous population having a PDI of zero.

[0378] Electrophoretic light scattering is used to characterize the surface charge of LNPs at a specified pH. Surface charge, or zeta potential, is a measure of the magnitude of electrostatic repulsion / attraction between particles in an LNP suspension.

[0379] Asymmetric flow field-flow fractionation-multiangle light scattering (AF4-MALS) is used to separate particles within a composition by hydrodynamic radius, and then the molecular weight, hydrodynamic radius, and root-mean-square radius of the fractionated particles are measured, allowing for the assessment of molecular weight and size distributions, as well as secondary properties such as Burchard-Stockmeyer plots (the ratio of root-mean-square ("rms") radius to hydrodynamic radius over time, which indicates the particle's inner core density), and rms conformation plots (the logarithm of rms radius versus the logarithm of molecular weight, with the slope of the resulting linear fit providing a measure of compactness versus elongation).

[0380] Nanoparticle Tracking Analysis (NTA, Malvern Nanosight) can be used to determine particle size distribution and particle concentration. LNP samples are appropriately diluted and injected onto a microscope slide. A camera records scattered light as particles are slowly injected through the field of view. After capturing a video, Nanoparticle Tracking Analysis processes the video by tracking pixels and calculating the diffusion coefficient, which can be converted to the hydrodynamic radius of the particle. The instrument also counts the number of individual particles counted in the analysis, yielding the particle concentration.

[0381] Cryo-electron microscopy ("cryo-EM") can be used to determine the particle size, morphology, and structural characteristics of LNPs.

[0382] Lipid composition analysis of LNPs can be determined from liquid chromatography followed by charged aerosol detection (LC-CAD), which can provide a comparison of actual lipid content with theoretical lipid content.

[0383] LNP compositions are analyzed for mean particle size, polydispersity index (PDI), total RNA content, RNA encapsulation efficiency, and zeta potential. LNP compositions may be further characterized by lipid analysis, AF4-MALS, NTA, and / or cryo-EM. Mean particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument or a Wyatt NanoStar. LNP samples are diluted with PBS buffer and then measured by DLS. The Z-average diameter, an intensity-based measure of mean particle size, is reported along with the number-average diameter and PDI. The Malvern Zetasizer or Wyatt NanoStar instrument is also used to measure the zeta potential of LNPs. Samples are diluted 1:17 (50 μL to 800 μL) with 0.1x PBS, pH 7.4, prior to measurement.

[0384] Total RNA concentration and free RNA are determined using a fluorescence-based assay (Ribogreen®, ThermoFisher Scientific). Encapsulation efficiency is calculated as (total RNA - free RNA) / total RNA. LNP samples are appropriately diluted with 1x TE buffer containing 0.2% Triton-X100 to determine total RNA or 1x TE buffer to determine free RNA. A standard curve is generated using the starting RNA solution used to make the composition and diluted with 1x TE buffer ± 0.2% Triton-X100. Diluted RiboGreen® dye (according to the manufacturer's instructions) is then added to each standard and sample and incubated for approximately 10 minutes at room temperature, protected from light. Samples are read using a SpectraMax M5 microplate reader (Molecular Devices) with excitation, auto-cutoff, and emission wavelengths set at 488 nm, 515 nm, and 525 nm, respectively. Total RNA and free RNA are determined from the appropriate standard curve.

[0385] The encapsulation efficiency is calculated as (total RNA - free RNA) / total RNA. The same procedure can be used to determine the encapsulation efficiency of DNA-based cargo components. Fluorescence-based assays can use Oligreen Dye for single-stranded DNA and Picogreen Dye for double-stranded DNA. Alternatively, total RNA concentration can be determined by reverse-phase ion-pairing (RP-IP) HPLC. Triton X-100 is used to disrupt the LNPs, releasing the RNA. The RNA is then chromatographically separated from the lipid components by RP-IP HPLC and quantified against a standard curve using UV absorbance at 260 nm.

[0386] AF4-MALS is used to investigate molecular weight and size distributions, as well as second-order statistics from their calculations. LNPs are diluted as needed and injected into the AF4 separation channel using an HPLC autosampler, where they are focused and eluted with an exponential gradient of cross-flow across the channel. All fluids are driven by an HPLC pump and a Wyatt Eclipse Instrument. Particles eluting from the AF4 channel pass through a UV detector, a multi-angle light scattering detector, a quasi-elastic light scattering detector, and a differential refractive index detector. Raw data are processed using the Debye model to determine molecular weight and rms radius from the detector signals.

[0387] The lipid components of the LNPs are quantitatively analyzed by HPLC coupled to a charged aerosol detector (CAD). Chromatographic separation of the four lipid components is achieved by reversed-phase HPLC. The CAD is a destructive mass-based detector that detects all nonvolatile compounds, ensuring a consistent signal regardless of the analyte structure.

[0388] Cas9 mRNA and gRNA cargo Cas9 mRNA (e.g., SEQ ID NO: 3) cargo was prepared by in vitro transcription. Capped, polyadenylated Cas9 mRNA containing a 1X NLS was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing a T7 promoter and a 100-nt poly(A / T) tract was linearized by incubating at 37°C for 2 hours with 200 ng / μL of plasmid, 2 U / μL of XbaI (NEB), and 1X reaction buffer. XbaI was inactivated by heating the reaction to 65°C for 20 minutes. The linearized plasmid was purified from enzyme and buffer using a silica maxi spin column (Epoch Life Sciences) and analyzed by agarose gel to confirm linearization. The IVT reaction to generate Cas9-modified mRNA was performed by incubating at 37°C for 4 hours with the following conditions: 50 ng / μL linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methylpseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U / μL T7 RNA polymerase (NEB); 1 U / μL mouse RNase inhibitor (NEB); 0.004 U / μL inorganic E. coli pyrophosphatase (NEB); and 1x reaction buffer. After the 4-hour incubation, TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U / μL, and the reaction was incubated for an additional 30 minutes to remove the DNA template. Cas9 mRNA was purified using TFF or a method involving LiCl precipitation.

[0389] Capped, polyadenylated Cas9 mRNA containing SEQ ID NO:6 and 1X NLS was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing a T7 promoter and a 90-100 nt poly(A / T) tract was linearized to completion by incubation with XbaI at 37°C. The linearized plasmid was purified from enzymes and buffer salts. The IVT reaction to generate Cas9-modified mRNA was performed by incubating at 37°C for 1.5 or 2 hours under the following conditions: 50 ng / µL linearized plasmid; 5 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP (Trilink); 25 mM ARCA (Trilink); 5 U / µL T7 RNA polymerase; 1 U / µL mouse RNase inhibitor; 0.004 U / µL inorganic E. coli pyrophosphatase; and 1X reaction buffer. TURBO DNase (ThermoFisher) is then added to remove the DNA template.

[0390] mRNA was purified from enzymes and nucleotides using the RNeasy Maxi kit (Qiagen) according to the manufacturer's protocol. Alternatively, mRNA was purified using the MEGAclear kit (Invitrogen) according to the manufacturer's protocol. Alternatively, mRNA was purified using LiCl precipitation, ammonium acetate precipitation, and sodium acetate precipitation. Alternatively, mRNA was purified by LiCl precipitation, followed by further purification by tangential flow filtration. Alternatively, RNA was purified by LiCl precipitation in combination with tangential flow filtration. Transcript concentrations were determined by measuring light absorbance at 260 nm (Nanodrop), and transcripts were analyzed by capillary electrophoresis using a Fragment Analyzer (Agilent).

[0391] The sgRNAs in the following examples were chemically synthesized by known methods using phosphoramidites.

[0392] In vivo LNP delivery Mouse studies Six- to ten-week-old CD-1 female mice were used for each study. Animals were weighed and divided into groups according to their weight to prepare dosing solutions based on the group's average weight. LNP was administered via the lateral tail vein at a volume of 0.2 mL per animal (approximately 10 mL per kilogram of body weight). Animals were observed regularly for adverse effects for at least 24 hours after dosing.

[0393] For studies measuring in vivo editing in the liver, CD-1 female mice were dosed at 0.1 mg / kg unless otherwise indicated. Animals were euthanized on day 6 or 7 by exsanguination via cardiac puncture under isoflurane anesthesia. Liver tissue was collected from each animal for DNA extraction and analysis. Blood was collected into serum separator tubes or tubes containing buffered sodium citrate for plasma, as described herein. Cohorts of mice were measured for editing by next-generation sequencing (NGS).

[0394] Rat studies Six- to seven-week-old female Sprague-Dawley rats were used in each study. Each animal was weighed and the dosing solution was prepared based on the animal's weight. LNP was administered via the lateral tail vein at a volume of 0.35 mL per animal (approximately 2 mL per kilogram of body weight). Animals were observed periodically for adverse effects for at least 24 hours after dosing.

[0395] For studies measuring in vivo editing in the liver, animals were dosed at 0.1 mg / kg or 0.3 mg / kg. Six days after administration, animals were euthanized by CO2 asphyxiation. At necropsy, livers were collected for editing analysis by NGS, and blood was collected into serum separator tubes for serum TTR measurement.

[0396] NGS sequencing Briefly, to quantitatively determine the editing efficiency at the target location in the genome, genomic DNA was isolated and deep sequencing was used to identify the presence of insertions and deletions introduced by gene editing.

[0397] PCR primers were designed around the target site (e.g., within B2M) to amplify the genomic region of interest. An additional PCR was performed according to the manufacturer's protocol (Illumina) to add the necessary sequencing chemistry. The amplicons were sequenced on an Illumina MiSeq instrument. Reads were aligned to a mouse or rat-related reference genome (e.g., GRCm38) after removing reads with low quality scores. The resulting file containing the reads was mapped to the reference genome (BAM file), where reads overlapping the target region of interest were selected, and the number of wild-type reads was calculated relative to the number of reads containing insertions, substitutions, or deletions.

[0398] The editing percentage (e.g., "indel efficiency" or "percent indels") was defined as the total number of sequence reads with insertions or deletions relative to the total number of sequence reads containing the wild type.

[0399] Transthyretin (TTR) ELISA analysis Blood was collected and serum isolated as indicated. Total mouse TTR serum levels were measured using a Mouse Prealbumin (Transthyretin) ELISA kit (Aviva Systems Biology, catalog number OKIA00111). Briefly, serum was serially diluted with the kit's sample diluent to a final dilution of 10,000:00 for a 0.1 mg / kg dose and 10,000:00 and 2,500:00 for a 0.3 mg / kg dose. The diluted samples were then added to an ELISA plate, and the assay was performed according to the manufacturer's instructions.

[0400] Example 121 - Evaluation of lipid efficacy by in vivo editing The in vivo editing efficiency of materials delivered by formulations containing various amine lipid compounds was evaluated. Editing was measured using either G282 (SEQ ID NO: 1), which targets the mouse TTR gene, or G650 (SEQ ID NO: 2), which targets the mouse B2M gene. The lipids were evaluated for efficacy through in vivo editing experiments. LNPs were formulated with a 1:1 or 1:1.3 sgRNA-to-Cas9 mRNA w / w ratio and an N / P ratio of approximately 6. The molar concentration of lipids in the lipid component of the LNPs was used in the following molar percentages: amine lipid / DSPC / cholesterol / PEG-2k-DMG / 50 / 9 / 38 / 3. The final LNPs were characterized to determine encapsulation efficiency, polydispersity index, and average particle size according to the analytical methods described above. Analysis of the average particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation efficiency is shown in Table 2.

[0401] [Table 2] JPEG2025066792000291.jpg253169JPEG2025066792000292.jpg253169JPEG2025066792000293.jpg205169

[0402] Table 3 shows the editing percentage in mouse liver as measured by NGS.

[0403] [Table 3] JPEG2025066792000295.jpg254162JPEG2025066792000296.jpg255155JPEG2025066792000297.j pg255155JPEG2025066792000298.jpg255155JPEG2025066792000299.jpg255155JPEG20250667920 00300.jpg255155JPEG2025066792000301.jpg255155JPEG2025066792000302.jpg255155JPEG202 5066792000303.jpg255155JPEG2025066792000304.jpg255155JPEG2025066792000305.jpg148156

[0404] Example 122 - Dose response of editing in the liver To assess whether editing was dose-responsive, in vivo experiments were performed at various LNP dose levels. The Cas9 mRNA of Example 120 was formulated as LNPs with an sgRNA targeting TTR (G282, SEQ ID NO: 1; or G502, SEQ ID NO: 4). Alternatively, the Cas9 mRNA of Example 120 was formulated as LNPs with an sgRNA targeting B2M (G650, SEQ ID NO: 2). These LNPs were formulated with the w / w ratios of sgRNA and Cas9 mRNA shown in Table 4. The LNPs were formulated using a cross-flow procedure with a lipid molar composition of 50 / 9 / 38 / 3 amine lipid / DSPC / cholesterol / PEG-2k-DMG and an N / P ratio of 6.0.

[0405] The LNP compositions were analyzed for mean particle size, polydispersity (PDI), total RNA content, and RNA encapsulation efficiency as described in Example 120.

[0406] The analysis of mean particle size, polydispersity (PDI), total RNA content, and RNA encapsulation efficiency is shown in Table 4.

[0407] [Table 4]

[0408] CD-1 female mice were intravenously administered the compounds as specified in Table 5 and assessed for editing in the liver. The results are shown in Figures 1A-1D and Table 5.

[0409] [Table 5] JPEG2025066792000308.jpg255169JPEG2025066792000309.jpg159169

[0410] Example 123 - TTR Liver Editing and Serum TTR Levels Providing % Indel Formation in the G502 Experiment In vivo delivery was evaluated by measuring the editing efficiency of materials delivered by formulations containing various amine lipid compounds. Cas9 mRNA (SEQ ID NO: 6) was formulated as LNPs with an sgRNA targeting the TTR mouse gene (SEQ ID NO: 4). The LNPs were formulated with a 1:2 w / w ratio of sgRNA to Cas9 mRNA and an N / P ratio of approximately 6. The molar concentrations of lipids in the lipid component of the LNPs were used in the following mole %: amine lipid / DSPC / cholesterol / PEG-2k-DMG: 50 / 9 / 38 / 3. The final LNP compositions were characterized and analyzed as described in Example 120. Analysis of the mean particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation efficiency are shown in Table 6.

[0411] [Table 6] JPEG2025066792000311.jpg128165

[0412] CD-1 female mice were administered 0.1 mg / kg intravenously and assessed for editing in the liver and circulating TTR levels. The results are shown in Figures 2 and 3 and Table 7.

[0413] [Table 7] JPEG2025066792000313.jpg233166

[0414] Example 124 TTR Liver Editing and Serum TTR Levels Providing % Indel Formation in the G650 Experiment In vivo delivery to the liver, spleen, and bone marrow was evaluated by measuring the editing efficiency of material delivered by formulations containing various amine lipid compounds. Cas9 mRNA (SEQ ID NO: 6) was formulated as LNPs with an sgRNA targeting the TTR rat gene (G650, SEQ ID NO: 2). LNPs were formulated with a 1:2 w / w ratio of sgRNA to Cas9 mRNA and an N / P ratio of approximately 6. The molar concentrations of lipids in the lipid component of the LNPs were used in the following molar percentages: amine lipid / DSPC / cholesterol / PEG-2k-DMG: 50 / 9 / 38 / 3. The final LNP compositions were characterized and analyzed as described in Example 120. Analysis of mean particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation efficiency are shown in Table 8.

[0415] [Table 8]

[0416] CD-1 female mice were administered 0.1, 0.3, or 1 mg / kg intravenously and assessed for editing in the liver, spleen, and bone marrow. The results are shown in Table 9.

[0417] [Table 9]

[0418] Example 125 Delivery in Rat Studies In vivo delivery was evaluated in a rat model by measuring the editing efficiency of materials delivered by formulations containing various amine lipid compounds. Cas9 mRNA (SEQ ID NO: 6) was formulated as LNPs with sgRNA targeting the TTR rat gene (G534, SEQ ID NO: 5). LNPs were formulated and characterized as described in Example 123. Analysis of mean particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation efficiency are shown in Table 10.

[0419] [Table 10]

[0420] Female Sprague Dawley rats were administered 0.1 mg / kg or 0.3 mg / kg intravenously and assessed for editing in the liver. The results are shown in Figure 4 and Table 11.

[0421] [Table 11] JPEG2025066792000318.jpg124168

[0422] Sequence Listing JPEG2025066792000319.jpg122168JPEG2025066792000320.jpg251169JPEG20250667920 00321.jpg253169JPEG2025066792000322.jpg253169JPEG2025066792000323.jpg254163 JPEG2025066792000324.jpg253167JPEG2025066792000325.jpg253167JPEG20250667920 00326.jpg253167JPEG2025066792000327.jpg253167JPEG2025066792000328.jpg129165

[0423] 2'-O-methyl modifications and phosphorothioate linkages are indicated as follows (m=2'-OMe; *=phosphorothioate):

[0424] Sequence Listing SEQUENCE LISTING <110> INTELLIA THERAPEUTICS, INC. <120> IONIZABLE AMINE LIPIDS AND LIPID NANOPARTICLES <130> PA25-014 <141> 2020-04-24 <150> US 62 / 843,854 <151> 2019-05-06 <150> US 62 / 838,551 <151> 2019-04-25 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 100 <212> RNA <213> Artificial Sequence <220> <2​​​​​​​​​​​​​ <210> 2 <211> 100 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 2 gacaagcacc agaaagacca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> 3 <211> 4516 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 3 gggucccgca gucggcgucc agcggcucug cuuguucgug ugugugucgu ugcaggccuu 60 auucggaucc gccaccaugg acaagaagua cagcaucgga cuggacaucg gaacaaacag 120 cgucggaugg cgagucauca cagacgaaua caagguccg agcaagaagu ucaagguccu 180 gggaaacaca gacagacaca caucaagaa gaaccugauc ggagcacugc uguucgacag 240 cggagaaaca cgagaagcaa caacacugaa gagaacagca agaagaagau acacaagaag 300 aaagaacaga aucugcuacc ugcaggaaau cuucagcaac gaauggcaa aggucgacga 360 cagcuuuc cacagacugg aagaaagcuu ccuggucgaa gaacaaga agcacgaaag 420 acaccgauc uucggaaaca ucgucgacga aguucgcauac cacgaaaagu acccgacaau 480 cuaccaccug agaaagaagc uggucgacag cacagacaag cgagaccuga gacugauucua 540 600 660 guucgaagaa aacccgauca acgcaagcgg agucgacgca aaggcaaucc ugagcgcaag 720 acugagcaag aggagaac uggaaaccu gaucgcacag cugccgggag aaaagaaa 780 cggacuguuc ggaaccuga ucgcacugag ccugggacug acaccgacu ucaaccuga 840 cuckgaccug gshahagacg caagcugca gcugagghagg gacauacg cuckoo 900 ggacaaccug cuggshaaga ucggagacca guacgcagac cuguuccugg cugghaugg 960 ccugagcgac gcaauccugc ugagcgacau ccugagaguc aacacagaaa ucacaaggc 1020 accgcugagc gcaagcauga ucaagagaua cgacgacac caccaggacc ugacacugcu 1080 gaaggcacug gagacacugc agcugccgga aaaguacaag gaaacuucu ucgaccagag 1140 chaggaaaaaaaaaaaaaaaaaaaaaaaaacaaaaaaaaaaacaaaaaaacaaaaaaux 1200 caucaagccg auccuggaaa agauggacgg aahagaaa cuggcuggca agcuggaacag 1260 agagaaccug cugagaaagc agagaacauu cgacaacgga agcaucccgc accagaucca 1320 ccugggagaa cugcacgcaa ucuggagaag acaggaagac uucuacccgu ucugaaagga 1380 caacagagaa aagaucgaaa agauccugac auucagaauc ccguacuacg ucggaccgcu 1440 ggcaagga aacagcagau ucgcauggau gacaagaaag agcgaaaaa caucacacc 1500 guggaacuuc gaagugcg ucgacaaggg agcaagcgca cagagcuuca ucgaaagaau 1560 gacaacuuc gacaagaacc ugccgaacga aaagguccug ccgaagcaca gccugcugua 1620 cgaauacuuc acagucuaca acgaacugac aaaggucaag uacgucacag aaaggaugag 1680 aaagccggca uuccugagcg gagaacagaa gaaggcaauc gucgaccugc uguucaagac 1740 aaacagaaag gucacaguca agcagcugaa ggagaacuac uucaagaa ucgaaugcuu 1800 cgacagcguc gaaucagcg gagucgaaga cagauucaac cgaagccugg gaacauacca 1860 cgaccugcug aaguauca agacaagga cuucuggac aacgaaa acgaacau 1920 ccuggaaac aucguccuga caucugacacu guucgaaca agaaauga ucgaaaaag 1980 acugaagaca uacgcacacc uguucgacga caaggucaug aagcagcuga agagaagaag 2040 aucacagga uggggagaac ugagcagaaa cgugauucaac ggaaucagag acaagcagag 2100 cggaaagaca auccuggacu uccugaagag cgacggauuc gcaacagaa acuucaugca 2160 gcugauccac gacgacagcc ugacauucaa ggagacauc cagaaggcac aggucagcgg 2220 acagggacac agccugcacg aacacaucgc aaaccuggca ggaagcccgg caaucaagaa 2280 gggaauccug cagacaguca aggucgucga cgaacugguc aaggucaugg gaacacaa 2340 gccggaaaac aucgucaucg aaauggcaag agaaaaccag acaacacaga aggcacagaa 2400 gacagcaga gaagaauga agaaucga agaaggaauc agaacugg gaagcagaug 2460 ccugaaggaa caccggugcg aaaacacaca gcugcaac gaaaagcugu accuguacua 2520 chuggshack frequency frequency frequency frequency 2580 quacgacguc gaccahaucg ucccggag chuccugag cuccugag ucgahac 2640 gguccugaca agaagcgaca agacagagg aagagcgac aacgucccga gcgaagaaugu 2700 cgucaagaag augagacu acuggagaca gcugcugaac gcaagcuga ucacacagag 2760 aaaguucgac accugachaa agcagagag aggacug agcgaacugg aaaggcagg 2820 auucaucaag agcagcugg ucgaacaag acagaucaca aagcacgucg cacagauccu 2880 ggshackaaaaaaaaaaaaaaaaaackaagg 2940 shake shake shake shake shake shake shake 3000 cagagaauc aaaacuacc accacgcaca cgacgcauac cugaacgcag ucgucggaac 3060 agcaacgaac aagaaaccc cgaagcugga aagcgaauuc agquaacggag acachaaggu 3120 cuacgacguc agaaagauga ucgcaaagag cgaacaggaa aucggaagg caacagcaaa 3180 guacuucuuc uacagcaaca ucaugaacuu cuucaagaca gaaucacac uggcaaacgg 3240 agaaucaga aagagaccgc ugaucgaaac aacggagaa acaggagaaa ucgucuggga 3300 caaggaaga gacuucgcaa cagucagaaa gguccugagc augccgcagg ucaacaucgu 3360 caagagaca gaaguccaga caggaggauu cagcaaggaa agcauccugc cgaagagaaa 3420 cagcgacaag cugaucgcaa gaagaagga cugggaccg aagaaguacg gaggauucga 3480 cagcccgaca gucgcauaca gcguccuggu cgucgcaaag gucgaaaagg gaagagcaa 3540 gaagcugaag agcgucaagg aacugcuggg aaucacaauc auggaaagaa ccagcuucga 3600 aaagaacccg aucgacuucc uggaagcaaa gggauacaag gaagucaaga aggaccugau 3660 caucaagcug ccgaaguaca gccuguucga acuggaaaac ggagaaaga gaaugcuggc 3720 aagcgcagg aggaagg aggaacug ccgagghagg acguacuu 3780 ccuguaccug gcaagccacu acgaaagcu gaaggaagc ccgaaaac acgaaaagaa 3840 gcagcuguuc gucgacagc acaagcacua ccuggacga aucaucgaac agaucacgacga 3900 auucagcaag agagucaucc uggcagacgc aaaccuggac aagguccuga gcgcauacaa 3960 caagcacaga ghaagccga ucagagaca ggcagaaaac aucauccacc uguucacacu 4020 gaaaccug ggagcaccgg cagcaucauaa guacuucgac ahaaaacg ahaaaagg 4080 auacacaagc aaaaggaag uccuggacgc aaacacugauc shaccagagca uhahaggaa 4140 guacgaaaca agaacgacc ugagccagcu gggaggagac ggaggaggaa gcccgaagaa 4200 gagagaaag gucuagcuag acauaaaaaaaaaaaaaaaaaa 4260 gagaaagaaaaagauca auagcuuauu caucucuuuu ucuuuuucgu uggguaaag 4320 ccaacacccu 4380 ucauaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 4440 aaaaaaaaaaaaaaaaaaaaaaaaa AAAAAAAAAAAAAA 4500 Aaaaaaaaaaaaaaaaag 4516 <210> 4 <211> 100 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 4 acacaaauac caguccagcg guuuuagagc uagaauagc aaguaaaau aaggcuaguc 60 cguaucaac ugaaaagu ggcaccgagu cggugcuuuu 100 <210> 5 <211> 100 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 5 acgcaaauau caguccagcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> 6 <211> 4140 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 6 auggacaaga aguacuccau cggccuggac aucggcacca acuccguggg cugggccgug 60 aucaccgacg aguacaaggu gcccuccaag aaguucaagg ugcugggcaa caccgaccgg 120 cacuccauca agaagaaccu gaucggcgcc cugcuguucg acuccggcga gaccgccgag 180 gccacccggc ugaagcggac cgcccggcgg cgguacaccc ggcggaagaa ccggaucugc 240 uaccugcagg agaucuucuc caacgagaug gccaaggugg acgacuccuu cuccaccgg 300 cuggaggagu cuccuccuggu ggaggaggac aagaagcacg agcggcaccc cuccuucggc 360 aacaucgugg acgagguggc cuaccacgag aaguacccca ccaucuacca ccugcggaag 420 aagcuggugg cucuccaccga cuggccgac cugcggcuga cucuaccuggc cuggcuccac 480 augaucaagu uccggggcca cuccugauc gaggggcgacc ugaaccccga cucuccgac 540 guggacaagc uguucaucca gcuggugcag accuaacc agcuguucga ggagaacccc 600 aucaacgccu ccggcgugga cgccaaggcc auccuguccg cccggcuguc caagucccgg 660 cggcuggaga accugaucgc ccagcugccc ggcgagaga agaacggccu guucggcac 720 cugaucgccc ugucccuggg cugaccccc aacuucaagu cugacuucga ccuggccgag 780 gacgccaagc ugcagcuguc 840 cagaucggcg accaguacgc cgaccuguuc cggccgcca agaaccuguc cgacgccauc 900 cugcuguccg acauccugcg ggugaacacc gagaucacca aggcccccccu guccgcccucc 960 1020 cagcagcugc ccgagaagua caaggaac uucuucgacc aguccaagaa cggcuacgcc 1080 ggcuacaucg acggcggcgc cucccaggag gaguacuaca aguucaua gcccauccug 1140 1200 1260 gccauccugc ggcggcagga ggacuucuac cccuuccuga aggacaaccg ggagaauc 1320 gagagaucc ugaccuuccg gaucccccuac uacguggcc cccuggcccg gggcaacucc 1380 cgguucgccu ggaugacccg gaaguccgag gagaccauca cccccuggaa cuucgaggag 1440 gugguggaca agggcgccuc cgcccagucc uucaucgagc ggaugaccaa cuucgacaag 1500 aaccugccca acgagaaggu gcugcccaag cacucccugc uguacgagua cuucaccgug 1560 uacaacgagc ugaccaaggu gaaguacgug accgagggca ugcggaagcc cgccuuccug 1620 uccggcgagc agaagaaggc caucguggac cugcuguuca agaccaaccg gaaggugacc 1680 gugaagcagc ugaaggagga cuacuucaag aagaucgagu gcuucgacuc cguggagauc 1740 uccggcgugg aggaccgguu caacgccucc cugggcaccu accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgag gagaacgagg acauccugga ggacaucgug 1860 cugacccuga cccuguucga ggaccgggag augaucgagg agcggcugaa gaccuacgcc 1920 caccuguucg acgacaaggu gaugaagcag cugaagcggc ggcgguacac cggcuggggc 1980 cggcuguccc ggaagcugau caacggcauc cgggacaagc aguccggcaa gaccauccug 2040 gacuuccuga aguccgacgg cucgccaac cggaacuca uggcugcuga cucgacgac 2100 cuckoo cuckoo cuckaggagg cuckaggag cuckaggugu ccggcugggg cuckoo cuckoog 2160 cacgagcaca ucgccaaccu ggccggcucc cccgccauca agaagggcucc ccugcagacc 2220 gugaaggugg uggacgagcu ggugaaggug augggccggc acaagcccga gaacaucgug 2280 aucgagaugg cccgggagaa ccagaccacc cagaagggcc agagaacuc ccgggagcgg 2340 augaagcgga ucgaggagggg caucaggag cuggcuccc agauccugaa ggaggcucccc 2400 guggagaa cccagcugca gaacgagaag cuguaccugca gaacggccgg 2460 galuuguacg uggaccagga gcuggacauc aaccggcugu ccgacuacga cguggaccac 2520 aucgugcccc aguccuuccu gaggcgac ahaggugcu gaccgucc 2580 gagaacc ggggcaaguc cgacaacgug cccuccgagg agggugaa gagaagaag 2640 aacuacuggc ggcagcugcu gaacgccaag cugaucaccc agcggaaguu cgacaaccug 2700 accaaggccg agcggggcgg ccuguccgag cuggacaagg ccggcuucau caagcggcag 2760 cugguggaga cccggcagau caccaagcac guggcccaga uccuggacuc ccggaugaac 2820 accaaguacg acgagaacga caagcugauc cgggagguga aggugaucac ccugaagucc 2880 aagcuggugu ccgacuuccg gaaggacuuc caguucuaca aggugcggga gaucaacaac 2940 uaccaccacg cccacgacgc cuaccugaac gccguggugg gcaccgcccu gaucaagaag 3000 uaccccaagc uggaguccga guucguguac ggcgacuaca agguguacga cgugcggaag 3060 augaucgcca aguccgagca ggagaucggc aaggccaccg ccaaguacuu cuucuacucc 3120 aacaucauga acuucuucaa gaccgagauc acccuggcca acggcgagau ccggaagcgg 3180 ccccugaucg agaccaacgg cgagaccggc gagaucgugu gggacaaggg ccgggacuuc 3240 gccaccgugc ggaaggugcu guccaugccc caggugaaca ucgugaagaa gaccgaggug 3300 3360 gcccggagaa agcauggga ccccaagaag uacggcggcu ucgacucccc caccguggcc 3420 uacuccgugc uggugguggc caaguggag aagggcaagu ccaagaagcu gaaguccgug 3480 aaggagcugc ugggcaucac caucauggag cgguccuccu ucgagaagaa cccaucgacgac 3540 uuccuggagg ccaagggcua caaggaggug aagaaggacc ugaucaucaa cguccccaag 3600 uacucccugu ucgagcugga gaacggccgg aagcgggaugc uggccuccgc cggcgagcug 3660 cagaagggca acgagcuggc ccugcccucc aaguacguga acuuccugua ccuggccucc 3720 3780 3840 auccuggccg acgccaaccu ggacaaggug cuguccgccu acaacaagca ccgggacaag 3900 cccauccggg agcaggccga gaacaucauc caccuguuca cccugaccaa ccugggcgcc 3960 cccgccgccu ucaaguacuu cgacaccacc aucgaccgga agcgguacac cuccaccaag 4020 gaggugcugg acgccacccu gauccaccag uccaucaccg gccuguacga gacccggauc 4080 gaccuguccc agcugggcgg cgacggcggc ggcuccccca agaagaagcg gaagguguga 4140

Claims

1. A compound of formula II, 【Chemistry 1】 During the ceremony, X １ But, O, NR １ or a direct bond, X ２ But, C ２－５ is alkylene, X ３ is C(=O) or a direct bond, R １ is H or Me; R ３ But, C １－３ is alkyl, R ２ But, C １－３ is alkyl, or R ２ is bonded to the nitrogen atom, and X ２ together with 1 to 3 carbon atoms of X １ But, N.R. １ , R １ , and R ２ together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R ２ But, R ３ and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y １ But, C ２－１２ is alkylene, Y ２ but, 【Chemistry 2】 is selected from n is 0 to 3; R ４ But, C １－１５ is alkyl, Z １ But, C １－６ is an alkylene or a direct bond; Z ２ but, 【Chemistry 3】 (in either direction) or absent, except that Z １ is a direct bond, Z ２ is not present, R ５ But, C ５－９ Alkyl or C ６－１０ is an alkoxy; R ６ But, C ５－９ Alkyl or C ６－１０ is an alkoxy; W is methylene or a direct bond; R ７ is H or Me; However, R ３ and R ２ But, C ２ is alkyl, and X １ is O and X ２ But linear C ３ is alkylene, and X ３ is C(=O), and Y １ But linear C ６ alkylene, (Y ２ ) ｎ -R ４ but, 【Chemistry 4】 and R ４ But linear C ５ is alkyl; Z １ But, C ２ alkylene; Z ２ is absent, W is methylene, and R ７ is H, then R ５ and R ６ But, C ８ The compound or a salt thereof, which is not alkoxy.

2. The compound is of formula I, 【Chemistry 5】 During the ceremony, X １ But, O, NR １ or a direct bond, X ２ But, C ２－５ is alkylene, X ３ is C(=O) or a direct bond, R １ is H or Me; R ３ But, C １－３ is alkyl, R ２ But, C １－３ is alkyl, or R ２ is bonded to the nitrogen atom, and X ２ together with 1 to 3 carbon atoms of X １ But, N.R. １ , R １ , and R ２ together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R ２ But, R ３ and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y １ But, C ２－１２ is alkylene, Y ２ but, 【Chemistry 6】 is selected from R ４ But, C ３－１５ is alkyl, Z １ But, C １－６ is an alkylene or a direct bond; Z ２ but, 【Chemistry 7】 (in either direction) or absent, except that Z １ is a direct bond, Z ２ is not present, R ５ But, C ５－９ Alkyl or C ６－１０ is an alkoxy; R ６ But, C ５－９ Alkyl or C ６－１０ is an alkoxy; W is methylene or a direct bond; R ７ is H or Me; However, R ３ and R ２ But, C ２ is alkyl, and X １ is O and X ２ But linear C ３ is alkylene, and X ３ is C(=O), and Y １ But linear C ６ is alkylene, and Y ２ but, 【Chemistry 8】 and R ４ But linear C ４ is alkyl; Z １ But, C ２ alkylene; Z ２ is absent, W is methylene, and R ７ is H, then R ５ and R ６ But, C ８ 2. The compound of claim 1, or a salt thereof, which is not alkoxy.

3. X ２ But linear C ２ 3. The compound of claim 1 or claim 2 which is an alkylene.

4. X ２ But linear C ３ 3. The compound of claim 1 or claim 2 which is an alkylene.

5. X ２ But linear C ４ 3. The compound of claim 1 or claim 2 which is an alkylene.

6. R ３ But, C １ 2. A compound according to any one of the preceding claims, which is alkyl.

7. R ３ But, C ２ The compound according to any one of claims 1 to 5, which is alkyl.

8. R ２ But, C １ 2. A compound according to any one of the preceding claims, which is alkyl.

9. R ２ But, C ２ The compound according to any one of claims 1 to 7, which is alkyl.

10. R ２ The nitrogen atom and X ２ 8. The compound according to claim 1, wherein, together with one or two carbon atoms of,

11. R ２ The nitrogen atom and X ２ 8. The compound according to claim 1, wherein, together with 1 to 3 carbon atoms of, form a 6-membered ring.

12. R ２ and R ３ The compound according to claims 1 to 5, wherein, together with the nitrogen atom, forms a five-membered ring.

13. X １ A compound according to any one of the preceding claims, wherein is NH.

14. X １ The compound according to claims 1 to 12, wherein is a direct bond.

15. Y １ But linear C ３－１０ 2. A compound according to any one of the preceding claims which is alkylene.

16. Y １ But linear C ４－８ 2. A compound according to any one of the preceding claims which is alkylene.

17. Y １ But linear C ５－７ 2. A compound according to any one of the preceding claims which is alkylene.

18. R ４ But linear C ４－１４ 2. A compound according to any one of the preceding claims, which is alkyl.

19. R ４ But linear C ６－１２ 2. A compound according to any one of the preceding claims, which is alkyl.

20. Z １ But linear C ２－４ 2. A compound according to any one of the preceding claims which is alkylene.

21. R ５ and R ６ each independently represents a linear C ５－９ 2. A compound according to any one of the preceding claims, which is alkyl.

22. R ５ and R ６ each independently represents a linear C ６－８ 2. A compound according to any one of the preceding claims, which is alkyl.

23. R ５ and R ６ each independently represents a linear C ７－９ 2. A compound according to any one of the preceding claims which is alkoxy.

24. Y ２ but, 【Chemistry 9】 2. A compound according to any one of the preceding claims,

25. Y １ But linear C ７ is alkylene, and Y ２ but, 【Chemistry 10】 where n=1 and R ４ But linear C １０ 2. A compound according to any one of the preceding claims, which is alkyl.

26. Z ２ but, 【Chemistry 11】 2. A compound according to any one of the preceding claims,

27. Z １ , Z ２ , and R ５ A compound according to any one of the preceding claims, wherein is selected to form a linear chain of 6 to 18 atoms, including the carbon and oxygen atoms of the ester and acetal.

28. Y １ , Y ２ , and R ４ 4. A compound according to any one of the preceding claims, wherein, when present, is selected to form a linear chain of from 14 to 24 atoms including the carbon and oxygen atoms of the ester(s).

29. The compound is 【Chemistry 12】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 2. The compound of claim 1, selected from:

30. The compound according to any one of claims 1 to 29, wherein the salt is a pharma- ceutically acceptable salt.

31. 2. The compound according to any one of the preceding claims, wherein the pKa of the protonated form of the compound is from about 5.1 to about 8.

0.

32. 2. The compound according to any one of the preceding claims, wherein the pKa of the protonated form of the compound is from about 5.7 to about 7.

6.

33. 2. The compound according to any one of the preceding claims, wherein the pKa of the protonated form of the compound is from about 6 to about 7.

6.

34. 13. A composition comprising a compound according to any one of the preceding claims in a lipid component.

35. The composition according to claim 34, wherein the composition comprises a lipid component comprising about 50% of a compound according to any one of claims 1 to 31.

36. The composition of claim 34 or 35, wherein the composition is an LNP composition.

37. The composition according to any one of claims 34 to 36, wherein the lipid component comprises a helper lipid and a PEG lipid.

38. The composition according to any one of claims 34 to 37, wherein the lipid components comprise a helper lipid, a PEG lipid, and a neutral lipid.

39. The composition of any one of claims 34 to 38, further comprising a cryoprotectant.

40. The composition of any one of claims 34 to 39, further comprising a buffer.

41. The composition of any one of claims 34 to 40, wherein the composition comprises an aqueous component that includes a biologically active agent.

42. 42. The composition of claim 41, wherein the biologically active agent comprises a polypeptide.

43. The composition of claim 41 , wherein the biologically active agent comprises a nucleic acid.

44. The composition of claim 43 , wherein the nucleic acid component comprises RNA.

45. 45. The composition of claim 43 or 44, wherein the composition has an N / P ratio of about 3 to 10.

46. 46. ​​The composition of claim 45, wherein the N / P ratio is about 6±1.

47. 46. ​​The composition of claim 45, wherein the N / P ratio is about 6±0.

5.

48. 46. ​​The composition of claim 45, wherein the N / P ratio is about 6.

49. 49. The composition of any one of claims 34 to 48, comprising an RNA component, said RNA component comprising mRNA.

50. 50. The composition of claim 49, wherein the RNA component comprises a sequence encoding an RNA-guided DNA-binding agent, such as a Cas nuclease mRNA.

51. 51. The composition of claim 49 or 50, wherein the RNA component comprises a class 2 Cas nuclease mRNA.

52. 52. The composition of any one of claims 49-51, wherein the RNA component comprises a Cas9 nuclease mRNA.

53. 53. The composition of any one of claims 49 to 52, wherein the RNA component comprises modified RNA.

54. 54. The composition of any one of claims 49 to 53, wherein the RNA component comprises a gRNA nucleic acid.

55. 55. The composition of claim 54, wherein the gRNA nucleic acid is a gRNA.

56. 56. The composition of any one of claims 49 to 55, wherein the RNA component comprises a class 2 Cas nuclease mRNA and a gRNA.

57. 57. The composition of any one of claims 54-56, wherein the gRNA nucleic acid is or encodes a dual guide RNA (dgRNA).

58. 57. The composition of any one of claims 54-56, wherein the gRNA nucleic acid is or encodes a single guide RNA (sgRNA).

59. The composition of any one of claims 54 to 58, wherein the gRNA is a modified gRNA.

60. 60. The composition of Claim 59, wherein the modified gRNA comprises a modification in one or more of the first five nucleotides at the 5' end.

61. 61. The composition of Claims 59 or 60, wherein the modified gRNA comprises a modification in one or more of the last five nucleotides at the 3' end.

62. The composition of any one of claims 41 to 61, further comprising at least one template nucleic acid.

63. 63. A method of delivering a biologically active agent to a cell, comprising contacting said cell with a composition according to any one of claims 34 to 62.

64. 64. The method of claim 63, wherein the cell is a liver cell, such as a hepatocyte.

65. 63. A method of gene editing comprising contacting a cell with a composition according to any one of claims 34 to 62.

66. 63. A method for cleaving DNA, comprising contacting a cell with a composition according to any one of claims 34 to 62.

67. 67. The method of claim 66, wherein the contacting step generates a single-stranded DNA nick.

68. 67. The method of claim 66, wherein the contacting step produces a double-stranded DNA break.

69. The method of any one of claims 63 to 67, wherein the composition comprises a Class 2 Cas mRNA and a gRNA nucleic acid.

70. 70. The method of any one of claims 63 to 69, further comprising introducing at least one template nucleic acid into the cell.

71. 71. The method of claim 70, comprising contacting the cell with a composition comprising a template nucleic acid.

72. 72. The method of any one of claims 63 to 71, comprising administering the composition to an animal.

73. 72. The method of any one of claims 63 to 71, comprising administering the composition to a human.

74. 72. The method of any one of claims 63 to 71, comprising administering the composition to a cell.

75. 75. The method of claim 74, wherein the cell is a eukaryotic cell.

76. 76. The method of any one of claims 63-75, comprising administering an mRNA formulated in a first LNP composition and a second LNP composition comprising one or more of an mRNA, a gRNA, a gRNA nucleic acid, and a template nucleic acid.

77. 77. The method of claim 76, wherein the first and second LNP compositions are administered simultaneously.

78. 77. The method of claim 76, wherein the first and second LNP compositions are administered sequentially.

79. 77. The method of claim 76, comprising administering the mRNA and the gRNA nucleic acid formulated in a single LNP composition.

80. 80. The method of any one of claims 63-79, wherein the gene editing results in a gene knockout.

81. 80. The method of any one of claims 63-79, wherein the gene editing results in gene correction.

82. 82. The method of any one of claims 63 to 81, comprising administering the composition to a liver cell.

83. 83. The method of any one of claims 63 to 82, comprising administering the composition to a hepatocyte.

84. 84. The method of any one of claims 63 to 83, comprising administering the composition to the liver.

Images