ionizable amine lipids

Abstract

The present disclosure provides ionizable lipids and lipid nanoparticle (LNP) compositions comprising ionizable lipids, helper lipids, neutral lipids, and PEG lipids, which can be used to deliver biologically active agents, for example, to deliver biologically active agents to cells to make engineered cells. The LNP compositions disclosed herein can be used in methods of gene editing and methods of delivering biologically active agents and methods of modifying or cleaving DNA.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Nos. 63 / 467,691, filed May 19, 2023; 63 / 610,628, filed December 15, 2023; and 63 / 648,469, filed May 16, 2024, the entire contents of each of which are incorporated herein by reference. Background of the Invention

[0003] Lipid nanoparticles formulated with ionizable lipids can serve as cargo carriers for delivering bioactive agents, particularly polynucleotides, such as those used in RNA interference, RNAi therapy, mRNA therapy, RNA drugs, antisense therapy, gene therapy, and nucleic acid vaccines (e.g., RNA vaccines). Lipid nanoparticles can include one or more small nucleic acid molecules, RNAi agents, short interfering nucleic acids (siNA), messenger ribonucleic acid (messenger RNA, mRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA (shRNA) molecules, peptide nucleic acids (PNA), locked nucleic acid ribonucleotides (LNA), morpholinonucleotides, threonine nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internal fragment interfering RNA), aiRNA (asymmetric interfering RNA), and siRNAs with one, two, or more mismatches between the sense and antisense strands targeting relevant cells and / or tissues, such as in cell cultures, subjects, or organisms. The delivery of mRNA and / or guide RNA is of particular interest. LNP compositions containing ionizable lipids can facilitate the delivery of oligonucleotide agents across cell membranes and can be used to introduce components and compositions for gene editing into living cells. Bioactive agents that are particularly difficult to deliver to cells include proteins, nucleic acid-based drugs and their derivatives, especially drugs comprising relatively large oligonucleotides (such as mRNA). Compositions for delivering promising gene editing technologies into cells (such as delivering components of the CRISPR / Cas9 system) are of particular interest (e.g., mRNA encoding nucleases and associated guide RNA (gRNA)).

[0004] There is a need for compositions to improve the in vivo and in vitro delivery of nucleic acids (such as RNA). As an example, there is a need for compositions for delivering CRISPR / Cas components into eukaryotic cells (such as human cells). In particular, there is particular interest in compositions for delivering mRNA encoding CRISPR protein components and for delivering CRISPR gRNA. Also of particular interest are compositions having useful properties for in vitro and in vivo delivery, said compositions being able to stabilize and deliver RNA components. Summary of the Invention

[0005] In some embodiments, this disclosure relates to a compound represented by structural formula I.

[0006] (I),

[0007] or its salt, wherein:

[0008] A is either O or NH.

[0009] X 1 C 1-5 Alkylene

[0010] R 1 and R 2 Each independently is C 1-3 alkyl, or

[0011] R 1 and R 2 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring, and

[0012] Z 1 C 1-5 Alkylene

[0013] Y 1 and Y 2 Each independently is C 3-10 Alkoxy or O(C) 3-10 (alkynyl group),

[0014] Z 2 C 1-5 Alkylene or direct bond, and

[0015] Y 3 and Y 4 Each independently is C 3-10 Alkoxy or O(C) 3-10 acetylenic group), or

[0016] Y 3 and Y 4 Each independently is C 3-10 Alkyl or C 3-10 alkynyl group,

[0017] The premise is that if Y 1 Y 2 Y 3 and Y 4 Each independently is C 3-10 alkoxy group, then R 1 and R 2 Not a C2 alkyl group, and R 1 and R 2 It does not form a 6-membered ring with the nitrogen atom it is attached to.

[0018] In some embodiments, this disclosure relates to a compound represented by structural formula II.

[0019] (II),

[0020] or its salt,

[0021] in,

[0022] Q is CR 3 or ,

[0023] A can be O, NH, or a direct bond.

[0024] X 1 C 1-5 Alkylene

[0025] R 1 and R 2 Each independently is C 1-3 alkyl, or

[0026] R 1 The nitrogen atom and X it is attached to 1 One to three carbon atoms together form a 4-, 5-, or 6-membered ring, or

[0027] R 1 and R 2 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring, and

[0028] R 3 For H or C 1-3 alkyl,

[0029] Z 1 and Z 2 Each independently is C 1-5 Alkylene

[0030] Z 3 and Z 4 Each is independently -C(=O)O- in any direction.

[0031] Z 5 and Z 6 Each is independently a direct key or C. 1-3 Alkylene

[0032] Y 1 Selected from H, C 1-10 Alkyl, C 3-10 alkenyl and C 3-10 alkynyl group,

[0033] Y 2 Y3 and Y 4 Each is independently selected from C 3-10 Alkyl, C 3-10 alkenyl and C 3-10 alkynyl group, and

[0034] n is 0 or 1.

[0035] In some embodiments, this disclosure relates to a compound represented by structural formula III.

[0036] (III),

[0037] or its salt,

[0038] in:

[0039] Q is CH or ,

[0040] A is either O or NH.

[0041] X 1 C 1-5 Alkylene

[0042] R 1 and R 2 Each independently is C 1-3 alkyl, or

[0043] R 1 The nitrogen atom and X it is attached to 1 One to three carbon atoms together form a 4-, 5-, or 6-membered ring, or

[0044] R 1 and R 2 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring, and

[0045] Z 1 C 2-9 Alkylene

[0046] Z 2 C 1-3 Alkylene or direct bond, and

[0047] Y 1 and Y 2 Each is independently selected from C 3-10 Alkyl, C 3-10 alkenyl and C 3-10 Alkyne group.

[0048] In some embodiments, this disclosure relates to a compound represented by structural formula IV.

[0049] (IV),

[0050] or its salt,

[0051] in:

[0052] Q is CH or ,

[0053] A can be O, NH, or a direct bond.

[0054] X 1 and X 2 Each independently is C 1-5 Alkylene

[0055] R 1 Selected from C 3-9 Alkyl, C 3-9 alkenyl and C 3-9 alkynyl group,

[0056] R 2 and R 3 Each independently is C 1-3 alkyl, or

[0057] R 2 and R 3 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring, and

[0058] Z 1 C 6-10 alkylene, and

[0059] Y 1 and Y 2 Each is independently selected from C 3-10 Alkyl, C 3-10 alkenyl and C 3-10 Alkyne group.

[0060] In some embodiments, this disclosure relates to a compound represented by one of the following structural formulas:

[0061] ,

[0062] ,

[0063] ,

[0064] ,

[0065] ,

[0066] ,

[0067] ,

[0068] ,

[0069] ,

[0070] ,

[0071] ,

[0072] ,

[0073] ,

[0074] ,

[0075] ,

[0076] ,

[0077] and

[0078] ,

[0079] Or its salt.

[0080] In some embodiments, this disclosure relates to a compound represented by one of the following structural formulas:

[0081] ,

[0082] ,

[0083] ,

[0084] ,

[0085] ,

[0086] ,

[0087] ,

[0088] ,

[0089] ,

[0090] ,

[0091] ,

[0092] ,

[0093] and

[0094] ,

[0095] Or its salt.

[0096] In some embodiments, the present invention relates to a composition comprising compounds of formulas (I)-(IV) or Table 1 and a lipid component.

[0097] In some embodiments, this disclosure relates to a method of lysing DNA, which includes contacting cells with a composition as described herein.

[0098] In some embodiments, this disclosure relates to a method of gene editing, which includes contacting cells with a composition as described herein. Attached Figure Description

[0099] Figure 1A This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 53, compound 54, compound 58, or compound 59 (Experiment 1).

[0100] Figure 1B This is a graph showing serum TTR (μg / mL) after delivery with an LNP containing compound 53, compound 54, compound 58 or compound 59 (Experiment 1).

[0101] Figure 2A This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compounds 53, 56, 57, 2, 3, 4, 5, 58, or 59 (Experiment 2).

[0102] Figure 2B The figure shows the serum TTR (μg / mL) after delivery with LNP containing compound 53, compound 56, compound 57, compound 2, compound 3, compound 4, compound 5, compound 58 or compound 59 (Experiment 2).

[0103] Figure 3 This is a graph showing the percentage of TTR edited in rat livers after delivery with an LNP containing compound 53, compound 1, compound 7, compound 4, compound 9, compound 57, compound 8, or compound 2 (Experiment 3).

[0104] Figure 4This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 53, compound 9, compound 11, compound 10, compound 14, compound 13, or compound 12 (Experiment 4).

[0105] Figure 5A This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 53, compound 24, compound 25, compound 26, or compound 27 (Experiment 5).

[0106] Figure 5B This is a graph showing serum TTR (μg / mL) after delivery with an LNP containing compound 53, compound 24, compound 25, compound 26 or compound 27 (Experiment 5).

[0107] Figure 6 This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 53 or compound 23 (Experiment 6).

[0108] Figure 7 This is a graph (Experiment 7) showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 53, compound 41, compound 43, compound 45, compound 47, compound 42, compound 44, compound 46, or compound 48.

[0109] Figure 8 This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 53 or compound 22 (Experiment 8).

[0110] Figure 9 This is a graph (Experiment 9) showing the percentage of TTR edited in rat livers after delivery with LNP containing compounds 53, 9, 15, 16, 17, 18, 19, or 20.

[0111] Figure 10 This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compounds 53, 56, 58, 49, or 50 (Experiment 10).

[0112] Figure 11 This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 53, compound 55, compound 23, or compound 51 (Experiment 11).

[0113] Figure 12This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 6, compound 28, compound 29, compound 30, compound 31, compound 32, compound 33, compound 36 or compound 37 (Experiment 12).

[0114] Figure 13 This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 53, compound 6, compound 60, compound 52, or compound 61 (Experiment 13).

[0115] Figure 14 This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 6, compound 2, compound 35, compound 39, compound 40, compound 38, or compound 34 (Experiment 14).

[0116] Figure 15 This is a graph showing the percentage of TTR edited in rat livers after LNP delivery containing one of compound 53, compound 6, or compound 60 and one of PEG2K-DMG, C13 ether, or C14 ether.

[0117] Figure 16 This is a graph showing the percentage of TTR edited in rat livers after delivery with LNP containing compound 6, compound 52, compound 62, or compound 63.

[0118] Figure 17 This is a graph showing the editing efficiency in rat livers after delivery of LNPs containing compounds 53, 6, 68, 66, 33, 69, 64, 65, 67, or 52, measured by the percentage of editing. Detailed Implementation

[0119] This disclosure provides lipids, particularly ionizable lipids, and lipid compositions that can be used to deliver bioactive agents, including nucleic acids, such as CRISPR / Cas component RNA (mRNA and / or gRNA) (“cargo”), to cells, as well as methods for preparing and using such lipids and compositions. The lipid compositions include ionizable lipids, neutral lipids, PEG lipids, and cofactor lipids. In some embodiments, the ionizable lipid is a compound of formulas (I)–(IV) as defined herein or a compound selected from the compounds in Table 1, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0120] Table 1.

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] In some embodiments, the lipid composition may comprise a bioactive agent, such as an RNA component. In some embodiments, the RNA component comprises mRNA. In some embodiments, the mRNA is mRNA encoding a class 2 Cas nuclease. In some embodiments, the RNA component comprises gRNA and optionally mRNA encoding a class 2 Cas nuclease. In some embodiments, the lipid composition is a lipid nanoparticle (LNP) composition. "Lipid nanoparticle" or "LNP" refers to a particle containing multiple (i.e., more than one) lipid components physically associated with each other by intermolecular forces, without limitation of the meaning.

[0131] Methods for gene editing using these lipid compositions and methods for preparing engineered cells are also provided. In some embodiments, the LNP compositions can be used to deliver bioactive agents to cells, tissues, or animals. In some embodiments, the cells are eukaryotic cells, particularly human cells. In some embodiments, the cells are hepatocytes. In some embodiments, the cells are cell types that can be used in therapies such as adoptive cell therapy (ACT), such as autologous and allogeneic cell therapies. In some embodiments, the cells are stem cells, such as hematopoietic stem cells, induced pluripotent stem cells, or other multipotent or pluripotent cells. In some embodiments, the cells are stem cells, such as mesenchymal stem cells that can develop into bone, cartilage, muscle, or fat cells. In some embodiments, the stem cells include ocular stem cells. In some embodiments, the cells are selected from mesenchymal stem cells, hematopoietic stem cells (HSCs), monocytes, endothelial progenitor cells (EPCs), neural stem cells (NSCs), limbal stem cells (LSCs), tissue-specific primary cells or cells derived therefrom (TSCs), induced pluripotent stem cells (iPSCs), ocular stem cells, pluripotent stem cells (PSCs), embryonic stem cells (ESCs), and cells for organ or tissue transplantation.

[0132] In some embodiments, the cells are immune cells, such as leukocytes or lymphocytes. In a preferred embodiment, the immune cells are lymphocytes. In some embodiments, the lymphocytes are T cells, B cells, or NK cells. In a preferred embodiment, the lymphocytes are T cells. In some embodiments, the lymphocytes are activated T cells. In some embodiments, the lymphocytes are inactivated T cells.

[0133] Ionizable lipids

[0134] This disclosure provides ionizable lipids that can be used in LNP compositions.

[0135] The compounds of formulas (I)-(IV) or Table 1 of this disclosure can form salts depending on the pH of the medium in which they are situated. For example, in a slightly acidic medium, the compounds of formulas (I)-(IV) or Table 1 can be protonated and thus become positively charged. Conversely, in a slightly alkaline medium (such as blood with a pH of approximately 7.35), the compounds of formulas (I)-(IV) or Table 1 can remain unprotonated and thus be uncharged. In some embodiments, the compounds of formulas (I)-(IV) or Table 1 of this disclosure can be predominantly protonated at a pH of at least about 9. In some embodiments, the compounds of formulas (I)-(IV) or Table 1 of this disclosure can be predominantly protonated at a pH of at least about 10.

[0136] The pH at which the compounds of formulas (I)-(IV) or Table 1 are primarily protonated is related to their inherent pKa. In some embodiments, the pKa of salts of the compounds of formulas (I)-(IV) or Table 1 of this disclosure is in the range of about 5.1 to about 8.0, or even more preferably in the range of about 5.5 to about 7.6. In some embodiments, the pKa of salts of the compounds of formulas (I)-(IV) or Table 1 of this disclosure is in the range of about 5.7 to about 8, about 5.7 to about 7.6, about 6 to about 8, about 6 to about 7.5, about 6 to about 7, about 6 to about 6.9, about 6 to about 6.5, about 6.1 to about 6.9, or about 6 to about 6.85. In some embodiments, the pKa of salts of the compounds of formulas (I)-(IV) or Table 1 of this disclosure is about 6.0, about 6.1, about 6.1, about 6.2, about 6.3, about 6.4, about 6.6, about 6.7, about 6.8, or about 6.9. Alternatively, the pKa of the salts of formulas (I)-(IV) or the compounds in Table 1 of this disclosure is in the range of about 6 to about 8. The pKa of the salts of formulas (I)-(IV) or the compounds in Table 1 may be an important consideration when formulating LNPs, as LNPs formulated with certain lipids in the range of about 5.5 to about 7.0 have been found to be efficient in vivo for delivery (e.g., to the liver). Furthermore, LNPs formulated with certain lipids in the range of about 5.3 to about 6.4 have been found to be efficient in vivo for delivery (e.g., to tumors). See, for example, WO 2014 / 136086. In some embodiments, the ionizable lipids are positively charged at an acidic pH but neutral in the blood.

[0137] Other lipids

[0138] The term "neutral lipid" applicable to the lipid compositions of this disclosure includes, for example, a variety of neutral, uncharged, or zwitterionic lipids. Examples of neutral phospholipids applicable to this disclosure include (but are not limited to) dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphatidylcholine (PLPC), 1,2-disarachidoyl-sn-glycerol-3-phosphatidylcholine (DAPC), phosphatidylethanolamine (PE), lecithinylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), and 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC). PC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-dibenzyl-sn-glycerol-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-bis(eicosanyl)-sn-glycerol-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearateoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In some embodiments, the neutral phospholipid may be selected from distearylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE), preferably distearylphosphatidylcholine (DSPC).

[0139] "Assisted lipids" include steroids, sterols, and alkylresorcinols. Assisted lipids suitable for use in this disclosure include (but are not limited to) cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In some embodiments, the assisted lipid may be cholesterol or a derivative thereof, such as cholesterol hemisuccinate.

[0140] In some embodiments, the LNP composition includes polymeric lipids, such as PEG lipids, which can affect the length of time that nanoparticles can exist in vivo or ex vivo (e.g., in blood or media). PEG lipids can facilitate the formulation process by, for example, reducing particle aggregation and controlling particle size. The PEG lipids used herein can modulate the pharmacokinetic properties of LNPs. Typically, PEG lipids comprise a lipid moiety and a PEG-based (sometimes referred to as poly(ethylene oxide)) polymer moiety (PEG moiety). Information on PEG lipids suitable for use in lipid compositions containing formulas (I)-(IV) or compounds in Table 1 of this disclosure, as well as on 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. Other suitable PEG lipids are disclosed, for example, in WO 2015 / 095340 (page 31, lines 14 to 37, line 6), WO2006 / 007712 and WO 2011 / 076807 (“Hidden Lipids”), each of which is incorporated herein by reference in its entirety.

[0141] In some embodiments, the lipid moiety may be derived from diacylglycerol or diacylglycerol amide, including those containing a dialkylglycerol or dialkylglycerol amide group having an alkyl chain length independently comprising about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may contain one or more functional groups, such as amides or esters. In some embodiments, the alkyl chain length comprises about C10 to C20. The dialkylglycerol or dialkylglycerol amide group may further comprise one or more substituted alkyl groups. The chain length may be symmetrical or asymmetrical.

[0142] Unless otherwise indicated, the term "PEG" as used herein means any polyethylene glycol or other polyalkylene ether polymer, such as ethylene glycol or ethylene oxide, optionally substituted linear or branched polymer. In some embodiments, the PEG moiety is not substituted. Alternatively, the PEG moiety may be substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxyl, or aryl groups. For example, the PEG moiety may comprise a PEG copolymer, such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); or the PEG moiety may be a PEG homopolymer. In some embodiments, the molecular weight of the PEG moiety is from about 130 to about 50,000, such as from about 150 to about 30,000 or even from about 150 to about 20,000. Similarly, the molecular weight of the PEG moiety can be about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, or even about 150 to about 5,000. In some preferred embodiments, the molecular weight of the PEG moiety is 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.

[0143] In some preferred embodiments, the PEG portion is “PEG-2K,” also known as “PEG 2000” or “PEG2K,” with an average molecular weight of about 2,000 Daltons. PEG-2K is expressed herein by formula (III). (III) indicates that n is about 45, meaning that the number-average degree of polymerization comprises about 45 subunits. However, other PEG embodiments known in the art can be used, including (e.g.) those with a number-average degree of polymerization comprising about 23 subunits (n=23) and / or 68 subunits (n=68). In some embodiments, n can be in the range of about 30 to about 60. In some embodiments, n can be in the range of about 35 to about 55. In some embodiments, n can be in the range of about 40 to about 50. In some embodiments, n can be in the range of about 42 to about 48. In some embodiments, n can be 45. In some embodiments, R can be selected from H, substituted alkyl groups, and unsubstituted alkyl groups. In some embodiments, R can be an unsubstituted alkyl group, such as methyl.

[0144] In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (e.g., 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (PEG2K-DMG) (e.g., catalog number GM-020, from NOF, Tokyo, Japan)), PEG-dipalmitoylglycerol, PEG-distearylglycerol (PEG-DSPE) (e.g., catalog number DSPE-020CN, NOF, Tokyo, Japan)), PEG-dilaurylglyceramide, PEG-dimyristoylglyceramide, PEG-dipalmitoylglyceramide and PEG-distearylglyceramide, PEG-cholesterol (1-[8'-(cholesterol-5-en-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DMPE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSPE) (e.g., catalog number 880120C, from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearyl-sn-glycerol, methoxy-polyethylene glycol (PEG2K-DSG; GS-020, NOF) The PEG lipids are: PEG2K-DMA (poly(ethylene glycol)-2000-dimethacrylate), 1,2-distearyloxypropyl-3-amine-N-[methoxy(poly(ethylene glycol)-2000)] (PEG2K-DSA), methoxy-PEG2000-carbamoyl-1,2-tetrateoxypropylamine (C13 ether), and methoxy-PEG2000-carbamoyl-1,2-tetradecyloxypropylamine (C14 ether). In some embodiments, the PEG lipid may be PEG2K-DMG. In some embodiments, the PEG lipid may be PEG2K-DSG. In other embodiments, the PEG lipid may be PEG2K-DSPE. In some embodiments, the PEG lipid may be PEG2K-DMA. In other embodiments, the PEG lipid may be PEG2K-C-DMA. In some embodiments, the PEG lipid may be compound S027 disclosed in WO2016 / 010840. (Paragraphs

[00240] to

[00244] ), which are incorporated herein by reference in their entirety. 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.

[0145] In a preferred embodiment, the PEG lipid comprises a glycerol group. In a preferred embodiment, the PEG lipid comprises a dimyristoylglycerol (DMG) group. In a preferred embodiment, the PEG lipid comprises PEG2K. In a preferred embodiment, the PEG lipid is PEG-DMG. In a preferred embodiment, the PEG lipid is PEG2K-DMG. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-racemic-glycerol-3-methoxypolyethylene glycol-2000. In a preferred embodiment, PEG2K-DMG is 1,2-dimyristoyl-racemic-glycerol-3-methoxypolyethylene glycol-2000.

[0146] lipid compositions

[0147] This document describes lipid compositions comprising at least one compound of formula (I)-(IV) or Table 1 or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), at least one cofactor lipid, at least one neutral lipid, and at least one polymeric lipid. In some embodiments, the lipid composition comprises at least one compound of formula (I)-(IV) or Table 1 or a salt thereof, at least one neutral lipid, at least one cofactor lipid, and at least one PEG lipid. In some embodiments, the neutral lipid is DSPC or DMPE. In some embodiments, the cofactor lipid is cholesterol, 5-heptadecanocyanate, or cholesterol hemisuccinate.

[0148] In some embodiments, the neutral lipid is DSPC. In some embodiments, the auxiliary lipid is cholesterol. In some embodiments, the PEG lipid is PEG2K-DMG, C13 ether, or C14 ether. In some embodiments, the PEG lipid is 1,2-dimyristoyl-racemic-glycerol-3-methoxypolyethylene glycol-2000, methoxy-PEG2000-carbamoyl-1,2-tetrateoxypropylamine, or methoxy-PEG2000-carbamoyl-1,2-tetradecyloxypropylamine.

[0149] In some embodiments, the lipid composition further comprises one or more other lipid components.

[0150] In some embodiments, the lipid composition is in the form of liposomes. In a preferred embodiment, the lipid composition is in the form of lipid nanoparticles (LNPs). In some embodiments, the lipid composition is suitable for in vivo delivery. In some embodiments, the lipid composition is suitable for delivery to organs, such as the liver. In some embodiments, the lipid composition is suitable for ex vivo delivery to tissues. In some embodiments, the lipid composition is suitable for in vitro delivery to cells.

[0151] Lipid compositions comprising lipids of formulas (I)-(IV) or pharmaceutically acceptable salts thereof, as described in Table 1, may be in various forms, including (but not limited to) particle-forming delivery agents, such as microparticles, nanoparticles, and transfection agents, for the delivery of various molecules to cells. Specific compositions are effective in transfecting or delivering bioactive agents. Preferred bioactive agents are nucleic acids, such as RNA. In other embodiments, the bioactive agent is selected from mRNA and gRNA. The gRNA may be dgRNA or sgRNA. In some embodiments, the cargo comprises mRNA, gRNA, or nucleic acid encoding gRNA, or a combination of mRNA and gRNA, encoding an RNA-guided DNA binder (e.g., Cas nuclease, class 2 Cas nuclease, or Cas9).

[0152] Compositions typically, but not necessarily, include one or more pharmaceutically acceptable excipients. The term "excipient" includes any component other than the compounds of this disclosure, other lipid components, and bioactive agents. Excipients may impart functional characteristics (e.g., drug release rate control) and / or non-functional characteristics (e.g., processing aids or diluents) to the composition. The selection of excipients will largely depend on factors such as the specific administration modality, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0153] Parenteral preparations are typically aqueous or oily solutions or suspensions. In the case of aqueous preparations, excipients such as sugars (including (but not limited to) glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffers (preferably up to pH 3 to 9) may be used, but for some applications, they may be more suitable to be formulated as sterile non-aqueous solutions or in a dry form for use with a suitable medium (such as sterile, pyrogen-free water (WFI)).

[0154] LNP Composition

[0155] Lipid compositions can be provided as LNP compositions, and the LNP compositions described herein can be provided as lipid compositions. Lipid nanoparticles can be, for example, microspheres (comprising monolayers and multilayers of vesicles, such as “liposomes”—layered lipid bilayers that are generally spherical in some embodiments and may contain an aqueous core (e.g., containing a large portion of RNA molecules)), dispersed phases in emulsions, micelles, or internal phases in suspensions.

[0156] This document describes LNP compositions comprising at least one compound of formula (I)-(IV) or Table 1 or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), at least one auxiliary lipid, at least one neutral lipid, and at least one polymeric lipid. In some embodiments, the LNP composition comprises at least one compound of formula (I)-(IV) or Table 1 or a pharmaceutically acceptable salt thereof, at least one neutral lipid, at least one auxiliary lipid, and at least one PEG lipid. In some embodiments, the neutral lipid is DSPC or DPME. In some embodiments, the auxiliary lipid is cholesterol, 5-heptadecanocyanate, or cholesterol hemisuccinate.

[0157] Embodiments of this disclosure provide lipid compositions described based on individual molar ratios of component lipids in the composition. All mol% numbers are given as a fraction of the lipid component in the lipid composition, or more specifically, the LNP composition. In some embodiments, the lipid mol% relative to the lipid component will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or actual mol% of the lipid. In some embodiments, the lipid mol% relative to the lipid component will be ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, ±0.25 mol%, or ±0.05 mol% of the specified, nominal, or actual mol% of the lipid. In some embodiments, the lipid mol% will vary from the specified, nominal, or actual mol% of the lipid by less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.5%. In some embodiments, the mol% numbers are based on nominal concentrations. As used herein, "nominal concentration" refers to a concentration based on the input amount of the substance used to form the resulting composition. For example, if 100 mg of solute is added to 1 L of water, the nominal concentration is 100 mg / L. In some embodiments, the mol% number is based on the actual concentration, such as the concentration determined by analytical methods. In some embodiments, the actual lipid concentration of the lipid component can be determined, for example, by chromatography (such as liquid chromatography), followed by detection methods (such as charged sol detection). In some embodiments, the actual lipid concentration of the lipid component can be characterized by lipid analysis, AF4-MALS, NTA, and / or cryogenic EM. All mol% numbers are given as a percentage of lipids in the lipid component.

[0158] Embodiments of this disclosure provide LNP compositions described according to individual molar ratios of lipids in the lipid fraction. In some embodiments, the amount of ionizable lipids is from about 25 mol% to about 75 mol%; the amount of neutral lipids is from about 5 mol% to about 20 mol%; the amount of accessory lipids is from about 25 mol% to about 50 mol%; and the amount of PEG lipids is from about 1.5 mol% to about 4 mol%. In some embodiments, the amount of ionizable lipids is from about 30-60 mol% of the lipid fraction; the amount of neutral lipids is from about 7.5-15 mol% of the lipid fraction; the amount of accessory lipids is from about 30-45 mol% of the lipid fraction; and the amount of PEG lipids is from about 2.3-3.5 mol% of the lipid fraction. In some embodiments, the amount of ionizable lipids is about 45-55 mol% of the lipid component; the amount of neutral lipids is about 8-12 mol% of the lipid component; the amount of accessory lipids is about 35-42 mol% of the lipid component; and the amount of PEG lipids is about 2.5-3.5 mol% of the lipid component. In some embodiments, the amount of ionizable lipids is about 47-52 mol% of the lipid component; the amount of neutral lipids is about 8-10 mol% of the lipid component; the amount of accessory lipids is about 37-40 mol% of the lipid component; and the amount of PEG lipids is about 2.7-3.3 mol% of the lipid component. In some embodiments, the amount of ionizable lipids is about 50 mol% of the lipid component; the amount of neutral lipids is about 9 mol% of the lipid component; the amount of accessory lipids is about 38 mol% of the lipid component; and the amount of PEG lipids is about 3 mol% of the lipid component.

[0159] In some embodiments, the amount of ionizable lipids is about 25-75 mol%, about 25-70 mol%, about 25-65 mol%, about 25-60 mol%, about 25-55 mol%, about 25-50 mol%, about 30-75 mol%, about 30-70 mol%, about 30-65 mol%, about 30-60 mol%, about 30-55 mol%, about 30-50 mol%, about 35-75 mol%, about 35-60 mol%, about 35-65 mol%, about 35-60 mol%, about 35-65 mol%, about 35-55 mol%, about 40-75 mol%, about 40-65 mol%, about 40-60 mol%, about 40-70 mol%, about 40-55 mol%, about 45-55 mol%, or about 45-60 mol%. In some embodiments, the mol% of ionizable lipids may be about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, or about 60 mol%. In some embodiments, the mol% of ionizable lipids relative to the lipid component will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or actual mol%. In some embodiments, the mol% of ionizable lipids relative to the lipid component will be ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, or ±0.25 mol% of the specified, nominal, or actual mol%. In some embodiments, the batch-to-batch variability of the LNP of the ionizable lipid mol% will be less than 15%, less than 10%, or less than 5%. In some embodiments, the mol% number is based on the nominal concentration. In some embodiments, the mol% number is based on the actual concentration.

[0160] In some embodiments, the amount of neutral lipids is about 5-20 mol%, about 5-15 mol%, about 5-10 mol%, about 7-10 mol%, or about 9 mol%. In other embodiments, the amount of neutral lipids may be about 5-30 mol%, about 5-28 mol%, about 5-25 mol%, about 5-23 mol%, about 5-20 mol%, about 5-18 mol%, about 5-23 mol%, about 5-20 mol%, about 5-18 mol%, about 5-15 mol%, about 5-13 mol%, or about 5-10 mol%. In some embodiments, the mol% of neutral lipids may be about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, or about 20 mol%. In some embodiments, the mol% of neutral lipids relative to the lipid component will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or actual mol% of neutral lipids. In some embodiments, the mol% of neutral lipids relative to the lipid component will be ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, or ±0.25 mol% of the specified, nominal, or actual mol% of neutral lipids. In some implementations, the batch-to-batch variability of LNP will be less than 15%, less than 10%, or less than 5%. In some implementations, the mol% number is based on the nominal concentration. In some implementations, the mol% number is based on the actual concentration.

[0161] In some embodiments, the amount of auxiliary lipids is about 30-50 mol%, about 30-65 mol%, about 30-55 mol%, about 33-50 mol%, about 32-55 mol%, about 32-65 mol%, about 35-50 mol%, about 35-55 mol%, about 35-40 mol%, about 35-45 mol%, or about 38 mol%. In other embodiments, the amount of auxiliary lipids may be about 25-65 mol%, about 28-65 mol%, about 30-65 mol%, about 32-65 mol%, about 35-65 mol%, about 25-62 mol%, about 28-62 mol%, about 30-62 mol%, about 32-62 mol%, about 35-62 mol%, about 38-62 mol%, about 40-62 mol%, about 42-62 mol%, about 45-62 mol%, about 48-62 mol%, about 50-62 mol%, about 52-62 mol%, about 55-62 mol%, about 58-62 mol%, about 60-62 mol%, about 25-60 mol%, about 28-60 mol%, about 30-60 mol%, about 32-60 mol%, about 35-60 mol%, about 38-60 mol%, about 40-60 mol%. mol%, approximately 42-60 mol%, approximately 45-60 mol%, approximately 48-60 mol%, approximately 50-60 mol%, approximately 52-60 mol%, approximately 55-60 mol%, approximately 58-60 mol%, approximately 25-58 mol%, approximately 28-58 mol%, approximately 30-58 mol%, approximately 32-58 mol%, approximately 35-58 mol%, approximately 38-58 mol%, approximately 40-58 mol%, approximately 42-58 mol%, approximately 45-58 mol%, approximately 48-58 mol%, approximately 50-58 mol%, approximately 52-58 mol%, approximately 55-58 mol%, approximately 25-55 mol%, approximately 28-55 mol%, approximately 30-55 mol%, approximately 32-55 mol%, approximately 35-55 mol%, approximately 38-55 mol%, approximately 40-55 mol%, approximately 42-55 mol%, approximately 45-55 mol%, approximately 48-55 mol%, approximately 50-55 mol%, approximately 52-55 mol%, approximately 25-53 mol%, approximately 28-53 mol%, approximately 30-53 mol%, approximately 32-53 mol%, approximately 35-53 mol%, approximately 38-53 mol%, approximately 40-53 mol%, approximately 42-53 mol%, approximately 45-53 mol%, approximately 48-53 mol%, approximately 50-53 mol%, approximately 25-50mol%, approximately 28-50 mol%, approximately 30-50 mol%, approximately 32-50 mol%, approximately 35-50 mol%, approximately 38-50 mol%, approximately 40-50 mol%, approximately 42-50 mol%, approximately 45-50 mol%, approximately 48-50 mol%, approximately 25-48 mol%, approximately 28-48 mol%, approximately 30-48 mol%, approximately 32-48 mol%, approximately 35-48 mol%, approximately 38-48 mol%, approximately 40-48 mol%, approximately 42-48 mol%, approximately 45-48 mol%, approximately 25-45 mol%, approximately 28-45 mol%, approximately 30-45 mol%, approximately 32-45 mol%, approximately 35-45 mol%, approximately 38-45 mol%, approximately 40-45 mol%, approximately 42-45 mol%, approximately 25-43 mol%, approximately 28-43 mol%, approximately 30-43 mol%, approximately 32-43 mol%, approximately 35-43 mol%, approximately 38-43 mol%, approximately 40-43 mol%, approximately 25-40 mol%, approximately 28-40 mol%, approximately 30-40 mol%, approximately 32-40 mol%, approximately 35-40 mol%, approximately 38-40 mol%, approximately 25-38 mol%, approximately 28-38 mol%, approximately 30-38 mol%, approximately 32-38 mol%, approximately 35-38 mol%, approximately 25-35 mol%, approximately 28-35 mol%, approximately 30-35 mol%, approximately 32-35 mol%, approximately 25-33 mol%, approximately 28-33 mol%, approximately 30-33 mol%, approximately 35-45 mol%, or approximately 35-40 mol%. In some embodiments, the amount of auxiliary lipids is adjusted based on the amount of ionizable lipids, neutral lipids, and / or PEG lipids to achieve a lipid component of approximately 100 mol%. In some embodiments, the mol% of auxiliary lipids relative to the lipid component will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or actual mol% of auxiliary lipids. In some embodiments, the mol% of auxiliary lipids relative to the lipid component will be ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, or ±0.25 mol% of the specified, nominal, or actual mol%. In some embodiments, the batch-to-batch variability of LNP will be less than 15%, less than 10%, or less than 5%. In some embodiments, the mol% number is based on the nominal concentration. In some embodiments, the mol% number is based on the actual concentration.

[0162] In some embodiments, the amount of PEG lipids is about 1.5-3.5 mol%, about 2.0-2.7 mol%, about 2.0-3.5 mol%, about 2.3-3.5 mol%, about 2.3-2.7 mol%, about 2.5-3.5 mol%, about 2.5-2.7 mol%, about 2.9-3.5 mol%, or about 2.7 mol%. In other embodiments, the amount of PEG lipids may be about 1.0-4.0 mol%, about 1.2-4.0 mol%, about 1.4-4.0 mol%, about 1.5-4.0 mol%, about 1.6-4.0 mol%, about 1.7-4.0 mol%, about 1.8-4.0 mol%, about 1.9-4.0 mol%, about 2.0-4.0 mol%, about 2.1-4.0 mol%, about 2.2-4.0 mol%, about 2.3-4.0 mol%, about 2.4-4.0 mol%, about 2.5-4.0 mol%, about 2.6-4.0 mol%, about 2.7-4.0 mol%, about 2.8-4.0 mol%, about 2.9-4.0 mol%, about 3.0-4.0 mol%, about 3.1-4.0 mol%, about 3.2-4.0 mol%. mol%, approximately 3.3-4.0 mol%, approximately 3.4-4.0 mol%, approximately 3.5-4.0 mol%, approximately 3.7-4.0 mol%, 1.0-3.7 mol%, approximately 1.2-3.7 mol%, approximately 1.4-3.7 mol%, approximately 1.5-3.7 mol%, approximately 1.6-3.7 mol%, approximately 1.7-3.7 mol%, approximately 1.8-3.7 mol%, approximately 1.9-3.7 mol%, approximately 2.0-3.7 mol%, approximately 2.1-3.7 mol%, approximately 2.2-3.7 mol%, approximately 2.3-3.7 mol%, approximately 2.4-3.7 mol%, approximately 2.5-3.7 mol%, approximately 2.6-3.7 mol%, approximately 2.7-3.7 mol%, approximately 2.8-3.7 mol%, approximately 2.9-3.7 mol%, approximately 3.0-3.7 mol%, approximately 3.1-3.7 mol%, approximately 3.2-3.7 mol%, approximately 3.3-3.7 mol%, approximately 3.4-3.7 mol%, approximately 3.5-3.7 mol%, 1.0-3.5 mol%, approximately 1.2-3.5 mol%, approximately 1.4-3.5 mol%, approximately 1.5-3.5 mol%, approximately 1.6-3.5 mol%, approximately 1.7-3.5 mol%, approximately 1.8-3.5 mol%, approximately 1.9-3.5 mol%, approximately 2.0-3.5 mol%, approximately 2.1-3.5 mol%, approximately 2.2-3.5 mol%, approximately 2.3-3.5 mol%, approximately 2.4-3.5 mol%, approximately 2.5-3.5 mol%, approximately 2.6-3.5 mol%, approximately 2.7-3.5 mol%, approximately 2.8-3.5 mol%, approximately 2.9-3.5 mol%, approximately 3.0-3.5 mol%, approximately 3.1-3.5 mol%, approximately 3.2-3.5 mol%, approximately 3.3-3.5 mol%, approximately 3.4-3.5 mol%, 1.0-3.4 mol%, approximately 1.2-3.4 mol%, approximately 1.4-3.4 mol%, approximately 1.5-3.4 mol%, approximately 1.6-3.4 mol%, approximately 1.7-3.4 mol%, approximately 1.8-3.4 mol%, approximately 1.9-3.4 mol%, approximately 2.0-3.4 mol%, approximately 2.1-3.4 mol%, approximately 2.2-3.4 mol%, approximately 2.3-3.4 mol%, approximately 2.4-3.4 mol%, approximately 2.5-3.4 mol%, approximately 2.6-3.4 mol%, approximately 2.7-3.4 mol%, approximately 2.8-3.4 mol%, approximately 2.9-3.4 mol%, approximately 3.0-3.4 mol%, approximately 3.1-3.4 mol%, approximately 3.2-3.4 mol%, approximately 3.3-3.4 mol%, 1.0-3.3 mol%, approximately 1.2-3.3 mol%, approximately 1.4-3.3 mol%, approximately 1.5-3.3 mol%, approximately 1.6-3.3 mol%, approximately 1.7-3.3 mol%, approximately 1.8-3.3 mol%, approximately 1.9-3.3 mol%, approximately 2.0-3.3 mol%, approximately 2.1-3.3 mol%, approximately 2.2-3.3 mol%, approximately 2.3-3.3 mol%, approximately 2.4-3.3 mol%, approximately 2.5-3.3 mol%, approximately 2.6-3.3 mol%, approximately 2.7-3.3 mol%, approximately 2.8-3.3 mol%, approximately 2.9-3.3 mol%, approximately 3.0-3.3 mol%, approximately 3.1-3.3 mol%, approximately 3.2-3.3 mol%, 1.0-3.2 mol%, approximately 1.2-3.2 mol%, approximately 1.4-3.2 mol%, approximately 1.5-3.2 mol%, approximately 1.6-3.2 mol%, approximately 1.7-3.2 mol%, approximately 1.8-3.2 mol%, approximately 1.9-3.2 mol%, approximately 2.0-3.2 mol%, approximately 2.1-3.2 mol%, approximately 2.2-3.2 mol%, approximately 2.3-3.2 mol%, approximately 2.4-3.2 mol%, approximately 2.5-3.2 mol%, approximately 2.6-3.2 mol%, approximately 2.7-3.2 mol%, approximately 2.8-3.2 mol%, approximately 2.9-3.2 mol%, approximately 3.0-3.2 mol%, approximately 3.1-3.2 mol%, 1.0-3.1 mol%, approximately 1.2-3.1 mol%, approximately 1.4-3.1 mol%, approximately 1.5-3.1 mol%, approximately 1.6-3.1 mol%, approximately 1.7-3.1 mol%, approximately 1.8-3.1 mol%, approximately 1.9-3.1 mol%, approximately 2.0-3.1 mol%, approximately 2.1-3.1 mol%, approximately 2.2-3.1 mol%, approximately 2.3-3.1 mol%, approximately 2.4-3.1 mol%, approximately 2.5-3.1 mol%, approximately 2.6-3.1 mol%, approximately 2.7-3.1 mol%, approximately 2.8-3.1 mol%, approximately 2.9-3.1 mol%, approximately 3.0-3.1 mol%, 1.0-3.0 mol%, approximately 1.2-3.0 mol%. mol%, approximately 1.4-3.0 mol%, approximately 1.5-3.0 mol%, approximately 1.6-3.0 mol%, approximately 1.7-3.0 mol%, approximately 1.8-3.0 mol%, approximately 1.9-3.0 mol%, approximately 2.0-3.0 mol%, approximately 2.1-3.0 mol%, approximately 2.2-3.0 mol%, approximately 2.3-3.0 mol%, approximately 2.4-3.0 mol%, approximately 2.5-3.0 mol%, approximately 2.6-3.0 mol%, approximately 2.7-3.0 mol%, approximately 2.8-3.0 mol%, approximately 2.9-3.0 mol%, 1.0-2.9 mol%, approximately 1.2-2.9 mol%, approximately 1.4-2.9 mol%, approximately 1.5-2.9 mol%, approximately 1.6-2.9 mol%, approximately 1.7-2.9 mol%, approximately 1.8-2.9 mol%, approximately 1.9-2.9 mol%, approximately 2.0-2.9 mol%, approximately 2.1-2.9 mol%, approximately 2.2-2.9 mol%, approximately 2.3-2.9 mol%, approximately 2.4-2.9 mol%, approximately 2.5-2.9 mol%, approximately 2.6-2.9 mol%, approximately 2.7-2.9 mol%, approximately 2.8-2.9 mol%, 1.0-2.8 mol%, approximately 1.2-2.8 mol%, approximately 1.4-2.8 mol%, approximately 1.5-2.8 mol%, approximately 1.6-2.8 mol%, approximately 1.7-2.8 mol%, approximately 1.8-2.8 mol%, approximately 1.9-2.8 mol%, approximately 2.0-2.8 mol%, approximately 2.1-2.8 mol%, approximately 2.2-2.8 mol%, approximately 2.3-2.8 mol%, approximately 2.4-2.8 mol%, approximately 2.5-2.8 mol%, approximately 2.6-2.8 mol%, approximately 2.7-2.8 mol%, 1.0-2.7 mol%, approximately 1.2-2.7 mol%, approximately 1.4-2.7 mol%, approximately 1.5-2.7 mol%, approximately 1.6-2.7 mol%, approximately 1.7-2.7 mol%, approximately 1.8-2.7 mol%, approximately 1.9-2.7 mol%, approximately 2.0-2.7 mol%, approximately 2.1-2.7 mol%, approximately 2.2-2.7 mol%, approximately 2.3-2.7 mol%, approximately 2.4-2.7 mol%, approximately 2.5-2.7 mol%, approximately 2.6-2.7 mol%, 1.0-2.6 mol%, approximately 1.2-2.6 mol%, approximately 1.4-2.6 mol%, approximately 1.5-2.6 mol%, approximately 1.6-2.6 mol%, approximately 1.7-2.6 mol%, approximately 1.8-2.6 mol%, approximately 1.9-2.6 mol%, approximately 2.0-2.6 mol%. mol%, approximately 2.1-2.6 mol%, approximately 2.2-2.6 mol%, approximately 2.3-2.6 mol%, approximately 2.4-2.6 mol%, approximately 2.5-2.6 mol%, 1.0-2.5 mol%, approximately 1.2-2.5 mol%, approximately 1.4-2.5 mol%, approximately 1.5-2.5 mol%, approximately 1.6-2.5 mol%, approximately 1.7-2.5 mol%, approximately 1.8-2.5 mol%, approximately 1.9-2.5 mol%, approximately 2.0-2.5 mol%, approximately 2.1-2.5 mol%, approximately 2.2-2.5 mol%, approximately 2.3-2.5 mol%, approximately 2.4-2.5 mol%, 1.0-2.4 mol%, approximately 1.2-2.4 mol%, approximately 1.4-2.4 mol%, approximately 1.5-2.4 mol%, approximately 1.6-2.4 mol%, approximately 1.7-2.4 mol%, approximately 1.8-2.4 mol%, approximately 1.9-2.4 mol%, approximately 2.0-2.4 mol%, approximately 2.1-2.4 mol%, approximately 2.2-2.4 mol%, approximately 2.3-2.4 mol%, 1.0-2.3 mol%, approximately 1.2-2.3 mol%, approximately 1.4-2.3 mol%, approximately 1.5-2.3 mol%, approximately 1.6-2.3 mol%, approximately 1.7-2.3 mol%, approximately 1.8-2.3 mol%, approximately 1.9-2.3 mol%, approximately 2.0-2.3 mol%, approximately 2.1-2.3 mol%, approximately 2.2-2.3 mol%, 1.0-2.2 mol%, approximately 1.2-2.2 mol%, approximately 1.4-2.2 mol%, approximately 1.5-2.2 mol%, approximately 1.6-2.2 mol%, approximately 1.7-2.2 mol%, approximately 1.8-2.2 mol%, approximately 1.9-2.2 mol%, approximately 2.0-2.2 mol%, approximately 2.1-2.2 mol%, approximately 2.2-2.2 mol%, approximately 2.3-2.2 mol%, approximately 2.4-2.2 mol%, 1.0-2.1 mol%, approximately 1.2-2.1 mol%, approximately 1.4-2.1 mol%, approximately 1.5-2.1 mol%, approximately 1.6-2.1 mol%, approximately 1.7-2.1 mol%, approximately 1.8-2.1 mol%, approximately 1.9-2.1 mol%, approximately 2.0-2.1 mol%, 1.0-2.0 mol%, approximately 1.2-2.0 mol%, approximately 1.4-2.0 mol%, approximately 1.5-2.0 mol%, approximately 1.6-2.0 mol%, approximately 1.7-2.0 mol%, approximately 1.8-2.0 mol%, approximately 1.9-2.0 mol%, 1.0-1.9 mol%, approximately 1.2-1.9 mol%, approximately 1.4-1.9 mol%, approximately 1.5-1.9 mol%, approximately 1.6-1.9 mol%, approximately 1.7-1.9 mol%, approximately 1.8-1.9 mol%, 1.0-1.8 mol%, approximately 1.2-1.8 mol%, approximately 1.4-1.8 mol%, approximately 1.5-1.8 mol%, approximately 1.6-1.8 mol%, approximately 1.7-1.8 mol%, 1.0-1.7 mol%, approximately 1.2-1.7 mol%, approximately 1.4-1.7 mol%, approximately 1.5-1.7 mol%, approximately 1.6-1.7 mol%, 1.0-1.6 mol%, approximately 1.2-1.6 mol%, approximately 1.4-1.6 mol%, approximately 1.5-1.6 mol%, 1.0-1.5 mol%, approximately 1.2-1.5 mol%, approximately 1.4-1.5 mol%, approximately 1.5-1.5 mol%, approximately 1.6-1.5 mol%, approximately 1.7-1.5 mol%, approximately 1.8-1.5 mol%, approximately 1.9-1.5 mol%, 1.0-1.4 mol%, approximately 1.2-1.4 mol% or 1.0-1.2 mol%. In some embodiments, the mol% of PEG lipids may be about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, or about 3.5 mol%. In some embodiments, the mol% of PEG lipids relative to the lipid component will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or actual PEG lipid mol%. In some embodiments, the mol% of PEG lipids relative to the lipid component will be ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, or ±0.25 mol%. In some embodiments, the batch-to-batch variability of LNP will be less than 15%, less than 10%, or less than 5%. In some embodiments, the mol% number is based on the nominal concentration. In some embodiments, the mol% number is based on the actual concentration.

[0163] In some embodiments, the amount of auxiliary lipid is about 30-40 mol%, and the amount of PEG lipid is about 2.5-3.5 mol%; the amount of auxiliary lipid is about 35-40 mol%, and the amount of PEG lipid is about 2.9-3.3 mol%; or the amount of auxiliary lipid is about 38 mol%, and the amount of PEG lipid is about 3 mol%. In some embodiments, the amount of auxiliary lipid is about 37-47 mol%, and the amount of PEG lipid is about 2.0-3.0 mol%; the amount of auxiliary lipid is about 40-45 mol%, and the amount of PEG lipid is about 2.2-2.8 mol%; or the amount of auxiliary lipid is about 42 mol%, and the amount of PEG lipid is about 2.2-2.8 mol%. In some embodiments, the amount of auxiliary lipid is about 47-57 mol%, and the amount of PEG lipid is about 2.0-3.0 mol%; the amount of auxiliary lipid is about 50-55 mol%, and the amount of PEG lipid is about 2.3-2.7 mol%; or the amount of auxiliary lipid is about 52 mol%, and the amount of PEG lipid is about 2.3-2.7 mol%.

[0164] In some embodiments, the lipid composition (such as the LNP composition) comprises a lipid component and a nucleic acid component (also referred to as an aqueous component), such as an RNA component, and the molar ratio of the compounds of formulas (I)-(IV) or Table 1 to the nucleic acid can be measured. Embodiments of this disclosure also provide lipid compositions having a defined molar ratio between a positively charged amino group (N) of a pharmaceutically acceptable salt of a compound of formulas (I)-(IV) or Table 1 and a negatively charged phosphate group (P) of the nucleic acid to be encapsulated. This can be mathematically represented by the equation N / P. In some embodiments, the lipid composition (such as the LNP composition) may comprise a lipid component comprising a compound of formulas (I)-(IV) or Table 1 or a pharmaceutically acceptable salt thereof; and a nucleic acid component, wherein the N / P ratio is about 3 to 10. In some embodiments, the LNP composition may comprise a lipid component comprising a compound of formulas (I)-(IV) or Table 1 or a pharmaceutically acceptable salt thereof; and an RNA component, wherein the N / P ratio is about 3 to 10. For example, the N / P ratio can be about 4-7, about 5-7, or about 6 to 7. In some embodiments, the N / P ratio can be about 6, such as 6 ± 1 or 6 ± 0.5. In some embodiments, the N / P ratio can be about 7, such as 7 ± 1 or 7 ± 0.5.

[0165] In some embodiments, the aqueous component contains a bioactive agent. In some embodiments, the aqueous component contains a polypeptide, optionally combined with a nucleic acid. In some embodiments, the aqueous component contains a nucleic acid, such as RNA. In some embodiments, the aqueous component is a nucleic acid component. In some embodiments, the nucleic acid component contains DNA, and it may be referred to as a DNA component. In some embodiments, the nucleic acid component contains RNA. In some embodiments, the aqueous component (such as an RNA component) may contain mRNA, such as mRNA encoding an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is a Cas nuclease. In some embodiments, the aqueous component may contain mRNA encoding a Cas nuclease (such as Cas9). In some embodiments, the bioactive agent is Cas nuclease mRNA. In some embodiments, the bioactive agent is class 2 Cas nuclease mRNA. In some embodiments, the bioactive agent is Cas9 nuclease mRNA. In some embodiments, the aqueous component may contain modified RNA. In some embodiments, the aqueous component may contain a guide RNA nucleic acid. In some embodiments, the aqueous component may contain gRNA. In some embodiments, the aqueous component may contain dgRNA. In some embodiments, the aqueous component may contain modified gRNA. In some compositions containing mRNA encoding an RNA-guided DNA binder, the composition further contains gRNA nucleic acid, such as gRNA. In some embodiments, the aqueous component contains an RNA-guided DNA binder and gRNA. In some embodiments, the aqueous component contains Cas nuclease mRNA and gRNA. In some embodiments, the aqueous component contains two types of Cas nuclease mRNA and gRNA.

[0166] In some embodiments, the lipid composition (such as the LNP composition) may comprise mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), compounds of formulas (I)-(IV) or Table 1 or pharmaceutically acceptable salts thereof, an accessory lipid, a neutral lipid optionally present, and a PEG lipid. In some compositions comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), the accessory lipid is cholesterol. In other compositions comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease), the neutral lipid is DSPC. In other embodiments comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease, e.g., Cas9), the PEG lipid is PEG2K-DMG. In specific compositions comprising mRNA encoding a Cas nuclease (such as a class 2 Cas nuclease) and compounds of formulas (I)-(IV) or Table 1 or pharmaceutically acceptable salts thereof. In some compositions, the composition further comprises gRNA, such as dgRNA or sgRNA.

[0167] In some embodiments, the lipid composition (such as an LNP composition) may comprise gRNA. In some embodiments, the composition may comprise a compound of formula (I)-(IV) or Table 1 or a pharmaceutically acceptable salt thereof, gRNA, an accessory lipid, optionally a neutral lipid, and a PEG lipid. In some LNP compositions comprising gRNA, the accessory lipid is cholesterol. In some compositions comprising gRNA, the neutral lipid is DSPC. In other embodiments comprising gRNA, the PEG lipid is PEG2K-DMG. In some compositions, the gRNA is selected from dgRNA and sgRNA.

[0168] In some embodiments, the lipid composition (such as the LNP composition) comprises mRNA encoding an RNA-guided DNA binder and gRNA, which may be sgRNA, in the aqueous component, and compounds of formulas (I)-(IV) or Table 1 in the lipid component. For example, the LNP composition may comprise compounds of formulas (I)-(IV) or Table 1 or pharmaceutically acceptable salts thereof, mRNA encoding a Cas nuclease, gRNA, accessory lipids, neutral lipids, and PEG lipids. In some compositions containing mRNA and gRNA encoding a Cas nuclease, the accessory lipid is cholesterol. In some compositions containing mRNA and gRNA encoding a Cas nuclease, the neutral lipid is DSPC. In other embodiments containing mRNA and gRNA encoding a Cas nuclease, the PEG lipid is PEG2K-DMG.

[0169] In some embodiments, the lipid composition (such as the LNP composition) comprises an RNA-guided DNA binder (such as class 2 Cas mRNA) and at least one gRNA. In some embodiments, the gRNA is sgRNA. In some embodiments, the RNA-guided DNA binder is Cas9 mRNA. In some embodiments, the LNP composition comprises gRNA and RNA-guided DNA binder mRNA (such as class 2 Cas nuclease mRNA) in a ratio of about 1:1 or about 1:2. In some embodiments, the weight 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, about 2:1 to about 1:2, about 2:1 to 1:4, or about 1:1 to about 1:2.

[0170] The lipid compositions disclosed herein (such as LNP compositions) can be used in the methods disclosed herein to deliver CRISPR / Cas9 components thereby inserting template nucleic acids, such as DNA templates. The template nucleic acid can be delivered separately from the lipid composition comprising compounds of formulas (I)-(IV) or those in Table 1, or pharmaceutically acceptable salts thereof. In some embodiments, the template nucleic acid can be single-stranded or double-stranded, depending on the desired repair mechanism. The template can have regions homologous to the target DNA, such as within the target DNA sequence, and / or regions homologous to sequences adjacent to the target DNA.

[0171] In some embodiments, an LNP composition is formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. For example, the organic solvent may be 100% ethanol. Pharmaceutically acceptable buffers, such as those used for in vivo administration of the LNP composition, can be used. In some embodiments, the pH of the LNP-containing composition is maintained at pH 6.5 or higher using a buffer. In some embodiments, the pH of the LNP-containing composition is maintained at pH 7.0 or higher using a buffer. In some embodiments, the pH of the composition is in the range of about 7.2 to about 7.7. In other embodiments, the pH of the composition is in the range of about 7.3 to about 7.7 or in the range of about 7.4 to about 7.6. In other embodiments, the pH of the composition is about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of the composition can be measured using a micro pH probe. In some embodiments, the composition includes a cryoprotectant. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as sucrose. In some embodiments, the composition may contain tris-saline sucrose (TSS). In some embodiments, the LNP composition may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% cryoprotectant. In some embodiments, the LNP composition may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% sucrose. In some embodiments, the LNP composition may include a buffer. In some embodiments, the buffer may contain phosphate-buffered saline (PBS), Tris buffer, citrate buffer, and mixtures thereof. In some exemplary embodiments, the buffer contains NaCl. In some embodiments, the buffer lacks NaCl. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts 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. Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts 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 contains NaCl and Tris. Certain exemplary embodiments of the LNP composition 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 (pH 7.5). The amounts of salt, buffer, and cryoprotectant can be varied to maintain the overall osmolality of the composition. For example, the final osmolality can be maintained at less than 450 mOsm / L. In other embodiments, the osmolality is between 350 mOsm / L and 250 mOsm / L. In some embodiments, the final osmolality is 300 + / - 20 mOsm / L or 310 + / - 40 mOsm / L.

[0172] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing of aqueous RNA and lipid solutions in an organic solvent is used. In some aspects, flow rate, adapter size, adapter geometry, adapter shape, tube diameter, and solution and / or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be buffer-exchanged, concentrated, or purified, for example by dialysis, centrifugal filtration, tangential flow filtration, chromatography, or size exclusion chromatography. LNP compositions may be stored, for example, as suspensions, emulsions, or lyophilized powders. In some embodiments, LNP compositions are stored at 2°C–8°C; in some aspects, LNP compositions are stored at room temperature. In other embodiments, LNP compositions are frozen and stored, for example, at -20°C or -80°C. In other embodiments, LNP compositions are stored at temperatures ranging from about 0°C to about -80°C. Frozen LNP compositions may be thawed before use, for example, on ice, at room temperature, or at 25°C, preferably at room temperature.

[0173] Preferred lipid compositions (such as LNP compositions) are biodegradable, for example, because they do not accumulate in vivo to cytotoxic levels at therapeutically effective doses. In some embodiments, the compositions do not induce innate immune responses that would lead to substantial adverse effects at therapeutic dose levels. In some embodiments, the compositions provided herein do not induce toxicity at therapeutic dose levels.

[0174] In some embodiments, the concentration of LNP in the LNP composition is about 1-10 μg / mL, about 2-10 μg / mL, about 2.5-10 μg / mL, about 1-5 μg / mL, about 2-5 μg / mL, about 2.5-5 μg / mL, about 0.04 μg / mL, about 0.08 μg / mL, about 0.16 μg / mL, about 0.25 μg / mL, about 0.63 μg / mL, about 1.25 μg / mL, about 2.5 μg / mL, or about 5 μg / mL.

[0175] In some embodiments, dynamic light scattering (“DLS”) can be used to characterize the polydispersity index (PDI) and size of the LNP of this disclosure. DLS measures the light scattering produced when a sample is illuminated by a light source. As determined by DLS measurements, PDI represents the particle size distribution in a population (near the average particle size), where the PDI is 0 for a completely homogeneous population.

[0176] In some embodiments, the PDI of the LNP disclosed herein is from about 0.005 to about 0.75. In some embodiments, the PDI of the LNP disclosed herein is from about 0.005 to about 0.1. In some embodiments, the PDI of the LNP disclosed herein is from about 0.005 to about 0.09, from about 0.005 to about 0.08, from about 0.005 to about 0.07, or from about 0.006 to about 0.05. In some embodiments, the PDI of the LNP is from about 0.01 to about 0.5. In some embodiments, the PDI of the LNP is from about 0 to about 0.4. In some embodiments, the PDI of the LNP is from about 0 to about 0.35. In some embodiments, the LNP PDI may be in the range of from about 0 to about 0.3. In some embodiments, the LNP PDI may be in the range of from about 0 to about 0.25. In some embodiments, the LNP PDI may be in the range of from about 0 to about 0.2. In some embodiments, the LNP PDI is from about 0 to about 0.05. In some implementations, the PDI of the LNP is about 0 to about 0.01. In some implementations, the PDI of the LNP is less than about 0.01, about 0.02, about 0.05, about 0.08, about 0.1, about 0.15, about 0.2 or about 0.4.

[0177] LNP size can be measured using various analytical methods known in the art. In some embodiments, LNP size can be measured using asymmetric flow field flow grading-multi-angle light scattering (AF4-MALS). In some embodiments, LNP size can be measured by separating particles in the composition according to their hydrodynamic radii, and then measuring the molecular weight, hydrodynamic radius, and root mean square radius of the graded particles. In some embodiments, LNP size and particle concentration can be measured using nanoparticle tracking analysis (NTA, Malvern Nanosight). In some embodiments, the LNP sample is appropriately diluted and injected onto a microscope slide. As the particles slowly cross the field of view, the camera records the scattered light. After capturing the film, nanoparticle tracking analysis processes the film by tracking pixels and calculating the diffusion coefficient. This diffusion coefficient can be converted into the hydrodynamic radius of the particles. Such methods can also count the number of individual particles to obtain the particle concentration. In some embodiments, LNP size, morphology, and structural characteristics can be determined using cryo-electron microscopy (“cryo-EM”).

[0178] The LNPs in the LNP compositions disclosed herein have an LNP size (e.g., Z-average diameter or number-average diameter) of about 1 to about 250 nm. In some embodiments, the LNP size is about 10 to about 200 nm. In other embodiments, the LNP size is about 20 to about 150 nm. In some embodiments, the LNP size is about 50 to about 150 nm or about 70 to 130 nm. In some embodiments, the LNP size is about 50 to about 100 nm. In some embodiments, the LNP size is about 50 to about 120 nm. In some embodiments, the LNP size is about 60 to about 100 nm. In some embodiments, the LNP size is about 75 to about 150 nm. In some embodiments, the LNP size is about 75 to about 120 nm. In some embodiments, the LNP size is about 75 to about 100 nm. In some embodiments, the size of the LNP is about 50 to about 145 nm, about 50 to about 120 nm, about 50 to about 120 nm, about 50 to about 115 nm, about 50 to about 100 nm, about 60 to about 145 nm, about 60 to about 120 nm, about 60 to about 115 nm, or about 60 to about 100 nm. In some embodiments, the size of the LNP is less than about 145 nm, less than about 120 nm, less than about 115 nm, less than about 100 nm, or less than about 80 nm. In some embodiments, the size of the LNP is greater than about 50 nm or greater than about 60 nm. In some embodiments, the particle size is the Z-mean particle size. In some embodiments, the particle size is the number-mean particle size. In some embodiments, the particle size is the size of an individual LNP. Unless otherwise indicated, all sizes mentioned herein are the average size (diameter) of the fully formed nanoparticles, as measured by dynamic light scattering on a Malvern Zetasizer or Wyatt NanoStar. The nanoparticle sample was diluted in phosphate-buffered saline (PBS) to achieve a count rate of approximately 200–400 kcps.

[0179] In some embodiments, the LNP composition is formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNP composition is formed with an average encapsulation efficiency ranging from about 50% to about 95%. In some embodiments, the LNP composition is formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNP composition is formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNP composition is formed with an average encapsulation efficiency ranging from about 95% to about 100%. In some embodiments, the LNP composition is formed with an average encapsulation efficiency ranging from about 98% to about 100%. In some embodiments, the LNP composition is formed with an average encapsulation efficiency ranging from about 99% to about 100%.

[0180] goods

[0181] The goods delivered via the LNP compositions described herein include bioactive agents. These bioactive agents may be nucleic acids, such as mRNA or gRNA. In some embodiments, the cargo is or comprises one or more bioactive agents, such as mRNA, gRNA, expression vectors, RNA-guided DNA binders, antibodies (e.g., monoclonal, chimeric, humanized, nanobodies and fragments thereof), cholesterol, hormones, peptides, proteins, chemotherapeutic agents and other types of antitumor agents, low molecular weight drugs, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzyme nucleic acids, antisense nucleic acids, triple-stranded oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allylases, aptamers, ribozymes, baits and their analogues, plasmids and other types of vectors, as well as small nucleic acid molecules, RNAi agents, short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and "self-replicating RNA" molecules (encoding replicase activity and capable of guiding its own replication or amplification in vivo), peptide nucleic acids (PNA), locked nucleic acid ribonucleotides (LNA), morpholinonucleotides, threonine nucleic acids (TNA), glycol nucleic acids (GNA), and sisiRNA. (Small internal segment interfering RNA) and iRNA (asymmetric interfering RNA). The above list of bioactive agents is merely illustrative and is not intended to be limiting. Such compounds can be purified or partially purified, can be natural or synthetic, and can be chemically modified.

[0182] The cargo delivered via the LNP composition may be RNA, such as an mRNA molecule encoding a protein of interest. For example, it may include mRNA for expressing proteins such as green fluorescent protein (GFP), RNA-guided DNA binders, or Cas nucleases. An LNP composition comprising Cas nuclease mRNA is provided, for example, a class 2 Cas nuclease mRNA that allows expression of class 2 Cas nucleases, such as Cas9 or Cpf1 (also known as Cas12a) proteins, in cells. Furthermore, the cargo may contain one or more gRNAs or nucleic acids encoding gRNAs. A template nucleic acid (e.g., 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 one sub-implementation, the cargo comprises mRNA encoding Streptococcus pyogenes Cas9, optionally and Streptococcus pyogenes gRNA. In another sub-implementation, the cargo comprises mRNA encoding Neisseria meningitidis Cas9, optionally and Nme (Neisseria meningitidis) gRNA.

[0183] “mRNA” refers to a polynucleotide containing an open reading frame that can be translated into a polypeptide (i.e., can be used as a translation substrate for ribosomes and aminoacylated tRNA). mRNA may contain a phosphate-sugar backbone, including ribose residues or analogues thereof, such as 2'-methoxyribose residues. In some embodiments, the sugars in the mRNA phosphate-sugar backbone consist primarily of ribose residues, 2'-methoxyribose residues, or combinations thereof. Generally, mRNA does not contain a large number of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). mRNA may contain modified uridine at some or all of its uridine sites.

[0184] Genome editing tools

[0185] In some embodiments, the LNP composition is a lipid nucleic acid assembly, also referred to as a lipid nucleic acid composition. In some embodiments, the lipid nucleic acid composition or LNP composition contains a genome editing tool or a nucleic acid encoding such a tool. As used herein, the term "genome editing tool" (or "gene editing tool") is any component of a "genome editing system" (or "gene editing system") necessary or helpful for producing editing in the cellular genome. In some embodiments, this disclosure provides a method for delivering a genome editing tool of a genome editing system (e.g., a zinc finger nuclease system, a TALEN system, a wide-range nuclease system, or a CRISPR / Cas system) to a cell (or cell population). Genome editing tools include, for example, nucleases capable of causing single-strand or double-strand breaks in the DNA or RNA of a cell (e.g., in the genome of a cell). Genome editing tools (e.g., nucleases) may optionally modify the genome of a cell without cleaving nucleic acids or nicking enzymes. Genome editing nucleases or nicking enzymes may be encoded by mRNA. Such nucleases include, for example, RNA-guided DNA binders and CRISPR / Cas components. Genome editing tools include fusion proteins, including, for example, nickases that fuse with effector domains (such as editing domains). Genome editing tools also include any items necessary or helpful in achieving genome editing goals, such as guide RNA, sgRNA, dgRNA, donor nucleic acids, etc.

[0186] This article describes various suitable gene editing systems, which include genome editing tools for delivery with lipid nucleic acid assembly compositions, including (but not limited to) CRISPR / Cas systems; zinc finger nuclease (ZFN) systems; and transcription activator-like effector nuclease (TALEN) systems. Typically, gene editing systems involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks (e.g., single-strand breaks or SSBs) in the target DNA sequence. Cleavage or nicks can occur by using specific nucleases (such as engineered ZFNs, TALENs) or by using CRISPR / Cas systems with engineered guide RNAs to guide specific cleavage or nicks of the target DNA sequence. Furthermore, targeted nucleases based on Argonaute systems (e.g., from *T. thermophilus*, called 'TtAgo', see Swarts et al. (2014) Nature 507(7491): 258-261) are being developed, which may also have potential for use in genome editing and gene therapy.

[0187] In some embodiments, the disclosed compositions comprise one or more DNA modifiers, such as DNA cleavage agents. The LNP compositions described herein may include a variety of DNA modifiers. For example, DNA modifiers include nucleases (sequence-specific and non-specific), topoisomerases, methyltransferases, acetyltransferases, chemicals, pharmaceuticals, and other agents. In some embodiments, a protein bound to a given DNA sequence or set of sequences can be used to induce DNA modifications, such as strand breaks. Proteins can be modified in various ways, such as by incorporation. 125 I. Radioactive decay of the protein-DNA leads to strand breaks, or modification with cross-linking agents, such as 4-azidobenzoylmethyl bromide, which cross-links with DNA upon exposure to UV light. Such protein-DNA cross-links can then be converted into double-stranded DNA breaks by treatment with piperidine. Another DNA modification method involves antibodies generated against specific proteins (such as transcription factors or structural chromatin proteins) that bind to one or more DNA sites, used to separate the DNA from the nucleoprotein complex.

[0188] In some embodiments, the disclosed compositions comprise one or more DNA cleaving agents. DNA cleaving agents include technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), mitochondrial TALENs, and large-scale nuclease systems. TALEN and ZFN technologies use a strategy of tethering the catalytic domain of a nuclease to a modularized DNA-binding protein to induce targeted DNA double-strand breaks (DSBs) at specific genomic loci. Other DNA cleaving agents include small interfering RNAs, microRNAs, anti-microRNAs, antagonists, small hairpin RNAs, and aptamers (based on RNA, DNA, or peptides, including affixes).

[0189] In some implementations, the gene editing system is a TALEN system. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific DNA sequences. They are prepared by fusing a TAL effector DNA-binding domain with a DNA-cleaving domain (a nuclease that cuts the DNA strand). Transcription activator-like effectors (TALEs) can be engineered to bind to a desired DNA sequence, promoting DNA cleavage at a specific location (e.g., see Boch, 2011, Nature Biotech). Restriction enzymes can be introduced into cells for gene editing or for in situ genome editing, a technique known as genome editing using engineered nucleases. Such methods and compositions used therein are known in the art. See, for example, WO2019147805, WO2014040370, and WO2018073393, the contents of which are incorporated herein by reference in their entirety.

[0190] In some implementations, the gene editing system is a zinc finger system. Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by fusing a zinc finger DNA-binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific desired DNA sequences, enabling the zinc finger nuclease to target unique sequences within a complex genome. Non-specific cleavage domains derived from the type IIs restriction endonuclease FokI are commonly used as cleavage domains in ZFNs. Cleavage is repaired by endogenous DNA repair mechanisms, allowing the ZFN to precisely alter the genome of higher organisms. Such methods and compositions used therein are known in the art. See, for example, WO2011091324, the contents of which are incorporated herein by reference in their entirety.

[0191] In a preferred embodiment, the disclosed composition comprises mRNA encoding an RNA-guided DNA binder (such as a Cas nuclease). In a particular embodiment, the disclosed composition comprises mRNA encoding a class 2 Cas nuclease (such as Streptococcus pyogenes Cas9).

[0192] As used herein, “RNA-guided DNA binder” means a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the RNA sequence. Exemplary RNA-guided DNA binders include Cas lysins / nicking enzymes and their inactivated forms (“dCas DNA binders”). As used herein, “Cas nuclease” encompasses Cas lysins, Cas nicking enzymes, and dCas DNA binders. Cas lysins / nicking enzymes and dCas DNA binders include the Csm or Cmr complex of the type III CRISPR system, its Cas10, Csm1, or Cmr2 subunits, the cascade complex of the type I CRISPR system, its Cas3 subunit, and class 2 Cas nucleases. As used herein, “class 2 Cas nuclease” is a single-stranded polypeptide having RNA-guided DNA binding activity. Class 2 Cas nucleases include two classes of Cas lysins / nicking enzymes (e.g., H840A, D10A, or N863A variants) that further possess RNA-guided DNA lysin or nicking enzyme activity; and two classes of dCas DNA binders in which the lysin / nicking enzyme activity is inactivated. Two classes of Cas nucleases that can be used with the LNP compositions described herein include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and their modifications. The Cpf1 protein (Zetsche et al., Cell, 163: 1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated by full reference. For example, see Zetsche, Tables 2 and 4. For example, see Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0193] Non-limiting exemplary species that can produce Cas nucleases include *Streptococcus pyogenes*, *Streptococcus thermophilus*, *Streptococcus* sp., *Staphylococcus aureus*, *Listeria innocua*, *Lactobacillus gasseri*, *Francisella novicida*, *Wolinella succinogene*, *Sutterella wadsworthensis*, *Gammaproteobacterium*, *Neisseria meningitidis*, *Campylobacter jejuni*, *Pasteurella multocida*, *Fibrobacter succinogene*, and *Rhodospirillum*. The following bacteria are listed: *Streptomyces rubrum*, *Nocardiopsis dassonvillei*, *Streptomyces pristinaespiralis*, *Streptomyces viridochromogene*, *Streptosporangium roseum*, *Streptosporangium roseum*, *Alicyclobacillus acidocaldarius*, *Bacillus pseudomycoides*, *Bacillus sselenitireducens*, *Exiguobacterium sibiricum*, *Lactobacillus delbrueckii*, *Lactobacillus salivarius*, *Lactobacillus buchneri*, *Treponema denticola*, and *Microscilla*. *Pseudomonas marina*, *Burkholderiales bacterium*, *Polaromonas naphthalenivorans*, and *Polaromonas sp.*Marine nitrogen-fixing cyanobacteria (Crocosphaera watsonii), Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrous thiobacillus *Ferrooxidans*, *Allochromatium vinosum*, *Marinobacter* sp., *Nitrosococcus halophilus*, *Nitrosococcus watsoni*, *Pseudoalteromonas haloplanktis*, *Ktedonobacter racemifer*, *Methanohalobium evestigatum*, *Anabaena variabilis*, *Nodularia spumigena*, *Nostoc* sp., *Arthrospira maxima*, *Arthrospira platensis*, *Arthrospira* sp., *Lyngbyasp.*, *Microcoleus chthonoplastes*, *Oscillatoria* sp.The following bacteria were observed: *Petrotoga mobilis*, *Thermosipho africanus*, *Streptococcus pasteurianus*, *Neisseria cinerea*, *Campylobacter lari*, *Parvibaculum lavamentivorans*, *Corynebacterium diphtheria*, *Acidaminococcus sp.*, *Lachnospiraceae bacterium* ND2006, and *Acaryochloris marina*.

[0194] In some embodiments, the Cas nuclease is a Cas9 nuclease from *Streptococcus pyogenes*. In other embodiments, the Cas nuclease is a Cas9 nuclease from *Streptococcus thermophilus*. In other embodiments, the Cas nuclease is a Cas9 nuclease from *Neisseria meningitidis*. In some embodiments, the Cas nuclease is a Cas9 nuclease from *Staphylococcus aureus*. In some embodiments, the Cas nuclease is a Cpf1 nuclease from *Francisella catarrhalis*. In other embodiments, the Cas nuclease is a Cpf1 nuclease from *Aminococcus*. In other embodiments, the Cas nuclease is a Cpf1 nuclease from *N. spp.* (a bacterium in the family *Trichophyton*). In other embodiments, the Cas nuclease is a Cpf1 nuclease from the following species: *Francisella tularensis*, bacteria of the family Trichophyceae, *Butyrivibrio proteoclasticus*, bacteria of the family Peregrinibacteriabacterium, bacteria of the family Parcubacteriabacterium, *Smithella*, aminococci, *Candidatus Methanoplasma termitum*, *Eubacterium eligens*, *Moraxella bovoculi*, *Leptospira inadai*, *Porphyromonas crevioricanis*, *Prevotella disiens*, or *Porphyromonas macacae*. In some implementations, the Cas nuclease is a Cpf1 nuclease derived from aminococci or spirochetalceae.

[0195] Wild-type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target DNA strand. In some embodiments, the Cas9 nuclease contains more than one RuvC domain and / or more than one HNH domain. In some embodiments, the Cas9 nuclease is wild-type Cas9. In some embodiments, Cas9 is capable of inducing double-strand breaks in the target DNA. In other embodiments, the Cas nuclease can cleave dsDNA, cleaves one strand of dsDNA, or may not have DNA cleavage or nicking enzyme activity.

[0196] In some embodiments, a chimeric Cas nuclease is used, wherein a domain or region of the protein is replaced by a portion of a different protein. In some embodiments, the Cas nuclease domain can be replaced with a domain from a different nuclease (such as Fok1). In some embodiments, the Cas nuclease can be a modified nuclease.

[0197] In other embodiments, the Cas nuclease or Cas cleavage enzyme may be derived from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of a cascade complex of a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0198] In some embodiments, the RNA-guided DNA binder has single-strand cleavage enzyme activity, meaning it can cleave one strand of DNA to create a single-strand break, also known as a "nick". In some embodiments, the RNA-guided DNA binder comprises a Cas cleavage enzyme. A cleavage enzyme is an enzyme that creates a nick in dsDNA, i.e., it cuts one strand of the DNA double helix but not the other. In some embodiments, the Cas cleavage enzyme is a form of Cas nuclease (e.g., the Cas nuclease discussed above), wherein the endonuclease activity site is inactivated, for example, by alteration (e.g., point mutation) in one or more catalytic domains. For a discussion of Cas cleavage enzymes and exemplary catalytic domain alterations, see, for example, U.S. Patent No. 8,889,356. In some embodiments, the Cas cleavage enzyme (such as the Cas9 cleavage enzyme) has an inactivated RuvC or HNH domain. In some embodiments, the RNA-guided DNA binder is modified to contain only one functional nuclease domain. For example, the protein can be modified such that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nicking enzyme having a reduced-activity RuvC domain is used. In some embodiments, a nicking enzyme having an inactive RuvC domain is used. In some embodiments, a nicking enzyme having a reduced-activity HNH domain is used. In some embodiments, a nicking enzyme having an inactive HNH domain is used.

[0199] In some embodiments, conserved amino acids within the Cas protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may contain amino acid substitutions in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the Streptococcus pyogenes Cas9 protein). See, for example, Zetsche et al. (2015) Cell Oct 22:163(3):759-771. In some embodiments, the Cas nuclease may contain amino acid substitutions in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the Streptococcus pyogenes Cas9 protein). See, for example, Zetsche et al. (2015). Other exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the novel culprit Francisella U112 Cpf1 (FnCpf1) sequence (UniProtKB - A0Q7Q2(CPF1_FRATN)).

[0200] In some embodiments, the mRNA encoding the nicking enzyme is provided in combination with a pair of guide RNAs, one with a sense strand and the other with an antisense strand complementary to the target sequence. In this embodiment, the guide RNAs guide the nicking enzyme to the target sequence and introduce the DSB by creating a nick on the opposing strand of the target sequence (i.e., a double nick). In some embodiments, using a double nick can improve specificity and reduce off-target effects. In some embodiments, the nicking enzyme is used with two separate guide RNAs targeting opposing DNA strands to create a double nick in the target DNA. In some embodiments, the nicking enzyme is used with two separate guide RNAs selected to be very close together to create a double nick in the target DNA.

[0201] In some embodiments, the RNA-guided DNA binder lacks lyase and nicking enzyme activity. In some embodiments, the RNA-guided DNA binder comprises a dCas DNA-binding polypeptide. The dCas polypeptide has DNA-binding activity but is substantially lacking in catalytic (lyase / nicking enzyme) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA binder or dCas DNA-binding polypeptide lacking lyase and nicking enzyme activity is a form of Cas nuclease (e.g., the Cas nucleases discussed above), wherein the endonuclease active site is inactivated, for example, by one or more alterations (e.g., point mutations) in the catalytic domain. See, for example, US 2014 / 0186958 A1; US ​​2015 / 0166980 A1.

[0202] In some embodiments, the RNA-guided DNA binder comprises APOBEC3 deaminase. In some embodiments, the APOBEC3 deaminase is APOBEC3A (A3A). In some embodiments, A3A is human A3A. In some embodiments, A3A is wild-type A3A.

[0203] In some embodiments, the RNA-guided DNA binder comprises an editor. An exemplary editor comprises human A3A fused with a Streptococcus pyogenes D10A Cas9 nickase. In some embodiments, the editor comprises human A3A fused with a Neisseria meningitidis D16A nickase. In some embodiments, a uracil glycosidase inhibitor (“UGI”) is provided to the editor. In some embodiments, the editor is fused with a UGI. In some embodiments, the UGI is not fused with the editor. In some embodiments, the mRNA encoding the editor and the mRNA encoding the UGI are formulated together in an LNP. In other embodiments, the editor and the UGI are provided in separate LNPs.

[0204] In some implementations, the RNA-guided DNA binder comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[0205] In some implementations, the heterologous functional domain can facilitate the transport of RNA-guided DNA binders into the cell nucleus. For example, the heterologous functional domain can be a nuclear localization signal (NLS).

[0206] In some embodiments, the heterofunctional domain can be able to alter the intracellular half-life of the RNA-guided DNA binder. In some embodiments, the half-life of the RNA-guided DNA binder can be prolonged. In some embodiments, the half-life of the RNA-guided DNA binder can be reduced. In some embodiments, the heterofunctional domain can be able to improve the stability of the RNA-guided DNA binder. In some embodiments, the heterofunctional domain can be able to reduce the stability of the RNA-guided DNA binder. In some embodiments, the heterofunctional domain can act as a signal peptide for protein degradation. In some embodiments, protein degradation can be mediated by proteases (such as proteasomes, lysosomal proteases, or calpases). In some embodiments, the heterofunctional domain can contain a PEST sequence. In some embodiments, the RNA-guided DNA binder can be modified by adding ubiquitin or polyubiquitin chains. In some embodiments, ubiquitin can be ubiquitin-like proteins (UBLs). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-associated modifier-1 (URM1), developmentally downregulated protein-8 expressed in neuronal progenitor cells (NEDD8, also known as Rub1 in Saccharomyces cerevisiae), human leukocyte antigen F-associated protein (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin folding modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

[0207] In some embodiments, the heterologous functional domain may be a labeling domain. Non-limiting examples of labeling domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the labeling domain may be a fluorescent protein. Suitable non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), and red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed). monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred) and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the labeled domain may be a purification tag and / or an epitope tag. Non-limiting exemplary tags include glutathione S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 8xHis, biotinylate carboxyl carrier protein (BCCP), polyHis, and calmodulin. Non-limiting exemplary reporter genes include glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, or fluorescent protein.

[0208] In other embodiments, the heterologous functional domain can target RNA-guided DNA binders to specific organelles, cell types, tissues, or organs. In some embodiments, the heterologous functional domain can target RNA-guided DNA binders to mitochondria.

[0209] In other embodiments, the heterologous functional domain may be an effector domain, such as an editing domain. When an RNA-guided DNA binder is directed to its target sequence, for example, when a Cas nuclease is directed to a target sequence by gRNA, the effector domain (such as an editing domain) may modify or influence the target sequence. In some embodiments, the effector domain (such as an editing domain) may be selected from nucleic acid-binding domains, nuclease domains (e.g., non-Cas nuclease domains), epigenetic modification domains, transcriptional activation domains, or transcriptional repression domains. In some embodiments, the heterologous functional domain is a nuclease, such as FokI nuclease. See, for example, U.S. Patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. For example, see Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression”, Cell 152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9-based transcription factors”, Nat. Methods 10:973-6 (2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering”, Nat. Biotechnol. 31:833-8 (2013); Gilbert et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes”, Cell 154:442-51 (2013). Therefore, RNA-guided DNA binders essentially become transcription factors, which can be guided by guide RNA to bind to desired target sequences. In some implementations, the DNA modification domain is a methylation domain, such as a demethylation or methyltransferase domain. In some implementations, the effector domain is a DNA modification domain, such as a base editing domain. In specific implementations, the DNA modification domain is a nucleic acid editing domain that introduces a specific modification into DNA, such as a deaminase domain. See, for example, WO 2015 / 089406; US2016 / 0304846.The nucleic acid editing domain, deaminase domain, and Cas9 variant described in WO 2015 / 089406 and US 2016 / 0304846 are each incorporated herein by reference in their entirety.

[0210] Nucleases may contain at least one domain that interacts with a guide RNA (“gRNA”). Additionally, the nuclease can be guided to a target sequence by the gRNA. In class 2 Cas nuclease systems, the gRNA interacts with both the nuclease and the target sequence, thereby guiding its binding to the target sequence. In some embodiments, the gRNA provides specificity for targeted cleavage, and the nuclease can be generic and pair with different gRNAs to cleave different target sequences. Class 2 Cas nucleases can pair with gRNA scaffold structures of the types, orthologs, and exemplary species listed above.

[0211] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to gRNA along with an RNA-guided DNA binder, such as a Cas nuclease, such as a Cas lyase, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the gRNA guides the RNA-guided DNA binder (such as Cas9) to a target sequence, the gRNA hybridizes to the target sequence, and the binder binds to the target sequence; where the binder is a lyase or nickase, cleavage or nick formation can occur after binding.

[0212] In some embodiments of this disclosure, the cargo for the LNP composition includes at least one gRNA containing a guide sequence for directing an RNA-guided DNA binder to target DNA, the RNA-guided DNA binder being a nuclease (e.g., a Cas nuclease, such as Cas9). The gRNA can direct a Cas nuclease or a class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to a class 2 Cas nuclease and provides specificity for cleavage by the class 2 Cas nuclease. In some embodiments, the gRNA and the Cas nuclease can form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex, such as a CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex can be a type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex can be a type V CRISPR / Cas complex, such as a Cpf1 / gRNA complex. The Cas nuclease and homologous gRNA can be paired. The gRNA scaffold structure paired with each class 2 Cas nuclease varies depending on the specific CRISPR / Cas system.

[0213] The terms “guide RNA,” “gRNA,” and simply “guide” are used interchangeably throughout this document to refer to a homologous guide nucleic acid that is an RNA-guided DNA binder. Guide RNA can include modified RNAs as described herein. gRNA can be, for example, a single guide RNA, or a combination of crRNA and trRNA (also known as tracrRNA). crRNA and trRNA can associate into a single RNA molecule (as a single guide RNA, sgRNA), or, for example, in two separate RNA strands (dgRNA). In some systems, gRNA can be crRNA (also known as CRISPR RNA). “Guide RNA” or “gRNA” refers to each type. trRNA can be a natural sequence or a trRNA sequence modified or altered compared to the natural sequence.

[0214] In some embodiments, mRNA encoding an RNA-guided DNA binder is formulated in a first LNP composition, and gRNA nucleic acid is formulated in a second LNP composition. In some embodiments, the first LNP composition and the second LNP composition are administered simultaneously. In other embodiments, the first LNP composition and the second LNP composition are administered sequentially. In some embodiments, the first LNP composition and the second LNP composition are combined prior to a pre-incubation step. In other embodiments, the first LNP composition and the second LNP composition are pre-incubated separately.

[0215] In some embodiments, the cargo may comprise a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding crRNA. In some embodiments, the nucleotide sequence encoding crRNA comprises a target sequence that sideways all or part of a repeating sequence derived from a natural CRISPR / Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding tracrRNA. In some embodiments, crRNA and tracrRNA may be encoded by two separate nucleic acids. In other embodiments, crRNA and tracrRNA may be encoded by a single nucleic acid. In some embodiments, crRNA and tracrRNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, crRNA and tracrRNA may be encoded by the same strand of a single nucleic acid. In some embodiments, the gRNA nucleic acid encodes sgRNA. In some embodiments, the gRNA nucleic acid encodes Cas9 nuclease sgRNA. In some embodiments, the gRNA nucleic acid encodes Cpf1 nuclease sgRNA.

[0216] The nucleotide sequence encoding the guide RNA can be operatively ligated to at least one transcriptional or regulatory control sequence, such as a promoter, 3' UTR, or 5' UTR. In one instance, the promoter can be a tRNA promoter, such as tRNALys3, or a tRNA chimera. See Mefferd et al., RNA. 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35: 2620-2628. In some embodiments, the promoter can be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters also include U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA can be operatively ligated to a mouse or human U6 promoter. In some embodiments, the gRNA nucleic acid is a modified nucleic acid. In some embodiments, the gRNA nucleic acid comprises a modified nucleoside or nucleotide. In some embodiments, the gRNA nucleic acid comprises a 5' end modification, such as a modified nucleoside or nucleotide, to stabilize and prevent nucleic acid integration. In other embodiments, the gRNA nucleic acid comprises double-stranded DNA with a 5' end modification on each strand. In some embodiments, the gRNA nucleic acid includes a reverse dideoxy-T or a reverse abase-free nucleoside or nucleotide as the 5' end modification. In some embodiments, the gRNA nucleic acid includes a label such as biotin, dethiobiotin-TEG, digoxigenin, and a fluorescent label including, for example, FAM, ROX, TAMRA, and AlexaFluor.

[0217] As used herein, a “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 and / or modification (e.g., cleavage) via an RNA-guided DNA binder. A “guide sequence” may also be referred to as a “target sequence” or a “spacer sequence.” The guide sequence can be 20 base pairs long, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences can also be used as guides, for example, sequences of 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the target sequence is located, for example, in a gene or on a chromosome and is complementary to the guide sequence. In some embodiments, the complementarity or similarity between the guide sequence and its corresponding target sequence can be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 adjacent nucleotides. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, wherein the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches, wherein the guide sequence contains at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches, wherein the guide sequence contains 20 nucleotides.

[0218] In some embodiments, multiple LNP compositions may be used synergistically and / or for individual purposes. In some embodiments, cells may be contacted with the first LNP composition and the second LNP composition described herein. In some embodiments, the first LNP composition and the second LNP composition each independently comprise one or more of mRNA, gRNA, and guide RNA nucleic acids. In some embodiments, the first LNP composition and the second LNP composition are administered simultaneously. In some embodiments, the first LNP composition and the second LNP composition are administered sequentially.

[0219] In some embodiments, a method is provided for generating multiple genome edits in cells (sometimes referred to herein and elsewhere as "multiplexing" or "multiplexed gene editing" or "multiplexed genome editing"). The ability to engineer multiple properties into a single cell depends on the ability to efficiently edit multiple target genes, including knockout and locus insertion, while maintaining viability and the desired cell phenotype. In some embodiments, the method includes culturing cells in vitro, contacting the cells with two or more lipid-nucleic acid assembly compositions, each lipid-nucleic acid assembly composition containing a nucleic acid genome editing tool capable of editing target sites, and expanding the cells in vitro. This method results in cells having more than one genome edit, wherein the genome edits are different. In some embodiments, a first LNP composition contains a first gRNA and a second LNP composition contains a second gRNA, wherein the first gRNA and the second gRNA contain different guide sequences complementary to different targets. In such embodiments, the LNP composition can accommodate multiplexed gene editing. In some embodiments, cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 lipid-nucleic acid assembly compositions. In some implementations, the cells are contacted with a composition of at least six lipid nucleic acid assemblies.

[0220] The target sequence of RNA-guided DNA-binding proteins (such as Cas proteins) includes both the positive and negative strands of genomic DNA (i.e., the given sequence and its inverse complement), because the nucleic acid substrate of Cas proteins is a double-stranded nucleic acid. Therefore, in cases where the guiding sequence is referred to as "complementary to the target sequence," it should be understood that the guiding sequence can guide gRNA to bind to the inverse complement of the target sequence. Thus, in some embodiments, where the guiding sequence binds to the inverse complement of the target sequence, the guiding sequence is identical to certain nucleotides of the target sequence (e.g., a target sequence excluding PAM), except that U is used instead of T in the guiding sequence.

[0221] In some embodiments, a method is provided for generating multiple genome edits in cells cultured in vitro, comprising the steps of: a) contacting the cells in vitro with at least a first lipid composition comprising a first nucleic acid, thereby generating contacted cells; b) contacting the cells in vitro with at least a second lipid composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and c) expanding the cells in vitro. In some embodiments, a method is provided for generating multiple genome edits in cells cultured in vitro, comprising the steps of: a) contacting the cells in vitro with at least a first lipid composition comprising a first nucleic acid, thereby generating contacted cells; b) culturing the contacted cells in vitro, thereby generating cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro. In other embodiments, the method further comprises contacting the cells in vitro with at least a third lipid composition comprising a third nucleic acid, wherein the third nucleic acid is different from the first and second nucleic acids. In other embodiments, the method further includes contacting cells in vitro with at least a fourth lipid composition comprising a fourth nucleic acid, wherein the fourth nucleic acid is different from the first, second, and third nucleic acids. In other embodiments, the method further includes contacting cells in vitro with at least a fifth lipid composition comprising a fifth nucleic acid, wherein the fifth nucleic acid is different from the first, second, third, and fourth nucleic acids. In other embodiments, the method further includes contacting cells in vitro with at least a sixth lipid composition comprising a sixth nucleic acid, wherein the sixth nucleic acid is different from the first, second, third, fourth, and fifth nucleic acids. In some embodiments, at least two of the lipid compositions are applied sequentially. In some embodiments, at least two of the lipid compositions are applied simultaneously. In some embodiments, the expanded cells exhibit an extended survival period.

[0222] In some embodiments, at least one of the aforementioned lipid compositions comprises a nucleic acid genome editing tool as described herein. In some embodiments, another lipid composition comprises an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is Cas9.

[0223] In some embodiments, the method of this disclosure further includes contacting the cell with a donor nucleic acid. In some embodiments, another lipid composition comprises a donor nucleic acid. The donor nucleic acid may be inserted into a target sequence. In some embodiments, the donor nucleic acid sequence is provided in vector form. In some embodiments, the donor nucleic acid encodes a target receptor. In some embodiments, the donor nucleic acid comprises a region homologous to a corresponding region of a T cell receptor sequence. A “target receptor” is present on the surface of a cell (e.g., a T cell) to allow the cell to bind to a polypeptide at a target site (e.g., a specific cell or tissue in an organism). In some embodiments, the target receptor is a CAR. In some embodiments, the target receptor is a universal CAR (UniCAR). In some embodiments, the target receptor is a TCR. In some embodiments, the target receptor is a T cell receptor fusion construct (TRuC). In some embodiments, the target receptor is a B cell receptor (BCR) (e.g., expressed on B cells). In some embodiments, the target receptor is a chemokine receptor. In some embodiments, the target receptor is a cytokine receptor.

[0224] The length of the target sequence can depend on the CRISPR / Cas system and the components used. For example, different classes of Cas nucleases from different bacterial species have different optimal target sequence lengths. Therefore, the target sequence can contain 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 nucleotides. In some embodiments, the target sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of the native CRISPR / Cas system. In some embodiments, the Cas nuclease and the gRNA scaffold are derived from the same CRISPR / Cas system. In some embodiments, the target sequence can contain 18-24 nucleotides, or consist of 18-24 nucleotides. In some embodiments, the target sequence can contain 19-21 nucleotides, or consist of 19-21 nucleotides. In some implementations, the target sequence may contain 20 nucleotides or consist of 20 nucleotides.

[0225] 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 some embodiments, the gRNA comprises crRNA and tracrRNA sufficient to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In some embodiments, the gRNA comprises crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0226] Some implementations also provide nucleic acids encoding the gRNAs described herein, such as expression cassettes. "Guide RNA nucleic acid" is used herein to refer to gRNAs (e.g., sgRNAs or dgRNAs) and gRNA expression cassettes, the latter being nucleic acids encoding one or more gRNAs.

[0227] Modified RNA

[0228] In some embodiments, the lipid composition (such as the LNP composition) contains modified nucleic acids, including modified RNA.

[0229] Modified nucleosides or nucleotides may be present in RNA, such as gRNA or mRNA. For example, gRNA or mRNA containing one or more modified nucleosides or nucleotides is referred to as “modified” RNA to describe the presence of one or more non-natural and / or natural components or conformations that replace or complement canonical A, G, C, and U residues. In some embodiments, modified RNA is synthesized using non-canonical nucleosides or nucleotides, referred to herein as “modified”.

[0230] Modified nucleosides and nucleotides may include one or more of the following: (i) alterations, such as replacing one or two non-linked phosphodiester oxygens and / or one or more linked phosphodiester oxygens in the phosphodiester backbone (exemplary backbone modification); (ii) alterations, such as replacing components of the ribose, for example replacing the 2' hydroxyl group on the ribose (exemplary sugar modification); (iii) bulk replacement of the phosphodiester portion with a "dephosphorylation" linker (exemplary backbone modification); (iv) modification or replacement of native nucleobases, including with non-canonical nucleobases (exemplary base modification); (v) replacement or modification of the ribose-phosphodiester backbone (exemplary backbone modification); (vi) modification of the 3' or 5' end of the polynucleotide, such as removing, modifying, or replacing the terminal phosphodiester group or binding portion, cap, or linker (such 3' or 5' cap modifications may include sugar and / or backbone modifications); and (vii) modification or replacement of sugars (exemplary sugar modification). Some embodiments include modifications to the 5' end of mRNA, gRNA, or nucleic acid. Some embodiments include modifications to mRNA, gRNA, or nucleic acid. Some embodiments include a 3' end modification of mRNA, gRNA, or nucleic acid. The modified RNA may contain both 5' and 3' end modifications. The modified RNA may contain one or more modified residues at non-terminal positions. In some embodiments, the gRNA includes at least one modified residue. In some embodiments, the mRNA includes at least one modified residue. In some embodiments, the modified gRNA contains a modification at one or more of the first five nucleotides at the 5' end. In some embodiments, the modified gRNA contains a modification at one or more of the first five nucleotides at the 5' end. In some embodiments, the modified gRNA contains a modification at one or more of the last five nucleotides at the 3' end.

[0231] Unmodified nucleic acids are readily degraded by, for example, intracellular nucleases or those found in serum. Nucleases, for instance, can hydrolyze phosphodiester bonds in nucleic acids. Therefore, on the one hand, 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 cell populations in vivo and in vitro. The term "innate immune response" includes cellular responses to exogenous nucleic acids (including single-stranded nucleic acids) involving the induction of cytokine (particularly interferon) expression and release, as well as cell death.

[0232] Therefore, in some embodiments, the RNA or nucleic acid contains at least one modification that confers increased or enhanced stability, including, for example, improved resistance to digestion by nucleases in vivo. As used herein, the terms “modified” and “modified” in relation to nucleic acids provided herein include at least one alteration that preferably enhances stability and makes the RNA or nucleic acid more stable than its wild-type or native form (e.g., resistant to nuclease digestion). As used herein, the terms “stable” and “stability” and such terms in relation to nucleic acids set forth herein, and particularly concerning RNA, refer to increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that are typically capable of degrading such RNA. Increased stability may include, for example, reduced sensitivity to hydrolysis or other damage caused by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within target cells or tissues, thus increasing or enhancing the retention of such RNA or nucleic acid in target cells, tissues, subjects, and / or cytoplasm. The stable RNA or nucleic acid molecules presented herein exhibit a longer half-life relative to their native, unmodified counterparts (e.g., the wild-type form of the molecule). The terms “modified” and “modified” also encompass, when used in relation to the mRNA of the LNP compositions disclosed herein, alterations that improve or enhance mRNA nucleic acid translation, including, for example, sequences that play a role in initiating protein translation (e.g., Kozak congruent sequences). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).

[0233] In some implementations, the RNA or nucleic acid has undergone chemical or biological modifications to make it more stable. Exemplary modifications to RNA or nucleic acids include base depletion (e.g., by deletion or by substituting one nucleotide for another) or base modifications, such as chemical modifications of the bases. As used herein, the phrase “chemical modification” includes modifications that introduce chemical properties different from those seen in native RNA or nucleic acids, such as covalent modifications, such as the introduction of modified nucleotides (e.g., nucleotide analogs, or side groups contained in such RNA that are not naturally present, such as deoxynucleosides or nucleic acid molecules).

[0234] In some embodiments of backbone modification, the phosphate groups of the modified residues can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, the modified residues (e.g., modified residues present in the modified nucleic acid) can include bulk replacement of unmodified phosphate moieties with modified phosphate groups as described herein. In some embodiments, backbone modification of the phosphate backbone can include alterations that produce uncharged joints or charged joints with asymmetric charge distributions.

[0235] Examples of modified phosphate groups include thiophosphates, selenophosphates, borate phosphates, boronic acid phosphates, hydrophosphonates, aminophosphates, alkyl or aryl phosphonates, and phosphate triesters. The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the aforementioned atoms or groups can make the phosphorus atom chiral. The stereosource phosphorus atom can have an "R" configuration (Rp herein) or an "S" configuration (Sp herein). The skeleton can also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate to the nucleoside) with nitrogen (bridging aminophosphate), sulfur (bridging thiophosphate), and carbon (bridging methylene phosphonate). Substitution can occur at any of the linked oxygens or at both linked oxygens. In some skeleton modifications, the phosphate group can be replaced by a phosphorus-free linking group. In some embodiments, the charged phosphate group can be replaced by a neutral portion. Examples of parts that can replace the phosphate ester group may include (but are not limited to) methylphosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide connector, sulfonate, sulfonamide, thiomethyl acetal, methyl acetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino.

[0236] mRNA

[0237] In some embodiments, the compositions or formulations disclosed herein comprise mRNA containing an open reading frame (ORF) encoding an RNA-guided DNA-binding agent (such as a Cas nuclease or a type 2 Cas nuclease as described herein). In some embodiments, mRNA is provided, used, or administered containing an ORF encoding an RNA-guided DNA-binding agent (such as a Cas nuclease or a type 2 Cas nuclease). The mRNA may contain one or more of a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenine tail. The mRNA may contain modified open reading frames, such as those encoding nuclear localization sequences or using alternative codons to encode proteins.

[0238] The mRNA in the disclosed LNP compositions may encode cell surface or intracellular polypeptides. The mRNA in the disclosed LNP compositions may encode, for example, normally secreted hormones, enzymes, receptors, polypeptides, peptides, or other proteins of interest. In some embodiments, the mRNA may optionally have chemical or biological modifications, such modifications being, for example, to improve the stability and / or half-life of such mRNA, or to improve or otherwise promote protein production.

[0239] Additionally, suitable modifications include changes to one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable than the codon found in the wild-type form of the mRNA. For example, an inverse relationship has been demonstrated between RNA stability and a higher number of cytidine (C) and / or uridine (U) residues, and RNA lacking C and U residues has been found to be stable 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 replacing another codon encoding the same or related amino acid with a codon encoding a specific amino acid. Modifications to the mRNA nucleic acid under consideration also include the incorporation of pseudouridine. In some embodiments, the modified uridine is a pseudouridine modified at position 1, for example, modified with halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or combinations thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl-pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine. Incorporating pseudouridine into mRNA nucleic acid can enhance stability and translational ability, as well as reduce in vivo immunogenicity. For example, see Karikó, K. et al., Molecular Therapy 16 (11): 1833-1840 (2008). Substitution and modification of mRNA can be carried out by methods readily known to those skilled in the art.

[0240] Compared to untranslated regions, the constraints on reducing the number of C and U residues in the coding regions of mRNA can be more stringent (i.e., it may be impossible to eliminate all C and U residues present in the message while preserving the ability to encode the desired amino acid sequence). However, the degeneracy of the genetic code provides an opportunity to reduce the number of C and / or U residues present in the sequence while maintaining the same coding capacity (i.e., the possibility of several different RNA sequence modifications depending on the amino acids encoded by the codon). The term "modification" also includes, for example, incorporating non-nucleotide-linked or modified nucleotides into the mRNA sequence (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secretory protein or enzyme). Such modifications include adding bases to the mRNA sequence (e.g., including a poly-A tail or a longer poly-A tail), altering the 3' UTR or 5' UTR, complexing the mRNA with an agent (e.g., a protein or complementary nucleic acid molecule), and including elements that alter the structure of the mRNA molecule (e.g., its formation of secondary structures).

[0241] Poly-A tails are believed to stabilize natural messengers. Therefore, long poly-A tails can be added to mRNA molecules, thereby making the mRNA more stable. Various well-established techniques can be used to add poly-A tails. For example, a long poly-A tail 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. Alternatively, poly-A tails can be added by transcription directly from PCR products. In some embodiments, the length of the poly-A tail is at least about 90, 200, 300, 400, or at least 500 nucleotides. In some embodiments, the length of the poly-A tail is adjusted to control the stability of the modified mRNA molecule, thereby controlling protein transcription. For example, since the length of the poly-A tail affects the half-life of the mRNA molecule, the length of the poly-A tail can be adjusted to improve the level of mRNA resistance to nucleases, thus controlling the timing of protein expression in cells. In some implementations, stable mRNA molecules are sufficiently resistant to in vivo degradation (e.g., by nucleases) to allow them to be delivered to target cells without a transfer medium.

[0242] In some embodiments, mRNA can be modified by incorporating 3' and / or 5' untranslated (UTR) sequences that are not naturally present in wild-type mRNA. In some embodiments, 3' and / or 5' flanking sequences of naturally flanked mRNA encoding a second unrelated protein can be incorporated into the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein to modify it. For example, 3' or 5' sequences from stable mRNA molecules (e.g., globulins, actin, GAPDH, tubulin, histiin, or citrate cycle enzymes) can be incorporated into the 3' and / or 5' regions of sense mRNA nucleic acid molecules to improve the stability of the sense mRNA molecule. See, for example, US2003 / 0083272.

[0243] A more detailed description of mRNA modifications can be found in US2017 / 0210698A1, pages 57-68, which are incorporated herein by reference.

[0244] Template Nucleic Acid

[0245] The methods disclosed herein may include the use of template nucleic acids. Templates can be used to alter or insert nucleic acid sequences at or near target sites of RNA-guided DNA-binding proteins, such as Cas nucleases, e.g., type 2 Cas nucleases. In some embodiments, the methods include introducing a template into a cell. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided, allowing editing to occur at two or more target sites. For example, different templates may be provided to edit a single gene or two different genes in a cell.

[0246] In some embodiments, the template can be used for homologous recombination. In some embodiments, homologous recombination can cause the template sequence or a portion of the template sequence to be integrated into the target nucleic acid molecule. In other embodiments, the template can be used for homology-directed repair, which involves DNA strand intrusion at a cleavage site in the nucleic acid. In some embodiments, homology-directed repair can result in the inclusion of the template sequence in the edited target nucleic acid molecule. In other embodiments, the template can be used for gene editing mediated by non-homologous end conjugation. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is incorporated. In some embodiments, the template includes side-attached inverted terminal repeats (ITRs).

[0247] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. It may also, or alternatively, correspond to, comprise, or consist of an exogenous sequence of the target cell. As used herein, the term "endogenous sequence" refers to a sequence that is natural for the cell. The term "exogenous sequence" refers to a sequence that is not natural for the cell, or a sequence whose natural location in the cell's genome is different. 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.

[0248] In some embodiments, the template contains ssDNA or dsDNA with inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided in the form of a vector, plasmid, microloop, nanoloop, or PCR product.

[0249] In some embodiments, nucleic acids are purified. In some embodiments, precipitation methods (e.g., LiCl precipitation, alcohol precipitation, or equivalent methods, such as those described herein) are used to purify nucleic acids. In some embodiments, chromatographic methods, such as HPLC-based methods, or equivalent methods (such as those described herein) are used to purify nucleic acids. In some embodiments, both precipitation methods (e.g., LiCl precipitation) and HPLC-based methods are used to purify nucleic acids. In some embodiments, nucleic acids are purified by tangential flow filtration (TFF).

[0250] Cell types

[0251] The ionizable lipid compounds and LNP compositions disclosed herein can be used for in vivo and in vitro gene editing. In one embodiment, one or more LNP compositions described herein may be administered to a subject in need. In one embodiment, one or more LNP compositions described herein may be contacted with cells. In one embodiment, a therapeutically effective amount of the composition described herein may be contacted with cells of a subject in need. In one embodiment, genetically engineered cells may be generated by contacting cells with the LNP composition described herein. In various embodiments, as stated above, the method includes introducing a template nucleic acid into cells or a subject. In some embodiments, the cells are in vivo. In some embodiments, the cells are hepatocytes. In a preferred embodiment, the cells are in vivo hepatocytes. In some embodiments, the cells are immune cells. As used herein, “immune cells” means cells of the immune system, including, for example, lymphocytes (e.g., T cells, B cells, natural killer cells (“NK cells” and NKT cells or iNKT cells)), monocytes, macrophages, basophils, dendritic cells, or granulocytes (e.g., neutrophils, eosinophils, and basophils). In some embodiments, the cells are primary immune cells. In some embodiments, the immune system cells may be selected from CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DCs). In some embodiments, the immune cells are allogeneic. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are adaptive immune cells. In some embodiments, the cells are T cells. In some embodiments, the cells are B cells. In some embodiments, the cells are NK cells.

[0252] Although the invention has been described in conjunction with the illustrated embodiments, it should be understood that the embodiments are not intended to limit the invention to those embodiments. Rather, this disclosure is intended to cover all alternatives, modifications, and equivalents that may be included within the invention as defined in the appended claims, including equivalents of specific features.

[0253] The foregoing general description and detailed description, as well as the following examples, are exemplary and illustrative only and do not limit the teaching content. Section headings used herein are for organizational purposes only and should not be construed as limiting the intended subject matter in any way. In the event of any conflict between any terminology defined in this specification and any reference incorporated herein by reference, this specification shall prevail. Unless otherwise stated, all scopes given herein cover all endpoints.

[0254] definition

[0255] It should be noted that, unless the context clearly indicates otherwise, the singular forms “a” and “the” as used herein include a plural of indicators. Thus, for example, a reference to “composition” includes multiple compositions, and a reference to “cell” includes multiple cells, etc. Unless otherwise stated, the use of “or” is inclusive and means “and / or”.

[0256] Unless expressly stated in the foregoing description, embodiments in this specification that "comprise" various components are also to be considered as "consisting of the listed components" or "essentially composed of the listed components"; embodiments in this specification that "consist of various components" are also to be considered as "containing the listed components" or "essentially composed of the listed components"; embodiments in this specification that "about" various components are also to be considered as "being in" the listed components; and embodiments in this specification that "essentially consist of various components" are also to be considered as "consisting of the listed components" or "containing the listed components" (this interchangeability does not apply to the use of these terms in the claims).

[0257] Numerical ranges include the numerical values ​​that define the range. Measured and measurable values ​​should be understood as approximate, taking into account significant figures and measurement-related errors. As used herein, the terms “about” and “approximately” have their meanings as understood in the art; the use of one relative to the other does not necessarily imply a different range. Unless otherwise indicated, as will be understood by one of ordinary skill in the art, the figures used herein, with or without the modifiers such as “about” or “approximately,” should be understood to cover normal divergence and / or fluctuations. In some embodiments, unless otherwise stated or otherwise clearly apparent from the context, the terms “about” or “approximately” may refer to a range of values ​​(greater than or less than) within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or less of the reference value in either direction (except where such values ​​would exceed 100% of the possible value).

[0258] As used herein, the term "contact" refers to establishing a physical connection between two or more entities. For example, contacting mammalian cells with a nanoparticle composition means that the mammalian cells and nanoparticles share a physical connection. Methods for contacting cells with external entities in vivo and in vitro are well known in biotechnology. For example, contacting a nanoparticle composition with mammalian cells distributed within a mammalian body can be performed via different routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve varying amounts of the nanoparticle composition. Furthermore, the nanoparticle composition can contact more than one type of mammalian cell.

[0259] As used herein, the term “delivery” means providing an entity to a destination. For example, delivering a therapeutic and / or preventative agent to a subject may involve administering to the subject a nanoparticle composition comprising the therapeutic and / or preventative agent (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes). Applying a nanoparticle composition to a mammal or mammalian cells may involve contacting one or more cells with the nanoparticle composition.

[0260] As used herein, “encapsulation efficiency” refers to the amount of therapeutic and / or preventive agents that become part of the nanoparticle composition relative to the initial total amount of therapeutic and / or preventive agents used in the preparation of the nanoparticle composition. For example, if 97 mg of a total of 100 mg of therapeutic and / or preventive agents initially provided to the composition are encapsulated in the nanoparticle composition, the encapsulation efficiency can be expressed as 97%. As used herein, “encapsulation” can mean complete, substantial, or partial encapsulation, closure, enclosure, or packaging.

[0261] As used herein, the terms “editing efficiency,” “editing percentage,” “insertion / deletion efficiency,” and “insertion / deletion percentage” refer to the total number of sequence reads with insertions or deletions relative to the total number of sequence reads. For example, editing efficiency at a target site in the genome can be measured by isolating genomic DNA and sequencing it to identify the presence of insertions and deletions introduced by gene editing. In some embodiments, editing efficiency is measured as the percentage of cells that no longer contain the gene (e.g., CD3) after treatment relative to the number of cells that initially contained the gene (e.g., CD3+ cells).

[0262] As used herein, “knockdown” refers to a reduction in the expression of a specific gene product, such as protein, mRNA, or both. Protein knockdown can be measured by detecting the total cellular amount of protein from a sample, such as a tissue, fluid, or cell population of interest. It can also be measured by measuring protein substitutes, labels, or activity. Methods for measuring mRNA knockdown are known and involve sequencing mRNA isolated from the sample of interest. In some embodiments, “knockdown” may refer to a certain loss of expression of a specific gene product, such as a reduction in the amount of transcribed mRNA or a reduction in the amount of protein expressed by a cell population, including in vivo cell populations such as those found in tissues.

[0263] As used in this article, "knockout" refers to the loss of expression of a specific gene or protein in a cell. Knockout can be measured by detecting the total amount of the protein in, for example, cells, tissues, or cell populations. It can also be detected at, for example, the genomic or mRNA level.

[0264] As used herein, the term "biodegradable" refers to a material that, when introduced into cells, breaks down into components that are reusable or disposable by the cells through cellular mechanisms (e.g., enzymatic degradation) or by hydrolysis without producing significant toxic effects on the cells. In some embodiments, the components resulting from the breakdown of the biodegradable material do not induce inflammation and / or other harmful effects in vivo. In some embodiments, the biodegradable material is enzymatically broken down. Or, additionally, in some embodiments, the biodegradable material is broken down by hydrolysis.

[0265] As used herein, “N / P ratio” is the molar ratio of ionizable nitrogen-containing lipids (e.g., compounds of formulas (I)-(IV)) to phosphate groups in RNA, for example in nanoparticle compositions comprising lipid components and RNA.

[0266] The composition may also include salts of one or more compounds. The salt may be a pharmaceutically acceptable salt. As used herein, a “pharmaceutically acceptable salt” refers to a derivative of the disclosed compound, wherein the parent compound is altered by converting an existing acid or base moiety into its salt form (e.g., by reacting the 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); basic or organic salts of acidic residues (such as carboxylic acids); etc. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, diglucuronate, dodecyl sulfate, ethanesulfonate, fumarate, glucohepanoate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including (but not limited to) ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. Pharmaceutically acceptable salts of this disclosure include conventionally non-toxic salts of parent compounds, for example, formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of this disclosure can be synthesized by conventional chemical methods from parent compounds containing a basic or acidic moiety. Typically, such salts are prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, in an organic solvent, or in a mixture of both; typically, a non-aqueous medium, such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, is preferred.A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, PH Stahl and CG 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.

[0267] As used herein, the "polydispersity index" is a ratio that describes the uniformity of the particle size distribution in a system. A small value (e.g., less than 0.3) indicates a narrow particle size distribution. In some embodiments, the polydispersity index may be less than 0.1.

[0268] As used in this article, "transfection" refers to the introduction of a substance (such as RNA) into a cell. Transfection can occur, for example, in vitro, in vivo, or in vivo.

[0269] The following section elaborates on the definitions of specific functional groups and chemical terms. Chemical elements are defined according to the periodic table (CAS version). Handbook of Chemistry and Physics The 75th edition uses the inner cover for identification, and the specific functional groups are generally defined as described herein. Furthermore, the general principles of organic chemistry, as well as specific functional groups and reactivity, are explained in... Organic Chemistry , Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry , 5th edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations , VCHPublishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis , 3rd edition, Cambridge University Press, Cambridge, 1987.

[0270] The compounds described herein may contain one or more asymmetric centers and therefore may exist in various stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. Isomers can be separated from mixtures by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation of chiral salts and crystallization; or preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al. Enantiomers, Racemates and Resolutions Wiley Interscience, New York, 1981; Wilen et al. Tetrahedron 33:2725 (1977); Eliel, EL Stereochemistry of Carbon Compounds, McGraw-Hill, NY, 1962; and Wilen, SH, Tables of Resolving Agents and Optical Resolutions Page 268, edited by EL Eliel, Univ. of Notre Dame Press, Notre Dame, IN 1972. This invention also covers compounds in the form of individual isomers that are substantially free of other isomers, or in the form of mixtures of various isomers.

[0271] Unless otherwise stated, the structures described herein are intended to include all isomers (e.g., enantiomers, diastereomers, and geometric (or configurations)) of the structure; for example, the R and S configurations of each stereocenter. Therefore, single stereochemical isomers of the compounds of the present invention, as well as mixtures of enantiomers, diastereomers, and geometric (or configurations), are within the scope of the present invention. Unless otherwise stated, all tautomers of the compounds of the present invention are within the scope of the present invention.

[0272] Unless otherwise stated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, those that replace hydrogen with deuterium or tritium, or those that use... 18 F replacement 19 F or use 13 C or 14 C Replacement 12 Compounds having the structure of this invention, other than C, are within the scope of this disclosure. Such compounds can be used as analytical tools or probes, for example, in bioassays.

[0273] When listing a range of values, the intention is to encompass every value within that range and its subranges. For example, "C 1-6The term "alkyl" is intended to encompass C1, C2, C3, C4, C5, C6, and C6 alkyl groups. 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0274] As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetradecyl, 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, an alkyl group can be substituted with one or more groups, including (but not limited to) alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfoxide, sulfonate, carboxylate, or thiol groups as described herein. "Low-carbon alkyl" refers to an alkyl group containing one to six (e.g., one to four) carbon atoms.

[0275] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one carbon-carbon double bond and 2 to 12 carbon atoms, and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter referring to an alkenyl moiety having a substituent replacing a hydrogen atom on one or more carbons of the alkenyl group. Such substituents may be present on one or more carbons, included or excluded from one or more double bonds. Furthermore, as discussed below, such substituents include all those considered for alkyl groups unless stability is suppressed. For example, an alkenyl group may be substituted with one or more of the considered alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups. Exemplary alkenyl groups include (but are not limited to) vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2). In some embodiments, the heteroalkenyl group has 3 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, the alkenyl group has 3 to 9 carbon atoms (“C…”). 3-9Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 3 to 8 carbon atoms (“C”). 3-8 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 3 to 7 carbon atoms (“C”). 3-7 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 3 to 6 carbon atoms (“C”). 3-6 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 3 to 5 carbon atoms (“C”). 3-5 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 3 to 4 carbon atoms (“C”). 3-4 The alkenyl group (“C3 alkenyl”) has three carbon atoms in some embodiments. The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). 2-4 Examples of alkenyl groups include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), etc. 2-6 Examples of alkenyl groups include the C group mentioned above. 2-4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Other examples of alkenyl groups include heptenyl (C7), octenyl (C8), octetrinyl (C8), etc. Unless otherwise specified, each example of an alkenyl group is independently unsubstituted (“unsubstituted alkenyl”) or substituted with one or more substituents (“substituted alkenyl”). In some embodiments, the alkenyl group is an unsubstituted C5 group. 3-10 Alkenyl group. In some embodiments, the alkenyl group is a substituted C-group. 3-10 Alkenyl. In alkenyl groups, the stereochemical C=C double bond is not specified (e.g., -CH=CHCH3 or...). It can be an (E)- or (Z)- double bond.

[0276] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one carbon-carbon triple bond, and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl," the latter referring to an alkynyl moiety having a substituent replacing a hydrogen atom on one or more carbons of the alkynyl group. Such substituents may be present on one or more carbons included or excluded in one or more double bonds. Furthermore, as discussed below, such substituents include all those considered for use with alkyl groups unless stability is inhibited. For example, an alkynyl group may be substituted with one or more of the considered alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups. The term "alkynyl" refers to a straight-chain or branched hydrocarbon group ("C-12") having 2 to 12 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds). 2-12 The alkynyl group (“C10”) is present in some embodiments. In some embodiments, the alkynyl group has 3 to 10 carbon atoms (“C10”). 3-10The alkynyl group (“C”) is present in some embodiments. In some embodiments, the alkynyl group has 3 to 9 carbon atoms (“C”). 3-9 The alkynyl group (“C”) is present in some embodiments. In some embodiments, the alkynyl group has 3 to 8 carbon atoms (“C”). 3-8 The alkynyl group (“C”) is present in some embodiments. In some embodiments, the alkynyl group has 3 to 7 carbon atoms (“C”). 3-7 The alkynyl group (“C6”) is present in some embodiments. In some embodiments, the alkynyl group has 3 to 6 carbon atoms (“C6”). 3-6 The alkynyl group (“C”) is present in some embodiments. In some embodiments, the alkynyl group has 3 to 5 carbon atoms (“C”). 3-5 The alkynyl group (“C”) is present in some embodiments. In some embodiments, the alkynyl group has 3 to 4 carbon atoms (“C”). 3-4 The alkynyl group has three carbon atoms (“C3 alkynyl”) in some embodiments. The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).

[0277] "Alkylene" refers to a divalent alkyl group, which can be branched or unbranched (i.e., straight-chain). Any of the monovalent alkyl groups mentioned above can be converted into an alkylene group by abstracting a second hydrogen atom from the alkyl group. Representative alkylene groups include C14 and C24. 2-4 Alkylene and C 2-3 Alkylenes. Typical alkylenes include (but are not limited to) -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc. Alkylenes may be substituted or unsubstituted. For example, an alkylene may be substituted with one or more groups, including (but not limited to) alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfoxide, sulfonate, carboxylate, or thiol groups as set forth herein.

[0278] The term "alkenyl" includes a divalent, straight-chain or branched, unsaturated, acyclic hydrocarbon group having at least one carbon-carbon double bond and, in one embodiment, no carbon-carbon triple bond. Any of the monovalent alkenyl groups mentioned above can be converted into an alkenyl group by abstracting a second hydrogen atom from the alkenyl group. Representative alkenyl groups include C... 2-6 Alkenyl group.

[0279] Term "C" x-y "When used in conjunction with a chemical moiety (such as an alkyl or alkylene group), it is intended to include a group containing x to y carbons in the chain. For example, the term 'C'..." x-y "Alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain and branched alkyl and alkylene groups containing x to y carbons in the chain.

[0280] The term "alkoxy" refers to an alkyl group that is attached to an oxygen-containing alkyl group, preferably an alkyl group with a low carbon number. Representative alkoxy groups include methoxy, ethoxy, propoxy, and tert-butoxy.

[0281] In some embodiments, this disclosure relates to compounds represented by structural formula I.

[0282] (I),

[0283] or its salt, wherein:

[0284] A is either O or NH.

[0285] X 1 C 1-5 Alkylene

[0286] R 1 and R 2 Each independently is C 1-3 alkyl, or

[0287] R 1 and R 2 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring, and

[0288] Z 1 C 1-5 Alkylene

[0289] Y 1 and Y 2 Each independently is C 3-10 Alkoxy or O(C) 3-10 (alkynyl group),

[0290] Z 2 C 1-5 Alkylene or direct bond, and

[0291] Y 3 and Y 4 Each independently is C 3-10 Alkoxy or O(C) 3-10 acetylenic group), or

[0292] Y 3 and Y 4 Each independently is C 3-10 Alkyl or C 3-10 alkynyl group,

[0293] The premise is that if Y 1 Y 2 Y 3 and Y 4 Each independently is C 3-10 alkoxy group, then R 1 and R 2Not a C2 alkyl group, and R 1 and R 2 It does not form a 6-membered ring with the nitrogen atom it is attached to.

[0294] In some implementations, A is O. Or, A is NH.

[0295] In some implementation schemes, X 1 C 1-4 Alkylene, C 1-3 Alkylene, C 1-5 Alkylene, C 1-2 Alkylene, C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene. For example, X 1 C 2-3 Alkylenes, such as C2-alkylenes or C3-alkylenes.

[0296] In some implementation schemes, Z 1 C 1-4 Alkylene, C 1-3 Alkylene, C 1-5 Alkylene, C 1-2 Alkylene, C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene. For example, Z 1 C 2-3 Alkylenes, such as C2-alkylenes or C3-alkylenes.

[0297] In some implementation schemes, Z 2 C 1-4 Alkylene, C 1-3 Alkylene, C 1-5 Alkylene, C 1-2 Alkylene, C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene. For example, Z 1 C 2-3 Alkylenes, such as C2-alkylenes or C3-alkylenes. In some embodiments, Z 2 For direct keys.

[0298] In some implementation schemes, Z 1 C 2-3 Alkylene, and Z 2 For direct keys.

[0299] In some implementation schemes, Y 1 and Y 2 Each independently is C 3-9 Alkyl groups. For example, Y 1and Y 2 Each independently is C 4-9 Alkoxy, C 5-9 Alkoxy, C 6-9 Alkoxy, C 7-9 Alkoxy, C 8-9 Alkoxy, C 3-8 Alkoxy, C 3-7 Alkoxy, C 3-6 Alkoxy, C 3-5 Alkoxy, C 3-4 Alkoxy, C 4-8 Alkoxy, C 4-7 Alkoxy, C 4-6 Alkoxy, C 4-5 Alkoxy, C 5-8 Alkoxy, C 5-7 Alkoxy, C 5-6 Alkoxy, C 6-8 Alkoxy, C 6-7 Alkoxy or C 7-8 Alkyl groups. For example, Y 1 and Y 2 Each independently is C 6-9 Alkyl group.

[0300] In some implementation schemes, Y 3 and Y 4 Each independently is C 3-9 Alkyl groups. For example, Y 3 and Y 4 Each independently is C 4-9 Alkoxy, C 5-9 Alkoxy, C 6-9 Alkoxy, C 7-9 Alkoxy, C 8-9 Alkoxy, C 3-8 Alkoxy, C 3-7 Alkoxy, C 3-6 Alkoxy, C 3-5 Alkoxy, C 3-4 Alkoxy, C 4-8 Alkoxy, C 4-7 Alkoxy, C 4-6 Alkoxy, C 4-5 Alkoxy, C 5-8 Alkoxy, C 5-7 Alkoxy, C 5-6 Alkoxy, C 6-8 Alkoxy, C 6-7 Alkoxy or C 7-8 Alkyl groups. For example, Y 3 and Y 4 Each independently is C 6-9 Alkyl group.

[0301] In some implementation schemes, Y 3 and Y 4 Each independently is C 3-9 Alkyl group. For example, Y 3 and Y 4 Each independently is C 4-9 Alkyl, C 5-9 Alkyl, C 6-9 Alkyl, C 7-9 Alkyl, C 8-9 Alkyl, C 3-8 Alkyl, C 3-7 Alkyl, C 3-6 Alkyl, C 3-5 Alkyl, C 3-4 Alkyl, C 4-8 Alkyl, C 4-7 Alkyl, C 4-6 Alkyl, C 4-5 Alkyl, C 5-8 Alkyl, C 5-7 Alkyl, C 5-6 Alkyl, C 6-8 Alkyl, C 6-7 Alkyl or C 7-8 Alkyl group. For example, Y 3 C 6-9 Alkyl, and Y 4 C 3-5 alkyl.

[0302] In some implementation schemes, R 1 and R 2 Each independently is C 1-3 Alkyl group. For example, R 1 and R 2 Each can be methyl, ethyl, propyl, or isopropyl.

[0303] In some embodiments, the compound of formula I is represented by one of the following structural formulas:

[0304] ,

[0305] ,

[0306] and

[0307] ,

[0308] Or its salt.

[0309] In some embodiments, this disclosure relates to compounds represented by structural formula II.

[0310] (II),

[0311] or its salt,

[0312] in,

[0313] Q is CR 3 or ,

[0314] A can be O, NH, or a direct bond.

[0315] X 1 C 1-5 Alkylene

[0316] R 1 and R 2 Each independently is C 1-3 alkyl, or

[0317] R 1 The nitrogen atom and X it is attached to 1 One to three carbon atoms together form a 4-, 5-, or 6-membered ring, or

[0318] R 1 and R 2 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring, and

[0319] R 3 For H or C 1-3 alkyl,

[0320] Z 1 and Z 2 Each independently is C 1-5 Alkylene

[0321] Z 3 and Z 4 Each is independently -C(=O)O- in any direction.

[0322] Z 5 and Z 6 Each is independently a direct key or C. 1-3 Alkylene

[0323] Y 1 Selected from H, C 1-10 Alkyl, C 3-10 alkenyl and C 3-10 alkynyl group,

[0324] Y 2 Y 3 and Y 4 Each is independently selected from C 3-10 Alkyl, C 3-10 alkenyl and C 3-10 alkynyl group, and

[0325] n is 0 or 1.

[0326] In some implementations, Q is .

[0327] In some embodiments, compounds of formula II are represented by structural formula IIa.

[0328] (IIa).

[0329] In some implementations, n is 0. Or, n is 1.

[0330] In some implementations, A is O. Or, A is NH. Or, A is a direct bond.

[0331] In some implementation schemes, X 1 C 1-4 Alkylene, C 1-3 Alkylene, C 1-5 Alkylene, C 1-2 Alkylene, C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene. For example, X 1 C 2-3 Alkylenes, such as C2-alkylenes or C3-alkylenes. For example, X 1 It is a C2 alkylene group.

[0332] In some implementation schemes, Z 1 C 1-4 Alkylene, C 1-3 Alkylene, C 1-5 Alkylene, C 1-2 Alkylene, C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene. For example, Z 1 C 2-3 Alkylenes, such as C2-alkylenes or C3-alkylenes.

[0333] In some implementation schemes, Z 2 C 1-4 Alkylene, C 1-3 Alkylene, C 1-5 Alkylene, C 1-2 Alkylene, C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene. For example, Z 1 C 2-3 Alkylenes, such as C2-alkylenes or C3-alkylenes.

[0334] In some implementation schemes, Z 1 and Z 2 Each is independently a C3 alkylene group. In some embodiments, Z 1 and Z 2 Each is independently a C4 alkylene group. In some embodiments, Z 1 and Z 2 Each is independently a C5 alkylene group.

[0335] In some implementations, Z 1 It is a C2 alkylene group; and Z 2 It is a C3 alkylene group.

[0336] In some implementation schemes, Z 1 and Z 2 Each is independently a C3 alkylene group, A is NH, and X is... 1 It is a C2 alkylene group.

[0337] In some implementation schemes, Z 3 and Z 4 Each for themselves , where 'a' indicates that Z is respectively 1 and Z 2 The connection point.

[0338] In some implementation schemes, Z 3 for Where b indicates Z 1 The connection point of Z; and Z 4 for Where b indicates Z 2 The connection point.

[0339] In some implementation schemes, Z 5 and Z 6 Each is a direct key independently.

[0340] In some implementation schemes, Z 5 It is a C1 alkylene group; and Z 6 For direct keys.

[0341] In some implementation schemes, Y 1 For H.

[0342] In some implementation schemes, Y 1 Y 2 Y 3 and Y 4 Each independently is C 3-9 Alkyl group. For example, Y 1 Y 2 Y 3 and Y 4 Each independently is C 4-9Alkyl, C 5-9 Alkyl, C 6-9 Alkyl, C 7-9 Alkyl, C 8-9 Alkyl, C 3-8 Alkyl, C 3-7 Alkyl, C 3-6 Alkyl, C 3-5 Alkyl, C 3-4 Alkyl, C 4-8 Alkyl, C 4-7 Alkyl, C 4-6 Alkyl, C 4-5 Alkyl, C 5-8 Alkyl, C 5-7 Alkyl, C 5-6 Alkyl, C 6-8 Alkyl, C 6-7 Alkyl or C 7-8 Alkyl group. For example, Y 1 Y 2 Y 3 and Y 4 Each independently is C 7-9 Alkyl groups, such as C8 alkyl groups.

[0343] In some implementation schemes, Y 1 and Y 2 Each independently is C 5-7 Alkyl, and Y 3 and Y 4 Each independently is C 3-5 alkyl.

[0344] In some implementation schemes, R 1 and R 2 Each independently is C 1-3 Alkyl group. For example, R 1 and R 2 Each can be independently methyl, ethyl, propyl, or isopropyl. For example, R 1 and R 2 Each is a C2 alkyl group.

[0345] In some implementation schemes, R 1 and R 2 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring. For example, R 1 and R 2 It forms a 5-membered ring together with the nitrogen atom it is attached to. Or, R 1 and R 2 It forms a 6-membered ring with the nitrogen atom it is attached to. Or, R 1 and R 2 Together with the nitrogen atom it is attached to, it forms a 7-membered ring.

[0346] In some embodiments, the compound of formula II is represented by one of the following structural formulas:

[0347] ,

[0348] ,

[0349] ,

[0350] ,

[0351] ,

[0352] ,

[0353] ,

[0354] ,

[0355] ,

[0356] ,

[0357] ,

[0358] ,

[0359] ,

[0360] ,

[0361] ,

[0362] ,

[0363] ,

[0364] ,

[0365] and

[0366] ,

[0367] Or its salt.

[0368] In some embodiments, the compound of formula II is represented by one of the following structural formulas:

[0369] ,

[0370] ,

[0371] ,

[0372] ,

[0373] ,

[0374] ,

[0375] ,

[0376] ,

[0377] ,

[0378] ,

[0379] ,

[0380] ,

[0381] ,

[0382] ,

[0383] and

[0384] ,

[0385] Or its salt.

[0386] In some embodiments, this disclosure relates to compounds represented by structural formula III.

[0387] (III),

[0388] or its salt,

[0389] in:

[0390] Q is CH or ,

[0391] A is either O or NH.

[0392] X 1 C 1-5 Alkylene

[0393] R 1 and R 2 Each independently is C 1-3 alkyl, or

[0394] R 1The nitrogen atom and X it is attached to 1 One to three carbon atoms together form a 4-, 5-, or 6-membered ring, or

[0395] R 1 and R 2 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring, and

[0396] Z 1 C 2-9 Alkylene

[0397] Z 2 C 1-3 Alkylene or direct bond, and

[0398] Y 1 and Y 2 Each is independently selected from C 3-10 Alkyl, C 3-10 alkenyl and C 3-10 Alkyne group.

[0399] In some implementations, Q is .

[0400] In some embodiments, compounds of formula III are represented by structural formula IIIa.

[0401] (IIIa).

[0402] In some implementations, A is O. Or, A is NH.

[0403] In some implementation schemes, X 1 C 1-4 Alkylene, C 1-3 Alkylene, C 1-5 Alkylene, C 1-2 Alkylene, C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene. For example, X 1 C 2-3 Alkylenes, such as C2-alkylenes or C3-alkylenes. For example, X 1 It is a C3 alkylene group.

[0404] In some implementation schemes, Z 1 C 3-9 Alkylene. For example, Z 1 C 4-9 Alkylene, C 5-9 Alkylene, C 6-9 Alkylene, C 7-9 Alkylene, C 8-9 Alkylene, C 3-8Alkylene, C 3-7 Alkylene, C 3-6 Alkylene, C 3-5 Alkylene, C 3-4 Alkylene, C 4-8 Alkylene, C 4-7 Alkylene, C 4-6 Alkylene, C 4-5 Alkylene, C 5-8 Alkylene, C 5-7 Alkylene, C 5-6 Alkylene, C 6-8 Alkylene, C 6-7 Alkylene or C 7-8 Alkyl group. For example, Z 1 C 3-5 Alkylene, C 5-7 Alkylene or C 7-9 Alkylene.

[0405] In some implementation schemes, Z 2 It is a direct key. In some implementations, Z 2 C 1-3 Alkylene.

[0406] In some implementation schemes, Y 1 and Y 2 Each independently is C 3-9 Alkyl group. For example, Y 1 and Y 2 Each independently is C 4-9 Alkyl, C 5-9 Alkyl, C 6-9 Alkyl, C 7-9 Alkyl, C 8-9 Alkyl, C 3-8 Alkyl, C 3-7 Alkyl, C 3-6 Alkyl, C 3-5 Alkyl, C 3-4 Alkyl, C 4-8 Alkyl, C 4-7 Alkyl, C 4-6 Alkyl, C 4-5 Alkyl, C 5-8 Alkyl, C 5-7 Alkyl, C 5-6 Alkyl, C 6-8 Alkyl, C 6-7 Alkyl or C 7-8 Alkyl group. In some embodiments, Y 1 and Y 2 Each independently is C 3-5 Alkyl, C 5-7 Alkyl or C 7-9 alkyl.

[0407] In some embodiments, the compound of formula III is represented by one of the following structural formulas:

[0408] ,

[0409] ,

[0410] ,

[0411] ,

[0412] ,

[0413] ,

[0414] ,

[0415] ,

[0416] ,

[0417] ,

[0418] ,

[0419] ,

[0420] and

[0421] ,

[0422] Or its salt.

[0423] In some embodiments, this disclosure relates to compounds represented by structural formula IV.

[0424] (IV),

[0425] or its salt,

[0426] in:

[0427] Q is CH or ,

[0428] A can be O, NH, or a direct bond.

[0429] X 1 and X 2 Each independently is C 1-5 Alkylene

[0430] R 1 Selected from C 3-9Alkyl, C 3-9 alkenyl and C 3-9 alkynyl group,

[0431] R 2 and R 3 Each independently is C 1-3 alkyl, or

[0432] R 2 and R 3 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring, and

[0433] Z 1 C 6-10 alkylene, and

[0434] Y 1 and Y 2 Each is independently selected from C 3-10 Alkyl, C 3-10 alkenyl and C 3-10 Alkyne group.

[0435] In some implementations, Q is .

[0436] In some implementations, compounds of formula IV are represented by the structural formula IVa.

[0437] (IVa).

[0438] In some implementations, A is O. Or, A is NH. Or, A is a direct bond.

[0439] In some implementation schemes, X 1 C 1-4 Alkylene, C 1-3 Alkylene, C 1-5 Alkylene, C 1-2 Alkylene, C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene. For example, X 1 C 2-3 Alkylenes, such as C2-alkylenes or C3-alkylenes.

[0440] In some implementation schemes, X 2 C 1-4 Alkylene, C 1-3 Alkylene, C 1-5 Alkylene, C 1-2 Alkylene, C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene. For example, X 1 C2-3 Alkylenes, such as C2-alkylenes or C3-alkylenes.

[0441] In some implementation schemes, Z 1 C 3-9 Alkylene. For example, Z 1 C 4-9 Alkylene, C 5-9 Alkylene, C 6-9 Alkylene, C 7-9 Alkylene, C 8-9 Alkylene, C 3-8 Alkylene, C 3-7 Alkylene, C 3-6 Alkylene, C 3-5 Alkylene, C 3-4 Alkylene, C 4-8 Alkylene, C 4-7 Alkylene, C 4-6 Alkylene, C 4-5 Alkylene, C 5-8 Alkylene, C 5-7 Alkylene, C 5-6 Alkylene, C 6-8 Alkylene, C 6-7 Alkylene or C 7-8 Alkyl group. For example, Z 1 C 7-9 Alkylene.

[0442] In some implementation schemes, Y 1 and Y 2 Each independently is C 3-10 Alkyl group. For example, Y 1 and Y 2 Each independently is C 4-10 Alkyl, C 5-10 Alkyl, C 6-10 Alkyl, C 7-10 Alkyl, C 8-10 Alkyl, C 9-10 Alkyl, C 4-9 Alkyl, C 5-9 Alkyl, C 6-9 Alkyl, C 7-9 Alkyl, C 8-9 Alkyl, C 3-8 Alkyl, C 3-7 Alkyl, C 3-6 Alkyl, C 3-5 Alkyl, C 3-4 Alkyl, C 4-8 Alkyl, C 4-7 Alkyl, C 4-6 Alkyl, C 4-5 Alkyl, C 5-8 Alkyl, C5-7 Alkyl, C 5-6 Alkyl, C 6-8 Alkyl, C 6-7 Alkyl or C 7-8 Alkyl group. In some embodiments, Y 1 and Y 2 Each independently is C 8-10 alkyl.

[0443] In some implementation schemes, R 1 C 3-9 Alkyl group. For example, R 1 C 4-9 Alkyl, C 5-9 Alkyl, C 6-9 Alkyl, C 7-9 Alkyl, C 8-9 Alkyl, C 3-8 Alkyl, C 3-7 Alkyl, C 3-6 Alkyl, C 3-5 Alkyl, C 3-4 Alkyl, C 4-8 Alkyl, C 4-7 Alkyl, C 4-6 Alkyl, C 4-5 Alkyl, C 5-8 Alkyl, C 5-7 Alkyl, C 5-6 Alkyl, C 6-8 Alkyl, C 6-7 Alkyl or C 7-8 Alkyl group. In some embodiments, R 1 C 3-5 Alkyl or C 7-9 alkyl.

[0444] In some implementation schemes, R 2 and R 3 Each independently is C 1-3 Alkyl group. Or, R 2 and R 3 Together with the nitrogen atom it is attached to, it forms a 5-, 6-, or 7-membered ring. For example, R 2 and R 3 It forms a 5-membered ring together with the nitrogen atom it is attached to. Or, R 2 and R 3 It forms a 6-membered ring with the nitrogen atom it is attached to. Or, R 2 and R 3 Together with the nitrogen atom it is attached to, it forms a 7-membered ring.

[0445] In some embodiments, the compound of formula IV is represented by one of the following structural formulas:

[0446] ,

[0447] ,

[0448] and

[0449] ,

[0450] Or its salt.

[0451] In some embodiments, this disclosure relates to compounds represented by one of the following structural formulas:

[0452] ,

[0453] ,

[0454] ,

[0455] ,

[0456] ,

[0457] ,

[0458] ,

[0459] ,

[0460] ,

[0461] ,

[0462] ,

[0463] ,

[0464] ,

[0465] ,

[0466] ,

[0467] ,

[0468] and

[0469] ,

[0470] Or its salt.

[0471] In some embodiments, this disclosure relates to compounds represented by one of the following structural formulas:

[0472] ,

[0473] ,

[0474] ,

[0475] ,

[0476] ,

[0477] ,

[0478] ,

[0479] ,

[0480] ,

[0481] ,

[0482] ,

[0483] ,

[0484] and

[0485] ,

[0486] Or its salt.

[0487] In some implementations, the salt is a pharmaceutically acceptable salt.

[0488] In some embodiments, the present invention relates to compositions comprising compounds of formulas (I)-(IV) or Table 1 and lipid components.

[0489] In some implementations, the lipid component further comprises accessory lipids and PEG lipids.

[0490] In some implementations, the lipid component further comprises neutral lipids.

[0491] In some embodiments, the PEG lipid is selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearylglycerol (PEG-DSPE), PEG-dilaurylglycerylamide, PEG-dimyristoylglycerylamide, PEG-distearylglycerylamide, PEG-distearylglycerylamide, 1-[8'-(cholesterol-5-en-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-bistetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DMPE), 1,2-dimyristoyl-racemic-glycerol-3-[methoxy(polyethylene glycol)-2000] (PEG2K-DMG), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSPE), 1,2-distearyl-sn-glycerol-[methoxy(polyethylene glycol)-2000] (PEG2K-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2K-DMA), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSA), and methoxy-PEG2000-carbamoyl-1,2-tetrazoloxypropylamine (C13 ether). For example, PEG lipids are selected from PEG2K-DMG, C13 ethers, and C14 ethers. For example, PEG lipids contain dimyristoylglycerol (DMG). The structures of C14 ethers, C13 ethers, and PEG2K-DMG are shown below:

[0492] (where the mean n is approximately 45, C14 ether);

[0493] (where the mean n is approximately 45, C13 ether);

[0494] (where the mean n is approximately 45, PEG2K-DMG).

[0495] In some embodiments, the neutral lipid is selected from dipalmitoylphosphatidylcholine (DPPC), distearatelphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphatidylcholine (PLPC), 1,2-dicarachidoyl-sn-glycerol-3-phosphatidylcholine (DAPC), phosphatidylethanolamine (PE), lecithinylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), and 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC). 1-Palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-dibenzyl-sn-glycerol-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-bis(eicosanyl)-sn-glycerol-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearateoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), and lysophosphatidylethanolamine, or combinations thereof. For example, neutral lipids are DSPC or DMPE. For example, neutral lipids are DSPC.

[0496] In some embodiments, the cofactor lipid is selected from cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. For example, the cofactor lipid is cholesterol.

[0497] In some embodiments, the lipid component comprises 45-55 mol% of any compound represented by structural formulas (I)-(IV) or selected from the compounds in Table 1, 35-40 mol% of accessory lipids, 7-10 mol% of neutral lipids, and 1-5 mol% of PEG lipids. For example, the lipid component comprises about 50 mol% of any compound represented by structural formulas (I)-(IV) or selected from the compounds in Table 1, about 38 mol% of accessory lipids, about 9 mol% of neutral lipids, and about 3 mol% of PEG lipids.

[0498] In some embodiments, the composition further comprises a cryoprotectant.

[0499] In some embodiments, the composition further comprises a buffer.

[0500] In some embodiments, the composition further comprises an aqueous component containing a bioactive agent. In some embodiments, the bioactive agent comprises a polypeptide. In some embodiments, the bioactive agent comprises or encodes a therapeutically active protein. In some embodiments, the bioactive agent comprises or encodes a genome editing tool. In some embodiments, the bioactive agent comprises or encodes one or more nucleases capable of causing single-stranded or double-stranded breaks in DNA or RNA.

[0501] In some embodiments, the bioactive agent comprises nucleic acid. In some embodiments, the nucleic acid comprises RNA.

[0502] In some embodiments, the N / P ratio of the composition is from about 5 to about 7. For example, the N / P ratio is about 6 ± 1. For example, the N / P ratio is about 6 ± 0.5. For example, the N / P ratio is about 6.

[0503] In some embodiments, the composition further comprises an RNA component, wherein the RNA component contains mRNA. In some embodiments, the RNA component contains a sequence encoding an RNA-guided DNA binder, such as Cas nuclease mRNA. In some embodiments, the RNA component contains Class 2 Cas nuclease mRNA. In some embodiments, the RNA component contains Cas9 nuclease mRNA.

[0504] In some implementations, the RNA component contains modified RNA.

[0505] In some embodiments, the RNA component comprises gRNA nucleic acid. In some embodiments, the gRNA nucleic acid is gRNA. In some embodiments, the RNA component comprises two types of Cas nuclease mRNA and gRNA. In some embodiments, the gRNA nucleic acid is or encodes dual guide RNA (dgRNA). In some embodiments, the gRNA nucleic acid is or encodes a single guide RNA (sgRNA).

[0506] In some embodiments, the gRNA is a modified gRNA. In some embodiments, the modified gRNA contains a modification at one or more of the first five nucleotides at the 5' end. In some embodiments, the modified gRNA contains a modification at one or more of the last five nucleotides at the 3' end.

[0507] In some embodiments, the composition comprises a guide RNA nucleic acid; the mRNA is a type 2 Cas nuclease mRNA; and the weight ratio of the mRNA to the guide RNA nucleic acid is about 2:1 to 1:4.

[0508] In some embodiments, the composition further comprises at least one template nucleic acid.

[0509] In some embodiments, this disclosure relates to a method of lysing DNA, which includes contacting cells with a composition as described herein.

[0510] In some embodiments, this disclosure relates to a method of gene editing, which includes contacting cells with a composition as described herein.

[0511] In some implementations, the contact step results in a nick in the single-stranded DNA. In some implementations, the contact step results in a break in the double-stranded DNA.

[0512] In some embodiments, the composition comprises two types of Cas mRNA and gRNA nucleic acids.

[0513] In some embodiments, the method further includes introducing at least one template nucleic acid into the cell. In some embodiments, the method includes contacting the cell with a composition containing the template nucleic acid.

[0514] In some embodiments, the method includes administering the composition to an animal. In some embodiments, the method includes administering the composition to a human. In some embodiments, the method includes administering the composition to cells. In some embodiments, the cells are eukaryotic cells.

[0515] In some embodiments, the method includes administering mRNA formulated in a first lipid nanoparticle (LNP) composition and a second LNP composition, the first LNP composition and the second LNP composition comprising one or more of mRNA, gRNA, gRNA nucleic acid, and template nucleic acid. In some embodiments, the first LNP composition and the second LNP composition are administered simultaneously. In some embodiments, the first LNP composition and the second LNP composition are administered sequentially.

[0516] In some embodiments, the method includes administering mRNA and gRNA nucleic acids formulated in a single LNP composition.

[0517] In some implementations, gene editing results in gene knockout. In other implementations, gene editing results in gene correction.

[0518] In some embodiments, cells are contacted with the lipid composition in vitro. In some embodiments, cells are contacted with the lipid composition ex vivo.

[0519] In some embodiments, the method includes contacting animal tissue with a lipid composition. In some embodiments, the tissue is liver tissue.

[0520] Incorporate by reference

[0521] The contents of any 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 as if each individual publication expressly and individually indicated that it is incorporated by reference. The applicant reserves the right to incorporate any and all materials and information entities from any such articles, patents, patent applications or other entities and electronic documents into this application.

[0522] Example

[0523] General information

[0524] All reagents and solvents were purchased from commercial suppliers and used as is, or synthesized according to the cited procedures. All intermediates and final compounds were purified using silica gel rapid column chromatography. NMR spectra were recorded on a Bruker or Varian 400 MHz spectrometer, and NMR data were collected in CDCl3 at ambient temperature. Chemical shifts were reported in parts per million (ppm) relative to CDCl3 (7.26). ¹H NMR data were reported as follows: chemical shifts, multiplicity (br = broad peak, s = singlet, d = doublet, t = triplet, q = quartet, dd = doubletie, dt = doubletie, m = multiplet), coupling constant, and integral. MS data were recorded on a Waters SQD2 mass spectrometer with an electrospray ionization (ESI) source. The purity of the final compound was determined by UPLC-MS-ELS using a Waters Acquity H-Class liquid chromatograph equipped with an SQD2 mass spectrometer with a photodiode array (PDA) and an evaporative light scattering (ELS) detector.

[0525] Example 1 - Compound 1

[0526] Intermediate 1a: 4,4-bis(octyloxy)butyronitrile

[0527]

[0528] Add 0.1-0.25 equivalents of 4-methylbenzenesulfonic acid monohydrate to a mixture of 20 g (1.0 equivalent) of 4,4-dimethoxybutyronitrile (2-3 equivalents) and oct-1-ol (2-3 equivalents) in toluene (0.5-2.0 M). Stir the mixture at 100-120 °C for at least 24 h under a nitrogen atmosphere. Concentrate the reaction mixture under reduced pressure to remove the solvent. Dry the residue with Na₂SO₄, filter, and concentrate the filtrate under reduced pressure to give a product (40%) as a colorless oil.

[0529] Intermediate 1b: 4,4-bis(octyloxy)butyric acid

[0530]

[0531] KOH (4.0-15.0 equivalents) was added to a solution of intermediate 1a (20 g, 1.0 equivalents) in 1:1 H₂O / EtOH (0.1-1.0 M). The mixture was stirred at 110 °C for at least 12 h under a N₂ atmosphere. The reaction mixture was then concentrated under reduced pressure to remove the solvent. 6 N HCl was added to adjust the pH of the residue to 5-7, and the residue was extracted 2-3 times with EtOAc. The combined organic layers were concentrated under reduced pressure to give a colorless oily residue (35%). 1 H NMR (400 MHz, CDCl3) δ4.46 (t, J = 5.4 Hz, 1H), 3.60 - 3.49 (m, 2H), 3.35 (dt, J = 9.3, 6.7 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 1.88 (td, J = 7.2, 5.3 Hz, 2H), 1.50 (p, J =6.9 Hz, 4H), 1.33 - 1.12 (m, 21H), 0.81 (t, J = 6.6 Hz, 6H).

[0532] Intermediate 1c: bis(4,4-bis(octyloxy)butyric acid)2-(hydroxymethyl)propane-1,3-dimethyl ester

[0533]

[0534] DMAP (0.1 equivalent), DIPEA (2.5 equivalent), and EDCI (1.2 equivalent) were added to a solution of intermediate 1b (7.5 g, 2.0 equivalent) and 2-(hydroxymethyl)propane-1,3-diol (1.0 equivalent) in a 3:1 DCM / DMF (0.1–0.5 M). The mixture was stirred at 20 °C for at least 12 h under a nitrogen atmosphere. The reaction mixture was diluted with water and extracted three times with DCM. The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give a product (36%) as a colorless oil.

[0535] Intermediate 1d: bis(4,4-bis(octyloxy)butyric acid)2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-1,3-dimethyl ester

[0536]

[0537] Pyridine (1.5-2.0 equivalents) was added dropwise to a mixture of intermediate 1c (3 g, 1.0 equivalents) and (4-nitrophenyl) chloroformate (1.5-2.0 equivalents) in DCM (0.1-0.5 M) at 0 °C. The mixture was then stirred at 15-20 °C for at least 1 h under a nitrogen atmosphere. The reaction mixture was diluted with hexane, filtered, and the filtrate was concentrated under reduced pressure to give a product (68%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 8.30 - 8.24 (m, 2H), 7.41 -7.35 (m, 2H), 4.47 (t, J = 5.5 Hz, 2H), 4.34 (d, J = 5.7 Hz, 2H), 4.19 (dd, J= 6.0, 2.7 Hz, 4H), 3.54 (dt, J = 9.4, 6.7 Hz, 4H), 3.38 (dt, J = 9.3, 6.7Hz, 4H), 2.50 (p, J = 6.0 Hz, 1H), 2.40 (t, J = 7.6 Hz, 4H), 1.92 (td, J =7.6, 5.5 Hz, 4H), 1.54 (q, J = 6.8 Hz, 8H), 1.32 - 1.20 (m, 40H), 0.93 - 0.78 (m, 12H).

[0538] Compound 1: bis(4,4-bis(octyloxy)butyric acid)2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl ester

[0539]

[0540] A mixture of intermediate 1d (2.5 g, 0.75–1.0 equivalents), 3-(diethylamino)prop-1-ol (2.0 equivalents), DMAP (0.1–2.0 equivalents), and pyridine (1.0–3.0 equivalents) in MeCN (0.05–0.5 M) was degassed and purged three times with N2. The mixture was then stirred at 20 °C for at least 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove MeCN. The residue was diluted with water and extracted three times with EtOAc. The combined organic layers were washed three times with aqueous NaHCO3 solution, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue, which was purified by column chromatography to give a colorless oily product. 1H NMR (400 MHz, CDCl3) δ 4.41 (t, J = 5.6 Hz, 2H), 4.16 -4.01 (m, 8H), 3.49 (dt, J = 9.3, 6.7 Hz, 4H), 3.33 (dt, J = 9.3, 6.7 Hz, 4H), 2.46 (q, J = 7.0 Hz, 6H), 2.34 (q, J = 7.3 Hz, 5H), 1.85 (td, J = 7.6, 5.4Hz, 4H), 1.75 (p, J = 6.9 Hz, 2H), 1.48 (q, J = 7.0 Hz, 8H), 1.33 - 1.06 (m,44H), 0.95 (t, J = 7.1 Hz, 6H), 0.81 (t, J = 6.7 Hz, 12H). MS: 916.5 m / z [M+H].

[0541] Example 2 - Compound 2

[0542] Intermediate 2a: Heptadecan-9-ol

[0543]

[0544] NaBH4 (1.5 equivalents) was added to a solution of heptadecanone (45 g, 1.0 equivalent) in 5:1 THF / MeOH (0.1–0.5 M) at 0 °C. The mixture was stirred at 25 °C for at least 1 h. The reaction mixture was quenched with saturated NH4Cl and diluted with H2O. The mixture was then extracted three times with EtOAc, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a product as a white solid. 1 H NMR (400 MHz, CDCl3) δ3.58 (dt, J = 7.3, 4.3 Hz, 1H), 1.56 - 1.14 (m, 29H), 0.88 (t, J = 6.7 Hz, 6H).

[0545] Intermediate 2b: 5-(heptadecane-9-yloxy)-5-oxovalerate

[0546]

[0547] (COCl)₂ (1.0-1.2 equivalents) and DMF (0.05-0.1 equivalents) were added to a solution of glutaric acid (5.0 g, 1.0 equivalent) in THF (0.5-1.0 M) at 0 °C. The mixture was stirred at 25 °C for 2 h. Then, intermediate 2a (1.0 equivalent) in THF (0.5-1.0 M) was added to the reaction mixture. The reaction mixture was stirred at 20-25 °C for at least 2 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with H₂O and extracted three times with EtOAc or DCM, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a product (27%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.87 (p, J = 6.3 Hz, 1H), 2.40 (dt, J = 21.5, 7.3 Hz, 4H), 1.97 (q, J = 7.4 Hz, 2H), 1.51 (q, J = 6.2 Hz, 4H), 1.25 (s, 23H), 0.97 - 0.76 (m, 6H).

[0548] Intermediate 2c: Di(heptadecane-9-yl) glutarate O,O'-(2-(hydroxymethyl)propane-1,3-diyl) ester

[0549]

[0550] Add DMAP (0.2 equivalents), EDCI (1.0-2.0 equivalents), and DIPEA (2.0 equivalents) to a solution of intermediate 2b (4 g, 0.5-1.0 equivalents) and 2-(hydroxymethyl)propane-1,3-diol (0.5-1.0 equivalents) in DCM (0.1-0.2 M). Stir the reaction mixture at 15-25°C for at least 5 h. Concentrate the reaction mixture under reduced pressure to remove the solvent, obtaining a residue. Dilute the residue with H₂O and extract three times with EtOAc or DCM, wash with brine, dry to Na₂SO₄, filter, and concentrate the filtrate under reduced pressure to obtain a residue. Purify the residue by column chromatography to give a product as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 4.87 (p, J = 6.3 Hz, 2H), 4.18 (dd, J = 6.1, 2.2Hz, 4H), 3.63 (d, J = 5.5 Hz, 2H), 2.38 (dt, J = 17.6, 7.3 Hz, 8H), 2.24 -2.16 (m, 2H), 1.96 (p, J = 7.4 Hz, 4H), 1.51 (q, J = 6.1 Hz, 8H), 1.26 (s,46H), 0.88 (t, J = 6.7 Hz, 12H).

[0551] Intermediate 2d: Di(heptadecyl-9-yl)diglutarate O,O'-(2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-1,3-diyl)ester

[0552]

[0553] Pyridine (2.0-3.0 equivalents) was added to a solution of intermediate 2c (1.1 g, 1.0 equivalents) and 4-nitrobenzene chloroformate (1.0-3.0 equivalents) in DCM (0.1-0.2 M). The reaction mixture was stirred at 25 °C for 1 h under N2. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a residue. The residue was diluted with H2O and extracted three times with hexane or EtOAc, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give a colorless oily product. 1 H NMR (400 MHz, CDCl3) δ 8.31 - 8.23 ​​(m, 2H), 7.43 -7.35 (m, 2H), 4.86 (p, J = 6.3 Hz, 2H), 4.35 (d, J = 5.8 Hz, 2H), 4.22 (dd, J= 6.1, 2.3 Hz, 4H), 2.51 (p, J = 5.9 Hz, 1H), 2.38 (dt, J = 22.6, 7.4 Hz,8H), 1.96 (p, J = 7.4 Hz, 4H), 1.50 (q, J = 6.1 Hz, 9H), 1.25 (s, 46H), 0.87(t, J = 6.8 Hz, 12H).

[0554] Compound 2: Diglutaric acid O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester di(heptadecano-9-yl) ester

[0555]

[0556] Pyridine (2.0-3.0 equivalents) and DMAP (0.1 equivalents) were added to a solution of intermediate 2d (1.8 g, 1.0 equivalents) and 3-(diethyl-amino)prop-1-ol (1.0-3.0 equivalents) in DCM (0.05-0.2 M). The reaction mixture was stirred at 25 °C under N2 for 2 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a residue. The residue was diluted with H2O and extracted three times with EtOAc, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a colorless oily product (95%). 1 H NMR (400 MHz, CDCl3) δ 4.79 (tt, J= 6.5, 3.2 Hz, 2H), 4.18 - 4.02 (m, 8H), 2.44 (qd, J = 7.0, 2.5 Hz, 6H), 2.31 (tdd, J = 15.2, 6.3, 2.0 Hz, 9H), 1.88 (pd, J = 7.4, 2.0 Hz, 4H), 1.75 (qt, J= 9.0, 4.6 Hz, 3H), 1.44 (dt, J = 11.4, 5.5 Hz, 8H), 1.29 - 1.12 (m, 46H), 0.94 (td, J = 7.1, 2.4 Hz, 6H), 0.81 (td, J = 6.8, 2.3 Hz, 12H). MS: 968.7 m / z[M+H].

[0557] Example 3 - Compound 3

[0558] Intermediate 3a: 7-(heptadecane-9-yloxy)-7-oxoheptanoic acid

[0559]

[0560] Intermediate 3a (36%) was synthesized from pimelic acid and heptadecanol using the method employed in the synthesis of intermediate 2b. 1H NMR (400 MHz, CDCl3) δ 4.86 (p, J = 6.3 Hz, 1H), 2.32 (dt, J = 24.7,7.5 Hz, 4H), 1.65 (dtt, J = 11.7, 7.5, 4.0 Hz, 4H), 1.50 (q, J = 6.0 Hz, 4H), 1.43 - 1.14 (m, 26H), 0.87 (t, J = 6.8 Hz, 6H).

[0561] Intermediate 3b: 7,7'-di(heptadecanoic acid) ester O'1,O1-(2-(hydroxymethyl)propane-1,3-diyl) ester

[0562]

[0563] Intermediate 3b (18%) was synthesized from intermediate 3a using the method employed in the synthesis of intermediate 2c. 1 H NMR(400 MHz, CDCl3) δ 4.79 (p, J = 6.3 Hz, 2H), 4.10 (h, J = 6.3, 5.9 Hz, 4H), 3.55 (d, J = 5.5 Hz, 2H), 2.24 (dt, J = 17.3, 7.5 Hz, 8H), 2.16 - 2.09 (m,1H), 1.58 (pd, J = 7.5, 5.1 Hz, 8H), 1.43 (d, J = 6.0 Hz, 8H), 1.33 - 1.25(m, 5H), 1.19 (s, 46H), 0.81 (t, J = 6.7 Hz, 12H).

[0564] Intermediate 3c: 7,7'-di(heptadecanoic acid) ester O'1,O1-(2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester

[0565]

[0566] Intermediate 3c (84%) was synthesized from intermediate 3b using the method employed in the synthesis of intermediate 2d. 1H NMR(400 MHz, CDCl3) δ 8.24 - 8.17 (m, 2H), 7.36 - 7.28 (m, 2H), 4.79 (p, J = 6.3Hz, 2H), 4.29 (d, J = 5.9 Hz, 2H), 4.14 (dd, J = 6.0, 2.3 Hz, 4H), 2.44 (p, J= 6.0 Hz, 1H), 2.24 (dt, J = 23.0, 7.5 Hz, 8H), 1.58 (h, J = 7.3 Hz, 9H), 1.43 (q, J = 6.1 Hz, 8H), 1.35 - 1.07 (m, 51H), 0.80 (t, J = 6.8 Hz, 12H).

[0567] Compound 3: Di(pimelic acid)O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)ester 7,7'-di(heptadecane-9-yl)ester

[0568]

[0569] Compound 3 (57%) was synthesized from intermediate 3c using the method employed in the synthesis of compound 2. 1 H NMR(400 MHz, CDCl3) δ 4.79 (p, J = 6.2 Hz, 2H), 4.15 - 4.04 (m, 8H), 2.43 (t, J= 7.0 Hz, 6H), 2.35 (p, J = 5.9 Hz, 1H), 2.23 (dt, J = 12.9, 7.5 Hz, 8H),1.75 (q, J = 6.9 Hz, 2H), 1.57 (p, J = 7.6 Hz, 9H), 1.43 (q, J = 5.9 Hz, 8H),1.32 - 1.11 (m, 53H), 0.94 (t, J = 7.1 Hz, 6H), 0.81 (t, J = 6.6 Hz, 12H). MS: 1024.8 m / z [M+H].

[0570] Example 4 - Compound 4

[0571] Intermediate 4a: 7-((2-Butyloctyl)oxy)-7-oxoheptanoic acid

[0572]

[0573] (COCl)₂ (1.0 equivalent) was added dropwise to a mixture of pimelic acid (20 g, 1.0 equivalent), DMF (0.2 equivalent), and THF (0.2–0.4 M) under N₂ atmosphere. The mixture was stirred at 20 °C for 2 h under N₂ atmosphere. Then, 2-Butyloct-1-ol (1.0 equivalent) was added dropwise to the reaction mixture, and the mixture was stirred at 20 °C for 2 h under N₂ atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent, poured into water, and extracted three times with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography to give a product (49%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 3.96 (d, J = 5.8 Hz, 2H), 2.33 (dt, J =16.4, 7.5 Hz, 4H), 1.66 (qd, J = 7.6, 2.7 Hz, 5H), 1.42 - 1.33 (m, 2H), 1.27 (t, J = 4.3 Hz, 16H), 0.88 (td, J = 6.6, 3.8 Hz, 6H).

[0574] Intermediate 4b: (9Z,12Z)-octadecyl-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester

[0575]

[0576] Add EDCI (1.0-2.0 equivalents), DMAP (0.1-0.2 equivalents), and DIPEA (2.0-3.0 equivalents) to a solution of 2-(hydroxymethyl)propane-1,3-diol (2.0-3.0 equivalents) and (9Z,12Z)-octadecyl-9,12-dienoic acid (20 g, 1.0 equivalents) in 2:1 DCM / DMF (0.2-0.5 M). Stir the reaction mixture under N2 at 15-25°C for at least 5 h. Concentrate the reaction mixture under reduced pressure to remove the solvent. Dilute the residue with H2O, extract three times with DCM or EtOAc, dry to Na2SO4, filter, and concentrate the filtrate under reduced pressure to obtain the residue. Purify the residue by column chromatography to give a product (76%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 5.47 - 5.05 (m,4H), 4.22 (d, J = 6.3 Hz, 2H), 3.74 (qd, J = 11.1, 5.2 Hz, 4H), 2.94 (s, 3H), 2.86 (s, 3H), 2.78 - 2.71 (m, 2H), 2.31 (t, J = 7.5 Hz, 2H), 2.02 (p, J =6.5, 6.0 Hz, 5H), 1.60 (p, J = 7.3 Hz, 2H), 1.41 - 1.12 (m, 13H), 0.91 - 0.75(m, 3H).

[0577] Intermediate 4c: 1-(2-Butyloctyl)pimelic acid ester 7-(3-hydroxy-2-((((9Z,12Z)-octadecyl-9,12-dienoyl)oxy)methyl)propyl) ester

[0578]

[0579] Intermediate 10a (1.0 equivalent), EDCI (1.2 equivalent), DMAP (0.1 equivalent), and DIPEA (2.0-3.0 equivalent) were added to a solution of intermediate 4b (9 g, 1.0 equivalent) in DCM (0.1-0.3 M). The mixture was stirred at least at 15-25°C under N2. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a residue. The residue was diluted with H2O and extracted three times with EtOAc or DCM. The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography to give a product (42%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 5.44 - 5.28 (m, 4H), 4.18 (pt, J = 6.4, 3.7 Hz, 4H), 3.98 (d, J =5.8 Hz, 2H), 3.63 (d, J = 5.6 Hz, 2H), 2.78 (t, J = 6.4 Hz, 2H), 2.39 - 2.28(m, 6H), 2.20 (hept, J = 5.8 Hz, 2H), 2.06 (q, J = 6.9 Hz, 4H), 1.66 (dtd, J= 15.5, 7.6, 4.1 Hz, 9H), 1.41 - 1.20 (m, 31H), 0.90 (td, J = 6.6, 3.1 Hz, 9H).

[0580] Intermediate 4d: 1-(2-Butyloctyl)pimelic acid ester 7-(3-(((4-nitrophenoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadecyl-9,12-dienoyl)oxy)methyl)propyl) ester

[0581]

[0582] To a solution of intermediate 4c (5 g, 1.0 equivalent) in DCM (0.1–0.2 M), chloroformate (4-nitrophenyl) ester (3.0 equivalent) and pyridine (3.0 equivalent) were added. The mixture was stirred at 25 °C under N2 for 2 h. The reaction mixture was concentrated under reduced pressure to remove DCM, yielding a residue, which was purified by column chromatography to give a product (64%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 8.32 - 8.23 ​​(m, 2H), 7.42 - 7.34 (m, 2H), 5.43 - 5.23 (m, 4H), 4.36 (d, J = 5.8 Hz, 2H), 4.27 - 4.13 (m, 4H), 3.97 (d,J = 5.8 Hz, 2H), 2.77 (t, J = 6.3 Hz, 2H), 2.52 (hept, J = 6.0 Hz, 1H), 2.39- 2.24 (m, 6H), 2.05 (q, J = 6.9 Hz, 4H), 1.65 (dtd, J = 15.2, 7.6, 5.0 Hz,8H), 1.43 - 1.19 (m, 32H), 0.89 (td, J = 6.6, 3.0 Hz, 9H).

[0583] Compound 4: 1-(2-Butyloctyl)pimelic acid ester 7-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadecyl-9,12-dienoyl)oxy)methyl)propyl) ester

[0584]

[0585] Compound 4 was synthesized from intermediate 4d using the method employed in the synthesis of compound 2. 1 H NMR (400MHz, CDCl3) δ 5.37 - 5.20 (m, 4H), 4.17 - 4.03 (m, 8H), 3.90 (d, J = 5.8 Hz, 2H), 2.70 (t, J = 6.4 Hz, 2H), 2.44 (q, J = 7.1 Hz, 6H), 2.35 (p, J = 6.0 Hz, 1H), 2.24 (td, J = 7.6, 4.7 Hz, 6H), 1.98 (q, J = 6.9 Hz, 4H), 1.74 (p, J =6.7 Hz, 2H), 1.55 (dt, J = 11.6, 6.0 Hz, 7H), 1.37 - 1.13 (m, 32H), 0.94 (t,J = 7.1 Hz, 6H), 0.82 (dp,J = 7.0, 3.1 Hz, 9H). MS: 836.6 m / z [M+H].

[0586] Example 5 - Compound 5

[0587] Intermediate 5a: 7,7'-bis(2-butyloctyl) ester O'1,O1-(2-(hydroxymethyl)propane-1,3-diyl) ester

[0588]

[0589] Intermediate 5a (68%) was synthesized from intermediate 4a using the method employed in the synthesis of intermediate 2c. 1H NMR(400 MHz, CDCl3) δ 4.19 (h, J = 5.9 Hz, 4H), 3.99 (d, J = 5.8 Hz, 4H), 3.65(t, J = 5.9 Hz, 2H), 2.42 - 2.30 (m, 9H), 2.22 (p, J = 5.9 Hz, 1H), 1.67 (ddp, J = 11.5, 7.7, 3.8 Hz, 10H), 1.44 - 1.23 (m, 36H), 0.91 (td, J = 6.6, 3.7 Hz, 12H).

[0590] Intermediate 5b: 7,7'-bis(2-butyloctyl) ester O'1,O1-(2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester

[0591]

[0592] Intermediate 5b (61%) was synthesized from intermediate 5a using the method employed in the synthesis of intermediate 2d. 1 H NMR(400 MHz, CDCl3) δ 8.24 - 8.17 (m, 2H), 7.36 - 7.28 (m, 2H), 4.29 (d, J = 5.8Hz, 2H), 4.20 - 4.08 (m, 4H), 3.95 - 3.84 (m, 4H), 2.45 (p, J = 6.0 Hz, 1H), 2.26 (dt, J = 14.7, 7.5 Hz, 8H), 1.58 (dtd, J = 15.3, 7.6, 5.1 Hz, 10H), 1.36- 1.09 (m, 36H), 0.81 (td, J = 6.6, 3.9 Hz, 12H).

[0593] Compound 5: 7,7'-bis(2-butyloctyl) ester O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester

[0594]

[0595] Compound 5 was synthesized from intermediate 5b using the method employed in the synthesis of compound 2. 1H NMR (400MHz, CDCl3) δ 4.24 - 4.09 (m, 8H), 3.97 (dd, J = 5.9, 2.2 Hz, 4H), 2.53 (q, J= 7.0 Hz, 6H), 2.46 - 2.39 (m, 1H), 2.32 (dt, J = 11.6, 5.7 Hz, 8H), 1.82 (p,J = 6.9 Hz, 2H), 1.72 - 1.57 (m, 10H), 1.46 - 1.18 (m, 34H), 1.02 (t, J = 7.2Hz, 6H), 0.89 (dq, J = 6.9, 3.3, 2.8 Hz, 12H). MS: 884.5 m / z [M+H].

[0596] Example 6 - Compound 6

[0597] Compound 6: Diglutaric acid O,O'-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl) ester di(heptadecano-9-yl) ester

[0598]

[0599] To a solution of intermediate 2d (3 g, 1.0 equivalent) in MeCN (0.05–0.25 M), N',N'-diethylethane-1,2-diamine (1.0–3.0 equivalent), pyridine (1.0–2.0 equivalent), and DMAP (0.1–1.0 equivalent) were added. The mixture was then stirred at 15–25 °C for at least 12 h under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with EtOAc and washed 2–5 times with 1 N NaHCO3 and 3 times with H2O. The organic layer was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by silica gel chromatography to give a product as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 5.35 (s, 1H), 4.85 (p, J = 6.3 Hz, 2H), 4.12(t, J = 6.0 Hz, 6H), 3.22 (q, J = 5.8 Hz, 2H), 2.53 (d, J = 6.8 Hz, 6H), 2.36(dt, J = 14.7, 7.4 Hz, 9H), 1.93 (p, J = 7.5 Hz, 4H), 1.49 (q, J = 5.9 Hz,8H), 1.24 (s, 48H), 1.00 (t, J = 7.1 Hz, 6H), 0.87 (t, J = 6.7 Hz, 12H). MS: 953.6 m / z [M+H].

[0600] Example 7 - Compound 7

[0601] Intermediate 7a: 2-Butyloctyl 9-bromononanoate

[0602]

[0603] A mixture of 9-bromononanoic acid (20 g, 1.0 equivalent), 2-butyloctyl-1-ol (1.0 equivalent), EDCI (1.0–2.0 equivalent), DIPEA (2.0–3.0 equivalent), and DMAP (0.1–0.5 equivalent) in DCM (0.1–0.5 M) was stirred at 15–25 °C for at least 12 h under a nitrogen atmosphere. The reaction mixture was poured into water and extracted three times with DCM or EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography to give a product (58%) as a colorless oil.

[0604] Intermediate 7b: 1,1-dibenzyl nonane-1,1,9-tricarboxylic acid ester 9-(2-butyloctyl) ester

[0605]

[0606] K₂CO₃ (1.0-5.0 equivalents) was added to a solution of intermediate 7a (9 g, 1.0 equivalents) and dibenzyl malonate (1.0-2.0 equivalents) in DMF (0.2-0.5 M) at 0 °C, and the mixture was stirred at 15 °C-60 °C for at least 12 h. The residue was poured into water and extracted three times with DCM or EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography to give a product as a colorless oil. 1 HNMR (400 MHz, CDCl3) δ 7.42 - 7.32 (m, 10H), 5.19 (s, 4H), 4.01 (d, J = 5.8Hz, 2H), 3.47 (t, J = 7.5 Hz, 1H), 2.33 (t, J = 7.5 Hz, 2H), 1.96 (q, J = 7.3Hz, 2H), 1.70 - 1.54 (m, 6H), 1.30 (dh, J = 10.1, 3.6, 3.2 Hz, 34H), 0.95 -0.90 (m, 6H).

[0607] Intermediate 7c: 2-(9-((2-Butyloctyl)oxy)-9-oxonyl)malonic acid

[0608]

[0609] Intermediate 7b (1.0 equivalent) was added to a suspension of Pd / C (0.1-2.0 equivalents) in THF (0.1-0.5 M). The reaction mixture was stirred at 15-25°C for at least 12 h under H2 atmosphere. The reaction mixture was filtered, and the filtrate was concentrated under vacuum and purified by column chromatography to give a colorless oily product (60%). 1 H NMR (400 MHz, CDCl3) δ 3.91 (d, J = 5.8 Hz, 2H), 3.37 (t, J = 7.3 Hz, 1H), 2.24 (t, J = 7.5Hz, 2H), 1.89 (q, J = 7.4 Hz, 2H), 1.54 (t, J = 7.3 Hz, 3H), 1.33 - 1.18 (m,24H), 0.82 (td, J = 6.7, 3.7 Hz, 6H).

[0610] Intermediate 7d: 2-Butyloctyl 11-hydroxy-10-(hydroxymethyl)undecanoic acid

[0611]

[0612] BH3·THF (2.5 equivalents) was added dropwise to a solution of intermediate 7c (4 g, 1.0 equivalent) in THF (0.2 M) at 0 °C, and the mixture was stirred at 20 °C for 12 h under a N2 atmosphere. The reaction mixture was poured into an aqueous solution of NH4Cl and stirred at 5 °C for 0.5 h. The mixture was then extracted 3 to 5 times with EtOAc. The organic layer was concentrated under vacuum, and the residue was purified by column chromatography to give a product as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 3.90 (d,J = 5.8 Hz, 2H), 3.75 (td, J = 12.5, 11.6, 4.8 Hz, 1H), 3.59 (dt, J = 9.5,6.5 Hz, 2H), 3.34 (q, J = 6.7, 6.1 Hz, 4H), 2.23 (t, J = 7.5 Hz, 2H), 1.57 (dtt, J = 10.2, 7.1, 3.5 Hz, 8H), 1.21 (d, J = 4.8 Hz, 26H), 0.82 (td, J =8.1, 6.9, 5.2 Hz, 6H).

[0613] Intermediate 7e: (9Z,12Z)-octadecyl-9,12-dienoic acid 11-((2-butyloctyl)oxy)-2-(hydroxymethyl)-11-oxoundecyl ester

[0614]

[0615] Add (9Z,12Z)-octadecyl-9,12-dienoic acid (1.0 equivalent) to a mixture of intermediate 7e (3.5 g, 1.0 equivalent), EDCI (1.0–2.0 equivalent), DMAP (0.1–0.5 equivalent), and DIPEA (2.0–4.0 equivalent) in DCM (0.1–0.5 M). Stir the reaction mixture at 15–25 °C for at least 12 h under N2 atmosphere. Concentrate the reaction mixture under reduced pressure to remove the solvent, and extract the residue three times with EtOAc or DCM. Wash the combined organic phases with brine, dry to anhydrous Na2SO4, filter, and concentrate the filtrate under vacuum. Purify the residue by column chromatography to give a product (43%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 5.38 - 5.16 (m, 4H), 4.24 (t, J =6.7 Hz, 1H), 4.15 (dd, J = 11.2, 4.3 Hz, 1H), 4.01 (dd, J = 11.2, 6.7 Hz, 1H), 3.90 (d, J = 5.8 Hz, 2H), 3.48 (dd, J = 32.6, 9.4 Hz, 2H), 2.70 (t, J =6.4 Hz, 2H), 2.24 (dt, J = 10.6, 7.5 Hz, 4H), 1.98 (q, J = 6.9 Hz, 4H), 1.54(ddd, J = 19.9, 9.5, 5.9 Hz, 7H), 1.24 (ddq, J = 13.9, 9.8, 5.9 Hz, 43H), 0.82 (td, J = 6.7, 3.4 Hz, 9H).

[0616] Intermediate 7f: (9Z,12Z)-octadecyl-9,12-dienoic acid 11-((2-butyloctyl)oxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-11-oxoundecyl ester

[0617]

[0618] Pyridine (1.0-3.0 equivalents) was added to a solution of (4-nitrophenyl) chloroformate (1.0-3.0 equivalents) and intermediate 7e (1.0 equivalent) in DCM (0.05-0.5 M), and the mixture was stirred at 15-25°C for at least 12 h under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography to give a product as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.25 - 8.17 (m, 2H), 7.36 - 7.28 (m, 2H), 5.38 - 5.16 (m, 4H), 4.31 - 4.14 (m, 3H), 4.10 (dd, J = 11.2, 4.7 Hz, 1H), 4.02 (dd, J = 11.3, 6.7 Hz, 1H), 3.95 - 3.88 (m, 2H), 2.70 (t, J = 6.3 Hz, 2H), 2.24 (dt, J = 9.7, 7.5 Hz, 4H), 2.07 - 1.91 (m, 5H), 1.55 (tt, J = 7.5,3.8 Hz, 5H), 1.39 - 1.12 (m, 42H), 0.81 (dh, J = 6.1, 3.4, 2.9 Hz, 9H).

[0619] Compound 7: (9Z,12Z)-octadecyl-9,12-dienoic acid 11-((2-butyloctyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-11-oxoundecyl ester

[0620]

[0621] Compound 7 (77%) was synthesized from intermediate 7f and 3-(diethylamino)prop-1-ol using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 5.28 (tt, J = 11.2, 5.4 Hz, 4H), 4.13 (t, J = 6.4 Hz, 2H), 4.06 - 3.93 (m, 4H), 3.90 (d, J = 5.8 Hz, 2H), 2.70 (t,J = 6.4 Hz, 2H), 2.66 - 2.46 (m, 5H), 2.23 (td, J = 7.6, 2.6 Hz, 4H), 1.98(q, J = 6.9 Hz, 4H), 1.94 - 1.82 (m, 2H), 1.54 (t, J = 7.2 Hz, 6H), 1.35 -1.14 (m, 45H), 1.04 (s, 6H), 0.82 (td, J = 6.6, 3.3 Hz, 9H). MS: 820.7 m / z [M+H].

[0622] Example 8 - Compound 8

[0623] Intermediate 8a: Heptadecan-9-yl ester of 7-bromoheptanoate

[0624]

[0625] Intermediate 8a (58%) was synthesized from 7-bromoheptanoic acid and heptadecan-9-ol using the method employed in the synthesis of intermediate 7a. 1 H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 1H), 3.33 (t, J = 6.8 Hz,2H), 2.21 (t, J = 7.5 Hz, 2H), 1.78 (p, J = 7.0 Hz, 2H), 1.55 (t, J = 7.2 Hz,2H), 1.47 - 1.16 (m, 36H), 0.81 (t, J = 6.7 Hz, 6H).

[0626] Intermediate 8b: 1,1-dibenzyl heptane-1,1,7-tricarboxylic acid ester 7-(heptadecane-9-yl) ester

[0627]

[0628] Intermediate 8b is synthesized from intermediate 8a using the method employed in the synthesis of intermediate 7b. 1 H NMR (400MHz, CDCl3) δ 7.30 - 7.19 (m, 10H), 5.07 (d, J = 1.3 Hz, 4H), 4.79 (p, J =6.2 Hz, 1H), 3.36 (t, J = 7.5 Hz, 1H), 2.18 (t, J = 7.5 Hz, 2H), 1.85 (d, J =7.1 Hz, 2H), 1.45 (dd, J = 16.0, 3.7 Hz, 8H), 1.28 - 1.05 (m, 30H), 0.80 (t,J = 6.7 Hz, 6H).

[0629] Intermediate 8c: 2-(7-(heptadecane-9-yloxy)-7-oxohepyl)malonic acid

[0630]

[0631] Intermediate 8c (69%) was synthesized from intermediate 8b using the method employed in the synthesis of intermediate 7c.

[0632] Intermediate 8d: Heptadecan-9-yl 9-hydroxy-8-(hydroxymethyl)nonanoic acid

[0633]

[0634] Intermediate 8d (47%) was synthesized from intermediate 8c using the method employed in the synthesis of intermediate 7d. 1 H NMR(400 MHz, CDCl3) δ 4.79 (p, J = 6.3 Hz, 1H), 3.90 - 3.69 (m, 2H), 3.59 (dt, J= 10.7, 7.8 Hz, 2H), 2.21 (td, J = 7.4, 1.7 Hz, 2H), 1.53 (q, J = 7.3 Hz, 2H), 1.43 (q, J = 6.1 Hz, 4H), 1.22 (d, J = 23.1 Hz, 32H), 0.86 - 0.76 (m,6H).

[0635] Intermediate 8e: Heptadecan-9-yl glutarate (9-(heptadecan-9-yloxy)-2-(hydroxymethyl)-9-oxonyl) ester

[0636]

[0637] Intermediate 8e (39%) was synthesized from intermediate 8d and intermediate 2b using the method employed in the synthesis of intermediate 7e.

[0638] Intermediate 8f: Heptadecan-9-yl glutarate (9-(heptadecan-9-yloxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-9-oxonyl) ester

[0639]

[0640] Intermediate 8f is synthesized from intermediate 8e using the method employed in the synthesis of intermediate 7f. 1H NMR (400MHz, CDCl3) δ 8.26 - 8.17 (m, 2H), 7.38 - 7.28 (m, 2H), 4.80 (p, J = 6.2 Hz,2H), 4.22 - 3.99 (m, 4H), 2.38 - 2.17 (m, 6H), 2.04 (q, J = 6.0 Hz, 1H), 1.89 (p, J = 7.4 Hz, 2H), 1.55 (q, J = 7.0 Hz, 3H), 1.43 (t, J = 6.2 Hz, 8H), 1.37- 1.13 (m, 58H), 0.80 (t, J = 6.7 Hz, 12H).

[0641] Compound 8: Glutaric acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-9-(heptadecane-9-yloxy)-9-oxonyl ester heptadecane-9-yl ester

[0642]

[0643] Compound 8 was synthesized from intermediate 8f using the method employed in the synthesis of compound 6. 1 H NMR (400MHz, CDCl3) δ 4.86 (pd, J = 6.3, 2.2 Hz, 2H), 4.18 (t, J = 6.5 Hz, 2H), 4.14- 4.01 (m, 4H), 2.52 (s, 5H), 2.36 (dt, J = 11.7, 7.4 Hz, 4H), 2.27 (t, J =7.6 Hz, 2H), 1.97 (dt, J = 22.2, 7.9 Hz, 3H), 1.83 (s, 2H), 1.60 (dd, J =10.1, 4.5 Hz, 2H), 1.49 (t, J 3= 6.1 Hz, 8H), 1.38 - 1.19 (m, 55H), 1.02 (d,J = 7.5 Hz, 6H), 0.87 (t,J = 6.7 Hz, 12H). MS: 952.8 m / z [M+H].

[0644] Example 9 - Compound 9

[0645] Intermediate 9a: Heptadecano-9-yl ester of 9-bromononanoic acid

[0646]

[0647] Intermediate 9a was synthesized from 9-bromononanoic acid and heptadecano-9-ol using the method employed in the synthesis of intermediate 7a. 1 H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 1H), 3.33 (t, J = 6.8 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.78 (p, J = 7.0 Hz, 2H), 1.55 (t, J = 7.2 Hz, 2H), 1.43 (q, J = 6.0 Hz, 4H), 1.41 - 1.30 (m, 3H), 1.22 (d, J = 23.8 Hz, 30H), 0.81 (t, J = 6.7 Hz, 6H).

[0648] Intermediate 9b: 1,1-dibenzyl nonane-1,1,9-tricarboxylic acid ester 9-(heptadecano-9-yl) ester

[0649]

[0650] Intermediate 9b (90%) was synthesized from intermediate 9a using the method employed in the synthesis of intermediate 7b. 1 H NMR(400 MHz, CDCl3) δ 7.34 - 7.20 (m, 10H), 5.07 (s, 3H), 4.80 (td, J = 6.3, 2.4Hz, 1H), 3.36 (t, J = 7.5 Hz, 1H), 2.20 (d, J = 14.9 Hz, 2H), 1.85 (q, J =7.3 Hz, 2H), 1.49 (dq, J = 38.6, 6.7, 6.1 Hz, 7H), 1.20 (d, J = 7.4 Hz, 36H), 0.81 (t, J = 6.6 Hz, 6H).

[0651] Intermediate 9C: 2-(9-(heptadecane-9-yloxy)-9-oxonyl)malonic acid

[0652]

[0653] Intermediate 9c (65%) was synthesized from intermediate 9b using the method employed in the synthesis of intermediate 7c. 1H NMR(400 MHz, CDCl3) δ 4.80 (p, J = 6.2 Hz, 1H), 3.35 (t, J = 7.3 Hz, 1H), 2.22(t, J = 7.5 Hz, 2H), 1.87 (q, J = 7.5 Hz, 2H), 1.54 (t, J = 7.3 Hz, 2H), 1.44(q, J = 6.2 Hz, 4H), 1.21 (d, J = 16.4 Hz, 36H), 0.81 (t, J = 6.7 Hz, 6H).

[0654] Intermediate 9d: Heptadecan-9-yl 11-hydroxy-10-(hydroxymethyl)undecanoate

[0655]

[0656] Intermediate 9d (35%) was synthesized from intermediate 9c using the method employed in the synthesis of intermediate 7d.

[0657] Intermediate 9e: (9Z,12Z)-octadec-9,12-dienoic acid 11-(heptadecane-9-yloxy)-2-(hydroxymethyl)-11-oxoundecyl ester

[0658]

[0659] Intermediate 9e (42%) was synthesized from intermediate 9d using the method employed in the synthesis of intermediate 7d. 1 H NMR(400 MHz, CDCl3) δ 5.38 - 5.21 (m, 4H), 4.80 (p, J = 6.3 Hz, 1H), 4.15 (dd, J= 11.2, 4.3 Hz, 1H), 4.01 (dd, J = 11.2, 6.7 Hz, 1H), 3.56 - 3.44 (m, 1H), 3.45 - 3.38 (m, 1H), 2.70 (t, J = 6.3 Hz, 2H), 2.23 (dt, J = 18.4, 7.5 Hz, 4H), 1.98 (q, J = 6.8 Hz, 4H), 1.72 (td, J = 6.6, 3.3 Hz, 1H), 1.54 (tt, J =7.3, 3.6 Hz, 4H), 1.43 (q, J = 6.0 Hz, 4H), 1.34 - 1.11 (m, 52H), 0.81 (q, J= 6.6 Hz, 9H).

[0660] Intermediate 9f: (9Z,12Z)-octadec-9,12-dienoic acid 11-(heptadecan-9-yloxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-11-oxoundecyl ester

[0661]

[0662] Intermediate 9f is synthesized from intermediate 9e using the method employed in the synthesis of intermediate 7e. 1 H NMR (400MHz, CDCl3) δ 8.27 - 8.15 (m, 2H), 7.37 - 7.28 (m, 2H), 5.38 - 5.19 (m, 4H), 4.80 (p, J = 6.3 Hz, 1H), 4.28 - 4.16 (m, 2H), 4.10 (dd, J = 11.3, 4.7 Hz, 1H), 4.02 (dd, J = 11.3, 6.6 Hz, 1H), 2.70 (t, J = 6.3 Hz, 2H), 2.23 (dt, J =17.9, 7.5 Hz, 4H), 2.06 - 1.91 (m, 5H), 1.59 - 1.48 (m, 4H), 1.43 (t, J = 6.2Hz, 4H), 1.38 - 1.14 (m, 51H), 0.81 (td, J = 6.8, 4.7 Hz, 9H).

[0663] Compound 9: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-11-(heptadecyl-9-yloxy)-11-oxoundecyl ester

[0664]

[0665] Compound 9 was synthesized from intermediate 9e using the method employed in the synthesis of compound 7. 1H NMR (400MHz, CDCl3) δ 5.38 - 5.18 (m, 4H), 4.79 (p, J = 6.3 Hz, 1H), 4.11 (t, J = 6.6Hz, 2H), 4.07 - 3.91 (m, 4H), 2.70 (t, J = 6.4 Hz, 2H), 2.47 - 2.40 (m, 5H), 2.22 (dt, J = 10.6, 7.5 Hz, 4H), 1.96 (dq, J = 11.9, 6.3, 5.9 Hz, 4H), 1.74(p, J = 6.8 Hz, 2H), 1.53 (d, J = 7.4 Hz, 4H), 1.43 (q, J = 5.9 Hz, 4H), 1.35- 1.11 (m, 48H), 0.94 (t, J = 7.1 Hz, 6H), 0.81 (h, J = 5.4 Hz, 9H). MS: 890.7m / z [M+H].

[0666] Example 10 - Compound 10

[0667] Compound 10: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)-11-(heptadecyl-9-yloxy)-11-oxoundecyl ester

[0668]

[0669] Compound 10 was synthesized from intermediate 9f and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 1. 1H NMR (400 MHz, CDCl3) δ 5.42 - 5.27 (m, 4H), 4.86 (p, J = 6.3Hz, 1H), 4.04 (tq, J = 11.0, 5.5, 4.6 Hz, 4H), 3.31 - 3.11 (m, 2H), 2.77 (t,J = 6.4 Hz, 2H), 2.53 (s, 5H), 2.28 (dt, J = 10.7, 7.5 Hz, 4H), 2.04 (q, J =6.9 Hz, 4H), 1.61 (p, J = 7.3 Hz, 4H), 1.50 (q, J = 5.9 Hz, 4H), 1.40 - 1.19(m, 52H), 1.01 (d, J = 10.1 Hz, 6H), 0.90 - 0.83 (m, 9H). MS: 875.5 m / z [M+H].

[0670] Example 11 - Compound 11

[0671] Compound 11: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)methyl)-11-(heptadecyl-9-yloxy)-11-oxoundecyl ester

[0672]

[0673] Compound 11 was synthesized from intermediate 9f and 3-[ethyl(methyl)amino]prop-1-ol using the method employed in the synthesis of compound 1. 1H NMR (400 MHz, CDCl3) δ 5.37 - 5.20 (m, 4H), 4.79 (p, J = 6.3Hz, 1H), 4.11 (t, J = 6.5 Hz, 2H), 4.09 - 3.92 (m, 4H), 2.70 (t, J = 6.4 Hz,2H), 2.50 - 2.29 (m, 4H), 2.27 - 2.10 (m, 7H), 1.98 (q, J = 6.8 Hz, 5H), 1.79(p, J = 7.1 Hz, 2H), 1.54 (p, J = 7.1 Hz, 4H), 1.43 (q, J = 5.8 Hz, 4H), 1.38- 1.05 (m, 49H), 0.99 (t, J = 7.2 Hz, 3H), 0.88 - 0.74 (m, 9H). MS: 876.5 m / z[M+H].

[0674] Example 12 - Compound 12

[0675] Intermediate 12a: Non-5-bromopentanoate

[0676]

[0677] Intermediate 12a was synthesized from 5-bromopentanoic acid and nonyl-5-ol using the method employed in the synthesis of intermediate 7a. 1 HNMR (400 MHz, CDCl3) δ 4.86 (p, J = 6.3 Hz, 1H), 3.39 (t, J = 6.6 Hz, 2H), 2.31 (t, J = 7.2 Hz, 2H), 1.94 - 1.84 (m, 2H), 1.82 - 1.71 (m, 2H), 1.57 -1.43 (m, 4H), 1.26 (tdd, J = 13.6, 10.2, 6.7 Hz, 8H), 0.87 (t, J = 6.9 Hz, 6H).

[0678] Intermediate 12b: 1,1-dibenzyl pentane-1,1,5-tricarboxylic acid ester 5-(non-5-yl) ester

[0679]

[0680] Intermediate 12b is synthesized from intermediate 12a using the method employed in the synthesis of intermediate 7b. 1H NMR (400MHz, CDCl3) δ 7.40 - 7.08 (m, 10H), 5.11 - 4.94 (m, 4H), 4.78 (p, J = 6.3 Hz, 1H), 3.36 (t, J = 7.5 Hz, 1H), 2.16 (t, J = 7.6 Hz, 2H), 1.87 (q, J = 7.6 Hz, 2H), 1.54 (q, J = 7.7 Hz, 2H), 1.47 - 1.34 (m, 4H), 1.32 - 1.08 (m, 10H), 0.81 (t, J = 6.9 Hz, 6H).

[0681] Intermediate 12c: 2-(5-(non-5-yloxy)-5-oxopentyl)malonic acid

[0682]

[0683] Intermediate 12c is synthesized from intermediate 12b using the method employed in the synthesis of intermediate 7c. 1 H NMR (400MHz, CDCl3) δ 4.87 (p, J = 6.3 Hz, 1H), 3.43 (t, J = 7.3 Hz, 1H), 2.32 (t, J= 7.5 Hz, 2H), 1.96 (q, J = 7.6 Hz, 2H), 1.68 (p, J = 7.6 Hz, 2H), 1.58 -1.39 (m, 6H), 1.27 (dddd, J = 19.7, 9.6, 7.8, 5.6 Hz, 9H), 0.88 (t, J = 7.0Hz, 6H).

[0684] Intermediate 12d: Non-5-yl 7-hydroxy-6-(hydroxymethyl)heptahydrate

[0685]

[0686] Intermediate 12d was synthesized from intermediate 12c using the method employed in the synthesis of intermediate 7d.

[0687] Intermediate 12e: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-(hydroxymethyl)-7-(non-5-yloxy)-7-oxohepyl ester

[0688]

[0689] Intermediate 12e was synthesized from intermediate 12d using the method employed in the synthesis of intermediate 7e. 1 H NMR (400MHz, CDCl3) δ 5.44 - 5.26 (m, 4H), 4.86 (p, J = 6.3 Hz, 1H), 4.20 (dd, J =11.3, 4.4 Hz, 1H), 4.07 (dd, J = 11.3, 6.6 Hz, 1H), 3.58 (dd, J = 11.3, 4.5Hz, 1H), 3.49 (dd, J = 11.3, 6.4 Hz, 1H), 2.76 (t, J = 6.4 Hz, 2H), 2.30 (q,J = 7.3 Hz, 4H), 2.04 (q, J = 6.9 Hz, 4H), 1.84 - 1.74 (m, 1H), 1.70 - 1.57 (m, 4H), 1.50 (h, J = 6.2 Hz, 4H), 1.44 - 1.20 (m, 26H), 0.96 - 0.81 (m, 9H).

[0690] Intermediate 12f: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-7-(non-5-yloxy)-7-oxohepyl ester

[0691]

[0692] Intermediate 12f was synthesized from intermediate 12e using the method employed in the synthesis of intermediate 7f. 1H NMR (400MHz, CDCl3) δ 8.32 - 8.23 ​​(m, 2H), 7.42 - 7.34 (m, 2H), 5.44 - 5.25 (m, 4H), 4.87 (p, J = 6.3 Hz, 1H), 4.27 (h, J = 5.9 Hz, 2H), 1.63 (ddd, J = 20.1, 16.1, 9.4 Hz, 4H), 1.56 -1.41 (m, 8H), 1.29 (tdd, J = 10.3, 7.6, 4.5 Hz, 23H), 0.88 (t, J = 6.8 Hz, 9H).

[0693] Compound 12: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)-7-(non-5-yloxy)-7-oxohepyl ester

[0694]

[0695] Compound 12 was synthesized from intermediate 12f and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 1. 1 H NMR (400 MHz, CDCl3) δ 5.28 (tt, J = 11.1, 5.6 Hz, 4H), 4.80 (p, J = 6.3 Hz, 1H), 3.98 (td, J = 13.6, 12.3, 6.6 Hz, 4H), 3.17 (s, 2H),2.70 (t, J = 6.5 Hz, 2H), 2.48 (s, 5H), 2.22 (td, J = 7.6, 4.0 Hz, 4H), 1.98(q, J = 6.8 Hz, 4H), 1.54 (p, J = 7.0 Hz, 4H), 1.50 - 1.41 (m, 4H), 1.39 -1.15 (m, 27H), 0.96 (s, 6H), 0.82 (t, J = 6.8 Hz, 9H). MS: 707.6 m / z [M+H].

[0696] Example 13 - Compound 13

[0697] Compound 13: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)methyl)-7-(non-5-yloxy)-7-oxoheptyl ester

[0698]

[0699] Compound 13 was synthesized from intermediate 12f and 3-[ethyl(methyl)amino]prop-1-ol using the method employed in the synthesis of compound 1. 1 H NMR (400 MHz, CDCl3) δ 5.38 - 5.18 (m, 4H), 4.80 (p, J = 6.3Hz, 1H), 4.12 (t, J = 6.5 Hz, 2H), 4.08 - 3.93 (m, 4H), 2.70 (t, J = 6.5 Hz,2H), 2.39 (s, 4H), 2.27 - 2.11 (m, 7H), 1.97 (p, J = 8.1, 7.4 Hz, 5H), 1.55(q, J = 7.5 Hz, 4H), 1.50 - 1.41 (m, 4H), 1.39 - 1.14 (m, 27H), 1.00 (t, J =7.4 Hz, 3H), 0.82 (td, J = 7.0, 1.6 Hz, 9H). MS: 708.6 m / z [M+H].

[0700] Example 14 - Compound 14

[0701] Compound 14: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-7-(non-5-yloxy)-7-oxohepyl ester

[0702]

[0703] Compound 14 was synthesized from intermediate 12f using the method employed in the synthesis of compound 1. 1H NMR (400MHz, CDCl3) δ 5.37 - 5.21 (m, 4H), 4.80 (p, J = 6.3 Hz, 1H), 4.11 (t, J = 6.6Hz, 2H), 4.07 - 3.94 (m, 4H), 2.70 (t, J = 6.4 Hz, 2H), 2.50 - 2.39 (m, 6H), 2.22 (td, J = 7.5, 3.1 Hz, 4H), 1.97 (p, J = 8.0, 7.4 Hz, 5H), 1.75 (p, J =6.8 Hz, 2H), 1.54 (t, J = 7.5 Hz, 4H), 1.44 (td, J = 7.8, 6.9, 4.4 Hz, 4H), 1.39 - 1.15 (m, 27H), 0.95 (t, J = 7.1 Hz, 6H), 0.87 - 0.74 (m, 9H). MS: 722.6m / z [M+H].

[0704] Example 15 - Compound 15

[0705] Intermediate 15a: Heptadecan-9-yl 5-bromopentanoate

[0706]

[0707] Intermediate 15a was synthesized from 5-bromopentanoic acid and heptadecano-9-ol using the method employed in the synthesis of intermediate 7a.

[0708] Intermediate 15b: 1,1-dibenzyl pentane-1,1,5-tricarboxylic acid ester 5-(heptadecano-9-yl) ester

[0709]

[0710] Intermediate 15b is synthesized from intermediate 15a using the method employed in the synthesis of intermediate 7b.

[0711] Intermediate 15c: 2-(5-(heptadecane-9-yloxy)-5-oxopentyl)malonic acid

[0712]

[0713] Intermediate 15c was synthesized from intermediate 15b using the method employed in the synthesis of intermediate 7c. 1H NMR (400MHz, CDCl3) δ 4.86 (p, J = 6.2 Hz, 1H), 3.42 (t, J = 7.3 Hz, 1H), 2.31 (t, J= 7.5 Hz, 2H), 1.96 (q, J = 7.7 Hz, 2H), 1.72 - 1.60 (m, 2H), 1.46 (dq, J =25.4, 4.9, 3.4 Hz, 6H), 1.25 (s, 24H), 0.94 - 0.77 (m, 6H).

[0714] Intermediate 15d: Heptadecano-9-yl ester of 7-hydroxy-6-(hydroxymethyl)heptadecanoate

[0715]

[0716] Intermediate 15d was synthesized from intermediate 15c using the method employed in the synthesis of intermediate 7d.

[0717] Intermediate 15e: (9Z,12Z)-octadecyl-9,12-dienoic acid 7-(heptadecan-9-yloxy)-2-(hydroxymethyl)-7-oxohepyl ester

[0718]

[0719] Intermediate 15e was synthesized from intermediate 15d using the method employed in the synthesis of intermediate 7e. 1 H NMR (400MHz, CDCl3) δ 5.45 - 5.24 (m, 4H), 4.86 (p, J = 6.2 Hz, 1H), 4.20 (dd, J =11.2, 4.4 Hz, 1H), 4.08 (dd, J = 11.3, 6.6 Hz, 1H), 3.58 (dd, J = 11.3, 4.5Hz, 1H), 3.49 (dd, J = 11.3, 6.4 Hz, 1H), 2.77 (t, J = 6.6 Hz, 2H), 2.30 (dt,J = 8.9, 7.5 Hz, 4H), 2.05 (q, J = 6.9 Hz, 4H), 1.79 (tt, J = 6.4, 4.3 Hz,1H), 1.62 (q, J = 7.2 Hz, 4H), 1.49 (t, J = 6.2 Hz, 4H), 1.43 - 1.20 (m,44H), 0.88 (td, J = 6.9, 5.1 Hz, 9H).

[0720] Intermediate 15f: (9Z,12Z)-octadecyl-9,12-dienoic acid 7-(heptadecan-9-yloxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-7-oxohepyl ester

[0721]

[0722] Intermediate 15f was synthesized from intermediate 15e using the method employed in the synthesis of intermediate 7f. 1 H NMR (400MHz, CDCl3) δ 8.28 - 8.14 (m, 2H), 7.40 - 7.28 (m, 2H), 5.37 - 5.18 (m, 4H), 4.80 (p, J = 6.3 Hz, 1H), 4.20 (h, J = 5.9 Hz, 2H), 4.13 - 3.96 (m, 2H), 2.70 (t, J = 6.4 Hz, 2H), 2.25 (td, J = 7.5, 3.6 Hz, 4H), 2.10 - 1.91 (m, 5H), 1.64 - 1.48 (m, 5H), 1.48 - 1.34 (m, 8H), 1.34 - 1.12 (m, 39H), 0.94 - 0.65(m, 9H).

[0723] Compound 15: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-7-(heptadecyl-9-yloxy)-7-oxohepyl ester

[0724]

[0725] Compound 15 was synthesized from intermediate 15f using the method employed in the synthesis of compound 1. ¹H NMR (400 MHz, CDCl₃) δ 5.34 (dddd, J = 12.1, 10.7, 8.8, 5.4 Hz, 4H), 4.85 (p, J = 6.3 Hz, 1H), 4.17 (t, J = 6.6 Hz, 2H), 4.12 - 3.98 (m, 4H), 2.76 (t, J = 6.7 Hz, 2H), 2.56 - 2.46 (m, 6H), 2.28 (td, J = 7.6, 5.0 Hz, 4H), 2.04 (q, J = 6.8 Hz, 5H), 1.81 (p, J = 6.7 Hz, 2H), 1.61 (dt, J = 12.4, 6.8 Hz, 4H), 1.49 (q,J = 6.2 Hz, 4H), 1.43 - 1.20 (m, 44H), 1.01 (t, J = 7.1 Hz, 6H), 0.87 (td, J= 6.8, 5.0 Hz, 9H). MS: 834.7 m / z [M+H].

[0726] Example 16 - Compound 16

[0727] Intermediate 16a: Heptadecan-9-yl ester of 7-bromoheptanoate

[0728]

[0729] Intermediate 16a was synthesized from intermediate 2a and 7-bromoheptanoic acid using the method employed in the synthesis of intermediate 7a. 1 HNMR (400 MHz, CDCl3) δ 4.86 (p, J = 6.3 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.86 (dt, J = 14.7, 6.9 Hz, 2H), 1.64 (p, J = 7.4Hz, 2H), 1.57 - 1.41 (m, 6H), 1.40 - 1.16 (m, 26H), 0.94 - 0.78 (m, 6H).

[0730] Intermediate 16b: 1,1-dibenzyl heptane-1,1,7-tricarboxylic acid ester 7-(heptadecane-9-yl) ester

[0731]

[0732] Intermediate 16b is synthesized from intermediate 16a using the method employed in the synthesis of intermediate 7b. 1 H NMR (400MHz, CDCl3) δ 7.46 - 7.14 (m, 10H), 5.15 (d, J = 1.3 Hz, 4H), 4.87 (p, J =6.3 Hz, 1H), 3.43 (t, J = 7.5 Hz, 1H), 2.25 (t, J = 7.5 Hz, 2H), 1.93 (q, J =7.2 Hz, 2H), 1.54 (dt, J = 31.6, 6.5 Hz, 6H), 1.28 (d, J = 15.4 Hz, 31H), 0.91 - 0.86 (m, 6H).

[0733] Intermediate 16C: 2-(7-(heptadecane-9-yloxy)-7-oxohepyl)malonic acid

[0734]

[0735] Intermediate 16c was synthesized from intermediate 16b using the method employed in the synthesis of intermediate 7c. 1 H NMR (400MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 1H), 3.35 (t, J = 7.4 Hz, 1H), 2.22 (t, J= 7.5 Hz, 2H), 1.88 (q, J = 7.4 Hz, 2H), 1.54 (t, J = 7.3 Hz, 2H), 1.44 (d, J= 6.1 Hz, 4H), 1.36 - 1.13 (m, 32H), 0.81 (t, J= 6.7 Hz, 6H).

[0736] Intermediate 16d: Heptadecan-9-yl 9-hydroxy-8-(hydroxymethyl)nonanoic acid

[0737]

[0738] Intermediate 16d was synthesized from intermediate 16c using the method employed in the synthesis of intermediate 7c.

[0739] Intermediate 16e: (9Z,12Z)-octadecyl-9,12-dienoic acid 9-(heptadecan-9-yloxy)-2-(hydroxymethyl)-9-oxonyl ester

[0740]

[0741] Intermediate 16e was synthesized from intermediate 16d using the method employed in the synthesis of intermediate 7e. 1 H NMR (400MHz, CDCl3) δ 5.29 (qd, J = 10.9, 4.4 Hz, 4H), 4.79 (p, J = 6.3 Hz, 1H), 4.14(dd, J = 11.2, 4.4 Hz, 1H), 4.01 (dd, J = 11.2, 6.6 Hz, 1H), 3.51 (dd, J =11.2, 4.5 Hz, 1H), 3.42 (dd, J = 11.3, 6.5 Hz, 1H), 2.70 (t, J = 6.4 Hz, 2H), 2.23 (dt, J = 17.1, 7.5 Hz, 4H), 1.98 (q, J = 6.9 Hz, 4H), 1.71 (p, J = 5.4Hz, 1H), 1.55 (t, J = 7.2 Hz, 4H), 1.43 (q, J = 6.2 Hz, 5H), 1.37 - 1.14 (m,48H), 0.81 (q, J = 6.9, 6.4 Hz, 9H).

[0742] Intermediate 16f: (9Z,12Z)-octadecyl-9,12-dienoic acid 9-(heptadecan-9-yloxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-9-oxonyl ester

[0743]

[0744] Intermediate 16f was synthesized from intermediate 16e using the method employed in the synthesis of intermediate 7f. 1H NMR (400MHz, CDCl3) δ 8.32 - 8.23 ​​(m, 2H), 7.41 - 7.35 (m, 2H), 5.44 - 5.26 (m, 3H), 4.86 (q, J = 6.3 Hz, 1H), 4.32 - 4.21 (m, 2H), 4.19 - 4.03 (m, 2H), 2.76 (dd,J = 7.3, 5.9 Hz, 2H), 2.30 (dt, J = 15.0, 7.5 Hz, 4H), 2.17 - 1.98 (m, 5H),1.62 (p, J = 6.9 Hz, 5H), 1.50 (q, J = 6.1 Hz, 4H), 1.46 - 1.18 (m, 48H), 0.88 (td, J = 6.8, 5.0 Hz, 9H).

[0745] Compound 16: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-9-(heptadecyl-9-yloxy)-9-oxonyl ester

[0746]

[0747] Compound 16 was synthesized from intermediate 16f using the method employed in the synthesis of compound 1. 1 H NMR (400MHz, CDCl3) δ 5.37 (tt, J = 11.3, 5.5 Hz, 4H), 4.88 (p, J = 6.3 Hz, 1H), 4.20(t, J = 6.6 Hz, 2H), 4.17 - 4.00 (m, 4H), 2.79 (t, J = 6.5 Hz, 2H), 2.53 (q,J = 6.9 Hz, 6H), 2.30 (dt, J = 11.0, 7.5 Hz, 4H), 2.05 (dq, J = 12.4, 6.3,5.8 Hz, 5H), 1.83 (p, J = 6.8 Hz, 2H), 1.64 (q, J = 7.1 Hz, 4H), 1.52 (q, J =6.0 Hz, 4H), 1.44 - 1.20 (m, 47H), 1.03 (t, J = 7.1 Hz, 6H), 0.90 (q, J = 6.5Hz, 9H). MS: 862.4 m / z [M+H].

[0748] Example 17 - Compound 17

[0749] Intermediate 17a: 5-bromopentanoic acid tridecane-7-yl ester

[0750]

[0751] Intermediate 17a was synthesized from 5-bromopentanoic acid and tridecane-7-ol using the method employed in the synthesis of intermediate 7a.

[0752] Intermediate 17b: 1,1-dibenzyl pentane-1,1,5-tricarboxylic acid ester 5-(tetane-7-yl) ester

[0753]

[0754] Intermediate 17b is synthesized from intermediate 17a using the method employed in the synthesis of intermediate 7b. 1 H NMR (400MHz, CDCl3) δ 7.46 - 7.22 (m, 10H), 5.20 - 5.08 (m, 4H), 4.85 (p, J = 6.3 Hz, 1H), 3.43 (t, J = 7.5 Hz, 1H), 2.23 (t, J = 7.6 Hz, 2H), 2.00 - 1.91 (m, 2H), 1.62 (p, J = 7.6 Hz, 2H), 1.50 (q, J = 7.2, 6.7 Hz, 4H), 1.38 - 1.19 (m,19H), 0.92 - 0.83 (m, 6H).

[0755] Intermediate 17c: 2-(5-oxo-5-(tetran-7-yloxy)pentyl)malonic acid

[0756]

[0757] Intermediate 17c is synthesized from intermediate 17b using the method employed in the synthesis of intermediate 7c. 1 H NMR (400MHz, CDCl3) δ 4.87 (p, J = 6.3 Hz, 1H), 3.43 (t, J = 7.3 Hz, 1H), 2.32 (t, J= 7.5 Hz, 2H), 1.96 (q, J = 7.6 Hz, 2H), 1.68 (q, J = 7.6 Hz, 2H), 1.57 -1.38 (m, 6H), 1.26 (h, J = 4.3, 3.5 Hz, 17H), 0.93 - 0.81 (m, 6H).

[0758] Intermediate 17d: 7-hydroxy-6-(hydroxymethyl)heptadecane-7-yl ester

[0759]

[0760] Intermediate 17d was synthesized from intermediate 17c using the method employed in the synthesis of intermediate 7d. 1 H NMR (400MHz, CDCl3) δ 4.86 (pd, J = 6.3, 2.2 Hz, 1H), 4.04 (ddd, J = 11.0, 7.8, 4.3Hz, 1H), 3.69 - 3.56 (m, 1H), 2.36 - 2.24 (m, 2H), 1.97 (dddt, J = 9.7, 7.4,4.5, 2.5 Hz, 1H), 1.64 (ddt, J = 15.1, 11.0, 7.6 Hz, 2H), 1.50 (d, J = 6.4Hz, 4H), 1.39 - 1.19 (m, 20H), 0.93 - 0.82 (m, 6H).

[0761] Intermediate 17e: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-(hydroxymethyl)-7-oxo-7-(tetran-7-yloxy)heptyl ester

[0762]

[0763] Intermediate 17e was synthesized from intermediate 17d using the method employed in the synthesis of intermediate 7e. 1H NMR (400MHz, CDCl3) δ 5.42 - 5.28 (m, 4H), 4.86 (p, J = 6.3 Hz, 1H), 4.20 (dd, J =11.2, 4.4 Hz, 1H), 4.08 (dd, J = 11.2, 6.6 Hz, 1H), 3.58 (dd, J = 11.3, 4.4Hz, 1H), 3.49 (dd, J = 11.3, 6.5 Hz, 1H), 2.77 (t, J = 6.4 Hz, 2H), 2.30 (q,J = 7.9 Hz, 4H), 2.04 (q, J = 6.9 Hz, 4H), 1.79 (ddt, J = 8.9, 6.5, 3.3 Hz,1H), 1.62 (q, J = 7.1 Hz, 4H), 1.50 (d, J = 6.4 Hz, 4H), 1.42 - 1.22 (m,34H), 0.92 - 0.83 (m, 9H).

[0764] Intermediate 17f: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-7-oxo-7-(tetran-7-yloxy)heptyl ester

[0765]

[0766] Intermediate 17f was synthesized from intermediate 17e using the method employed in the synthesis of intermediate 7f. 1 H NMR (400MHz, CDCl3) δ 8.28 - 8.15 (m, 2H), 7.41 - 7.28 (m, 2H), 5.41 - 5.16 (m, 4H), 4.80 (p, J = 6.3 Hz, 1H), 4.20 (h, J = 5.9 Hz, 2H), 4.15 - 3.96 (m, 2H), 2.70(t, J = 6.3 Hz, 2H), 2.24 (tt, J = 7.2, 3.7 Hz, 4H), 2.10 - 1.89 (m, 5H),1.56 (dq, J = 15.3, 7.5 Hz, 4H), 1.50 - 1.34 (m, 8H), 1.34 - 1.10 (m, 30H), 0.81 (q, J = 6.5 Hz, 9H).

[0767] Compound 17: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-7-oxo-7-(tetran-7-yloxy)heptyl ester

[0768]

[0769] Compound 17 was synthesized from intermediate 17f using the method employed in the synthesis of compound 1. 1 H NMR (400MHz, CDCl3) δ 5.37 (tt, J = 11.2, 5.6 Hz, 4H), 4.88 (p, J = 6.2 Hz, 1H), 4.20(t, J = 6.6 Hz, 2H), 4.16 - 4.01 (m, 4H), 2.79 (t, J = 6.4 Hz, 2H), 2.55 (q,J = 6.9 Hz, 5H), 2.31 (td, J = 7.5, 4.7 Hz, 4H), 2.06 (p, J = 8.0, 7.4 Hz,4H), 1.85 (p, J = 6.9 Hz, 2H), 1.63 (p, J = 7.2 Hz, 4H), 1.52 (t, J = 6.4 Hz, 4H), 1.47 - 1.21 (m, 34H), 1.04 (t, J = 7.1 Hz, 6H), 0.90 (q, J = 6.6 Hz, 9H). MS: 778.3 m / z [M+H].

[0770] Example 18 - Compound 18

[0771] Intermediate 24a: Tridecane-7-yl ester of 7-bromoheptanoate

[0772]

[0773] Intermediate 18a was synthesized from 7-bromoheptanoic acid and tridecane-7-ol using the method employed in the synthesis of intermediate 7a. 1H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.2 Hz, 1H), 3.46 (t, J = 6.7 Hz,1H), 3.34 (t, J = 6.8 Hz, 1H), 2.22 (t, J = 7.4 Hz, 2H), 1.79 (p, J = 6.9 Hz,1H), 1.71 (p, J = 6.9 Hz, 1H), 1.57 (p, J = 7.5 Hz, 2H), 1.42 (qd, J = 16.2,14.5, 9.2 Hz, 6H), 1.34 - 1.12 (m, 18H), 0.81 (t, J = 6.7 Hz, 6H).

[0774] Intermediate 18b: 1,1-dibenzyl heptane-1,1,7-tricarboxylic acid ester 7-(tetane-7-yl) ester

[0775]

[0776] Intermediate 18b is synthesized from intermediate 18a using the method employed in the synthesis of intermediate 7b. 1 H NMR (400MHz, CDCl3) δ 7.43 - 7.18 (m, 10H), 5.14 (d, J = 1.2 Hz, 4H), 4.86 (p, J =6.3 Hz, 1H), 3.43 (t, J = 7.6 Hz, 1H), 2.25 (t, J = 7.5 Hz, 2H), 1.92 (d, J =7.2 Hz, 2H), 1.63 - 1.43 (m, 7H), 1.27 (dd, J = 10.7, 4.6 Hz, 23H), 0.90 -0.84 (m, 6H).

[0777] Intermediate 18c: 2-(7-oxo-7-(tetran-7-yloxy)heptyl)malonic acid

[0778]

[0779] Intermediate 18c was synthesized from intermediate 18b using the method employed in the synthesis of intermediate 7c. 1H NMR (400MHz, CDCl3) δ 4.87 (p, J = 6.3 Hz, 1H), 3.42 (t, J = 7.4 Hz, 1H), 2.29 (t, J= 7.5 Hz, 2H), 1.94 (q, J = 7.4 Hz, 2H), 1.61 (t, J = 7.3 Hz, 2H), 1.56 -1.46 (m, 4H), 1.44 - 1.21 (m, 23H), 0.93 - 0.83 (m, 6H).

[0780] Intermediate 18d: Tridecane-7-yl ester of 9-hydroxy-8-(hydroxymethyl)nonanoic acid

[0781]

[0782] Intermediate 18d was synthesized from intermediate 18c using the method employed in the synthesis of intermediate 7d.

[0783] Intermediate 18e: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-(hydroxymethyl)-9-oxo-9-(tetran-7-yloxy)nonyl ester

[0784]

[0785] Intermediate 18e was synthesized from intermediate 18d using the method employed in the synthesis of intermediate 7e. 1 H NMR (400MHz, CDCl3) δ 5.44 - 5.26 (m, 4H), 4.86 (p, J = 6.3 Hz, 1H), 4.21 (dd, J =11.2, 4.3 Hz, 1H), 4.07 (dd, J = 11.2, 6.7 Hz, 1H), 3.58 (dd, J = 11.3, 4.5Hz, 1H), 3.49 (dd, J = 11.3, 6.5 Hz, 1H), 2.77 (t, J = 6.4 Hz, 2H), 2.30 (dt,J = 17.4, 7.5 Hz, 4H), 2.05 (q, J = 6.9 Hz, 4H), 1.78 (ddt, J = 10.8, 6.7,4.3 Hz, 1H), 1.67 - 1.57 (m, 4H), 1.50 (d, J = 6.4 Hz, 4H), 1.30 (tt, J =17.5, 14.4, 6.2 Hz, 39H), 0.88 (q, J = 6.8 Hz, 9H).

[0786] Intermediate 18f: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-9-oxo-9-(tetran-7-yloxy)nonyl ester

[0787]

[0788] Intermediate 18f was synthesized from intermediate 18e using the method employed in the synthesis of intermediate 7f. 1 H NMR (400MHz, CDCl3) δ 8.26 - 8.17 (m, 2H), 7.35 - 7.27 (m, 2H), 5.39 - 5.19 (m, 4H), 4.80 (p, J = 6.3 Hz, 1H), 4.27 - 4.14 (m, 2H), 4.10 (dd, J = 11.3, 4.7 Hz, 1H), 4.02 (dd, J = 11.3, 6.6 Hz, 1H), 2.70 (t, J = 6.4 Hz, 2H), 2.23 (dt, J =14.9, 7.5 Hz, 4H), 2.07 - 1.93 (m, 5H), 1.57 (q, J = 7.1 Hz, 4H), 1.52 - 1.41 (m, 5H), 1.41 - 1.12 (m, 39H), 0.87 - 0.75 (m, 9H).

[0789] Compound 18: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-9-oxo-9-(tetran-7-yloxy)nonyl ester

[0790]

[0791] Compound 18 was synthesized from intermediate 18f using the method employed in the synthesis of compound 1. 1H NMR (400MHz, CDCl3) δ 5.35 (tt, J = 11.2, 5.4 Hz, 4H), 4.86 (p, J = 6.3 Hz, 1H), 4.18(t, J = 6.5 Hz, 2H), 4.15 - 4.06 (m, 3H), 4.02 (dd, J = 11.2, 6.4 Hz, 1H), 2.77 (t, J = 6.5 Hz, 2H), 2.53 (s, 5H), 2.28 (dt, J = 10.6, 7.6 Hz, 4H), 2.03(dq, J = 12.3, 6.2, 5.6 Hz, 5H), 1.84 (s, 2H), 1.62 (q, J = 7.2 Hz, 4H), 1.50 (q, J = 6.3 Hz, 4H), 1.40 - 1.19 (m, 39H), 1.12 - 0.97 (m, 6H), 0.88 (q, J =6.6 Hz, 9H). MS: 806.3 m / z [M+H].

[0792] Example 19 - Compound 19

[0793] Intermediate 19a: Tridecane-7-yl ester of 9-bromononanoate

[0794]

[0795] Intermediate 19a was synthesized from 9-bromononanoic acid and tridecane-7-ol using the method employed in the synthesis of intermediate 7a.

[0796] Intermediate 19b: 1,1-dibenzyl nonane-1,1,9-tricarboxylic acid 9-(tetran-7-yl) ester

[0797]

[0798] Intermediate 19b is synthesized from intermediate 19a using the method employed in the synthesis of intermediate 7b. 1H NMR (400MHz, CDCl3) δ 7.36 - 7.14 (m, 10H), 5.07 (s, 4H), 4.80 (p, J = 6.3 Hz, 1H), 3.36 (t, J = 7.6 Hz, 1H), 2.19 (t, J = 7.5 Hz, 2H), 1.84 (q, J = 7.6, 5.2 Hz, 2H), 1.59 - 1.37 (m, 7H), 1.28 - 1.14 (m, 26H), 0.80 (t, J = 6.7 Hz, 6H).

[0799] Intermediate 19c: 2-(9-oxo-9-(tetran-7-yloxy)nonyl)malonic acid

[0800]

[0801] Intermediate 19c was synthesized from intermediate 19b using the method employed in the synthesis of intermediate 7c. 1 H NMR (400MHz, CDCl3) δ 4.81 (p, J = 6.2 Hz, 1H), 3.35 (t, J = 7.4 Hz, 1H), 2.22 (t, J= 7.5 Hz, 2H), 1.87 (q, J = 7.5 Hz, 2H), 1.54 (t, J = 7.1 Hz, 2H), 1.50 -1.38 (m, 4H), 1.39 - 0.97 (m, 26H), 0.80 (t, J = 6.8 Hz, 6H).

[0802] Intermediate 19d: 11-hydroxy-10-(hydroxymethyl)undecanoic acid tridecane-7-yl ester

[0803]

[0804] Intermediate 19d was synthesized from intermediate 19c using the method employed in the synthesis of intermediate 7d. 1H NMR (400MHz, CDCl3) δ 4.80 (p, J = 6.2 Hz, 1H), 3.75 (dd, J = 10.6, 3.7 Hz, 2H), 3.59(dd, J = 10.6, 7.6 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.69 (ddt, J = 10.4,6.9, 3.5 Hz, 1H), 1.54 (p, J = 7.2, 6.6 Hz, 2H), 1.44 (q, J = 6.4 Hz, 4H), 1.32 - 1.09 (m, 28H), 0.87 - 0.74 (m, 6H).

[0805] Intermediate 19e: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-(hydroxymethyl)-11-oxo-11-(tetran-7-yloxy)undecyl ester

[0806]

[0807] Intermediate 19e was synthesized from intermediate 19d using the method employed in the synthesis of intermediate 7e.

[0808] Intermediate 19f: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-11-oxo-11-(tetran-7-yloxy)undecyl ester

[0809]

[0810] Intermediate 19f was synthesized from intermediate 19e using the method employed in the synthesis of intermediate 7f. 1H NMR (400MHz, CDCl3) δ 8.36 - 8.23 ​​(m, 2H), 7.43 - 7.35 (m, 2H), 5.46 - 5.25 (m, 4H), 4.87 (p, J = 6.3 Hz, 1H), 4.37 - 4.24 (m, 2H), 4.24 - 4.12 (m, 1H), 4.09 (dd,J = 11.2, 6.6 Hz, 1H), 2.77 (t, J = 6.3 Hz, 2H), 2.30 (dt, J = 17.7, 7.5 Hz,4H), 2.15 - 2.00 (m, 5H), 1.62 (tt, J = 8.0, 4.0 Hz, 4H), 1.51 (q, J = 6.3Hz, 5H), 1.45 - 1.18 (m, 44H), 0.88 (td, J = 6.8, 5.1 Hz, 9H).

[0811] Compound 19: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-11-oxo-11-(tetran-7-yloxy)undecyl ester

[0812]

[0813] Compound 19 was synthesized from intermediate 19f using the method employed in the synthesis of compound 1. 1 H NMR (400MHz, CDCl3) δ 5.48 - 5.24 (m, 4H), 4.86 (p, J = 6.3 Hz, 1H), 4.18 (t, J = 6.5Hz, 2H), 4.14 - 4.00 (m, 4H), 2.77 (t, J = 6.5 Hz, 2H), 2.31 - 2.22 (m, 4H), 2.03 (dq, J = 12.1, 6.4, 6.0 Hz, 5H), 1.84 (s, 2H), 1.61 (p, J = 7.2 Hz, 4H), 1.50 (q, J = 6.2 Hz, 4H), 1.41 - 1.17 (m, 42H), 1.04 (s, 6H), 0.88 (q, J =6.7 Hz, 9H). MS: 834.7 m / z [M+H].

[0814] Example 20 - Compound 22

[0815] Intermediate 20a: Non-5-ol

[0816]

[0817] NaBH4 (2.0 equivalents) was slowly added to a solution of nonan-5-one (25 g, 1.0 equivalent) in MeOH (0.5–1.0 M) at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred at 20 °C for 5 h under a nitrogen atmosphere. The reaction mixture was slowly quenched under nitrogen with saturated NH4Cl, and stirred for another 60 min after the addition. The reaction mixture was poured into water and extracted twice with EtOAc. The organic phase was evaporated under reduced pressure to obtain the residue, which was used directly in the next step without further purification.

[0818] Intermediate 20b: Non-5-yl 7-bromoheptanoate

[0819]

[0820] Intermediate 20b was synthesized from intermediate 20a and 7-bromoheptanoic acid using the method employed in the synthesis of intermediate 7a.

[0821] Intermediate 20c: 1,1-dibenzyl heptane-1,1,7-tricarboxylic acid ester 7-(non-5-yl) ester

[0822]

[0823] Intermediate 20c is synthesized from intermediate 20b using the method employed in the synthesis of intermediate 7b. 1 H NMR (400MHz, CDCl3) δ 7.32 - 7.10 (m, 10H), 5.07 (d, J = 1.1 Hz, 4H), 4.79 (p, J =6.3 Hz, 1H), 3.36 (t, J = 7.5 Hz, 1H), 2.18 (t, J = 7.5 Hz, 2H), 1.85 (d, J =7.2 Hz, 2H), 1.54 - 1.39 (m, 6H), 1.30 - 1.13 (m, 14H), 0.81 (t, J = 6.9 Hz, 6H).

[0824] Intermediate 20d: 2-(7-(non-5-yloxy)-7-oxohepyl)malonic acid

[0825]

[0826] Intermediate 20d was synthesized from intermediate 20c using the method employed in the synthesis of intermediate 7c. 1H NMR (400MHz, CDCl3) δ 4.88 (p, J = 6.3 Hz, 1H), 3.43 (t, J = 7.3 Hz, 1H), 2.29 (t, J= 7.5 Hz, 2H), 1.99 - 1.91 (m, 2H), 1.63 (q, J = 7.2 Hz, 2H), 1.56 - 1.48 (m,4H), 1.44 - 1.20 (m, 15H), 0.88 (t, J = 6.9 Hz, 6H).

[0827] Intermediate 20e: Non-5-yl 9-hydroxy-8-(hydroxymethyl)nonanoic acid ester

[0828]

[0829] Intermediate 20e was synthesized from intermediate 20d using the method employed in the synthesis of intermediate 7d. 1 H NMR (400MHz, CDCl3) δ 4.87 (p, J = 6.3 Hz, 1H), 3.86 - 3.77 (m, 1H), 3.69 - 3.60 (m,1H), 2.28 (td, J = 7.5, 1.3 Hz, 2H), 1.61 (t, J = 7.2 Hz, 2H), 1.56 - 1.46 (m, 4H), 1.42 - 1.19 (m, 16H), 0.88 (t, J = 6.9 Hz, 6H).

[0830] Intermediate 26f: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-(hydroxymethyl)-9-(non-5-yloxy)-9-oxonyl ester

[0831]

[0832] Intermediate 20f was synthesized from intermediate 20e using the method employed in the synthesis of intermediate 7e. 1H NMR (400MHz, CDCl3) δ 5.29 (tp, J = 10.1, 5.0 Hz, 4H), 4.80 (p, J = 6.4 Hz, 1H), 4.15(dd, J = 11.2, 4.3 Hz, 1H), 4.00 (dd, J = 11.2, 6.7 Hz, 1H), 3.51 (dd, J =11.3, 4.5 Hz, 1H), 3.42 (dd, J = 11.3, 6.5 Hz, 1H), 2.70 (t, J = 6.4 Hz, 2H), 2.23 (dt, J = 15.3, 7.5 Hz, 5H), 1.98 (q, J = 6.9 Hz, 4H), 1.72 (t, J = 5.9Hz, 1H), 1.54 (d, J = 7.3 Hz, 6H), 1.45 (q, J = 7.0 Hz, 6H), 1.33 - 1.13 (m,35H), 0.82 (t, J = 6.8 Hz, 9H).

[0833] Intermediate 20g: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-9-(non-5-yloxy)-9-oxonyl ester

[0834]

[0835] Intermediate 20g was synthesized from intermediate 20f using the method employed in the synthesis of intermediate 7f. 1H NMR (400MHz, CDCl3) δ 8.33 - 8.25 (m, 2H), 7.42 - 7.36 (m, 2H), 5.44 - 5.28 (m, 4H), 4.87 (p, J = 6.3 Hz, 1H), 4.27 (h, J = 6.0 Hz, 2H), 4.17 (dd, J = 11.3, 4.8Hz, 1H), 4.09 (dd, J = 11.3, 6.7 Hz, 1H), 2.77 (t, J = 6.3 Hz, 2H), 2.31 (dt,J = 12.9, 7.5 Hz, 5H), 2.18 - 1.99 (m, 5H), 1.68 - 1.57 (m, 5H), 1.57 - 1.47 (m, 6H), 1.47 - 1.18 (m, 34H), 0.89 (t, J = 6.8 Hz, 9H).

[0836] Compound 20: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-9-(non-5-yloxy)-9-oxonyl ester

[0837]

[0838] Compound 20 was synthesized from intermediate 20g using the method employed in the synthesis of compound 1. 1 H NMR (400MHz, CDCl3) δ 5.36 - 5.21 (m, 4H), 4.80 (p, J = 6.3 Hz, 1H), 4.11 (t, J = 6.5Hz, 2H), 4.07 - 3.92 (m, 4H), 2.70 (t, J = 6.4 Hz, 2H), 2.46 (q, J = 6.9 Hz, 5H), 2.22 (q, J = 7.9 Hz, 4H), 2.03 - 1.90 (m, 5H), 1.77 (q, J = 7.2 Hz, 2H), 1.53 (q, J = 7.0 Hz, 5H), 1.44 (dq, J = 13.2, 6.4 Hz, 5H), 1.33 - 1.09 (m,31H), 0.96 (t, J = 7.2 Hz, 6H), 0.82 (t, J = 6.9 Hz, 9H). MS: 750.7 m / z [M+H].

[0839] Example 21 - Compound 21

[0840] Intermediate 27a: Non-5-yl 9-bromononanoate

[0841]

[0842] Intermediate 21a was synthesized from 9-bromononanoic acid and intermediate 20a using the method employed in the synthesis of intermediate 7a.

[0843] Intermediate 21b: 1,1-dibenzyl nonane-1,1,9-tricarboxylic acid ester 9-(non-5-yl) ester

[0844]

[0845] Intermediate 21b is synthesized from intermediate 21a using the method employed in the synthesis of intermediate 7b. 1 H NMR (400MHz, CDCl3) δ 7.34 - 7.16 (m, 10H), 5.07 (s, 4H), 4.80 (p, J = 6.3 Hz, 1H), 3.36 (t, J = 7.5 Hz, 1H), 2.20 (t, J = 7.5 Hz, 2H), 1.85 (d, J = 7.1 Hz, 2H), 1.48 (dq, J = 29.2, 7.0 Hz, 7H), 1.20 (ttt, J = 14.1, 11.2, 7.4 Hz, 19H), 0.81 (t, J = 6.9 Hz, 6H).

[0846] Intermediate 21c: 2-(9-(nonyl-5-yloxy)-9-oxonyl)malonic acid

[0847]

[0848] Intermediate 21c is synthesized from intermediate 21b using the method employed in the synthesis of intermediate 7c. 1 H NMR (400MHz, CDCl3) δ 4.88 (p, J = 6.3 Hz, 1H), 3.43 (t, J = 7.3 Hz, 1H), 2.29 (t, J= 7.5 Hz, 2H), 1.94 (q, J = 7.5 Hz, 2H), 1.61 (p, J = 7.2 Hz, 2H), 1.57 -1.46 (m, 4H), 1.43 - 1.21 (m, 19H), 0.88 (t, J = 6.9 Hz, 6H).

[0849] Intermediate 21d: Non-5-yl 11-hydroxy-10-(hydroxymethyl)undecanoate

[0850]

[0851] Intermediate 21d was synthesized from intermediate 21c using the method employed in the synthesis of intermediate 7d.

[0852] Intermediate 21e: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-(hydroxymethyl)-11-(non-5-yloxy)-11-oxoundecyl ester

[0853]

[0854] Intermediate 21e was synthesized from intermediate 21d using the method employed in the synthesis of intermediate 7e. 1 H NMR (400MHz, CDCl3) δ 5.48 - 5.23 (m, 4H), 4.87 (p, J = 6.3 Hz, 1H), 4.22 (dd, J =11.2, 4.3 Hz, 1H), 4.08 (dd, J = 11.2, 6.7 Hz, 1H), 3.59 (dd, J = 11.3, 4.5Hz, 1H), 3.49 (dd, J = 11.3, 6.5 Hz, 1H), 2.77 (t, J = 6.4 Hz, 2H), 2.30 (dt,J = 16.8, 7.5 Hz, 4H), 2.05 (q, J = 6.8 Hz, 4H), 1.83 - 1.74 (m, 1H), 1.63(ddt, J = 10.5, 7.2, 3.3 Hz, 5H), 1.58 - 1.46 (m, 5H), 1.40 - 1.21 (m, 38H), 0.89 (td, J = 6.9, 1.8 Hz, 9H).

[0855] Intermediate 21f: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((4-nitrophenoxy)carbonyl)oxy)methyl)-11-(non-5-yloxy)-11-oxoundecyl ester

[0856]

[0857] Intermediate 21f is synthesized from intermediate 21e using the method employed in the synthesis of intermediate 7f. 1H NMR (400MHz, CDCl3) δ 8.28 - 8.15 (m, 2H), 7.38 - 7.28 (m, 2H), 5.38 - 5.20 (m, 4H), 4.80 (p, J = 6.3 Hz, 1H), 4.26 - 4.14 (m, 2H), 4.10 (dd, J = 11.3, 4.8 Hz, 1H), 4.02 (dd, J = 11.3, 6.7 Hz, 1H), 2.70 (t, J = 6.3 Hz, 2H), 2.23 (dt, J =16.2, 7.5 Hz, 4H), 2.07 - 1.93 (m, 5H), 1.55 (tt, J = 7.2, 3.6 Hz, 4H), 1.52- 1.40 (m, 5H), 1.40 - 1.12 (m, 36H), 0.82 (t, J = 6.8 Hz, 9H).

[0858] Compound 21: (9Z,12Z)-octadecyl-9,12-dienoic acid 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-11-(non-5-yloxy)-11-oxoundecyl ester

[0859]

[0860] Compound 21 was synthesized from intermediate 21f using the method employed in the synthesis of compound 1. 1 H NMR (400MHz, CDCl3) δ 5.43 - 5.27 (m, 4H), 4.87 (p, J = 6.3 Hz, 1H), 4.19 (t, J = 6.5Hz, 2H), 4.15 - 4.00 (m, 4H), 2.77 (t, J = 6.5 Hz, 2H), 2.57 (s, 5H), 2.33 -2.22 (m, 4H), 2.03 (dq, J = 12.2, 6.3, 5.8 Hz, 5H), 1.61 (p, J = 7.1 Hz, 5H),1.56 - 1.45 (m, 5H), 1.40 - 1.20 (m, 35H), 1.06 (s, 6H), 0.89 (td, J = 6.9, 1.8 Hz, 9H). MS: 778.6 m / z [M+H].

[0861] Example 22 - Compound 22

[0862] Compound 22: bis(4,4-bis(octyloxy)butyric acid)2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl ester

[0863]

[0864] Compound 22 was synthesized from intermediate 1d and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 1. 1 H NMR (400 MHz, CDCl3) δ 5.21 (d, J = 5.4 Hz, 1H), 4.48 (t, J =5.5 Hz, 2H), 4.12 (d, J = 5.7 Hz, 6H), 3.56 (dt, J = 9.3, 6.7 Hz, 4H), 3.40(dt, J = 9.3, 6.7 Hz, 4H), 3.20 (q, J = 5.9 Hz, 2H), 2.51 (q, J = 7.1, 6.2Hz, 6H), 2.39 (t, J = 7.6 Hz, 5H), 1.92 (td, J = 7.6, 5.5 Hz, 4H), 1.54 (q, J= 6.9 Hz, 8H), 1.39 - 1.21 (m, 41H), 1.00 (t, J = 7.1 Hz, 6H), 0.92 - 0.82 (m, 12H). MS: 901.3 m / z [M+H].

[0865] Example 23 - Compound 23

[0866] Intermediate 23a: (9Z,12Z)-Octadeca-9,12-dien-1-yl methanesulfonate

[0867]

[0868] A mixture of (9Z,12Z)-octadecyl-9,12-dien-1-ol (16 g, 1.0 equivalent) and TEA (1.2 equivalent) in DCM (0.1–0.2 M) was degassed and purged three times with N2. MsCl (1.2 equivalent) was then slowly added to the mixture at 0 °C. The mixture was stirred at 20 °C for 2 h under N2 atmosphere. Saturated NaHCO3 was then slowly added to the reaction mixture under N2 atmosphere, followed by stirring for 20 min. The mixture was then poured into water and extracted with EtOAc. The organic phase was evaporated under reduced pressure to obtain the residue, which was used directly in the next step without further purification.

[0869] Intermediate 23b: (6Z,9Z)-18-bromooctadecyl-6,9-diene

[0870]

[0871] TBAB (1.5 equivalents) was added to a solution of intermediate 23a (16 g, 1.0 equivalent) in THF (0.2–0.4 M). The mixture was stirred at 80 °C for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with H₂O and extracted twice with EtOAc. The combined organic layers were washed twice with H₂O, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a colorless oily product (98%). 1 H NMR(400 MHz, CDCl3) δ 5.43 - 5.16 (m, 4H), 3.46 (t, J = 6.8 Hz, 2H), 2.70 (t, J= 6.6 Hz, 2H), 1.97 (t, J = 6.9 Hz, 4H), 1.75 - 1.65 (m, 2H), 1.40 - 1.18 (m,16H), 0.82 (t, J = 6.8 Hz, 3H).

[0872] Intermediate 23c: Diethyl 2-((9Z,12Z)-octadecyl-9,12-dien-1-yl)malonate

[0873]

[0874] K₂CO₃ (3.0 equivalents) and diethyl malonate (1.5 equivalents) were added to a solution of intermediate 23c (8 g, 1.0 equivalents) in DMF (0.2–0.4 M). The mixture was stirred at 60 °C for 24 h. The reaction mixture was diluted with H₂O and extracted three times with EtOAc. The combined organic layers were washed three times with brine, dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a colorless oily product (60%). 1H NMR (400MHz, CDCl3) δ 5.38 - 5.20 (m, 4H), 4.12 (qd, J = 7.1, 1.1 Hz, 4H), 3.24 (t, J= 7.6 Hz, 1H), 2.70 (t, J = 6.5 Hz, 2H), 1.98 (qd, J = 6.7, 2.4 Hz, 4H), 1.81(q, J = 7.5 Hz, 2H), 1.32 - 1.16 (m, 23H), 0.82 (t, J = 6.8 Hz, 3H).

[0875] Intermediate 23d: 2-((9Z,12Z)-octadec-9,12-dien-1-yl)propane-1,3-

[0876]

[0877] LiAlH4 (2.2 equivalents) was slowly added to a solution of intermediate 23c (5 g, 1.0 equivalent) in THF (0.2–0.4 M) at 0 °C under a nitrogen atmosphere. The mixture was stirred at 20 °C for 12 h. The reaction mixture was quenched by pouring it into saturated NH4Cl and stirring for 0.5 h, followed by extraction three times with EtOAc. The combined organic phases were washed three times with brine, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give a product (53%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.28 (tt, J = 11.1, 4.8 Hz, 4H), 3.75 (dd, J =10.6, 3.7 Hz, 2H), 3.59 (dd, J = 10.6, 7.6 Hz, 2H), 2.71 (t, J = 6.5 Hz, 2H), 1.98 (q, J = 7.0 Hz, 4H), 1.70 (ddq, J = 10.6, 7.1, 3.5 Hz, 1H), 1.34 - 1.12(m, 21H), 0.82 (t, J = 6.7 Hz, 3H).

[0878] Intermediate 23e: 1-(heptadecanoic acid-9-yl) ester 7-((11Z,14Z)-2-(hydroxymethyl)eicoseno-11,14-dien-1-yl) ester

[0879]

[0880] A mixture of intermediate 23d (4 g, 1.0 equivalent), DMAP (0.2 equivalent), EDCI (2.0 equivalent), and DIPEA (2.0 equivalent) in DCM (0.3 M) was degassed and purged three times with N2. Then, 7-(1-octylnonoxy)-7-oxo-heptanoic acid (1.0 equivalent) was slowly added to the mixture at 0 °C. The mixture was stirred at 20 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O and extracted twice with DCM. The combined organic layers were washed twice with brine, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a colorless oily product (48%). 1 H NMR (400 MHz, CDCl3) δ 5.28 (tt, J = 11.0, 4.9 Hz, 4H), 4.79 (p, J =6.3 Hz, 1H), 4.15 (dd, J = 11.2, 4.3 Hz, 1H), 4.01 (dd, J = 11.2, 6.7 Hz,1H), 3.52 (dt, J = 10.7, 5.0 Hz, 1H), 3.44 (q, J = 5.7 Hz, 1H), 2.70 (t, J =6.5 Hz, 2H), 2.24 (dt, J = 17.0, 7.5 Hz, 4H), 1.98 (q, J = 7.0 Hz, 5H), 1.72(ddt, J = 11.1, 6.6, 4.4 Hz, 1H), 1.58 (pd, J = 7.5, 5.1 Hz, 4H), 1.43 (q, J= 6.0 Hz, 4H), 1.36 - 1.13 (m, 47H), 0.91 - 0.73 (m, 9H).

[0881] Intermediate 23f: 1-(heptadecanoic acid-9-yl) ester 7-((11Z,14Z)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)eicoseno-11,14-dien-1-yl) ester

[0882]

[0883] A solution of intermediate 23e (4 g, 1.0 equivalent) in DCM (0.1–0.2 M) was added to a solution of chloroformate (4-nitrophenyl) ester (2.0 equivalent). Pyridine (2.0 equivalent) was then slowly added at 0 °C. The mixture was stirred at 20 °C for 1 h. The reaction mixture was diluted with hexane, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give a colorless oily product. 1 H NMR (400 MHz, CDCl3) δ 8.26 - 8.17 (m, 2H), 7.36- 7.27 (m, 2H), 5.37 - 5.22 (m, 4H), 4.79 (p, J = 6.3 Hz, 1H), 4.26 - 4.16(m, 2H), 4.10 (dd, J = 11.2, 4.7 Hz, 1H), 4.02 (dd, J = 11.3, 6.7 Hz, 1H), 2.70 (t, J = 6.5 Hz, 2H), 2.24 (dt, J = 20.3, 7.5 Hz, 4H), 2.00 (dq, J =13.9, 7.0 Hz, 5H), 1.56 (dq, J = 16.5, 9.3, 8.3 Hz, 4H), 1.43 (q, J = 6.2 Hz, 4H), 1.39 - 1.15 (m, 45H), 0.81 (q, J = 6.7 Hz, 9H).

[0884] Compound 23: 1-((11Z,14Z)-2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)eicosuccinate-11,14-dien-1-yl) ester-7-(heptadecane-9-yl) ester of pimecrolic acid

[0885]

[0886] Compound 23 was synthesized from intermediate 23f and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 1. 1H NMR (400 MHz, CDCl3) δ 5.29 (qd, J = 11.0, 5.0 Hz, 4H), 5.10(s, 1H), 4.79 (p, J = 6.3 Hz, 1H), 3.97 (dq, J = 16.0, 8.7, 5.8 Hz, 4H), 3.14(q, J = 5.9 Hz, 2H), 2.70 (t, J = 6.5 Hz, 2H), 2.45 (q, J = 7.1, 6.3 Hz, 6H), 2.23 (dt, J = 10.4, 7.6 Hz, 4H), 1.98 (q, J = 6.9 Hz, 4H), 1.88 (d, J = 7.6Hz, 1H), 1.62 - 1.52 (m, 5H), 1.43 (q, J = 6.1 Hz, 4H), 1.33 - 1.17 (m, 46H), 0.93 (t, J = 7.1 Hz, 6H), 0.85 - 0.77 (m, 9H). MS: 847.4 m / z [M+H].

[0887] Example 24 - Compound 24

[0888] Intermediate 24a: Octadecan-9-ol

[0889]

[0890] Magnesium bromo(octyl)bromo(1.0 equivalent) was added dropwise to a solution of decanal (50 g, 1.0 equivalent) in THF (0.1–0.2 M) at -40 °C under a nitrogen atmosphere. The mixture was stirred at 20 °C for 2 h. The reaction mixture was quenched by adding H₂O and extracted twice with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give the product as a white solid.

[0891] Intermediate 24b: 9-Bromononanoic acid

[0892]

[0893] Intermediate 24b (60%) was synthesized from intermediate 24a and 9-bromononanoic acid using the method employed in the synthesis of intermediate 7a. 1H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 1H), 3.46 (t, J = 6.8 Hz,1H), 3.33 (t, J = 6.9 Hz, 1H), 2.21 (t, J = 7.5 Hz, 2H), 1.78 (p, J = 7.0 Hz,1H), 1.69 (dt, J = 14.4, 6.9 Hz, 1H), 1.55 (p, J = 7.2 Hz, 2H), 1.43 (q, J =6.1 Hz, 4H), 1.39 - 1.31 (m, 2H), 1.22 (d, J = 24.2 Hz, 32H), 0.81 (t, J =6.8 Hz, 6H).

[0894] Intermediate 24c: 1,1-dibenzyl nonane-1,1,9-tricarboxylic acid ester 9-(octadecane-9-yl) ester

[0895]

[0896] Intermediate 24c (74%) was synthesized from intermediate 24b using the method employed in the synthesis of intermediate 7b. 1 HNMR (400 MHz, CDCl3) δ 7.35 - 7.10 (m, 10H), 5.07 (s, 4H), 4.79 (p, J = 6.3Hz, 1H), 3.36 (t, J = 7.5 Hz, 1H), 2.19 (t, J = 7.5 Hz, 2H), 1.85 (d, J = 7.2Hz, 2H), 1.58 - 1.47 (m, 3H), 1.43 (q, J = 6.1 Hz, 4H), 1.19 (d, J = 7.9 Hz, 36H), 0.80 (t, J = 6.7 Hz, 6H).

[0897] Intermediate 24d: 2-(9-(octadecane-9-yloxy)-9-oxonyl)malonic acid

[0898]

[0899] Intermediate 24d was synthesized from intermediate 24c using the method employed in the synthesis of intermediate 24c.

[0900] Intermediate 24e: 11-hydroxy-10-(hydroxymethyl)undecanoic acid octadecane-9-yl ester

[0901]

[0902] Intermediate 24e (30%) was synthesized from intermediate 24d using the method employed in the synthesis of intermediate 7d. 1 HNMR (400 MHz, CDCl3) δ 4.79 (p, J = 6.3 Hz, 1H), 3.75 (dd, J = 10.6, 3.8 Hz, 2H), 3.59 (dd, J = 10.6, 7.6 Hz, 2H), 2.21 (t, J = 7.5 Hz, 4H), 1.70 (ddq, J= 10.5, 7.0, 3.4 Hz, 1H), 1.54 (t, J = 7.2 Hz, 2H), 1.43 (q, J = 6.1 Hz, 4H), 1.20 (d, J = 12.6 Hz, 39H), 0.81 (t, J = 6.7 Hz, 6H).

[0903] Intermediate 24f: (9Z,12Z)-octadec-9,12-dienoic acid 2-(hydroxymethyl)-11-(octadecane-9-yloxy)-11-oxoundecyl ester

[0904]

[0905] Intermediate 24f (58%) was synthesized from intermediate 24e using the method employed in the synthesis of intermediate 7e. 1HNMR (400 MHz, CDCl3) δ 5.37 - 5.18 (m, 4H), 4.79 (p, J = 6.3 Hz, 1H), 4.15 (dd, J = 11.2, 4.3 Hz, 1H), 4.01 (dd, J = 11.2, 6.7 Hz, 1H), 1.98 (q, J = 6.8 Hz, 4H), 1.92 (t, J =6.2 Hz, 1H), 1.72 (ddd, J = 11.0, 6.7, 4.3 Hz, 1H), 1.54 (qd, J = 7.2, 4.1Hz, 4H), 1.43 (q, J = 5.9 Hz, 4H), 1.35 - 1.09 (m, 54H), 0.81 (td, J = 6.8, 4.9 Hz, 9H).

[0906] Intermediate 24g: Sodium 4-hydroxydodecanoate

[0907]

[0908] Add NaOH (1.0–1.1 equivalents) to a solution of 5-octyltetrahydrofuran-2-one (50 g, 1.0 equivalent) in H₂O (0.5 M). Degas the mixture, purge three times with N₂, and stir at 20 °C for 12 h. Concentrate the reaction mixture to give the crude product, which can be used directly in the next step (83%) without further purification.

[0909] Intermediate 24h: Benzyl 4-hydroxydodecanoate

[0910]

[0911] BnBr (1.0 equivalent) was added to a solution of 24 g (10 g, 1.0 equivalent) of the intermediate in DMSO (0.5–5.0 M) at 0 °C. The mixture was stirred at 20 °C for 10 min. The reaction mixture was then diluted with EtOAc / hexane at a 1:1 ratio. The combined organic layers were washed three times with NH4Cl, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue, which was used directly in the next step (78%) without further purification.

[0912] Intermediate 24i: Benzyl 4-(((4-nitrophenoxy)carbonyl)oxy)dodecanoate

[0913]

[0914] Pyridine (2.0 equivalent) was added dropwise to a solution of intermediate 24h (25 g, 1.0 equivalent) and (1.5 equivalent) chloroformate in DCM (0.2–0.5 M) at 0 °C. The mixture was stirred at 20 °C for 1 h under N2 atmosphere. The reaction mixture was diluted with hexane and filtered. The filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography to give a product (26%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 8.24 - 8.14 (m, 2H),7.38 - 7.27 (m, 7H), 5.06 (s, 2H), 4.80 (tdd, J = 7.6, 5.5, 4.0 Hz, 1H), 2.49- 2.39 (m, 2H), 2.04 (dtd, J = 15.5, 7.7, 4.1 Hz, 1H), 1.97 - 1.87 (m, 1H), 1.70 - 1.60 (m, 1H), 1.60 - 1.48 (m, 1H), 1.36 - 1.14 (m, 12H), 0.94 - 0.69(m, 3H).

[0915] Intermediate 24j: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)benzyl dodecanoate

[0916]

[0917] Pyridine (2.0 equivalents) and DMAP (0.1 equivalents) were added to a solution of intermediate 24i (8 g, 1.0 equivalents), N'-ethyl-N'-methyl-ethane-1,2-diamine hydrochloride (1.0 equivalents) in MeCN (0.1–0.2 M). The mixture was stirred at 20 °C for 12 h under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with EtOAc, washed three times with NaHCO3 and five times with H2O. The organic layer was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a product (54%) as a pale yellow oil. 1H NMR (400MHz, CDCl3) δ 7.34 - 7.21 (m, 5H), 5.07 (s, 2H), 4.69 (s, 1H), 3.17 (q, J =6.0 Hz, 2H), 2.44 - 2.30 (m, 6H), 2.15 (s, 3H), 1.86 (td, J = 7.8, 4.1 Hz,1H), 1.75 (dq, J = 14.9, 7.9 Hz, 1H), 1.57 - 1.35 (m, 2H), 1.29 - 1.10 (m,12H), 0.97 (t, J = 7.1 Hz, 3H), 0.85 - 0.74 (m, 3H).

[0918] Intermediate 24k: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoic acid

[0919]

[0920] Intermediate 24j (4 g, 1.0 equivalent) was added to a solution of Pd / C (1.0 equivalent) in THF (0.1–0.5 M). The mixture was degassed and purged three times with H2, and stirred at 20 °C for 5 h under H2 atmosphere. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a product (63%) as a brown oil. 1 H NMR (400 MHz, CDCl3) δ 6.14 (dd, J = 7.9, 4.0 Hz, 1H), 4.66 (qd, J =7.4, 3.9 Hz, 1H), 3.53 (dtd, J = 15.1, 7.8, 7.4, 3.9 Hz, 1H), 3.13 (ddt, J =14.8, 7.4, 3.9 Hz, 1H), 2.87 - 2.57 (m, 4H), 2.38 (s, 3H), 2.31 - 2.13 (m,2H), 1.95 (s, 1H), 1.73 (dt, J = 14.4, 7.5 Hz, 1H), 1.59 - 1.47 (m, 1H), 1.41 (dt, J = 13.8, 7.4 Hz, 1H), 1.30 - 0.97 (m, 15H), 0.80 (t, J = 6.8 Hz, 3H).

[0921] Compound 24: (9Z,12Z)-octadec-9,12-dienoic acid 3-methyl-15-(9-(octadecano-9-yloxy)-9-oxonyl)-9-octyl-7,12-dioxo-8,13-dioxa-3,6-diazahexadecane-16-yl ester

[0922]

[0923] A mixture of intermediates 24k (2.0 g, 1.0 equivalent), 24f (1.0 equivalent), EDCI (1.2 equivalent), DIPEA (2.5 equivalent), and DMAP (0.1 equivalent) in DCM (0.1–0.5 M) was degassed and purged three times with N2. The mixture was then stirred at 20 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O and extracted three times with DCM. The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography, diluted in MeCN, and washed three times with hexane to give a colorless oily product. 1 H NMR (400 MHz, CDCl3)δ 5.41 - 5.20 (m, 4H), 5.05 (d, J = 6.0 Hz, 1H), 4.80 (q, J = 6.2 Hz, 1H), 4.68 (s, 1H), 3.97 (qd, J = 11.1, 6.3 Hz, 4H), 3.17 (q, J = 6.0 Hz, 2H), 2.70 (t, J = 6.4 Hz, 2H), 2.42 - 2.25 (m, 6H), 2.22 (dt, J = 9.5, 7.5 Hz, 4H), 2.13 (s, 3H), 1.98 (q, J = 6.9 Hz, 4H), 1.92 - 1.69 (m, 3H), 1.60 - 1.39 (m,11H), 1.33 - 1.12 (m, 62H), 0.97 (t, J = 7.1 Hz, 3H), 0.86 - 0.78 (m, 12H). MS: 1074.0 m / z [M+H].

[0924] Example 25 - Compound 25

[0925] Intermediate 25a: 4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)benzyl dodecanoate

[0926]

[0927] Intermediate 25a was synthesized from intermediate 24i and 2-pyrrolidine-1-ylethylamine using the method employed in the synthesis of intermediate 24j.

[0928] Intermediate 25b: 4-(((2-(pyrrolid-1-yl)ethyl)carbamoyl)oxy)dodecanoic acid

[0929]

[0930] Intermediate 25b is synthesized from intermediate 25a using the method employed in the synthesis of intermediate 24k. 1 H NMR (400 MHz, CDCl3) δ 6.19 (s, 1H), 5.87 (s, 2H), 4.67 (p, J = 6.4 Hz, 1H), 3.65- 3.53 (m, 1H), 3.14 - 3.01 (m, 1H), 2.94 (qd, J = 16.2, 13.5, 5.5 Hz, 4H),2.76 (t, J = 9.6 Hz, 1H), 2.20 (hept, J = 7.3 Hz, 2H), 2.07 - 1.83 (m, 5H),1.71 (dq, J = 14.7, 7.4 Hz, 1H), 1.54 (dt, J = 14.2, 6.8 Hz, 1H), 1.40 (dt, J= 13.8, 6.9 Hz, 1H), 1.31 - 1.13 (m, 12H), 0.80 (t, J= 6.7 Hz, 3H).

[0931] Compound 25: (9Z,12Z)-octadecyl-9,12-dienoic acid 11-(octadecane-9-yloxy)-11-oxo-2-(((4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)dodecanoyl)oxy)methyl)undecyl ester

[0932]

[0933] Compound 25 was synthesized from intermediates 25b and 24f using the method employed in the synthesis of compound 24. 1H NMR (400 MHz, CDCl3) δ 5.28 (tt, J = 11.0, 5.4 Hz, 4H), 5.09 (s, 1H), 4.79 (p, J = 6.2 Hz, 1H), 4.70 (d, J = 11.8 Hz, 1H), 3.97 (qd, J = 11.2, 6.2 Hz, 4H), 3.18 (q, J = 6.1 Hz, 2H), 2.70 (t, J = 6.4 Hz, 2H), 2.38 (dt, J = 14.2, 6.4 Hz, 4H), 2.30 (dt, J = 8.8, 5.8 Hz, 2H), 2.22 (dt, J = 9.7, 7.6 Hz, 4H),2.14 (s, 3H), 1.99 (t, J = 6.8 Hz, 4H), 1.91 - 1.69 (m, 3H), 1.49 (dq, J =45.3, 6.5, 6.0 Hz, 13H), 1.36 - 1.06 (m, 58H), 0.81 (q, J = 6.8, 6.3 Hz, 12H). MS: 1085.9 m / z [M+H].

[0934] Example 26 - Compound 26

[0935] Intermediate 26a: Sodium 4-hydroxyoctanoate

[0936]

[0937] Intermediate 26a was synthesized from 5-butyltetrahydrofuran-2-one using the method employed in the synthesis of intermediate 24g.

[0938] Intermediate 26b: 4-hydroxybenzyl octanoate

[0939]

[0940] Intermediate 26b was synthesized from intermediate 26a using the method employed in the synthesis of intermediate 24h.

[0941] Intermediate 26c: 4-(((4-nitrophenoxy)carbonyl)oxy)benzyl octanoate

[0942]

[0943] Intermediate 26c is synthesized from intermediate 26b using the method employed in the synthesis of intermediate 24i. 1H NMR (400 MHz, CDCl3) δ 8.38 - 8.19 (m, 2H), 7.51 - 7.29 (m, 7H), 5.15 (s, 2H), 4.97 - 4.86 (m, 1H), 2.53 (dd, J = 8.1, 6.8 Hz, 2H), 2.14 (dtd, J = 15.4,7.7, 4.0 Hz, 1H), 2.08 - 1.95 (m, 1H), 1.82 - 1.72 (m, 1H), 1.66 (ddt, J =14.5, 8.9, 5.5 Hz, 1H), 1.48 - 1.32 (m, 4H), 1.04 - 0.85 (m, 3H).

[0944] Intermediate 26d: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)benzyl octanoate

[0945]

[0946] Intermediate 26d was synthesized from intermediate 26c using the method employed in the synthesis of intermediate 24j.

[0947] Intermediate 26e: 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)octanoic acid

[0948]

[0949] Intermediate 26e was synthesized from intermediate 26d using the method employed in the synthesis of intermediate 24k. 1H NMR(400 MHz, CDCl3) δ 6.16 (s, 1H), 4.76 (dq, J = 11.5, 6.6 Hz, 1H), 3.64 (dtd,J = 14.9, 7.5, 2.7 Hz, 1H), 3.20 (ddt, J = 14.7, 7.1, 3.7 Hz, 1H), 2.93 -2.71 (m, 4H), 2.47 (s, 3H), 2.41 - 2.20 (m, 2H), 2.05 (ddd, J = 18.5, 9.7,5.7 Hz, 1H), 1.81 (dq, J = 14.7, 7.5 Hz, 1H), 1.75 - 1.58 (m, 1H), 1.50 (d, J= 13.7 Hz, 1H), 1.40 - 1.25 (m, 4H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 - 0.73 (m, 3H).

[0950] Compound 26: (9Z,12Z)-octadec-9,12-dienoic acid 9-butyl-3-methyl-15-(9-(octadecano-9-yloxy)-9-oxonyl)-7,12-dioxo-8,13-dioxa-3,6-diazahexadecane-16-yl ester

[0951]

[0952] Compound 26 was synthesized from intermediates 26e and 24f using the method employed in the synthesis of compound 24. 1H NMR (400 MHz, CDCl3) δ 5.28 (tt, J = 11.0, 5.4 Hz, 4H), 5.09 (s, 1H), 4.79 (p, J = 6.2 Hz, 1H), 4.70 (d, J = 11.8 Hz, 1H), 3.97 (qd, J = 11.2, 6.2 Hz, 4H), 3.18 (q, J = 6.1 Hz, 2H), 2.70 (t, J = 6.4 Hz, 2H), 2.38 (dt, J = 14.2, 6.4 Hz, 4H), 2.30 (dt, J = 8.8, 5.8 Hz, 2H), 2.27 - 2.07 (m, 7H), 1.99 (t, J= 6.8 Hz, 4H), 1.94 - 1.79 (m, 2H), 1.73 (t, J = 7.8 Hz, 1H), 1.49 (dq, J =45.3, 6.5, 6.0 Hz, 13H), 1.37 - 1.09 (m, 57H), 0.98 (t, J = 7.1 Hz, 3H), 0.81 (q, J = 6.8, 6.3 Hz, 12H). MS: 1017.9 m / z [M+H].

[0953] Example 27 - Compound 27

[0954] Intermediate 27a: 4-(((2-(pyrrolidone-1-yl)ethyl)carbamoyl)oxy)benzyl octanoate

[0955]

[0956] Intermediate 27a was synthesized from intermediate 26c using the method employed in the synthesis of intermediate 25a.

[0957] Intermediate 27b: 4-(((2-(pyrrolid-1-yl)ethyl)carbamoyl)oxy)octanoic acid

[0958]

[0959] Intermediate 27b is synthesized from intermediate 27a using the method employed in the synthesis of intermediate 24k. 1H NMR (400 MHz, CDCl3) δ 6.49 (dd, J = 7.8, 4.0 Hz, 1H), 4.66 (tt, J = 10.4, 5.1Hz, 1H), 3.55 (dtd, J = 14.5, 7.1, 3.4 Hz, 1H), 3.14 (ddt, J = 15.1, 7.7, 3.6Hz, 1H), 3.05 - 2.81 (m, 5H), 2.80 - 2.59 (m, 1H), 2.32 - 2.09 (m, 2H), 2.00- 1.83 (m, 5H), 1.73 (dtd, J = 14.6, 8.8, 6.2 Hz, 1H), 1.62 - 1.34 (m, 2H), 1.22 (dddd, J = 14.2, 11.9, 8.9, 5.2 Hz, 4H), 0.80 (q, J = 5.0, 3.4 Hz, 3H).

[0960] Compound 27: (9Z,12Z)-octadecyl-9,12-dienoic acid 11-(octadecyl-9-yloxy)-11-oxo-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)methyl)undecyl ester

[0961]

[0962] Compound 27 was synthesized from intermediates 27b and 24f using the method employed in the synthesis of compound 24. 1H NMR (400 MHz, CDCl3) δ 5.38 - 5.20 (m, 4H), 5.06 (s, 1H), 4.79 (p, J = 6.3Hz, 1H), 4.69 (s, 1H), 3.97 (dtd, J = 17.5, 10.7, 4.3 Hz, 4H), 3.21 (q, J =5.9 Hz, 2H), 2.70 (t, J = 6.4 Hz, 2H), 2.51 (t, J = 6.1 Hz, 2H), 2.43 (d, J =6.0 Hz, 4H), 2.34 - 2.26 (m, 2H), 2.22 (dt, J = 9.7, 7.5 Hz, 4H), 1.98 (q, J= 6.9 Hz, 4H), 1.94 - 1.79 (m, 2H), 1.72 (td, J = 12.1, 10.3, 4.9 Hz, 5H), 1.54 (t, J = 7.1 Hz, 6H), 1.43 (q, J = 6.5 Hz, 5H), 1.34 - 1.13 (m, 58H), 0.81 (td, J = 6.7, 4.7 Hz, 12H). MS: 1029.9 m / z [M+H].

[0963] Example 28 - Compound 28

[0964] Compound 28: Di(heptadecane-9-yl) ester of diglutaric acid O,O'-(2-((((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl) ester

[0965]

[0966] Compound 28 was synthesized from intermediate 2d and 2-(1-piperidinyl)ethylamine using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 5.18 (s, 1H), 4.80 (p, J = 6.2 Hz, 2H), 4.06(t, J = 7.2 Hz, 6H), 3.19 (q, J = 5.8 Hz, 2H), 2.30 (dt, J = 14.9, 7.4 Hz, 15H), 1.87 (p, J= 7.4 Hz, 4H), 1.55 - 1.31 (m, 14H), 1.19 (s, 50H), 0.81 (t, J = 6.7 Hz, 12H). MS: 966.3 m / z [M+H].

[0967] Example 29 - Compound 29

[0968] Compound 29: Di(heptadecane-9-yl) glutarate O,O'-(2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl) ester

[0969]

[0970] Compound 29 was synthesized from intermediate 2d and 2-pyrrolidine-1-ylethylamine using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 5.18 (s, 1H), 4.80 (p, J = 6.3 Hz, 2H), 4.06(t, J = 6.6 Hz, 6H), 3.21 (q, J = 5.8 Hz, 2H), 2.51 (t, J = 6.1 Hz, 2H), 2.45(d, J = 7.2 Hz, 4H), 2.30 (dt, J = 14.7, 7.4 Hz, 9H), 1.87 (p, J = 7.5 Hz,4H), 1.71 (q, J = 3.4 Hz, 4H), 1.44 (q, J = 6.0 Hz, 8H), 1.19 (s, 49H), 0.81(t, J = 6.8 Hz, 12H). MS: 952.3 m / z [M+H].

[0971] Example 30 - Compound 30

[0972] Compound 30: Diglutaric acid O,O'-(2-((((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester di(heptadecano-9-yl) ester

[0973]

[0974] Compound 30 was synthesized from intermediate 2d and 3-[ethyl(methyl)amino]prop-1-ol using the method employed in the synthesis of compound 6. 1H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 2H), 4.16 - 4.00(m, 8H), 2.44 - 2.23 (m, 12H), 2.15 (s, 3H), 1.86 (q, J = 7.4 Hz, 4H), 1.79(q, J = 7.3 Hz, 2H), 1.44 (q, J = 5.9 Hz, 10H), 1.19 (s, 47H), 0.98 (t, J =7.2 Hz, 3H), 0.81 (t, J = 6.7 Hz, 12H). MS: 955.3 m / z [M+H].

[0975] Example 31 - Compound 31

[0976] Compound 31: Diglutaric acid O,O'-(2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester di(heptadecano-9-yl) ester

[0977]

[0978] Compound 31 was synthesized from intermediate 2d and 3-(dimethylamino)prop-1-ol using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 2H), 4.17 - 4.04 (m,8H), 2.38 - 2.26 (m, 11H), 2.16 (s, 6H), 1.86 (q, J = 7.4 Hz, 4H), 1.77 (p, J = 6.8 Hz, 2H), 1.44 (q, J = 6.1 Hz, 8H), 1.19 (s, 50H), 0.81 (t, J = 6.8 Hz, 12H). MS: 941.5 m / z [M+H].

[0979] Example 32 - Compound 32

[0980] Compound 32: Di(heptadecyl-9-yl) glutarate O,O'-(2-((((3-(piperidin-1-yl)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester

[0981]

[0982] Compound 32 was synthesized from intermediate 2d and 3-(1-piperidinyl)prop-1-ol using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 2H), 4.10 (dd, J = 14.7,6.0 Hz, 8H), 2.30 (dt, J = 15.3, 7.4 Hz, 14H), 1.87 (p, J = 7.5 Hz, 4H), 1.79(t, J = 7.3 Hz, 2H), 1.47 (dd, J = 29.0, 5.7 Hz, 15H), 1.19 (s, 49H), 0.81(t, J = 6.7 Hz, 12H). MS: 981.6 m / z [M+H].

[0983] Example 33 - Compound 33

[0984] Compound 33: Di(heptadecane-9-yl) ester of diglutaric acid O,O'-(2-((((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester

[0985]

[0986] Compound 33 was synthesized from intermediate 2d and 3-pyrrolidone-1-ylprop-1-ol using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 4.89 (p, J = 6.3 Hz, 2H), 4.27 - 4.12 (m,8H), 2.62 - 2.46 (m, 6H), 2.39 (dt, J = 15.3, 7.3 Hz, 9H), 1.95 (dp, J =12.8, 7.3 Hz, 6H), 1.53 (q, J = 6.0 Hz, 8H), 1.28 (s, 48H), 0.90 (t, J = 6.7Hz, 12H). MS: 967.6 m / z [M+H].

[0987] Example 34 - Compound 34

[0988] Compound 34: Diglutaric acid O,O'-(2-(((4-(dimethylamino)butyryl)oxy)methyl)propane-1,3-diyl) ester di(heptadecano-9-yl) ester

[0989]

[0990] A mixture of intermediate 2C (1.0 equivalent), 4-(dimethylamino)butyrate (2.0 equivalent), EDCI (1.2 equivalent), DMAP (0.1 equivalent), DIPEA (4.0 equivalent), and DCM (0.1–0.2 M) was degassed and purged three times with N2. The mixture was then stirred at 15 °C for 12 h under N2 atmosphere. After completion, the reactants were concentrated under reduced pressure, and the residue was diluted with water, extracted two to three times with EtOAc, and the combined organic layers were washed twice with saturated NaHCO3, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to obtain the residue, which was then purified by column chromatography to obtain the product. 1 HNMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 2H), 4.06 (d, J = 6.0 Hz, 6H), 2.30 (dt, J = 15.0, 7.3 Hz, 11H), 2.21 (t, J = 7.2 Hz, 2H), 1.87 (p, J = 7.4Hz, 4H), 1.71 (p, J = 7.4 Hz, 2H), 1.44 (q, J = 6.1 Hz, 8H), 1.19 (s, 49H), 0.81 (t, J = 6.7 Hz, 12H). MS: 925.4 m / z [M+H].

[0991] Example 35 - Compound 35

[0992] Intermediate 35a: Di(heptadecane-9-yl) ester of dipentanoate O,O'-(2-oxopropane-1,3-diyl) ester

[0993]

[0994] Intermediate 35a was synthesized from intermediate 2b and 1,3-dihydroxyprop-2-one using the method employed in the synthesis of intermediate 2c. 1 H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.2 Hz, 2H), 4.69 (s, 4H), 2.44 (t, J = 7.4 Hz, 4H), 2.32 (t, J = 7.3 Hz, 4H), 1.94 (dt, J = 14.7, 7.3 Hz,4H), 1.43 (t, J = 6.1 Hz, 8H), 1.19 (s, 49H), 0.81 (t, J = 6.7 Hz, 12H).

[0995] Intermediate 35b: Di(heptadecane-9-yl) glutarate O,O'-(2-hydroxy)propane-1,3-diyl) ester

[0996]

[0997] NaBH4 (5.0 equivalents) was added to a solution of intermediate 35b (1.0 equivalent) in THF / H2O / toluene (30:15:8, 0.02 M) at 0°C under a N2 atmosphere. The mixture was stirred at 5°C for 5 h under a N2 atmosphere. The reactants were then poured into cold saturated NH4Cl and stirred for another 30 min. The mixture was then poured into water and extracted twice with EtOAc. The combined organic phases were evaporated under reduced pressure, and the residue was purified by column chromatography to give a product as a white solid. 1 HNMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 2H), 4.18 - 3.95 (m, 5H), 2.33(dt, J = 25.4, 7.3 Hz, 8H), 1.90 (p, J = 7.4 Hz, 4H), 1.44 (q, J = 5.9 Hz, 8H), 1.19 (s, 48H), 0.81 (t, J = 6.7 Hz, 12H).

[0998] Intermediate 35c: Di(heptadecyl-9-yl) glutarate O,O'-(2-(((4-nitrophenoxy)carbonyl)oxy)propane-1,3-diyl) ester

[0999]

[1000] Intermediate 35c was synthesized from intermediate 35b using the method employed in the synthesis of intermediate 1d. 1H NMR (400MHz, CDCl3) δ 8.31 - 8.16 (m, 2H), 7.42 - 7.30 (m, 2H), 5.10 (td, J = 6.0,3.0 Hz, 1H), 4.80 (p, J = 6.3 Hz, 2H), 4.44 (dd, J = 12.3, 3.9 Hz, 2H), 4.18(dd, J = 12.3, 5.9 Hz, 2H), 2.33 (dt, J = 30.6, 7.4 Hz, 8H), 1.90 (p, J = 7.4Hz, 4H), 1.43 (q, J = 6.3 Hz, 8H), 1.18 (s, 51H), 0.80 (t, J = 6.7 Hz, 12H).

[1001] Compound 35: Diglutaric acid O,O'-(2-(((2-(diethylamino)ethyl)carbamoyl)oxy)propane-1,3-diyl) ester di(heptadecano-9-yl) ester

[1002]

[1003] Compound 35 was synthesized from intermediate 35c and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 5.24 (t, J = 5.2 Hz, 1H), 5.13 - 5.02(m, 1H), 4.79 (p, J = 6.3 Hz, 2H), 4.21 (dd, J = 11.9, 4.4 Hz, 2H), 4.11 (dd,J = 11.9, 5.7 Hz, 2H), 3.15 (q, J = 5.9 Hz, 2H), 2.45 (q, J = 7.2 Hz, 6H), 2.30 (dt, J = 19.3, 7.4 Hz, 8H), 1.87 (p, J = 7.4 Hz, 4H), 1.43 (q, J = 5.9Hz, 8H), 1.19 (s, 46H), 0.93 (t, J = 7.1 Hz, 6H), 0.81 (t, J = 6.7 Hz, 12H). MS: 940.4 m / z [M+H].

[1004] Example 36 - Compound 36

[1005] Intermediate 36a: Di(heptadecane-9-yl) glutarate O,O'-(2-(hydroxymethyl)-2-methylpropane-1,3-diyl) ester

[1006]

[1007] Intermediate 36a (36%) was synthesized from 2-(hydroxymethyl)-2-methyl-propane-1,3-diol using the method employed in the synthesis of intermediate 2c. 1 H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 2H), 3.96 (d, J = 1.6 Hz, 4H), 3.33 (s, 2H), 2.31 (dt, J = 21.4, 7.3 Hz, 8H), 1.90 (q,J = 7.3 Hz, 4H), 1.44 (q, J = 6.1 Hz, 8H), 1.19 (s, 49H), 0.88 (s, 3H), 0.81 (t, J = 6.7 Hz, 12H).

[1008] Intermediate 36b: Di(heptadecyl-9-yl) glutarate O,O'-(2-methyl-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester

[1009]

[1010] Intermediate 36b (53%) was synthesized from intermediate 36a using the method employed in the synthesis of intermediate 1d. 1 HNMR (400 MHz, CDCl3) δ 8.28 - 8.17 (m, 2H), 7.40 - 7.27 (m, 2H), 4.80 (p, J =6.3 Hz, 2H), 4.14 (s, 2H), 4.02 (s, 4H), 2.35 (t, J = 7.5 Hz, 4H), 2.29 (t, J= 7.3 Hz, 4H), 1.89 (p, J = 7.4 Hz, 4H), 1.44 (q, J = 6.2 Hz, 8H), 1.28 -1.12 (m, 49H), 1.02 (s, 3H), 0.80 (t, J = 6.8 Hz, 12H).

[1011] Compound 36: Diglutaric acid O,O'-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)-2-methylpropane-1,3-diyl) ester di(heptadecano-9-yl) ester

[1012]

[1013] Compound 36 was synthesized from intermediate 36b and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 5.14 (s, 1H), 4.80 (p, J = 6.3 Hz,2H), 3.93 (d, J = 3.3 Hz, 6H), 3.14 (d, J = 7.2 Hz, 2H), 2.45 (d, J = 7.6 Hz,6H), 2.30 (dt, J = 17.6, 7.4 Hz, 8H), 1.87 (t, J = 7.4 Hz, 4H), 1.43 (d, J =6.1 Hz, 10H), 1.19 (s, 50H), 0.93 (d, J = 4.9 Hz, 9H), 0.81 (t, J = 6.7 Hz, 12H). MS: 968.3 m / z [M+H].

[1014] Example 37 - Compound 37

[1015] Intermediate 37a: 5-oxo-5-(tetran-7-yloxy)valerate

[1016]

[1017] Intermediate 37a (29%) was synthesized from glutaric acid and tridecane-7-ol using the method employed in the synthesis of intermediate 2b. 1 H NMR (400 MHz, CDCl3) δ 4.81 (p, J = 6.3 Hz, 1H), 2.34 (dt, J = 22.0,7.3 Hz, 4H), 1.90 (q, J = 7.3 Hz, 2H), 1.44 (q, J = 6.2 Hz, 4H), 1.26 - 1.15 (m, 16H), 0.86 - 0.75 (m, 6H).

[1018] Intermediate 37b: Diglutaric acid O,O'-(2-(hydroxymethyl)propane-1,3-diyl) ester di(tetane-7-yl) ester

[1019]

[1020] A mixture of intermediate 37a (2.0 equivalents), 2-(hydroxymethyl)propane-1,3-diol (1.0 equivalents), EDCI (1.2 equivalents), DIPEA (2.5 equivalents), and DMAP (0.1 equivalents) in a 1:1 DCM / DMF (0.1 M) was degassed and purged three times with N2. The mixture was then stirred at 20 °C for 24 h under N2 atmosphere. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was diluted with water and extracted three times with EtOAc. The combined organic layers were washed three times with brine, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a colorless oily product (23%). 1 H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 2H), 4.11 (dd, J= 6.1, 2.6 Hz, 4H), 3.56 (t, J = 5.9 Hz, 2H), 2.31 (dt, J = 17.6, 7.3 Hz, 8H), 2.15 (q, J = 5.8 Hz, 2H), 1.88 (p, J = 7.4 Hz, 4H), 1.44 (q, J = 6.2 Hz,8H), 1.20 (p, J = 7.1 Hz, 32H), 0.87 - 0.77 (m, 12H).

[1021] Intermediate 37c: Diglutaric acid O,O'-(2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester di(tetane-7-yl) ester

[1022]

[1023] Intermediate 37c (65%) was synthesized from intermediate 37b using the method employed in the synthesis of intermediate 2d.

[1024] Compound 37: Diglutaric acid O,O'-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl) ester di(tetane-7-yl) ester

[1025]

[1026] Compound 37 was synthesized from intermediate 37c and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 6. 1H NMR (400 MHz, CDCl3) δ 5.16 (s, 1H), 4.80 (p, J = 6.3 Hz, 2H), 4.06 (t, J = 6.3 Hz, 6H), 3.14 (d, J = 6.3 Hz, 2H), 2.45 (q, J = 7.5,6.8 Hz, 6H), 2.30 (dt, J = 14.7, 7.4 Hz, 9H), 1.87 (p, J = 7.5 Hz, 4H), 1.44(q, J = 6.0 Hz, 8H), 1.20 (dd, J = 9.9, 5.2 Hz, 32H), 0.94 (t, J = 7.1 Hz,6H), 0.81 (t, J = 6.7 Hz, 12H). MS: 842.5 m / z [M+H].

[1027] Example 38 - Compound 38

[1028] Intermediate 38a: 7-(heptadecane-9-yloxy)-7-oxoheptanoic acid

[1029]

[1030] Intermediate 38a (40%) was synthesized from pimelic acid and intermediate 2a using the method employed in the synthesis of intermediate 2b. 1 H NMR (400 MHz, CDCl3) δ 4.80 (p, J = 6.3 Hz, 1H), 2.33 - 2.18 (m, 5H), 1.58 (ddt, J = 11.1, 7.5, 3.6 Hz, 6H), 1.43 (q, J = 6.1 Hz, 5H), 1.38 - 1.28(m, 4H), 1.20 (d, J = 7.6 Hz, 25H), 0.81 (t, J = 6.8 Hz, 6H).

[1031] Intermediate 38b: 7,7'-di(heptadecanoic acid) ester O'1,O1-(2-(hydroxymethyl)propane-1,3-diyl) ester

[1032]

[1033] Intermediate 38b (16%) was synthesized from intermediate 38a using the method employed in the synthesis of intermediate 2c. 1HNMR (400 MHz, CDCl3) δ 4.79 (p, J = 6.3 Hz, 2H), 4.10 (h, J = 6.3, 5.9 Hz, 4H), 3.55 (d, J = 5.6 Hz, 2H), 2.24 (dt, J = 17.1, 7.5 Hz, 9H), 2.12 (p, J =5.9 Hz, 1H), 1.57 (ddd, J = 15.3, 7.6, 5.1 Hz, 8H), 1.43 (q, J = 6.2 Hz, 8H),1.33 - 1.14 (m, 54H), 0.81 (t, J = 6.7 Hz, 12H).

[1034] Intermediate 38c: 7,7'-di(heptadecanoic acid) ester O'1,O1-(2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-1,3-diyl) ester

[1035]

[1036] Intermediate 38c (54%) was synthesized from intermediate 38b using the method employed in the synthesis of intermediate 8d. 1 HNMR (400 MHz, CDCl3) δ 8.28 - 8.16 (m, 2H), 7.39 - 7.26 (m, 2H), 4.79 (p, J =6.3 Hz, 2H), 4.29 (d, J = 5.8 Hz, 2H), 4.22 - 4.09 (m, 4H), 2.44 (h, J = 6.0Hz, 1H), 2.25 (dt, J = 21.3, 7.5 Hz, 8H), 1.63 - 1.52 (m, 9H), 1.43 (q, J =6.2 Hz, 9H), 1.38 - 1.06 (m, 60H), 0.80 (t, J = 6.7 Hz, 16H).

[1037] Compound 38: Di(pimelic acid)O'1,O1-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl) ester 7,7'-di(heptadecane-9-yl) ester

[1038]

[1039] Compound 38 was synthesized from intermediate 38c and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 6.1 H NMR (400 MHz, CDCl3) δ 5.15 (s, 1H), 4.79 (p, J = 6.2 Hz, 2H), 4.05 (d, J = 6.0 Hz, 6H), 3.18 - 3.06 (m, 2H), 2.46 (t, J = 6.8 Hz, 6H), 2.23 (dt, J = 12.2, 7.5 Hz, 9H), 1.56 (q, J = 7.3 Hz, 9H), 1.43 (q, J = 5.9Hz, 8H), 1.35 - 1.07 (m, 47H), 0.93 (t, J = 7.1 Hz, 6H), 0.81 (t, J = 6.7 Hz, 12H). MS: 1011.2 m / z [M+H].

[1040] Example 39 - Compound 39

[1041] Intermediate 39a: Ethyl 2-octyl-2-propionyldecanoate

[1042]

[1043] A solution of NaH (60% mineral oil, 1.2 equivalents) in THF (0.25–0.5 M) was slowly cooled to 0°C under a nitrogen atmosphere. Next, diethyl malonate (0.4 equivalents) was added to the mixture, and the reaction mixture was stirred at 0°C for 30 min, followed by the addition of 1-iodooctane (1.0 equivalents). The mixture was stirred at 20°C under a nitrogen atmosphere for 6 h, then quenched with water, extracted twice with EtOAc, washed with brine, dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated, and the residue was purified by column chromatography to give a yellow oily product (65%).

[1044] Intermediate 39b: 2-Octylated decanoic acid

[1045]

[1046] 10 M NaOH (7.4 equivalents) was added to a solution of intermediate 39a (70 g, 1.0 equivalents) in butan-1-ol (32 equivalents). The mixture was stirred at 120 °C for 6 h under a nitrogen atmosphere. The reaction mixture was then adjusted to pH 7 with 1 M HCl and extracted twice with EtOAc. The residue was dried over Na2SO4, filtered, and concentrated to obtain a residue. Acetic acid (19 equivalents) was then added to the residue, and the mixture was stirred at 120 °C for 12 h. The mixture was then quenched with water, extracted twice with EtOAc, dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography to give a product (55%) as a white solid.

[1047] Intermediate 39c: Pent-4-en-1-yl 2-octyldecanoate

[1048]

[1049] DIPEA (2.0 equivalents), EDCI (2.0 equivalents), and DMAP (0.2 equivalents) were added to a solution of intermediate 39b (1.0 equivalents) in DCM (0.4 M). Then, pent-4-en-1-ol (1.0 equivalent) was added to the mixture, and the mixture was stirred at 25°C for 12 h under a N2 atmosphere. The reaction mixture was then concentrated under reduced pressure, and the residue was diluted with water, extracted twice with EtOAc, and the combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated, and the residue was purified by column chromatography to give a colorless oily product (71%).

[1050] Intermediate 39d: 4-((2-octyldecanoyl)oxy)butyric acid

[1051]

[1052] A solution of NaIO4 (5.0 equivalents) in H2O (0.05 M) was added to a solution of intermediate 39c (1.0 equivalents) in 1:1 DCM / MeCN (0.05 M). The mixture was cooled to 10 °C and stirred for 30 min under a N2 atmosphere. Next, RuCl3 (0.04 equivalents) was added to the mixture, and it was stirred at 25 °C for 12 h. The mixture was quenched by adding saturated NaHCO3 at 0 °C, diluted with water, and extracted twice with EtOAc. The combined organic layers were then subjected to Na2... S The product was dried with O4, filtered, and the filtrate was concentrated to obtain a residue. The residue was then purified by column chromatography to obtain a product (57%) that was a colorless oil.

[1053] Intermediate 39e: bis(2-octyldecanoic acid) ((2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(4-oxobutane-4,1-diyl) ester

[1054]

[1055] Intermediate 39e (30%) was synthesized from intermediate 39d using the method employed in the synthesis of intermediate 2c. 1 HNMR (400 MHz, CDCl3) δ 4.19 - 4.08 (m, 4H), 4.04 (t, J = 6.4 Hz, 4H), 3.57(d, J = 5.5 Hz, 2H), 2.34 (t, J = 7.4 Hz, 4H), 2.25 (tt, J = 8.7, 5.4 Hz,3H), 2.17 - 2.11 (m, 1H), 1.90 (p, J = 6.8 Hz, 4H), 1.51 (td, J = 8.6, 4.2Hz, 4H), 1.46 - 1.29 (m, 5H), 1.18 (s, 49H), 0.81 (t, J = 6.8 Hz, 12H).

[1056] Intermediate 39f: bis(2-octyldecanoic acid) ((2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-1,3-diyl)bis(oxy))bis(4-oxobutane-4,1-diyl) ester

[1057]

[1058] Intermediate 39f (62%) was synthesized from intermediate 39e using the method employed in the synthesis of intermediate 2d. 1HNMR (400 MHz, CDCl3) δ 8.22 (d, J = 9.0 Hz, 2H), 7.33 (d, J = 8.9 Hz, 2H), 4.30 (d, J = 5.7 Hz, 2H), 4.21 - 4.10 (m, 4H), 4.05 (t, J = 6.4 Hz, 4H), 2.46(hept, J = 6.0 Hz, 1H), 2.37 (t, J = 7.5 Hz, 4H), 2.25 (tt, J = 8.9, 5.3 Hz,2H), 1.91 (p, J = 6.9 Hz, 4H), 1.51 (ddt, J = 15.7, 12.2, 6.5 Hz, 4H), 1.44 -1.29 (m, 5H), 1.18 (s, 49H), 0.80 (t, J = 6.7 Hz, 12H).

[1059] Compound 39: 2-Octydecanoic acid [4-[2-[2-(diethylamino)ethylcarbamoyloxymethyl]-3-[4-(2-octyldecyloxy)butyryloxy]propoxy]-4-oxo-butyl] ester

[1060]

[1061] Compound 39 was synthesized from intermediate 39f and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 5.16 (s, 1H), 4.05 (dt, J = 15.4, 6.4Hz, 10H), 3.15 (q, J = 6.0 Hz, 2H), 2.46 (t, J = 6.7 Hz, 6H), 2.34 (t, J =7.5 Hz, 5H), 2.25 (tt, J = 8.7, 5.4 Hz, 2H), 1.88 (q, J = 7.1 Hz, 4H), 1.51(tt, J = 12.2, 5.0 Hz, 4H), 1.37 (p, J = 6.9, 6.2 Hz, 5H), 1.18 (s, 49H),0.93 (t, J = 7.2 Hz, 6H), 0.81 (t, J = 6.7 Hz, 12H). MS: 955.2 m / z [M+H].

[1062] Example 40 - Compound 40

[1063] Intermediate 40a: bis(2-octyldecanoic acid)2-(hydroxymethyl)propane-1,3-dimethyl ester

[1064]

[1065] Intermediate 40a was synthesized from intermediate 39b and 2-(hydroxymethyl)propane-1,3-diol using the method employed in the synthesis of intermediate 2c. 1 H NMR (400 MHz, CDCl3) δ 4.11 (qd, J = 11.3, 6.0 Hz, 4H), 3.53 (t, J = 6.0 Hz, 2H), 2.39 - 2.22 (m, 3H), 2.13 (p, J = 5.9 Hz, 1H), 1.53 (ddd, J = 14.0, 9.2, 5.6 Hz, 4H), 1.38 (td, J = 8.1, 3.9 Hz, 5H), 1.19 (d, J= 4.4 Hz, 47H), 0.81 (t, J = 6.8 Hz, 12H).

[1066] Intermediate 40b: bis(2-octyldecanoic acid)2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-1,3-dimethyl ester

[1067]

[1068] Intermediate 40b (68%) was synthesized from intermediate 40a using the method employed in the synthesis of intermediate 2d.

[1069] Compound 40: bis(2-octyldecanoic acid)2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-1,3-dimethyl ester

[1070]

[1071] Compound 40 was synthesized from intermediate 40b and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 6. 1H NMR (400 MHz, CDCl3) δ 5.13 (s, 1H), 4.06 (dd, J = 6.2, 3.3Hz, 6H), 3.14 (d, J = 6.7 Hz, 2H), 2.46 (dd, J = 9.4, 4.9 Hz, 6H), 2.34 -2.22 (m, 3H), 1.60 (s, 1H), 1.50 (dd, J = 8.5, 5.3 Hz, 4H), 1.43 - 1.28 (m,4H), 1.18 (s, 46H), 0.93 (t, J = 7.1 Hz, 6H), 0.81 (t, J = 6.7 Hz, 12H). MS: 782.3 m / z [M+H].

[1072] Example 41 - Compound 41

[1073] Intermediate 41a: 3-hydroxy-2-(hydroxymethyl)propyl dodecanoate

[1074]

[1075] Intermediate 41a (13%) was synthesized from dodecanoic acid using the method employed in the synthesis of intermediate 4b.

[1076] Intermediate 41b: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(hydroxymethyl)propyl dodecanoate

[1077]

[1078] Intermediate 41b (29%) was synthesized from intermediate 41a and intermediate 1b using the method employed in the synthesis of intermediate 4c. 1H NMR (400 MHz, CDCl3) δ 4.42 (t, J = 5.5 Hz, 1H), 4.09 (ddd, J = 14.7,7.9, 5.1 Hz, 4H), 3.61 - 3.53 (m, 3H), 3.50 (dt, J = 9.3, 6.7 Hz, 2H), 3.33(dt, J = 9.3, 6.7 Hz, 2H), 2.33 (q, J = 7.9 Hz, 2H), 2.24 (q, J = 9.3, 8.5Hz, 3H), 2.13 (p, J = 5.8 Hz, 1H), 1.87 (td, J = 7.3, 5.3 Hz, 2H), 1.52 (dt,J = 24.7, 6.6 Hz, 7H), 1.32 - 1.13 (m, 40H), 0.81 (t, J = 6.7 Hz, 9H).

[1079] Intermediate 41c: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propyl dodecanoate

[1080]

[1081] Intermediate 41c (69%) was synthesized from intermediate 41b using the method employed in the synthesis of intermediate 2d. 1 HNMR (400 MHz, CDCl3) δ 8.31 - 8.25 (m, 2H), 7.43 - 7.35 (m, 2H), 4.49 (t, J =5.5 Hz, 1H), 4.36 (d, J = 5.8 Hz, 2H), 4.21 (dt, J = 6.1, 2.0 Hz, 4H), 3.56(dt, J = 9.3, 6.7 Hz, 2H), 3.40 (dt, J = 9.3, 6.7 Hz, 2H), 2.52 (p, J = 5.9Hz, 1H), 2.42 (t, J = 7.6 Hz, 2H), 2.39 - 2.30 (m, 2H), 1.98 - 1.92 (m, 2H), 1.67 - 1.51 (m, 8H), 1.34 - 1.23 (m, 38H), 0.91 - 0.85 (m, 9H).

[1082] Compound 41: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl dodecanoate

[1083]

[1084] Compound 41 was synthesized from intermediate 41c and 3-(diethylamino)prop-1-ol using the method employed in the synthesis of compound 6. 1 H NMR (400 MHz, CDCl3) δ 4.41 (t, J = 5.6 Hz, 1H), 4.12 (dt, J =6.5, 3.5 Hz, 4H), 4.08 (dd, J = 6.1, 1.9 Hz, 4H), 3.49 (dt, J = 9.3, 6.7 Hz,2H), 3.33 (dt, J = 9.3, 6.7 Hz, 2H), 2.40 - 2.28 (m, 4H), 2.23 (t, J = 7.6Hz, 2H), 1.85 (td, J = 7.6, 5.5 Hz, 2H), 1.50 (dq, J = 20.9, 7.1 Hz, 7H),1.30 - 1.12 (m, 37H), 0.96 (s, 6H), 0.85 - 0.77 (m, 9H). MS: 773.0 m / z [M+H].

[1085] Example 42 - Compound 42

[1086] Compound 42: Undecanoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propyl ester

[1087]

[1088] Compound 42 was synthesized from intermediate 41c and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 6. 1H NMR (400 MHz, CDCl3) δ 4.48 (t, J = 5.6 Hz, 1H), 4.12 (dd, J= 6.2, 2.9 Hz, 6H), 3.56 (dt, J = 9.3, 6.7 Hz, 2H), 3.40 (dt, J = 9.3, 6.7Hz, 2H), 3.30 (s, 2H), 2.59 (d, J = 40.7 Hz, 5H), 2.38 (q, J = 6.8, 5.9 Hz, 3H), 2.30 (t, J = 7.6 Hz, 2H), 1.92 (td, J = 7.6, 5.4 Hz, 2H), 1.58 (dt, J =20.2, 7.3 Hz, 8H), 1.40 - 1.18 (m, 41H), 1.09 (s, 6H), 0.88 (t, J = 6.7 Hz, 9H). MS: 758.6 m / z [M+H].

[1089] Example 43 - Compound 43

[1090] Intermediate 43a: 2-Butyloctyl mesylate

[1091]

[1092] MsCl (1.5 equivalents) was added dropwise to a solution of 2-butyloct-1-ol (20 g, 1.0 equivalent) and Et3N (1.2 equivalent) in DCM (0.5 M) at 0 °C under N2. The mixture was stirred at 0 °C for 2 h. The reaction mixture was quenched by adding saturated NaHCO3, diluted with water, and extracted three times with DCM. The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give a white solid, which was used directly in the next step without further purification.

[1093] Intermediate 43b: 3-Butylnononitrile

[1094]

[1095] NaCN (5.0 equivalents) was added to a solution of intermediate 43a (20 g, 1.0 equivalents) in DMF (0.3–0.4 M) under N2 conditions. The reaction mixture was heated to 60 °C and stirred for 12 h. The reaction mixture was then poured into an aqueous NaOH solution until pH > 11, and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to remove the solvent, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a colorless oily product (81%). 1 H NMR (400 MHz, CDCl3) δ 2.32 (d, J = 5.8 Hz, 2H), 1.67 (ddd, J = 13.1,7.1, 5.9 Hz, 1H), 1.48 - 1.18 (m, 16H), 0.89 (dt, J = 8.6, 6.9 Hz, 6H).

[1096] Intermediate 43C: 3-Butylnonanoic acid

[1097]

[1098] A mixture of intermediate 43b (11 g, 1.0 equivalent) and KOH (10.0 equivalent) in a 1:1 EtOH and H2O (1 M) was degassed and purged three times with N2. The mixture was then stirred at 120 °C for 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove the solvent. The reaction mixture was adjusted to pH 5 with 1 M HCl, diluted with H2O, and extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a yellow oily product (66%). 1 H NMR (400 MHz, CDCl3) δ 2.21 (d, J = 6.9 Hz, 2H), 1.78 (d, J = 6.1 Hz, 1H), 1.29 - 1.16 (m, 16H), 0.82 (td, J =6.7, 4.0 Hz, 6H).

[1099] Intermediate 43d: 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(octyloxy)butyrate

[1100]

[1101] A mixture of intermediate 1b (10 g, 1.0 equivalent), 2-(hydroxymethyl)propane-1,3-diol (1.0 equivalent), EDCI (2.0 equivalent), DMAP (0.2 equivalent), and DIPEA (2.0 equivalent) in DCM (0.2 M) was degassed and purged three times with N2. The mixture was then stirred at 15 °C for 12 h under N2 atmosphere. The reaction mixture was diluted with H2O and extracted three times with DCM. After drying with Na2SO4, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography to give a product (52%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 4.42 (t, J = 5.5 Hz,1H), 4.27 - 4.03 (m, 2H), 3.76 - 3.61 (m, 4H), 3.57 (t, J = 6.7 Hz, 1H), 3.50(dt, J = 9.6, 6.7 Hz, 2H), 3.33 (dt, J = 9.3, 6.7 Hz, 2H), 2.34 (q, J = 7.4Hz, 2H), 2.15 - 2.00 (m, 1H), 2.00 - 1.92 (m, 1H), 1.87 (td, J = 7.2, 5.1 Hz,2H), 1.49 (p, J = 6.5 Hz, 4H), 1.32 - 1.11 (m, 20H), 0.81 (t, J = 6.7 Hz, 6H).

[1102] Intermediate 43e: 3-Butylnonanoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(hydroxymethyl)propyl ester

[1103]

[1104] The mixture of intermediates 43d (1.0 equivalent), 43c (1.0 equivalent), DMAP (0.2 equivalent), EDCI (2.0 equivalent), and DIPEA (1.0 equivalent) in DCM (0.25 M) was degassed and purged three times with N2. The mixture was then stirred at 0°C for 6 h under N2 atmosphere. The reaction mixture was diluted with H2O, extracted three times with DCM, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue (53%) was purified by column chromatography to give a colorless oily product. 1H NMR (400 MHz, CDCl3) δ 4.48 (t, J = 5.5 Hz, 1H), 4.22 - 4.10 (m, 4H), 3.68- 3.49 (m, 4H), 3.39 (dt, J = 9.3, 6.7 Hz, 2H), 2.40 (q, J = 7.9 Hz, 2H), 2.25 (d, J = 6.8 Hz, 2H), 2.22 - 2.14 (m, 1H), 1.93 (td, J = 7.4, 5.4 Hz, 2H), 1.83 (p, J = 5.8 Hz, 1H), 1.56 (q, J = 7.2, 6.4 Hz, 4H), 1.26 (dt, J =11.5, 6.8 Hz, 35H), 0.88 (td, J = 6.8, 3.3 Hz, 12H).

[1105] Intermediate 43f: 3-Butylnonanoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propyl ester

[1106]

[1107] Intermediate 43f (69%) was synthesized from intermediate 43e using the method employed in the synthesis of intermediate 2d.

[1108] Compound 43: 3-Butylnonanoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester

[1109]

[1110] Compound 43 was synthesized from intermediate 43f and 3-(diethylamino)prop-1-ol using the method employed in the synthesis of compound 6. 1H NMR (400 MHz, CDCl3) δ 4.48 (t, J = 5.5 Hz, 1H), 4.21 - 4.09 (m,8H), 3.55 (dt, J = 9.3, 6.7 Hz, 2H), 3.39 (dt, J = 9.3, 6.7 Hz, 2H), 2.52 (q,J = 7.0 Hz, 6H), 2.40 (q, J = 7.6, 7.0 Hz, 3H), 2.24 (d, J = 6.8 Hz, 2H), 1.92 (td, J = 7.6, 5.4 Hz, 2H), 1.82 (p, J = 6.8 Hz, 3H), 1.56 (q, J = 6.9Hz, 4H), 1.39 - 1.18 (m, 37H), 1.01 (t, J = 7.1 Hz, 6H), 0.88 (td, J = 6.9, 2.6 Hz, 12H). MS: 787.0 m / z [M+H].

[1111] Example 44 - Compound 44

[1112] Compound 44: 3-Butylnonanoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propyl ester

[1113]

[1114] Compound 44 was synthesized from intermediate 43f and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 6. 1H NMR (400 MHz, CDCl3) δ 5.14 (s, 1H), 4.41 (t, J = 5.6 Hz, 1H), 4.06 (t, J = 5.1 Hz, 6H), 3.49 (dt, J = 9.3, 6.7 Hz, 2H), 3.33 (dt, J =9.3, 6.7 Hz, 2H), 3.14 (q, J = 5.9 Hz, 2H), 2.45 (q, J = 7.1, 6.1 Hz, 6H), 2.32 (q, J = 7.9 Hz, 3H), 2.17 (d, J = 6.8 Hz, 2H), 1.85 (td, J = 7.6, 5.4Hz, 2H), 1.55 - 1.43 (m, 6H), 1.22 (dd, J = 18.6, 8.2 Hz, 35H), 0.93 (t, J =7.1 Hz, 6H), 0.81 (td, J = 6.9, 2.4 Hz, 12H). MS: 772.5 m / z [M+H].

[1115] Example 45 - Compound 45

[1116] Intermediate 45a: 3-hydroxy-2-(hydroxymethyl)propyl nonanoate

[1117]

[1118] Intermediate 45a (32%) was synthesized from 2-(hydroxymethyl)propane-1,3-diol and nonanoic acid using the method employed in the synthesis of intermediate 4b. 1 H NMR (400 MHz, CDCl3) δ 4.28 (d, J = 6.3 Hz, 2H), 3.79 (qd, J = 11.1, 5.1 Hz, 4H), 2.35 (t, J = 7.6 Hz, 2H), 2.28 (s, 2H), 2.05 (p,J = 5.9 Hz, 1H), 1.68 - 1.56 (m, 2H), 1.39 - 1.24 (m, 10H), 0.96 - 0.83 (m, 3H).

[1119] Intermediate 45b: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(hydroxymethyl)propyl nonanoate

[1120]

[1121] Intermediate 45b (76%) was synthesized from intermediate 45a and intermediate 1b using the method employed in the synthesis of intermediate 4c. 1 H NMR (400 MHz, CDCl3) δ 4.42 (t, J = 5.5 Hz, 1H), 4.18 - 4.01 (m, 4H), 3.60 - 3.46 (m, 4H), 3.33 (dt, J = 9.3, 6.7 Hz, 2H), 2.33 (q, J = 7.8 Hz,2H), 2.28 - 2.18 (m, 3H), 2.13 (h, J = 5.9 Hz, 1H), 1.87 (td, J = 7.3, 5.3Hz, 2H), 1.52 (dt, J = 25.0, 7.1 Hz, 6H), 1.33 - 1.17 (m, 32H), 0.81 (t, J =6.7 Hz, 9H).

[1122] Intermediate 45c: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propyl nonanoic acid

[1123]

[1124] Intermediate 45c was synthesized from intermediate 45b using the method employed in the synthesis of intermediate 2d.

[1125] Compound 45: Nonanoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester

[1126]

[1127] Compound 45 was synthesized from intermediate 45c and 3-(diethylamino)prop-1-ol using the method employed in the synthesis of compound 6. 1H NMR (400 MHz, CDCl3) δ 4.48 (t, J = 5.5 Hz, 1H), 4.20 (t, J =6.0 Hz, 4H), 4.14 (dd, J = 6.0, 1.9 Hz, 4H), 3.58 - 3.51 (m, 2H), 3.40 (dt, J= 9.3, 6.7 Hz, 2H), 2.61 (s, 5H), 2.46 - 2.36 (m, 4H), 2.30 (t, J = 7.6 Hz, 2H), 1.92 (td, J = 7.6, 5.5 Hz, 4H), 1.63 - 1.49 (m, 7H), 1.34 - 1.25 (m,31H), 1.08 (s, 6H), 0.89 - 0.85 (m, 9H). MS: 732.0 m / z [M+H].

[1128] Example 46 - Compound 46

[1129] Compound 46: Nonanoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propyl ester

[1130]

[1131] Compound 46 was synthesized from intermediate 45c and N',N'-diethylethane-1,2-diamine using the method employed in the synthesis of compound 6. 1H NMR (400 MHz, CDCl3) δ 5.25 - 5.11 (m, 1H), 4.42 (t, J = 5.5Hz, 1H), 4.13 - 4.00 (m, 6H), 3.49 (dt, J = 9.3, 6.7 Hz, 2H), 3.33 (dt, J =9.1, 6.6 Hz, 2H), 3.15 (q, J = 5.8 Hz, 2H), 2.46 (q, J = 7.1, 6.1 Hz, 6H), 2.32 (q, J = 7.6 Hz, 3H), 2.23 (t, J = 7.6 Hz, 2H), 1.91 - 1.80 (m, 2H), 1.51(dp, J = 20.9, 7.1 Hz, 6H), 1.32 - 1.12 (m, 32H), 0.94 (t, J = 7.1 Hz, 6H), 0.81 (t, J = 6.7 Hz, 9H). MS: 717.0 m / z [M+H].

[1132] Example 47 - Compound 47

[1133] Intermediate 47a: 3-hydroxy-2-(hydroxymethyl)propyl 2-butyloctanoate

[1134]

[1135] Intermediate 47a (35%) was synthesized from 2-(hydroxymethyl)propane-1,3-diol an...

Claims

1. A compound, said compound being selected from: (Compound 10) (Compound 12) (Compound 23) (Compound 49), and (Compound 50); Or its salt.

2. The compound of claim 1, wherein the compound is compound 10: ; or its salt.

3. The compound of claim 1, wherein the compound is compound 12: ; or its salt.

4. The compound of claim 1, wherein the compound is compound 23: ; or its salt.

5. The compound of claim 1, wherein the compound is compound 49: ; or its salt.

6. The compound of claim 1, wherein the compound is compound 50: ; or its salt.

7. A lipid nanoparticle (LNP) composition comprising the compound of claim 1, encapsulating nucleic acid.

8. The LNP composition of claim 7, wherein the nucleic acid is messenger RNA (mRNA).

9. The LNP composition of claim 8, wherein the mRNA encodes an RNA-guided DNA binder.

10. The LNP composition of claim 9, wherein the RNA-guided DNA binder comprises two types of Cas nucleases.

11. The LNP composition of claim 10, wherein the type 2 Cas nuclease is Streptococcus pyogenes (… S. pyogenes Cas9.

12. The LNP composition of claim 9, further comprising guide RNA (gRNA).

13. The LNP composition of claim 7, further comprising: (a) a neutral lipid; (b) an accessory lipid; and (c) a PEG lipid.

14. The LNP composition of claim 13, wherein: (a) The neutral lipid is DSPC; (b) The auxiliary lipid is cholesterol; and (c) The PEG lipid is 1,2-dimyristoyl-racemic-glycerol-3-[methoxy(polyethylene glycol)-2000] (PEG2K-DMG), methoxy-PEG2000-carbamoyl-1,2-tetrateoxypropylamine (C13 ether) or methoxy-PEG2000-carbamoyl-1,2-tetradecyloxypropylamine (C14 ether).

15. The LNP composition of claim 7, wherein the compound is compound 10: ; or its salt.

16. The LNP composition of claim 7, wherein the compound is compound 12: ; or its salt.

17. The LNP composition of claim 7, wherein the compound is compound 23: ; or its salt.

18. The LNP composition of claim 7, wherein the compound is compound 49: ; or its salt.

19. The LNP composition of claim 7, wherein the compound is compound 50: ; or its salt.

20. The LNP composition of claim 7, further comprising: (a) Neutral lipids, wherein the neutral lipids are DSPC; (b) Auxiliary lipids, wherein the auxiliary lipid is cholesterol; (c) PEG lipids, wherein the PEG lipid is 1,2-dimyristoyl-racemic-glycerol-3-[methoxy(polyethylene glycol)-2000] (PEG2K-DMG), methoxy-PEG2000-carbamoyl-1,2-tetrateoxypropylamine (C13 ether), or methoxy-PEG2000-carbamoyl-1,2-tetradecyloxypropylamine (C14 ether); and (d) Guide RNA (gRNA); The nucleic acid contained in the nucleic acid includes mRNA encoding a type 2 Cas nuclease.

21. Use of the LNP composition according to any one of claims 7 to 20 in the preparation of a medicament for lysing DNA.

22. Use of the LNP composition according to any one of claims 7 to 20 in the preparation of a medicament for gene editing.

23. The use as described in claim 22, wherein the drug is intended for administration to humans.

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