nanomaterials

Abstract

Lipid nanoparticle compositions for nucleic acid delivery are described. In various embodiments, the lipid nanoparticles comprise an ionizable lipid of formula (I). Also provided is a method for using such lipid nanoparticle compositions to achieve targeted delivery of therapeutic cargo without the need for targeting ligands. [Formula 1] JPEG2023510778000295.jpg4480

Description

[Technical Field]

[0001] Information about related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 958,876, filed January 9, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Field This application relates to the fields of chemistry, biology, and medicine. Disclosed herein are drug delivery systems and methods of their use. More particularly, disclosed herein are nanoparticle compositions for the delivery of nucleic acids to cells.

[0003] explanation Hepatocytes help maintain homeostasis and produce many secreted proteins, and are therefore implicated in many genetic diseases. Although nanoparticles carrying RNA have been targeted to hepatocytes, systemic delivery to hepatocytes without a targeting ligand remains a challenge. Summary of the Invention

[0004] Some embodiments described herein include compounds of formula (I):

[0005] [ka] Regarding the compound (In the formula, R 1 is C9~C 20 Alkyl or C9-C with 1-3 units of unsaturation 20 is alkenyl; X 1 and X 2 are each independently absent, -O-, or -NR 2 - and

[0006] [ka] where each R 2 are independently hydrogen or C1-C6 alkyl; each a is independently an integer between 1 and 6; X 3 and X 4 are each independently absent or selected from a 4- to 8-membered heterocyclyl optionally substituted with one or two C1-C6 alkyl groups, a 5- to 6-membered heteroaryl optionally substituted with one or two C1-C6 alkyl groups, a 5- to 6-membered aryl optionally substituted with one or two C1-C6 alkyl groups, a 4- to 7-membered cycloalkyl optionally substituted with one or two C1-C6 alkyl groups, -O-, and -NR 3 -, wherein each R 3 is a hydrogen atom or a C1-C6 alkyl, where X 1 -X 2 -X 3 -X 4 does not contain any oxygen-oxygen, oxygen-nitrogen, or nitrogen-nitrogen bonds; X 5 Ha-(CH2) b where b is an integer between 0 and 6; X 6 is hydrogen, C1-C6 alkyl, 5-6 membered heteroaryl optionally substituted with one or two C1-C6 alkyl groups, or -NR 4 R 5 where R 4 and R 5 are each independently hydrogen or C1-C6 alkyl; or alternatively, R 4 and R 5 together with the nitrogen to which they are attached form a 4-7 membered heterocyclyl optionally substituted with 1 or 2 C1-C6 alkyl groups, wherein the heterocyclyl optionally contains additional heteroatoms selected from oxygen, sulfur, and nitrogen; each X 7 are independently hydrogen, hydroxyl, or -NR 6 R7 where R 6 and R 7 are each independently hydrogen or C1-C6 alkyl; or alternatively, R 6 and R 7 together with the nitrogen to which they are attached form a 4-7 membered heterocyclyl optionally substituted with 1 or 2 C1-C6 alkyl groups, wherein the heterocyclyl optionally contains additional heteroatoms selected from oxygen, sulfur, and nitrogen; X 1 , X 2 , X 3 , X 4 , and X 5 At least one of the following is present; A 1 and A 2 are independently C5 to C 12 Haloalkyl, C5-C 12 Alkenyl, C5-C 12 Alkynyl, (C5-C 12 Alkoxy)-(CH2) n2 -, optionally ring-substituted with one or two halo groups, C1-C6 alkyl groups, C1-C6 haloalkyl groups, or C1-C6 alkoxy groups (C5-C 10 Aryl)-(CH2) n3 - and (C3-C8 cycloalkyl)-(CH2) optionally ring-substituted with one or two C1-C6 alkyl groups n4 -; or alternatively, A 1 and A 2 together with the atoms to which they are attached, form one or two C4-C 10 Forms 5-6 membered cyclic acetals substituted with alkyl groups; n1, n2, and n3 are each independently an integer between 1 and 4; n4 is an integer between 0 and 4).

[0007] Some embodiments feature a lipid nanoparticle composition comprising an ionizable lipid; a phospholipid; a polyethylene glycol-lipid; cholesterol; and optionally a nucleic acid, as described herein. Further embodiments are directed to a method of delivering a nucleic acid to a subject in need thereof, comprising administering a lipid nanoparticle composition to a subject in need thereof.

[0008] These and other embodiments are described in more detail below. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 illustrates a reaction scheme for preparing compounds of formula (I). [Figure 2-1] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-2] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-3] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-4] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-5] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-6] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-7] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-8] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-9] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-10] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-11] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-12] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-13]1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-14] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-15] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-16] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-17] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-18] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-19] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-20] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-21] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-22] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-23] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-24] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-25] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-26] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-27] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-28] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-29] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-30] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-31] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-32] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. [Figure 2-33] 1 is a table summarizing the structures of the compounds of Examples 1 to 148. DETAILED DESCRIPTION OF THE INVENTION

[0010] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications referenced herein are incorporated by reference in their entirety unless otherwise indicated. In the event that there are multiple definitions for terms herein, those in this section prevail unless otherwise indicated.

[0011] As used herein, any "R" or "X" group, such as, but not limited to, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , A 1 , and A 2 represents a substituent that may be attached to the indicated atom. Such R, X, and A groups may be collectively referred to herein as an "R" group. The R group may be substituted or unsubstituted. When two "R" groups are described as "together," it will be understood that the R groups and the atoms to which they are attached can form a ring structure, such as a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocycle. For example, but not limited to, NR a R b Group R a and R b When the term "together" is used, they are covalently bonded to each other to form a ring:

[0012] [ka] In addition, when two "R" groups are "together" with the atoms to which they are attached to alternatively form a ring, the R groups are not limited to the variables or substituents defined above.

[0013] Whenever a group is described as "optionally substituted," the group can be unsubstituted or substituted with one or more of the specified substituents. Similarly, when a group is described as "unsubstituted or substituted," if the group is substituted, the substituents may be selected from one or more of the specified substituents. If no substituents are indicated, the indicated "optionally substituted" or "substituted" group means that the group can be substituted with one or more groups individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, acylalkyl, hydroxy, alkoxy, alkoxyalkyl, aminoalkyl, amino acid, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxyalkyl, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanate, thiocyanate, isothiocyanate, azido, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, monosubstituted amino, and disubstituted amino.

[0014] As used herein, "C" is a set of integers where "a" and "b" are integers. a ~C b" refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or in the ring of a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heteroalicyclyl group. That is, an alkyl, alkenyl, alkynyl, cycloalkyl ring, cycloalkenyl ring, aryl ring, heteroaryl ring, or heteroalicyclyl ring can contain "a" through "b," inclusive. Thus, for example, a "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-. When "a" and "b" are not specified with respect to an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heteroalicyclyl group, the broadest range described by these definitions is intended.

[0015] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain, including fully saturated (no double or triple bonds) hydrocarbon groups. The alkyl group can have 1 to 20 carbon atoms. (Whenever it appears herein, a numerical range, such as "1 to 20," refers to each integer within the given range; for example, "1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, and so on, up to and including 20 carbon atoms, but this definition also covers the occurrence of the term "alkyl" when no numerical range is specified.) The alkyl group can also be a medium-sized alkyl having 1 to 10 carbon atoms. The alkyl group can also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group of a compound can be designated as "C1-C4 alkyl" or similar designation. By way of example only, "C1-C4 alkyl" indicates that the alkyl chain has 1 to 4 carbon atoms, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl. Alkyl groups can be substituted or unsubstituted.

[0016] As used herein, "alkenyl" refers to an alkyl group containing one or more double bonds in a straight or branched hydrocarbon chain. Examples of alkenyl groups include allenyl, vinylmethyl, and ethenyl. Alkenyl groups can be unsubstituted or substituted.

[0017] As used herein, "alkynyl" refers to an alkyl group containing one or more triple bonds in a straight or branched hydrocarbon chain. Examples of alkynyl include ethynyl and propynyl. Alkynyl groups can be unsubstituted or substituted.

[0018] As used herein, "cycloalkyl" refers to a fully saturated (no double or triple bonds) monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings can be joined together in a fused, bridged, or spiro fashion. As used herein, the term "fused" refers to two rings that share two atoms and one bond in common. As used herein, the term "bridged cycloalkyl" refers to a compound in which a cycloalkyl contains a linkage of one or more atoms connecting non-adjacent atoms. As used herein, the term "spiro" refers to two rings that share one atom in common, but the two rings are not connected by a bridge. Cycloalkyl groups can contain 3 to 30 atoms in the rings, 3 to 20 atoms in the rings, 3 to 10 atoms in the rings, 3 to 8 atoms in the rings, or 3 to 6 atoms in the rings. Cycloalkyl groups can be unsubstituted or substituted. Exemplary monocycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of fused cycloalkyl groups are decahydronaphthalenyl, dodecahydro-1H-phenalenyl, and tetradecahydroanthracenyl. Examples of bridged cycloalkyl groups are bicyclo[1.1.1]pentyl, bicyclo[2.1.1]heptane, adamantanyl, and norbornanyl. Examples of spirocycloalkyl groups include spiro[3.3]heptane and spiro[4.5]decane.

[0019] As used herein, "cycloalkenyl" refers to a monocyclic or polycyclic hydrocarbon ring system containing one or more double bonds in at least one ring; however, if there are two or more, the double bonds cannot form a completely delocalized π-electron system throughout all rings (otherwise the group is an "aryl" as defined herein). Cycloalkenyl groups can contain 3 to 10 atoms in the rings, or 3 to 8 atoms in the rings. When composed of two or more rings, the rings can be connected together in a fused fashion. Cycloalkenyl groups can be unsubstituted or substituted.

[0020] As used herein, "aryl" refers to a carbocyclic (all carbon) monocyclic or polycyclic aromatic ring system (including fused ring systems in which two carbocyclic rings share a chemical bond) having a completely delocalized pi-electron system throughout all rings. The number of carbon atoms in an aryl group can vary. For example, an aryl group can be any group from C6 to C6. 14 Even if it is an aryl group, it is C6-C 10 It may be an aryl group or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene, and azulene. The aryl group may be substituted or unsubstituted.

[0021] As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic ring system (a ring system having a fully delocalized π-electron system) containing one, two, three, or more heteroatoms, i.e., elements other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur. The number of atoms in the ring of a heteroaryl group can vary. For example, a heteroaryl group can contain 4 to 14 atoms in the ring, 5 to 10 atoms in the ring, or 5 to 6 atoms in the ring. Additionally, the term "heteroaryl" includes fused ring systems, in which two rings, e.g., at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings, share at least one chemical bond. Examples of heteroaryl rings include, but are not limited to, those described herein and the following: furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, and triazine. Heteroaryl groups can be substituted or unsubstituted.

[0022] As used herein, "heterocyclyl" or "heteroalicyclyl" refers to 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, and up to 18-membered monocyclic, bicyclic, and tricyclic ring systems, in which carbon atoms, together with one to five heteroatoms, comprise the ring system. Heterocycles can optionally contain one or more unsaturated bonds, provided that the unsaturated bonds are positioned such that a fully delocalized π-electron system does not exist throughout all rings. Heteroatoms are elements other than carbon, including, but not limited to, oxygen, sulfur, and nitrogen. Heterocycles can further contain one or more carbonyl or thiocarbonyl functional groups, such that the definition includes oxo and thio systems, such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of more than one ring, the rings can be joined together in a fused or spiro fashion, as described herein for "cycloalkyl." Additionally, any nitrogen in a heterocyclyl may be quaternized.A heterocyclyl or heteroalicyclyl group may be unsubstituted or substituted.Examples of such "heterocyclyl" or "heteroalicyclyl" groups include, but are not limited to, those described herein and the following: 1,3-dioxine, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiine, 1,3,4-oxadiazol-2(3H)-one, 1,2,3-oxadiazol-5(2H)-one, 1,3-oxathiolane, 1,3-dithiole, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 1,3-thiazinane, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydroxybenzoate ... Dantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, and their benzo-fused analogs (e.g., benzimidazolidinone, tetrahydroquinoline, and 3,4-methylenedioxyphenyl).

[0023] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl group connected as a substituent via a lower alkylene group. The lower alkylene group and the aryl group of an aralkyl can be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

[0024] As used herein, "heteroaralkyl" and "heteroaryl(alkyl)" refer to a heteroaryl group connected as a substituent via a lower alkylene group. The lower alkylene group and heteroaryl group of a heteroaralkyl can be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, imidazolylalkyl, and their benzo-fused analogs.

[0025] "Heteroalicyclyl(alkyl)" and "heterocyclyl(alkyl)" refer to a heterocyclyl group or a heteroalicyclyl group connected as a substituent via a lower alkylene group. The lower alkylene and heterocyclyl of a heteroalicyclyl(alkyl) may be substituted or unsubstituted. Examples include, but are not limited to, tetrahydro-2H-pyran-4-yl(methyl), piperidin-4-yl(ethyl), piperidin-4-yl(propyl), tetrahydro-2H-thiopyran-4-yl(methyl), and 1,3-thiazinan-4-yl(methyl).

[0026] A "lower alkylene group" is a straight-chain -CH- tethered group that forms a bond connecting molecular fragments through its terminal carbon atom. Examples include, but are not limited to, methylene (-CH-), ethylene (-CHCH-), propylene (-CHCHCH-), and butylene (-CHCHCHCHCH-). A lower alkylene group can be substituted by replacing one or more hydrogens of the lower alkylene group with a substituent listed under the definition of "substituted."

[0027] As used herein, "alkoxy" refers to a group of formula -OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. A non-limiting list of alkoxy is methoxy, ethoxy, n-propoxy, 1-methylethoxy(isopropoxy), cyclopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, cyclobutoxy, phenoxy, and benzoxy. Alkoxy can be substituted or unsubstituted.

[0028] As used herein, "acyl" refers to a hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl) connected as a substituent through a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl, and acryl. Acyl can be substituted or unsubstituted.

[0029] As used herein, "acylalkyl" refers to an acyl connected as a substituent through a lower alkylene group. Examples include aryl-C(=O)-(CH) n - and heteroaryl-C(=O)-(CH2) n - (wherein n is an integer ranging from 1 to 6).

[0030] As used herein, "alkoxyalkyl" refers to an alkoxy group connected as a substituent via a lower alkylene group. Examples include C 1~4 Alkyl-O-(CH2) n - (wherein n is an integer ranging from 1 to 6).

[0031] As used herein, "aminoalkyl" refers to an optionally substituted amino group connected as a substituent via a lower alkylene group. Examples include HN(CH) n - (wherein n is an integer ranging from 1 to 6).

[0032] As used herein, "hydroxyalkyl" refers to an alkyl group in which one or more of the hydrogen atoms have been replaced by a hydroxy group. Exemplary hydroxyalkyl groups include, but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. Hydroxyalkyl can be substituted or unsubstituted.

[0033] As used herein, "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced by halogen (e.g., monohaloalkyl, dihaloalkyl, and trihaloalkyl). Such groups include, but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloro-fluoroalkyl, chloro-difluoroalkyl, and 2-fluoroisobutyl. Haloalkyl can be substituted or unsubstituted.

[0034] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more of the hydrogen atoms have been replaced by halogen (e.g., monohaloalkoxy, dihaloalkoxy, and trihaloalkoxy). Such groups include, but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloro-fluoroalkyl, chloro-difluoroalkoxy, and 2-fluoroisobutoxy. Haloalkoxy can be substituted or unsubstituted.

[0035] A "sulfenyl" group refers to a "-SR" group, where R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The sulfenyl can be substituted or unsubstituted.

[0036] A "sulfinyl" group refers to a "-S(=O)-R" group, where R can be the same as defined for sulfenyl. The sulfinyl can be substituted or unsubstituted.

[0037] A "sulfonyl" group refers to a "SO2R" group, where R can be the same as defined for sulfenyl. The sulfonyl can be substituted or unsubstituted.

[0038] An "O-carboxy" group refers to an "RC(=O)O-" group, where R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl), as defined herein. The O-carboxy can be substituted or unsubstituted.

[0039] The terms "ester" and "C-carboxy" refer to the group "-C(=O)OR" where R can be the same as defined for O-carboxy. Esters and C-carboxy can be substituted or unsubstituted.

[0040] A "thiocarbonyl" group refers to a "-C(=S)R" group, where R can be the same as defined for O-carboxy. The thiocarbonyl can be substituted or unsubstituted.

[0041] A "trihalomethanesulfonyl" group refers to an "X3CSO2-" group where each X is a halogen.

[0042] The "trihalomethanesulfonamide" group is "X3CS(O)2N(R A )— group, where each X is a halogen and R A is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).

[0043] As used herein, the term "amino" refers to the group --NH.sub.2.

[0044] As used herein, the term "hydroxy" refers to an --OH group.

[0045] A "cyano" group refers to a "-CN" group.

[0046] As used herein, the term "azido" refers to the group --N3.

[0047] An "isocyanate" group refers to a "-NCO" group.

[0048] A "thiocyanate" group refers to a "-CNS" group.

[0049] An "isothiocyanate" group refers to a "-NCS" group.

[0050] A "carbonyl" group refers to a C=O group.

[0051] The "S-sulfonamide" group is "-SO2N(R A R B ) group, where R A and R Bcan be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). S-sulfonamides can be substituted or unsubstituted.

[0052] The "N-sulfonamide group" is "RSO2N(R A )— group, where R and R A can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-sulfonamides can be substituted or unsubstituted.

[0053] The "O-carbamyl" group is defined as "-OC(=O)N(R A R B ) group, where R A and R B can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). O-carbamyl can be substituted or unsubstituted.

[0054] The "N-carbamyl" group is "ROC(=O)N(R A )— group, where R and R A can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-carbamyl can be substituted or unsubstituted.

[0055] The "O-thiocarbamyl" group is defined as "-OC(=S)-N(R A R B ) group, where R A and R B can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). O-thiocarbamyl can be substituted or unsubstituted.

[0056] The "N-thiocarbamyl group" is "ROC(=S)N(R A )— group, where R and R A can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-thiocarbamyl can be substituted or unsubstituted.

[0057] A "C-amide" group is a group consisting of -C(=O)N(R A R B ) group, where R A and R B can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). C-amides can be substituted or unsubstituted.

[0058] The "N-amide" group is defined as "RC(=O)N(R A )— group, where R and R Acan be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). N-amides can be substituted or unsubstituted.

[0059] The "urea" group is defined as "N(R)-C(=O)-NR A R B " group, where R can be hydrogen or alkyl, and R A and R B can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The urea can be substituted or unsubstituted.

[0060] The "oxime" group is "-C(=N-OH)R A " (wherein R A can independently be alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The oxime can be substituted or unsubstituted.

[0061] "Acylhydrozone" means "-C(=N-NH-acyl)-R A " where the acyl moiety has the structure provided herein for "acyl" and R A can independently be alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The acylhydrozone can be substituted or unsubstituted.

[0062] "Hydrazine" is "-NHNR A R B " (wherein R A and R B can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl). The hydrazine can be substituted or unsubstituted.

[0063] The term "halogen atom" or "halogen" as used herein means any one of the radiostable atoms in column 7 of the periodic table of the elements, such as fluorine, chlorine, bromine and iodine.

[0064] As used herein,

[0065] [ka] indicates a single or double bond unless otherwise indicated.

[0066] Where the number of substituents is not specified (e.g., haloalkyl), one or more substituents can be present. For example, "haloalkyl" can include one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" can include one or more of the same or different alkoxy groups containing 1, 2, or 3 atoms.

[0067] As used herein, abbreviations for all protecting groups, amino acids and other compounds follow their common usage, accepted abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. 11:942-944 (1972)) unless otherwise indicated.

[0068] As used herein, the terms "protecting group" and "protecting groups" (and the abbreviation "PG") refer to any atom or group of atoms added to a molecule to prevent an existing group in the molecule from undergoing an undesired chemical reaction. Examples of protecting group moieties are described in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3. Ed. John Wiley & Sons, 1999, and in J.F.W. McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, both of which are incorporated herein by reference for the limited purpose of disclosing suitable protecting groups. Protecting group moieties can be selected so that the protecting group is stable to certain reaction conditions and is easily removed at a convenient stage using methodologies known in the art.A non-limiting list of protecting groups includes: benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., t-butoxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl, or t-butyldiphenylsilyl); esters (e.g., benzoate esters); carbonates (e.g., methoxymethylcarbonate cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein); acyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane); orthoesters (e.g., those described herein), and triarylmethyl groups (e.g., trityl; monomethoxytrityl (MMTr); 4,4'-dimethoxytrityl (DMTr); 4,4',4"-trimethoxytrityl (TMTr); and those described herein).

[0069] As used herein, the term "leaving group" (and the abbreviation "LG") refers to any atom or moiety that can be replaced by another atom or moiety in a chemical reaction. More specifically, in some embodiments, a "leaving group" refers to an atom or moiety that is displaced in a nucleophilic substitution reaction. In some embodiments, a "leaving group" is any atom or moiety that is the conjugate base of a strong acid. Examples of suitable leaving groups include, but are not limited to, tosylate, mesylate, trifluoroacetate, and halogens (e.g., I, Br, and Cl). Non-limiting characteristics and examples of leaving groups can be found, for example, in Organic Chemistry, 2nd ed., Francis Carey (1992), pages 328-331; Introduction to Organic Chemistry, 2nd ed., Andrew Streitwieser and Clayton Heathcock (1981), pages 169-171; and Organic Chemistry, 5th ed., John McMurry (2000), pages 398 and 408; all of which are incorporated herein by reference for the limited purpose of disclosing characteristics and examples of leaving groups.

[0070] The term "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not neutralize the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with an inorganic acid such as a hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid. Pharmaceutical salts can also be obtained by reacting a compound with an organic acid such as an aliphatic or aromatic carboxylic or sulfonic acid, for example, formic acid, acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form salts such as ammonium salts, alkali metal salts such as sodium salts or potassium salts, alkaline earth metal salts such as calcium salts or magnesium salts, salts of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamines, cyclohexylamine, triethanolamine, ethylenediamine, and the like, and salts with amino acids such as arginine and lysine.

[0071] Terms and phrases used in this application, and variations thereof, should be construed as open-ended, as opposed to limiting, unless expressly stated otherwise, particularly in the appended claims. As examples above, the term "including" should be read to mean "including, without limitation," "including but not limited to," etc.; the term "comprising," as used herein, is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term "having" should be interpreted as "having at least"; the term "includes" should be interpreted as "includes but is not limited to." The term "example" is used to provide an illustrative example of the matter under discussion, rather than an exhaustive or limiting enumeration thereof; the use of terms such as "preferably," "preferred," "desired," or "desirable," and words of similar import, should not be understood to imply that a particular characteristic is critical, essential, or even important to structure or function, but instead is merely intended to highlight alternative or additional characteristics that may or may not be utilized in a particular embodiment. Additionally, the term "comprising" should be interpreted as synonymous with the phrase "having at least" or "including at least."When used in the context of a method, the term "comprising" means that the method includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. Similarly, a group of items joined by the conjunction "and" should not be read as requiring each and every one of such items to be present in the group, but rather should be read as "and / or" unless the context dictates otherwise. Similarly, a group of items joined by the conjunction "or" should not be read as requiring mutual exclusivity within the group, but rather should be read as "and / or" unless the context dictates otherwise.

[0072] With respect to the use of virtually any plural and / or singular term herein, those skilled in the art can convert from plural to singular and / or from singular to plural where appropriate for the context and / or application. Various singular / plural permutations may be expressly indicated herein for clarity. The indefinite article "a" or "an" does not exclude plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0073] In any compound described herein having one or more chiral centers, unless the absolute stereochemistry is explicitly indicated, it is understood that each center can independently be in the R-configuration, the S-configuration, or a mixture thereof. Thus, the compounds provided herein can be enantiomerically pure, enantiomerically enriched, racemic, diastereomerically pure, diastereomerically enriched, or a mixture of stereoisomers. Additionally, in any compound described herein having one or more double bonds that produce geometric isomers that can be defined as E or Z, it is understood that each double bond can independently be E, Z, or a mixture thereof.

[0074] Likewise, it is understood that in any compound described, all tautomeric forms are intended to be included.

[0075] It is understood that when the compounds disclosed herein have open valencies, the valencies will be filled with hydrogen or its isotopes, such as hydrogen-1 (protium) and hydrogen-2 (deuterium).

[0076] It is understood that the compounds described herein can be isotopically labeled. Substitution with isotopes such as deuterium can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Each chemical element represented in a compound structure can include any isotope of said element. For example, in a compound structure, a hydrogen atom may be explicitly disclosed as being present in the compound or may be understood as being present. At any position in the compound where a hydrogen atom can be present, the hydrogen atom may be any isotope of hydrogen, including, but not limited to, hydrogen-1 (protium) and hydrogen-2 (deuterium). Thus, unless the context dictates otherwise, reference to a compound herein encompasses all possible isotopic forms.

[0077] It is understood that the methods and combinations described herein include crystalline forms (also known as polymorphs, which include different crystalline packing arrangements of compounds with the same elemental composition), amorphous phases, salts, solvates, and hydrates. In some embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In other embodiments, the compounds described herein exist in unsolvated forms. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and can be formed during the crystallization process using pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. In addition, the compounds provided herein can exist in unsolvated and solvated forms. In general, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0078] Where a range of values ​​is provided, it is understood that the upper and lower limits, and each intervening value between the upper and lower limits of the range, are encompassed within the embodiment.

[0079] As used herein, "RNA" refers to naturally occurring or non-naturally occurring ribonucleic acid. For example, RNA can contain modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA can contain a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA can have a nucleotide sequence encoding a polypeptide of interest. For example, RNA can be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, for example, in vivo translation of the mRNA inside a mammalian cell, can produce the encoded polypeptide. The RNA can be selected from the non-limiting group consisting of small interfering RNA (siRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single guide RNA (sgRNA), cas9 mRNA, and mixtures thereof.

[0080] The terms "polypeptide," "peptide," and "protein" can be used interchangeably to refer to a string of at least three amino acids linked together by peptide bonds. Peptide can refer to an individual peptide or a collection of peptides. Peptides can contain natural amino acids, unnatural amino acids (i.e., compounds that do not occur in nature but can be incorporated into a polypeptide chain), and / or amino acid analogs. One or more of the amino acids in a peptide can also be modified by the addition of a chemical entity, such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification. Modifications can include cyclization of the peptide, incorporation of D-amino acids, and the like.

[0081] As used herein, the terms "treat," "treating," "treatment," and "therapeutic use" refer to the elimination, reduction, or amelioration of one or more symptoms of a disease or disorder. As used herein, a "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to mediate a clinically significant elimination, reduction, or amelioration of such symptoms. An effect is clinically significant if its magnitude is sufficient to affect the health or prognosis of the recipient subject. A therapeutically effective amount can refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, for example, to delay or minimize the spread of cancer. A therapeutically effective amount can also refer to the amount of a therapeutic agent that provides a therapeutic effect in the treatment or management of a disease.

[0082] The compositions described herein are preferably provided in unit dosage form. As used herein, a "unit dosage form" refers to a composition containing an amount of a compound or composition suitable for administration in a single dose to an animal, preferably a mammalian subject, in accordance with good medical practice. However, the preparation of a single dosage form or unit dosage form does not imply that the dosage form is administered once daily or once per course of treatment. While a single dose is not specifically excluded, such dosage forms are contemplated for administration once, twice, three times, or more times per day, and may be administered as an infusion over a period of time (e.g., from 30 minutes to about 2-6 hours), or as a continuous infusion, and may be given multiple times over the course of treatment. Those skilled in the art will recognize that the formulation does not specifically contemplate an entire course of treatment, and such a determination is left to those skilled in the art of treatment rather than formulation. As used herein, the term "prophylactic agent" refers to an agent that can be used to prevent a disorder or disease prior to the detection of any symptoms associated with the disorder or disease. A "prophylactically effective" amount is the amount of a prophylactic agent sufficient to mediate such prevention. A prophylactically effective amount may also refer to the amount of a prophylactic agent that provides a prophylactic benefit in the prevention of disease.

[0083] The above-described useful compositions can be in any of a variety of forms suitable for various routes of administration, such as oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intraarterial, intravenous, intramuscular, or other parenteral routes. Those skilled in the art will understand that oral and nasal compositions include compositions administered by inhalation and prepared using available methodologies. Depending on the particular route of administration desired, various pharmaceutically acceptable carriers well known in the art can be used. Pharmaceutically acceptable carriers include, for example, solid or liquid fillers, diluents, hydrotropes, surfactants, and encapsulating materials. Optional pharmaceutically active materials can be included that do not substantially interfere with the inhibitory activity of the compound. The amount of carrier used in conjunction with the compound is sufficient to provide a practical amount of the substance for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods described herein are described in the following references, all of which are incorporated herein by reference: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

[0084] As used herein, the terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans, rodents such as mice and rats, and other laboratory animals.

[0085] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline, water and emulsions such as oil / water or water / oil emulsions, and various types of wetting agents.

[0086] The term "PEG-lipid" refers to a lipid modified with polyethylene glycol. Exemplary PEG-lipids include, but are not limited to, C 14 PEG 350 , C 14 PEG 1000 , C 14 PEG 2000 , C 14 PEG 3000 , and C 18 PEG 2000 Includes:

[0087] The term "oligonucleotide" refers to a short DNA, RNA, or DNA / RNA molecule or oligomer containing a relatively small number of nucleotides.

[0088] A. Lipid Nanoparticles Effective targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids is an ongoing challenge in the field of medicine.The delivery of nucleic acids is particularly difficult due to the relative instability and poor cell permeability of nucleic acids.It has been discovered that lipid nanoparticles with ionizable lipids as described herein can more effectively deliver nucleic acids to specific tissues in the body.In one embodiment, lipid nanoparticles can be formulated by mixing nucleic acids with ionizable lipids, PEG-lipids, phospholipids, cholesterol, and optionally nucleic acids.In some embodiments, lipid nanoparticles do not contain targeting ligands.In some embodiments, the disclosed lipid nanoparticles preferentially target T cells over hepatocytes in the absence of targeting ligands.

[0089] Lipid nanoparticles vary in size. In one embodiment, the lipid nanoparticles can have an average hydrodynamic diameter of between about 30 and about 170 nm. The lipid nanoparticles can have an average hydrodynamic diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, or any range having endpoints defined by any two of the foregoing values. For example, in one embodiment, the nanoparticles have an average hydrodynamic diameter of between 50 nm and 100 nm.

[0090] 1.Compound Some embodiments described herein include compounds of formula (I):

[0091] [ka] The present invention relates to the compound

[0092] In various embodiments, the compound of formula (I) is an ionizable lipid, as described elsewhere herein. In various embodiments, R of formula (I) 1 is C9~C 20 Alkyl or C9-C with 1-3 units of unsaturation 20 For example, in some embodiments, R in formula (I) is alkenyl. 1 is the expression

[0093] [ka] C 17 C9-C with two units of unsaturation, such as alkenyl groups 20 It is alkenyl.

[0094] In various embodiments, X in formula (I) 1 and X 2are each independently absent, -O-, or -NR 2 - and

[0095] [ka] where R 2 is hydrogen or C1-C6 alkyl, a is an integer between 1 and 6, and X 7 are independently hydrogen, hydroxyl, or -NR 6 R 7 and R 6 and R 7 are each independently hydrogen or C1-C6 alkyl; or alternatively, taken together with the nitrogen to which they are attached, form a 4-7 membered heterocyclyl optionally substituted with 1 or 2 C1-C6 alkyl groups, where the heterocyclyl optionally contains additional heteroatoms selected from oxygen, sulfur, and nitrogen. In some embodiments, X 1 is absent or X 2 is absent or X 1 and X 2 As described elsewhere herein, X 1 -X 2 -X 3 -X 4 do not contain any oxygen-oxygen, oxygen-nitrogen, or nitrogen-nitrogen bonds. Therefore, X 1 and X 2 cannot both be -O- and both be -NR 2 - should not be the same. Similarly, X 1 and X 2 are -O- and -NR, respectively. 2 - and -NR respectively. 2 - and -O-.

[0096] In various embodiments, X 1 is —O—. In various embodiments, X 2 is —O—. In some embodiments, X 1 is -(CH2)a -, -CH(OH)-, or -(CH2) a-1 CH(OH)-, etc.

[0097] [ka] In some embodiments, X 2 is -(CH2) a -, -CH(OH)-, or -(CH2) a-1 CH(OH)-, etc.

[0098] [ka] In various embodiments, each a is independently 1, 2, 3, 4, 5, or 6. In various embodiments, X 1 is -NR 6 R 7 In various embodiments, X 2 is -NR 6 R 7 In some embodiments, R 6 is hydrogen or C1-C6 alkyl. In some embodiments, R 7 is hydrogen or C1-C6 alkyl. In other embodiments, R 6 and R 7 together with the nitrogen to which they are attached form a 4-7 membered heterocyclyl optionally substituted with one or two C1-C6 alkyl groups. In some embodiments, R 6 and R 7 The 4- to 7-membered heterocyclyl formed by combining contains an additional heteroatom selected from oxygen, sulfur, and nitrogen.

[0099] In various embodiments, X in formula (I) 3 and X 4 are each independently absent, or (1) 4- to 8-membered heterocyclyl optionally substituted with 1 or 2 C1-C6 alkyl groups; (2) 5- to 6-membered heteroaryl optionally substituted with one or two C1-C6 alkyl groups; (3) 5- to 6-membered aryl optionally substituted with one or two C1-C6 alkyl groups; (4) 4- to 7-membered cycloalkyl optionally substituted with one or two C1-C6 alkyl groups; (5) -O-; or (6)-NR 3 -(In the formula, each R 3 are independently a hydrogen atom or a C1-C6 alkyl. is selected from.

[0100] In some embodiments, X 3 is absent or X 4 is absent or X 3 and X 4 As described elsewhere herein, X 1 -X 2 -X 3 -X 4 do not contain any oxygen-oxygen, oxygen-nitrogen, or nitrogen-nitrogen bonds. Therefore, X 2 and X 3 and X cannot both be -O-. 2 is -O- or -NR 2 -If X 3 is -NR 3 - should not be the same. Similarly, X 3 is -O- or -NR 3 -If X 2 is -NR 2 - should not be. Similarly, X 3 and X 4 cannot both be -O- and both be -NR 3 - should not be the same. Similarly, X 3 and X 4 are -O- and -NR, respectively. 3 - and -NR respectively. 3 - and -O-.

[0101] In various embodiments, X in formula (I) 3 and X 4 are each independently 4-8 membered heterocyclyl optionally substituted with 1 or 2 C1-C6 or C1-C3 alkyl groups. For example, in various embodiments, X 3 and X 4 are each independently azetidinyl, methylazetidinyl, pyrrolidinyl, methylpyrrolidinyl, piperidinyl, methylpiperidinyl, piperazinyl, methylpiperazinyl, dimethylpiperazinyl, morpholinyl, diazepanyl, methyldiazepanyl, octahydro-2H-quinolidinyl, azabicyclo[3.2.1]octyl, methyl-azabicyclo[3.2.1]octyl, diazaspiro[3.5]nonyl, or methyldiazaspiro[3.5]nonyl.

[0102] In various embodiments, X in formula (I) 3 and X 4 are each independently a 5- to 6-membered heteroaryl optionally substituted with one or two C1-C6 or C1-C3 alkyl groups. For example, in various embodiments, X 3 and X 4 are each independently pyrrolyl, methylpyrrolyl, imidazolyl, methylimidazolyl, pyridinyl, or methylpyridinyl.

[0103] In various embodiments, X in formula (I) 3 and X 4 are each independently a 5- to 6-membered aryl optionally substituted with one or two C1-C6 or C1-C3 alkyl groups. For example, in various embodiments, X 3 and X 4 are each independently phenyl, methylphenyl, naphthyl, or methylnaphthyl.

[0104] In various embodiments, X in formula (I) 3 and X 4are each independently 4-7 membered cycloalkyl optionally substituted with 1 or 2 C1-C6 or C1-C3 alkyl groups. For example, in various embodiments, X 3 and X 4 are each independently cyclopentyl, methylcyclopentyl, cyclohexyl, or methylcyclohexyl.

[0105] In various embodiments, X in formula (I) 3 In another embodiment, X in formula (I) is -O-. 4 is —O—. In various embodiments, X 3 Ha-NR 3 -, where R 3 is a hydrogen atom or a C1-C6 alkyl, such as a C1-C3 alkyl. For example, in various embodiments, X 3 is -N(CH3)-, -N(CH2CH3)-, or N(CH2CH2CH3)-. In other embodiments, X 4 Ha-NR 3 -, where R 3 is a hydrogen atom or a C1-C6 alkyl, such as a C1-C3 alkyl. For example, in various embodiments, X 4 is -N(CH3)-, -N(CH2CH3)-, or N(CH2CH2CH3)-.

[0106] In various embodiments, X in formula (I) 5 Ha-(CH2) b -, where b is an integer between 0 and 6. In some embodiments, b is 0, in which case X 5 In other embodiments, b is 1, 2, 3, 4, 5, or 6.

[0107] In various embodiments, X in formula (I) 6 is hydrogen, C1-C6 alkyl, 5- to 6-membered heteroaryl optionally substituted with one or two C1-C6 alkyl groups, or -NR 4 R 5 In some embodiments, R 4 and R 5are each independently hydrogen or C1-C6 alkyl. Alternatively, in other embodiments, R 4 and R 5 together with the nitrogen to which they are attached form a 4-7 membered heterocyclyl optionally substituted with one or two C1-C6 alkyl groups, where the 4-7 membered heterocyclyl optionally contains additional heteroatoms selected from oxygen, sulfur, and nitrogen.

[0108] In various embodiments of formula (I), X 1 , X 2 , X 3 , X 4 , and X 5 For example, in various embodiments, Formula (I) contains at least one of X 1 , X 2 , X 3 , X 4 , and X 5 In other embodiments, at least two of X 1 , X 2 , X 3 , X 4 , and X 5 For example, in some embodiments, Formula (I) contains at least three of X 1 , X 2 , X 3 , X 4 , and X 5 In other embodiments, at least four of X 1 , X 2 , X 3 , X 4 , and X 5 All of these exist.

[0109] In some embodiments, X 6 is hydrogen. In other embodiments, X 6 is C1-C6 alkyl, such as C1-C3 alkyl (e.g., methyl, ethyl, or propyl). 6is a 5-6 membered heteroaryl optionally substituted with one or two C1-C6 alkyl groups. For example, in various embodiments, X 6 is pyrrolyl, methylpyrrolyl, imidazolyl, methylimidazolyl, pyridinyl, or methylpyridinyl. 6 Ha-NR 4 R 5 For example, in some embodiments, X 6 is -NH, -NHCH, -NHCHCH, -NHCHCHCH, -N(CH), -N(CHCH), or -N(CHCHCH). Alternatively, in other embodiments, R 4 and R 5 taken together with the nitrogen to which they are attached form a 4-7 membered heterocyclyl. The 4-7 membered heterocyclyl may be optionally substituted with one or two C1-C6 alkyl groups, such as C1-C3 alkyl, and / or the 4-7 membered heterocyclyl may optionally contain additional heteroatoms selected from oxygen, sulfur, and nitrogen. For example, in some embodiments, X 6 is azetidinyl, methylazetidinyl, pyrrolidinyl, methylpyrrolidinyl, piperidinyl, methylpiperidinyl, piperazinyl, methylpiperazinyl, dimethylpiperazinyl, morpholinyl, diazepanyl, or methyldiazepanyl.

[0110] In various embodiments, each X in formula (I) 7 is hydrogen. In other embodiments, each X 7 is hydroxyl. In other embodiments, each X 7 Ha-NR 6 R 7 For embodiments where a is between 2 and 6, each X 7 may be the same or different. For example, in various embodiments, X 7 is -(CH2) a-1 CH(X 7 )-, where a is 2, 3, 4, 5, or 6. X 7 Ga-NR 6 R7 In some embodiments, R 6 and R 7 are each independently hydrogen or C1-C6 alkyl, such as C1-C3 alkyl. For example, in some embodiments, X 7 is -NH, -NHCH, -NHCHCH, -NHCHCHCH, -N(CH), -N(CHCH), or -N(CHCHCH). Alternatively, X 7 Ga-NR 6 R 7 In some embodiments, R 6 and R 7 together with the nitrogen to which they are attached form a 4-7 membered heterocyclyl optionally substituted with one or two C1-C6 alkyl groups. Alternatively, X 7 Ga-NR 6 R 7 In other embodiments, R 6 and R 7 taken together with the nitrogen to which they are attached form a 4-7 membered heterocyclyl. The 4-7 membered heterocyclyl may be optionally substituted with one or two C1-C6 alkyl groups, such as C1-C3 alkyl, and / or the 4-7 membered heterocyclyl may optionally contain additional heteroatoms selected from oxygen, sulfur, and nitrogen. For example, in some embodiments, X 6 is azetidinyl, methylazetidinyl, pyrrolidinyl, methylpyrrolidinyl, piperidinyl, methylpiperidinyl, piperazinyl, methylpiperazinyl, dimethylpiperazinyl, morpholinyl, diazepanyl, or methyldiazepanyl.

[0111] In various embodiments, A in formula (I) 1 and A 2 are each independently (1) C5~C 12 Haloalkyl; (2) C5~C 12 Alkenyl; (3) C5~C 12 Alkynyl; (4) (C5~C12 Alkoxy)-(CH2) n2 -; (5) (C5-C) optionally ring-substituted with one or two halo groups, C1-C6 alkyl groups, C1-C6 haloalkyl groups, or C1-C6 alkoxy groups. 10 Aryl)-(CH2) n3 -; and (6) (C3-C8 cycloalkyl)-(CH2) optionally ring-substituted with one or two C1-C6 alkyl groups n4 -; Selected from or or alternatively, A 1 and A 2 together with the atoms to which they are bonded form one or two C4-C 10 It forms a 5- to 6-membered cyclic acetal substituted with an alkyl group.

[0112] In various embodiments of Formula (I), n1, n2, and n3 are each independently an integer between 1 and 4 (i.e., 1, 2, 3, or 4), and n4 is an integer between 0 and 4 (i.e., 0, 1, 2, 3, or 4). 1 and A 2 have the same chemical structure.

[0113] In various embodiments of formula (I), A 1 and A 2 are C5 to C 12 For example, in various embodiments, C5-C 12 Haloalkyl is C6 fluoroalkyl, C7 fluoroalkyl, C8 fluoroalkyl, C9 fluoroalkyl, C 10 Fluoroalkyl, C 11 Fluoroalkyl, or C 12 C5-C, such as fluoroalkyl 12 Fluoroalkyl. C5~C 12 The number of halogen atoms attached to a haloalkyl can vary over a wide range, depending on the length of the alkyl chain and the degree of halogenation. For example, in various embodiments, C5 to C12 Haloalkyl contains between 1 and 25 halogen atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 halogen atoms. In various embodiments, C5-C 12 Haloalkyl is CF3(CF2) n5 - (where n5 is an integer ranging from 0 to 5), 12 For example, in various embodiments, C5 to C 12 Fluoroalkyl is CF3(CF2) n5 (CH2) n6 In the formula, n5 is an integer ranging from 0 to 5, n6 is an integer ranging from 0 to 11, and n5+n6+1 is a group selected from C5 to C 12 Equal to the number of carbons in the fluoroalkyl.

[0114] In various embodiments of formula (I), A 1 and A 2 are independently C5 to C 12 The position of the alkenyl double bond may vary. For example, in various embodiments, C5 to C 12 Alkenyl is CH3CH2CH=CH(CH2) such as CH3CH2CH=CH(CH2)4-. n7 - (wherein n7 is an integer ranging from 1 to 8). In some embodiments, C5 to C 12 Alkenyl is, for example, (CH3)2C=CH(CH2) n8 -CH(CH3)-(CH2) n9 - (wherein n8 and n9 are each independently 1, 2, or 3).

[0115] In various embodiments of formula (I), A 1 and A 2 are independently C5 to C 12 The position of the alkynyl triple bond may vary. For example, in various embodiments, C5 to C 12 Alkynyl is CH3CH2C≡C(CH2) such as CH3CH2C≡C(CH2)4-. n10- (wherein n10 is an integer ranging from 1 to 8). In some embodiments, C5 to C 12 Alkynyl is, for example, (CH3)2CHC≡C(CH2) n11 -CH(CH3)-(CH2) n12 - (wherein n11 and n12 are each independently 1, 2, or 3, and n11+n12 is in the range of 2 to 5), and the like.

[0116] In various embodiments of formula (I), A 1 and A 2 are each independently (C5~C 12 Alkoxy)-(CH2) n2 In various embodiments, each n2 is independently an integer ranging from 1 to 4 (i.e., 1, 2, 3, or 4). The position of the oxygen may vary. For example, in various embodiments, (C5 to C 12 Alkoxy)-(CH2) n2 - is CH3O(CH2) such as CH3O(CH2)7- n13 -(CH2) n2 - (wherein n13 is an integer ranging from 1 to 11). In another embodiment, (C5 to C 12 Alkoxy)-(CH2) n2 - is CH3(CH2)7-O-(CH2)2-(CH2) n2 - CH3 (CH2) n14 -O-(CH2) n15 -(CH2) n2 - (wherein n14 and n15 are each independently an integer between 1 and 8, and n14 + n15 is an integer ranging from 4 to 11). 12 Alkoxy is, for example, CH3O(CH2) n16 -CH(CH3)-(CH2) n17 -(CH2) n2 - (wherein n16 and n17 are each independently 1, 2, 3, 4, or 5, and n16+n17 is an integer ranging from 2 to 9), and the like.

[0117] In various embodiments of formula (I), A 1and A 2 are each independently optionally ring-substituted with one or two halo groups, C1-C6 alkyl groups, C1-C6 haloalkyl groups, or C1-C6 alkoxy groups (C5-C 10 Aryl)-(CH2) n3 In various embodiments, each n3 is independently an integer between 1 and 4 (i.e., 1, 2, 3, or 4). In some embodiments, C5 to C 10 Aryl is phenyl. For example, in various embodiments, (C5-C 10 Aryl)-(CH2) n3 - is C6H5-(CH2) optionally ring-substituted with one or two halo groups, C1-C6 alkyl groups, C1-C6 haloalkyl groups, or C1-C6 alkoxy groups. n3 In one embodiment, an optionally ring-substituted (C5-C 10 Aryl)-(CH2) n3 - is CF3-C6H4-(CH2), such as CF3-C6H4-CH2- or CF3-C6H4-(CH2)2-. n3 In another embodiment, an optionally ring-substituted (C5-C 10 Aryl)-(CH2) n3 - is CH3-(CH2), such as CH3(CH2)3-C6H4-CH2- or CH3(CH2)3-C6H4-(CH2)2-. n18 -C6H4-(CH2) n2 - (wherein n18 is 1, 2, or 3, and n2 is 1, 2, 3, or 4).

[0118] In various embodiments of formula (I), A 1 and A 2 are each independently (C3-C8 cycloalkyl)-(CH2) optionally ring-substituted with one or two C1-C6 alkyl groups. n4 In various embodiments, each n4 is independently an integer between 0 and 4 (i.e., 0, 1, 2, 3, or 4). In some embodiments, C3-C8 cycloalkyl is cyclohexyl or cyclopentyl. For example, in various embodiments, (C3-C8 cycloalkyl)-(CH2)n4 - C6H 11 -(CH2)2-, CH 11 -(CH2)3-, or CH3-C6H 10 CH optionally ring-substituted with one or two C1-C6 alkyl groups, such as -(CH2)3- 11 -(CH2) n4 -It is.

[0119] Alternatively, in other embodiments of formula (I), A 1 and A 2 together with the atoms to which they are attached, form one or two C4-C 10 Forms a 5-6 membered cyclic acetal substituted with an alkyl group. For example, in one embodiment, A 1 and A 2 together with the atoms to which they are attached form a six-membered cyclic acetal ring-substituted with two C8 alkyl groups, as shown below.

[0120] [ka] In another embodiment, A 1 and A 2 together with the atoms to which they are attached form a 5-membered cyclic acetal ring-substituted with two C8 alkyl groups, as shown below.

[0121] [ka]

[0122] 2. Ionizable lipids In one embodiment, the disclosed lipid nanoparticles comprise an ionizable lipid. The ionizable lipid typically comprises an amine-containing group on the head group. In various embodiments, the ionizable lipid is a compound of Formula (I). In some embodiments, the ionizable lipid is present in the lipid nanoparticle at 35, 45, 50, or 65 mole percent, based on the total moles of the components of the lipid nanoparticle. In another embodiment, the ionizable lipid is present at about 33 mol% to about 36 mol%, based on the total moles of the components of the lipid nanoparticle. In yet another embodiment, the ionizable lipid is present at about 35 mol%, based on the total moles of the components of the lipid nanoparticle.

[0123] Further embodiments relate to lipid nanoparticle compositions comprising an ionizable lipid; a phospholipid; a polyethylene glycol-lipid; cholesterol; and optionally, a nucleic acid. In some embodiments, the ionizable lipid comprises a structure according to any one of formulas (I), (Ia), (II), (IIa), (IIb), and (IIc). In some embodiments, the amount of ionizable lipid is present in a range of about 35 to 65 mole percent, based on the total moles of the components of the lipid nanoparticle.

[0124] 3. Sterols In some embodiments, the disclosed lipid nanoparticles comprise one or more sterols. In one embodiment, the sterol is cholesterol, or a variant or derivative thereof. In some embodiments, the cholesterol is modified, e.g., oxidized. Unmodified cholesterol can be enzymatically acted upon to form side-chain-oxidized or ring-oxidized variants. Cholesterol may be oxidized on the beta ring structure or on the hydrocarbon tail structure. Exemplary cholesterols contemplated for use in the disclosed lipid nanoparticles include, but are not limited to, 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-25-dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or a combination thereof. In one embodiment, side-chain-oxidized cholesterol can enhance cargo delivery compared to other cholesterol variants. In one embodiment, the cholesterol is unmodified cholesterol.

[0125] 4. PEG-lipids In some embodiments, the disclosed nanoparticle compositions also include one or more PEG or PEG-modified lipids. Such lipids can alternatively be referred to as PEGylated lipids or PEG-lipids. The inclusion of PEGylated lipids can be used to enhance the stability of lipid nanoparticle colloids in vitro and their circulation time in vivo. In some embodiments, PEGylation is reversible in that the PEG moiety is gradually released in the blood circulation. Exemplary PEG-lipids include, but are not limited to, C6-C 20The PEG lipids include PEG conjugated to saturated or unsaturated alkyl chains having a length of 0.01 to 0.01. PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DAG), PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG, or PEG-DSPE.

[0126] 5. Phospholipids The phospholipid component of the nanoparticles can include one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids can be assembled into one or more lipid bilayers. In some embodiments, the phospholipids can include a phospholipid moiety and one or more fatty acid moieties.

[0127] In some embodiments, the phospholipid moiety includes, but is not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. In some embodiments, the fatty acid moiety includes, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species, including natural species, with modifications and substitutions, including branching, oxidation, cyclization, and alkynes, are also contemplated. For example, the phospholipid can be functionalized with or crosslinked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azides. Such reactions can be useful in functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane penetration or cellular recognition, or in conjugating nanoparticle compositions to useful components such as targeting or imaging moieties (e.g., dyes).

[0128] Exemplary phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl l)-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 lysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16).0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE) ), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE). In a preferred embodiment, the phospholipid is DSPC. In another embodiment, the phospholipid is DMPC.

[0129] E. Cargo In one embodiment, the disclosed lipid nanoparticle composition comprises a therapeutic or prophylactic agent for delivery to a subject. In some embodiments, the therapeutic or prophylactic agent is encapsulated by the lipid nanoparticle. In one embodiment, the lipid nanoparticle is loaded with one or more nucleic acids.

[0130] Representative nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), RNA, DNA, single-stranded RNA, single-stranded DNA, double-stranded RNA, double-stranded DNA, triple-stranded DNA, siRNA, shRNA, sgRNA, mRNA, miRNA, and antisense DNA. In one embodiment, the nucleic acid is an siRNA, miRNA, mRNA, expressed DNA, an antisense oligonucleotide, or an immunostimulatory oligonucleotide.

[0131] CRISPR (clustered regularly interspaced short palindromic repeats)-based gene editing requires two components: a guide RNA and a CRISPR-associated endonuclease protein (Cas). The guide RNA directs the Cas nuclease to a specific target DNA sequence. The Cas then creates a double-strand break in the DNA at that site. In one embodiment, the disclosed lipid nanoparticles can be used to carry the components required for CRISPR-based gene editing. In one lipid nanoparticle, the nucleic acid cargo is a guide RNA. In such an embodiment, a second lipid nanoparticle can contain a nucleic acid cargo encoding an RNA-guided endonuclease. The two types of lipid nanoparticles can be administered together. Exemplary RNA-guided endonucleases include, but are not limited to, Cas9, CasX, CasY, Cas13, or Cpf1.

[0132] In one embodiment, the cargo is siRNA. Short interfering RNA (siRNA) is a double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby reducing or even suppressing gene expression. In one example, siRNA induces specific degradation of homologous RNA molecules, such as mRNA, within the region of sequence identity between both siRNA and target RNA. For example, International Publication No. 02 / 44321 discloses siRNA that can sequence-specifically degrade target mRNA when base-pairing with 3' overhanging end, and the method for producing these siRNAs is incorporated herein by reference. Sequence-specific gene silencing can be achieved in mammalian cells using synthetic short double-stranded RNA that mimics the siRNA generated by the enzyme Dicer (Elbashir, et al. (2001) Nature, 411:494-498) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82).

[0133] In one embodiment, cargo is messenger RNA (mRNA).Messenger RNA is a single-stranded RNA that can be translated into the protein it codes for by using the cell's protein synthesis machinery after cargo reaches the cytoplasm.The translation and stability of mRNA can be affected by the modification of 5'UTR and 3'UTR, which plays a role in recruiting RNA binding protein and microRNA, and RNA binding protein and microRNA can all affect translation activity (Sahin, et al (2014) Nat Rev Drug Disco 13: 759-580) (Kariko et al (2008) Mol Ther 11:1833-1840).

[0134] In one embodiment, the lipid nanoparticles contain less than 1.0 mg / kg of inhibitory nucleic acid. The nanoparticles can contain 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 mg / kg of inhibitory nucleic acid. In another embodiment, the lipid nanoparticles contain 0.5 mg / kg of inhibitory nucleic acid. This is an advantage over current technology, where nanoparticles require high doses of nucleic acid (>1 mg / kg) to achieve gene silencing, a dose that is not approved for human delivery. The disclosed technology can achieve gene silencing using 0.5 mg / kg of inhibitory nucleic acid in lipid nanoparticles that do not contain a targeting ligand.

[0135] In some embodiments, nucleic acids, including but not limited to oligonucleotides, are modified or contain another modified nucleotide to improve stability, half-life, and / or nuclease stability. To limit nuclease susceptibility, natural phosphodiester oligodeoxyribonucleotides, natural phosphodiester oligoribonucleotides, ribonucleotide polymers, and deoxyribonucleotide polymers can contain different other modifications. Exemplary modifications include, but are not limited to, phosphorothioate (PS) linkages, 2'-O-methyl (2'OMe), 2' fluoro bases, inverted dT and ddT, phosphorylation of the 3' end of the oligonucleotide, locked nucleic acids, and phosphoramidite C3 spacers.

[0136] Phosphorothioate bond substitutes sulfur atom for non-bridging oxygen in the phosphate backbone of oligonucleotide. Approximately 50% (due to the two resulting stereoisomers that can be formed) PS modification makes internucleotide linkage more resistant to nuclease degradation. In some embodiments, nucleic acid comprises one or more PS bonds, for example, at least three PS bonds at the 5' and 3' ends of oligonucleotide, to suppress exonuclease degradation. Some nucleic acids also comprise PS bonds throughout the oligonucleotide to help reduce endonuclease attack.

[0137] 2'OMe, a naturally occurring post-transcriptional modification of RNA, is found in tRNA and other small RNAs. In some embodiments, nucleic acids or oligonucleotides are directly synthesized to contain 2'OMe. This modification improves the Tm of the RNA:RNA duplex but results in only minor changes in RNA:DNA stability. This modification prevents attack by single-stranded endonucleases but does not prevent exonuclease digestion. In some embodiments, these nucleic acids or oligonucleotides are also end-blocked. DNA oligonucleotides containing this modification are typically 5-10 times less susceptible to DNases than unmodified DNA. 2'OMe modifications are commonly used in antisense oligonucleotides as a means to improve stability and binding affinity to target transcripts.

[0138] The 2'-fluoro base has a fluorine-modified ribose, which improves binding affinity (Tm) and also confers some relative nuclease resistance compared to natural RNA. In some embodiments, the nucleic acid or oligonucleotide comprises a 2'-fluoro base in combination with a PS-modified linkage.

[0139] Inverted dT can be incorporated at the 3' end of an oligonucleotide, resulting in a 3'-3' linkage that inhibits degradation by 3' exonucleases and extension by DNA polymerases. In addition, placing an inverted 2',3' dideoxy-dT base (5' inverted ddT) at the 5' end of an oligonucleotide prevents spurious ligation and protects against some forms of enzymatic degradation.

[0140] Some embodiments provide nucleic acids or oligonucleotides that contain a phosphoramidite C3 spacer. The phosphoramidite C3 spacer can be incorporated internally or at either end of the oligo to introduce a long hydrophilic spacer arm for attachment of a fluorophore or other pendant group. The C3 spacer can also be used to suppress degradation by 3' exonucleases.

[0141] In some embodiments, nucleic acid or oligonucleotide comprises locked nucleic acid.Locked nucleic acid comprises modified RNA nucleotide in which 2'-O atom and 4'-C atom of ribose are linked through methylene bridge.This additional bridge restricts the flexibility that is usually associated with the ring, and essentially locks the structure into a rigid conformation.LNA can be inserted into both RNA and DNA oligonucleotides.

[0142] Other types of cargo that can be delivered via the disclosed nanoparticles include, but are not limited to, chemotherapeutic agents, cytotoxic drugs, radioactive ions, small molecules, proteins, polynucleotides, and nucleic acids.

[0143] Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, and loxacin. Icovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. Exemplary pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

[0144] Some embodiments relate to a method of delivering a nucleic acid to a subject in need thereof, comprising administering to the subject a lipid nanoparticle composition described herein, hi some embodiments, the nucleic acid is an siRNA, miRNA, mRNA, expressed DNA, an antisense oligonucleotide, or an immunostimulatory oligonucleotide.

[0145] B. Exemplary Lipid Nanoparticle Formulations In one embodiment, the lipid nanoparticle formulation comprises about 30 mol% to about 70 mol% ionizable lipid, about 5 mol% to about 25 mol% phospholipid, about 25 mol% to about 45 mol% cholesterol, and about 0 mol% to about 5 mol% PEG-lipid. In another embodiment, the lipid nanoparticle formulation comprises about 45 mol% ionizable lipid, about 9 mol% phospholipid, about 44 mol% cholesterol, and about 2 mol% PEG-lipid. In another embodiment, the lipid nanoparticle formulation comprises about 50 mol% ionizable lipid, about 9 mol% phospholipid, about 38 mol% cholesterol, and about 3 mol% PEG-lipid.

[0146] One embodiment is a lipid composition comprising, based on the total moles of the following four components, about 40 mol % to about 60 mol % of an ionizable lipid of formula (I), about 5 mol % to about 15 mol % of 1-2-distearoyl-sn-glycero-3-phosphocholine, and C 14 PEG 2000 A lipid nanoparticle formulation is provided that comprises about 1 mol % to about 5 mol % of cholesterol, and about 30 mol % to about 47 mol % of cholesterol.

[0147] Another embodiment is a lipid composition comprising, based on the total moles of the following four components: 50 mol % of an ionizable lipid of formula (I), 9 mol % of 1-2-distearoyl-sn-glycero-3-phosphocholine, C 14 PEG 2000 3 mol % and cholesterol 38 mol %.

[0148] Another embodiment provides a lipid nanoparticle formulation having a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):mRNA of between about 2:1 and 50:1.

[0149] C. Pharmaceutical Compositions Pharmaceutical compositions comprising the disclosed lipid nanoparticles are provided. The lipid nanoparticle compositions can be formulated, in whole or in part, as pharmaceutical compositions. The pharmaceutical compositions can include one or more nanoparticle compositions. For example, the pharmaceutical compositions can include one or more nanoparticle compositions containing one or more different therapeutic and / or prophylactic agents, including, but not limited to, one or more nucleic acids of different types, or can encode different agents. In some embodiments, the pharmaceutical compositions include one or more pharmaceutically acceptable excipients or accessory ingredients, including, but not limited to, a pharmaceutically acceptable carrier.

[0150] Pharmaceutical compositions containing nanoparticles can be formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration, or by using bioerodible inserts, and can be formulated in dosage forms appropriate for each route of administration.

[0151] In some in vivo approaches, the nanoparticle compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to treat, suppress, or alleviate one or more symptoms of the disorder being treated, or to provide the desired pharmacological and / or physiological effect. The exact dosage will vary depending on various factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being performed.

[0152] As further research is conducted on the disclosed nanoparticles, information will emerge regarding appropriate dosage levels for treating various conditions in various patients, and those skilled in the art will be able to ascertain appropriate dosing, taking into account the recipient's treatment status, age, and overall health. The selected dosage will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. For the disclosed nanoparticles, dosage levels of 0.001 mg to 5 mg of nucleic acid per kg of body weight per day are generally administered to mammals. More specifically, preferred doses of the disclosed nanoparticles are 0.01 mg / kg to 0.25 mg / kg. For the disclosed nanoparticles, dosage levels of 0.2 mg to 100 mg of the four components (ionizable lipid, cholesterol, PEG-lipid, and phospholipid) per kg of body weight are generally administered to mammals. More specifically, preferred doses of the disclosed nanoparticles are 0.05 mg / kg to 0.5 mg / kg of the four components per kg of body weight.

[0153] In certain embodiments, the lipid nanoparticle composition is administered locally, for example, by direct injection into the treatment site. Typically, injection results in a greater local concentration of the lipid nanoparticle composition than can be achieved by systemic administration. The lipid nanoparticle composition, when combined with the matrix described above, can help to create a higher local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide out of the treatment site.

[0154] 1. Formulations for parenteral administration In some embodiments, the nanoparticle compositions disclosed herein, including those containing lipid nanoparticles, are administered in aqueous solution via parenteral injection. The formulations can also be in the form of suspensions or emulsions. Generally, pharmaceutical compositions are provided containing an effective amount of lipid nanoparticles, and optionally contain pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. These compositions optionally contain one or more of the following: diluents, sterile water, various buffer contents (e.g., Tris-HCl, acetate, phosphate), buffered saline solutions of various pH and ionic strengths; and additives such as surfactants and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimerosal, benzyl alcohol), and bulking agents (e.g., lactose, mannitol). Examples of non-aqueous solvent or vehicle are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.Preparation can be lyophilized and redissolved / resuspended immediately before use.Preparation can be sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating sterilizing agent into composition, by irradiating composition, or by heating composition.

[0155] 2. Controlled Delivery Polymer Matrix The lipid nanoparticles disclosed herein can also be administered in controlled-release formulations. Controlled-release polymer devices can be fabricated for systemic, prolonged release after implantation or injection (microparticles) of the polymer device (rods, cylinders, films, disks). The matrix can be in the form of microparticles, such as microspheres, in which the drug is dispersed within a solid polymer matrix or microcapsules, where the core is made of a different material from the polymer shell, and the peptide is dispersed or suspended within the core, which may be liquid or solid in nature. Unless otherwise defined herein, the terms microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer can be cast as thin slabs or films ranging from a few nanometers to 4 centimeters, powders prepared by milling or other standard techniques, or even gels, such as hydrogels.

[0156] Either non-biodegradable or biodegradable matrices can be used to deliver lipid nanoparticles, although biodegradable matrices are preferred in some embodiments. These can be natural or synthetic polymers, although synthetic polymers are preferred in some embodiments due to their better characterization in terms of degradation and release profiles. The polymer is selected based on the desired period of release. In some cases, linear release may be most useful, while in other cases, pulsed or "bulk" release may provide more effective results. The polymer can be in the form of a hydrogel (typically absorbing up to about 90% water by weight) and, if necessary, can be crosslinked with multivalent ions or polymers.

[0157] The matrix can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed to make microspheres for drug delivery, such as those described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0158] The devices can be formulated for local release to treat the area of ​​implantation or injection, which usually delivers a much smaller dosage than that for systemic treatment, or for systemic delivery: they can be implanted or injected subcutaneously into muscle, fat, or swallowed.

[0159] D. Methods for Producing Lipid Nanoparticles Methods for producing lipid nanoparticles are known in the art. In one embodiment, the disclosed lipid nanoparticles are produced using microfluidics. For exemplary methods of forming lipid nanoparticles using microfluidics, see Leung, AKK, et al., J Phys Chem, 116:18440-18450 (2012); Chen, D., et al., J Am Chem Soc, 134:6947-6951 (2012); and Belliveau, NM, et al., Molecular Therapy- Nucleic Acids, 1: e37 (2012). In summary, cargoes such as oligonucleotides and siRNAs are prepared in one buffer. Other lipid nanoparticle components (ionizable lipids, PEG-lipids, cholesterol, and DSPC) are prepared in another buffer. The two solutions are introduced into the microfluidic device by a syringe pump. The two solutions are brought into contact within the microfluidic device to form lipid nanoparticles that encapsulate the cargo.

[0160] A method for screening the disclosed lipid nanoparticles is discussed in International Patent Application No. PCT / US / 2018 / 058171, which is incorporated by reference in its entirety. The screening method characterizes vehicle delivery formulations to identify formulations that have the desired tropism and deliver functional cargo to the cytoplasm of specific cells. The screening method uses a reporter that has a detectable function when delivered to a cell. Detecting reporter function within the cell indicates that formulating the delivery vehicle will deliver a functional cargo to the cell. A chemical composition identifier is included in each different delivery vehicle formulation to keep track of the unique chemical composition of each different delivery vehicle formulation. In one embodiment, the chemical composition identifier is a nucleic acid barcode. By pairing the sequence of the nucleic acid barcode with the chemical moiety used to formulate the delivery vehicle into which it is loaded, the chemical composition of the delivery vehicle that delivered the barcode is identified when the nucleic acid barcode is sequenced. Exemplary reporters include, but are not limited to, siRNA, mRNA, nuclease proteins, nuclease mRNA, small molecules, epigenetic modifiers, and phenotypic modifiers.

[0161] E. Method of Use Disclosed herein is a method for using the disclosed lipid nanoparticles to deliver cargo, such as nucleic acid, to specific cells or organs.In some embodiments, the nanoparticles deliver therapeutic or prophylactic drugs to specific cells or organs in the subject in need thereof without targeting ligand.In another embodiment, the disclosed lipid nanoparticles are useful for treating or preventing disease in the subject in need thereof.

[0162] In some embodiments, the disclosed nanoparticles are directly delivered to the subject.In other embodiments, lipid nanoparticles are contacted with cells ex vivo, and the treated cells are administered to the subject.Cells can be autologous cells, such as immune cells, including but not limited to T cells or cells that differentiate into T cells.In some embodiments, the disclosed lipid nanoparticles can be used as a vehicle for adoptive cell transfer.

[0163] 1. Methods for delivering cargo into cells Provided herein are methods for delivering therapeutic and / or prophylactic nucleic acids to a subject in need thereof.

[0164] In some embodiments, the disclosed lipid nanoparticle compositions target a specific type or class of cells (e.g., cells of a specific organ or system). For example, nanoparticle compositions containing a therapeutic and / or prophylactic agent of interest can be delivered specifically to a subject's immune cells. Exemplary immune cells include, but are not limited to, CD8+, CD4+, or CD8+CD4+ cells. In other embodiments, lipid nanoparticles can be formulated to be delivered to mammalian liver hepatocytes, liver immune cells, splenic T cells, or lung endothelial cells in the absence of a targeting ligand. Specific delivery to a particular class or type of cell dictates that a higher percentage of lipid nanoparticles are delivered to the target type or class of cells. In some embodiments, specific delivery can result in a 2-fold, 5-fold, 10-fold, 15-fold, or greater than 20-fold increase in the amount of therapeutic and / or prophylactic agent per gram of targeted tissue.

[0165] 2. Methods of gene regulation The present specification provides a method for using the disclosed lipid nanoparticles for gene regulation.In one embodiment, lipid nanoparticles can be used to reduce gene expression in the target cells of a subject that requires it.The lipid nanoparticles can deliver inhibitory nucleic acid to the target cells of a subject without targeting ligand.The inhibitory nucleic acid can be siRNA.

[0166] Another embodiment provides a method of using the disclosed lipid nanoparticles to edit genes in cells of a subject in need thereof.

[0167] In one embodiment, the cell targeted for gene regulation is an immune cell.The immune cell can be a T cell, such as CD8+ T cell, CD4+ T cell, or regulatory T cell.Other exemplary immune cells for gene editing include, but are not limited to, macrophages, dendritic cells, B cells, or natural killer cells.In some embodiments, the cell targeted for gene regulation is a hepatocyte.

[0168] Exemplary genes that can be targeted include, but are not limited to, T cell receptor, B cell receptor, CTLA4, PD1, FOXO1, FOXO3, AKT, CCR5, CXCR4, LAG3, TIM3, killer immunoglobulin-like receptor, GITR, BTLA, LFA-4, T4, LFA-1, Bp35, CD27L receptor, TNFRSF8, TNFRSF5, CD47, CD52, ICAM-1, LFA-3, L-selectin, Ki-24, MB1, B7, B70, M-CSFR, TNF R-II, IL-7R, OX-40, CD137, CD137L, CD30L, CD40L, FasL, TRAIL, CD257, LIGHT, TRAIL-R1, TRAILR2, TRAIL-R4, TWEAK-R, TNFR, BCMA, B7DC, BTLA, B7-H1, B7-H2, B7-H3, ICOS, VEGFR2, NKG2D, JAG1, GITR, CD4, CCR2, GATA-3, MTORC1, MTORC2, RAPTOR, GATOR, FOXP3, NFAT, IL2R, and IL7. Other exemplary genes that can be targeted include, but are not limited to, OCT, G6Pase, Mut, PCCA, PCCB, and PAH.

[0169] Exemplary tumor-associated antigens that can be recognized by T cells and are contemplated for targeting include, but are not limited to, MAGE1, MAGE3, MAGE6, BAGE, GAGE, NYESO-1, MART1 / Melan A, MC1R, GP100, tyrosinase, TRP-1, TRP-2, PSA, CEA, Cyp-B, Her2 / Neu, hTERT, MUC1, PRAME, WT1, RAS, CDK-4, MUM-1, KRAS, MSLN, and β-catenin.

[0170] 3. Subject to treatment In some embodiments, the subject to be treated is a mammalian animal suffering from cancer, autoimmune disease, infectious disease, organ transplant, organ failure, protein deficiency, or a combination thereof.In one embodiment, the subject is a human.In some embodiments, the method described herein can cause hepatocytes to translate certain proteins.In some embodiments, the method described herein can be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to hepatocytes. [Example]

[0171] General notes: All reactions were carried out in flasks or vials under a nitrogen atmosphere with magnetic stirring using anhydrous-grade solvents unless otherwise noted. Anhydrous solvents were purchased from Sigma-Aldrich and used as received. Flash column chromatography was performed using a Biotage Selekt or Teledyne-Isco Combiflash Nextgen 300+ equipped with prepacked Biotage Sfar silica gel cartridges. Thin-layer chromatography was performed using Merck silica gel 60 plates, and compounds were visualized using iodine. Nuclear magnetic resonance (NMR) spectroscopy was performed using a Varian INOVA 500 MHz spectrometer; chemical shifts are expressed in δ parts per million (ppm) upfield of tetramethylsilane relative to the residual solvent peak of CHCl at δ = 7.26 ppm. Liquid chromatography-mass spectrometry (LCMS) was performed using a Waters Acquity UPLC H-class Plus equipped with a QDa detector (ESI) equipped with a Waters Acquity UPLC BEH C18 column (130 Å, 1.7 μM, 2.1 mm × 50 mm). Compounds were analyzed using one of the following basic LCMS methods: Method A: Solvent A = water + 0.1% formic acid, Solvent B = acetonitrile; gradient from 90% A, 10% B to 5% A, 95% B over 3 min, then hold at 95% B for 2 min, then ramp back to 10% B over 1 min; flow rate = 0.5 mL / min. Method B: Column—XTERRA RP18 (4.6×50 mm), 5μ, (Mobile phase: Initially 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF]; then 2% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN:THF] at 2.65 min, this mobile phase composition was held until 3.75 min, and finally returned to the initial condition, i.e., 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF] at 4.90 min, and this mobile phase composition was held until 5.10 min. Flow rate=1.2 ml / min.

[0172] List of abbreviations DCM: dichloromethane DIPEA: N,N-diisopropylethylamine DMAP: 4-(dimethylamino)pyridine DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine EDC: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride Eq: equivalent ESI: electrospray ionization LCMS: Liquid chromatography-mass spectrometry LNP: lipid nanoparticles NMR: nuclear magnetic resonance PPTS: Pyridinium p-toluenesulfonate RT: retention time

[0173] Exemplary lipids were prepared according to the synthetic scheme illustrated in FIG.

[0174] A representative synthetic procedure is exemplified using Example 1: Preparation of 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate.

[0175] Step 1: 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile (Intermediate Ia)

[0176] [ka]

[0177] The following is representative of general procedure A: To a vial containing pyridinium p-toluenesulfonate (0.12 g, 0.48 mmol, 0.05 Eq) was added 4,4-diethoxybutanenitrile (1.5 g, 9.5 mmol, 1 equiv.) and cis-5-octen-1-ol (3.7 g, 29 mmol, 3 Eq.). The vial was tightly capped, and the resulting mixture was heated at 105 °C for 72 h. After this time, the mixture was cooled to room temperature. The crude material was purified by flash column chromatography (100 g silica, 0–100% dichloromethane in hexanes over 25 min). 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile (1.14 g, 37%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.27 (m, 4H), 4.56 (t, J = 5.3 Hz, 1H), 3.61 (dt, J = 9.3, 6.6 Hz, 2H), 3.44 (dt, J = 9.3, 6.6 Hz, 2H), 2.42 (t, J = 7.4 Hz, 2H), 2.12 - 1.91 (m, 9H), 1.66 - 1.54 (m, 5H), 1.49 - 1.36 (m, 4H), 0.96 (t, J = 7.6 Hz, 6H).

[0178] Step 2: 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (Intermediate IIa)

[0179] [ka]

[0180] The following is representative of general procedure B: To a vial containing 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile (Intermediate Ia, 1.14 g, 3.54 mmol, 1 Eq) was added potassium hydroxide (0.60 g, 10.6 mmol, 3 Eq), followed by ethanol (3.5 mL) and water (3.5 mL). The vial was tightly capped, and the reaction mixture was heated to 110 °C for 18 h. After this time, the mixture was cooled to room temperature. The mixture was diluted with ethyl acetate (20 mL), and the pH was adjusted to approximately 5 by adding 1 M HCl. The resulting biphasic mixture was separated, and the aqueous phase was extracted twice more with ethyl acetate (2 × 20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated to give 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (1.16 g, 96% yield) as a sticky white solid. 1 H NMR (400 MHz, chloroform-d) δ 5.41 - 5.24 (m, 4H), 4.45 (t, J = 5.6 Hz, 1H), 3.51 (dt, J = 9.0, 6.7 Hz, 2H), 3.39 (dt, J = 9.0, 6.7 Hz, 2H), 2.17 (t, J = 7.6 Hz, 2H), 2.08 - 1.98 (m, 8H), 1.81 (q, J = 7.3 Hz, 2H), 1.59 - 1.52 (m, 4H), 1.44 - 1.32 (m, 4H), 0.94 (t, J = 7.5 Hz, 6H).

[0181] Step 3: 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Intermediate III)

[0182] [ka]

[0183] To a mixture of trimethylolmethane (3.0 g, 1 eq, 28 mmol) in dichloromethane (100 mL) was added linoleic acid (7.9 g, 1 eq, 28 mmol), DIPEA (5.5 g, 7.4 mL, 1.5 eq, 42 mmol), and DMAP (0.69 g, 0.2 eq, 5.7 mmol). Finally, EDC (8.1 g, 1.5 eq, 42 mmol) was added and stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (200 g silica, 0–90% ethyl acetate in hexanes over 20 min). 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (2.6 g, 25%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.27 (m, 5H), 4.24 (d, J = 6.3 Hz, 2H), 3.76 (ddt, J = 21.1, 11.1, 5.3 Hz, 4H), 2.77 (d, J = 6.8 Hz, 2H), 2.61 - 2.55 (m, 2H), 2.36 - 2.29 (m, 2H), 2.10 - 1.98 (m, 6H), 1.66 - 1.58 (m, 2H), 1.41 - 1.21 (m, 12H), 0.92 - 0.85 (m, 3H).

[0184] Step 4: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (intermediate IVa).

[0185] [ka]

[0186] The following is representative of general procedure C: To a mixture of 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (Intermediate IIa, 591 mg, 1 eq, 1.74 mmol) in dichloromethane (10 mL), 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Intermediate III, 640 mg, 1 eq, 1.74 mmol), DIPEA (673 mg, 904 μL, 3 eq, 5.21 mmol), and DMAP (42.4 mg, 0.2 eq, 347 μmol) were added. Finally, EDC (666 mg, 2 eq, 3.47 mmol) was added and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (50 g silica, 0 to 40% ethyl acetate in hexanes over 12 column volumes) to give 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (450 mg, 38%) as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.27 (m, 8H), 4.49 (t, J = 5.5 Hz, 1H), 4.23 - 4.12 (m, 4H), 3.65 - 3.60 (m, 2H), 3.58 (dt, J = 9.3, 6.6 Hz, 2H), 3.41 (dt, J = 9.3, 6.6 Hz, 2H), 2.81 - 2.73 (m, 2H), 2.41 (t, J = 7.5 Hz, 2H), 2.32 (dd, J = 7.9, 7.2 Hz, 2H), 2.25 - 2.15 (m, 2H), 2.12 - 1.97 (m, 12H), 1.94 (ddd, J = 8.0, 7.2, 5.5 Hz, 2H), 1.67 - 1.53 (m, 8H), 1.45 - 1.26 (m, 17H), 0.96 (t, J = 7.6 Hz, 5H), 0.92 - 0.87 (m, 3H)

[0187] Step 5: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (Example 1)

[0188] [ka]

[0189] The following is representative of general procedure D: To a solution of 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Intermediate IVa, 100 mg, 1 Eq, 145 μmol) in dichloromethane (1 mL) was added pyridine (22.9 mg, 23.3 μL, 2 Eq, 289 μmol), DMAP (4.42 mg, 0.25 Eq, 36.2 μmol), and 4-nitrophenyl chloroformate (58.3 mg, 2 Eq, 289 μmol). The resulting mixture was stirred at 23° C. for 1 hour. After this time, DIPEA (74.8 mg, 101 μL, 4 eq, 579 μmol) and 3-(diethylamino)propan-1-ol (76.0 mg, 85.9 μL, 4 eq, 579 μmol) were added to it. The resulting mixture was stirred at 23 °C for an additional 18 h. After this time, the reaction mixture was diluted with dichloromethane (10 mL) and washed with 0.75 M aqueous sodium carbonate (3 × 10 mL), water (10 mL), and aqueous saturated sodium chloride (10 mL). The resulting organic layer was dried over sodium sulfate and concentrated, and the residue was purified by flash column chromatography (silica 10 g, 0–25% methanol in dichloromethane over 12 min). Obtained 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (81 mg, 66%) as a pale yellow oil. 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.27 (m, 8H), 4.49 (t, J = 5.5 Hz, 1H), 4.22 - 4.11 (m, 8H), 3.57 (dt, J = 9.3, 6.6 Hz, 2H), 3.41 (dt, J = 9.3, 6.6 Hz, 2H), 2.81 - 2.74 (m, 2H), 2.51 (q, J = 7.1 Hz, 6H), 2.45 - 2.36 (m, 3H), 2.31 (dd, J = 8.0, 7.2 Hz, 2H), 2.11 - 1.97 (m, 14H), 1.92 (ddd, J = 8.2, 7.1, 5.5 Hz, 2H), 1.86 - 1.77 (m, 2H), 1.65 - 1.53 (m, 8H), 1.49 - 1.23 (m, 14H), 1.01 (t, J = 7.1 Hz, 6H), 0.95 (t, J = 7.5 Hz, 6H), 0.92 - 0.86 (m, 3H). LCMS (Method A): Actual value of (M+H) m / z = 848.7, RT = 3.73 min.

[0190] [Example 2] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0191] [ka]

[0192] The following is representative of general procedure E: To a mixture of 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (Intermediate IVa, 78 mg, 1 Eq, 0.11 mmol) in dichloromethane (1 mL) was added 4-(pyrrolidin-1-yl)butanoic acid hydrochloride (26 mg, 1.2 Eq, 0.14 mmol), DIPEA (73 mg, 98 μL, 5 Eq, 0.56 mmol), and DMAP (2.8 mg, 0.2 Eq, 23 μmol). Finally, EDC (43 mg, 2 Eq, 0.23 mmol) was added and stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (10 g silica, 0–20% methanol in DCM over 12 column volumes). 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (74 mg, 78%) was obtained as a colorless oil. 1H NMR (500 MHz, chloroform-d) δ 5.42 - 5.28 (m, 8H), 4.49 (t, J = 5.6 Hz, 1H), 4.15 - 4.10 (m, 6H), 3.57 (dt, J = 9.3, 6.6 Hz, 2H), 3.41 (dt, J = 9.3, 6.6 Hz, 2H), 2.77 (tdq, J = 7.1, 1.4, 0.8 Hz, 2H), 2.57 - 2.43 (m, 6H), 2.43 - 2.35 (m, 5H), 2.30 (dd, J = 8.0, 7.2 Hz, 2H), 2.11 - 1.97 (m, 12H), 1.92 (ddd, J = 8.3, 7.2, 5.6 Hz, 2H), 1.89 - 1.73 (m, 6H), 1.68 - 1.52 (m, 8H), 1.48 - 1.24 (m, 16H), 0.95 (t, J = 7.6 Hz, 6H), 0.92 - 0.85 (m, 3H). LCMS (Method A): Actual value of (M+H) m / z = 830.7, RT = 3.75 min

[0193] The following intermediates Ib-Ih were prepared according to general procedure A, but with variations in the alcohol building blocks.

[0194] [ka]

[0195] Intermediate Ib, 4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanenitrile: Prepared from rac-citronellol on a 9.5 mmol scale. Yield 0.87 g (24%).

[0196] [ka]

[0197] Intermediate Ic, 4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanenitrile: Prepared from 4,4,5,5,5-pentafluoropentan-1-ol on a 9.5 mmol scale. Yield 1.8 g (45%).

[0198] [ka]

[0199] Intermediate Id, 4,4-bis(5,5,6,6,6-pentafluorohexyl)oxy)butanenitrile: Prepared from 5,5,6,6,6-pentafluorohexan-1-ol on a 9.5 mmol scale. Yield 0.70 g (16%).

[0200] [ka]

[0201] Intermediate Ie, 4,4-bis((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)butanenitrile: Prepared from 4,4,5,5,6,6,7,7,7-nonafluoroheptan-1-ol on a 9.5 mmol scale. Yield 2.0 g (34%).

[0202] [ka]

[0203] Intermediate If, ​​4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanenitrile: Prepared from 7,7,8,8,8-pentafluorooctan-1-ol on a 9.5 mmol scale. Yield 1.6 g (33%). 1H NMR (500 MHz, chloroform-d) δ 4.57 (t, J = 5.3 Hz, 1H), 3.61 (dt, J = 9.2, 6.5 Hz, 2H), 3.45 (dt, J = 9.3, 6.5 Hz, 2H), 2.43 (t, J = 7.3 Hz, 2H), 2.09 - 1.91 (m, 6H), 1.65 - 1.53 (m, 8H), 1.47 - 1.35 (m, 8H).

[0204] [ka]

[0205] Intermediate Ig, 4,4-bis(4-(trifluoromethyl)phenethoxy)butanenitrile: Prepared from (4-trifluoromethylphenyl)ethanol on a 9.5 mmol scale. Yield 2.2 g (52%).

[0206] [ka]

[0207] Intermediate Ih, 4,4-bis(3-cyclohexylpropoxy)butanenitrile: Prepared from 3-cyclohexylpropan-1-ol on a 9.5 mmol scale. Yield 2.3 g (68%).

[0208] The following intermediates IIb-IIh were prepared from the corresponding intermediates Ib-Ih according to general procedure B.

[0209] [ka]

[0210] Intermediate IIb, 4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoic acid: Prepared from 4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanenitrile (Intermediate Ib) on a 2.3 mmol scale. Yield 0.88 g (97%).1 H NMR (500 MHz, chloroform-d) δ 5.09 (dddd, J = 7.1, 5.7, 2.9, 1.4 Hz, 2H), 4.51 (t, J = 5.5 Hz, 1H), 3.62 (dddd, J = 16.2, 14.0, 6.9, 1.5 Hz, 2H), 3.45 (ddt, J = 16.7, 9.2, 6.7 Hz, 2H), 2.45 (t, J = 7.3 Hz, 2H), 2.06 - 1.89 (m, 6H), 1.68 (d, J = 1.4 Hz, 6H), 1.66 - 1.52 (m, 8H), 1.42 - 1.29 (m, 5H), 1.16 (dddd, J = 13.4, 9.8, 7.8, 5.8 Hz, 3H), 0.89 (dd, J = 6.6, 1.7 Hz, 6H).

[0211] [ka]

[0212] Intermediate IIc, 4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanoic acid: Prepared from 4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanenitrile (Intermediate Ic) on a 4.3 mmol scale. Yield 1.6 g (83%). 1 H NMR (500 MHz, chloroform-d) δ 4.55 (t, J = 5.4 Hz, 1H), 3.65 (dt, J = 9.5, 6.1 Hz, 2H), 3.49 (dt, J = 9.5, 6.1 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 2.22 - 2.06 (m, 4H), 1.92 - 1.82 (m, 6H).

[0213] [ka]

[0214] Intermediate IId, 4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanoic acid: Prepared from 4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanenitrile (Intermediate Id) on a 1.6 mmol scale. Yield 0.53 g (73%).

[0215] [ka]

[0216] Intermediate IIe, 4,4-bis((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)butanoic acid: Prepared from 4,4-bis((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)butanenitrile (Intermediate Ie) on a 3.3 mmol scale. Yield 1.9 g (91%). 1 H NMR (500 MHz, chloroform-d) δ 4.55 (t, J = 5.5 Hz, 1H), 3.65 (dt, J = 9.5, 6.1 Hz, 2H), 3.50 (dt, J = 9.5, 6.1 Hz, 2H), 2.43 (td, J = 7.2, 3.5 Hz, 2H), 2.18 (tt, J = 18.5, 7.8 Hz, 4H), 2.02 - 1.83 (m, 6H).

[0217] [ka]

[0218] Intermediate IIf, 4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoic acid: Prepared from 4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanenitrile (Intermediate If) on a 3.1 mmol scale. Yield 1.6 g (96%). 1H NMR (500 MHz, chloroform-d) δ 4.50 (t, J = 5.5 Hz, 1H), 3.57 (dt, J = 9.4, 6.6 Hz, 2H), 3.41 (dt, J = 9.4, 6.6 Hz, 2H), 2.38 (t, J = 7.5 Hz, 2H), 2.08 - 1.87 (m, 6H), 1.69 - 1.51 (m, 8H), 1.41 - 1.38 (m, J = 3.5 Hz, 8H).

[0219] [ka]

[0220] Intermediate IIg, 4,4-bis(4-(trifluoromethyl)phenetoxy)butanoic acid: Prepared from 4,4-bis(4-(trifluoromethyl)phenetoxy)butanenitrile (Intermediate Ig) on ​​a 5.0 mmol scale. Yield 1.7 g (74%). 1 H NMR (500 MHz, chloroform-d) δ 7.52 (d, J = 7.6 Hz, 4H) 7.27 (d, J = 7.6 Hz, 4H), 4.49 (t, J = 5.5 Hz, 1H), 3.66 (dt, J = 9.4, 6.6 Hz, 2H), 3.53 (dt, J = 9.4, 6.6 Hz, 2H), 2.84 (t, J = 6.6 Hz, 4H), 2.31 (t, J = 7.3 Hz, 2H), 1.88 (td, J = 7.4, 5.6 Hz, 2H).

[0221] [ka]

[0222] Intermediate IIh, 4,4-bis(3-cyclohexylpropoxy)butanoic acid: Prepared from 4,4-bis(3-cyclohexylpropoxy)butanenitrile (Intermediate Ih) on a 6.5 mmol scale. Yield 1.9 g (79%). 1H NMR (500 MHz, chloroform-d) δ 4.50 (t, J = 5.5 Hz, 1H), 3.55 (dt, J = 9.3, 6.8 Hz, 2H), 3.40 (dt, J = 9.3, 6.8 Hz, 2H), 2.41 (t, J = 7.3 Hz, 2H), 1.92 (td, J = 7.4, 5.4 Hz, 2H), 1.76 - 1.49 (m, 14H), 1.33 - 1.06 (m, 12H), 0.94 - 0.73 (m, 4H).

[0223] The following intermediates IVb-IVh were prepared from the corresponding intermediates IIb-IIh and intermediate III according to general procedure C.

[0224] [ka]

[0225] Intermediate IVb, 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoic acid (Intermediate IIb) on a 1.1 mmol scale. Yield 0.37 g (45%). 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 5.13 - 5.06 (m, 2H), 4.49 (t, J = 5.5 Hz, 1H), 4.23 - 4.12 (m, 4H), 3.68 - 3.55 (m, 4H), 3.50 - 3.38 (m, 2H), 2.80 - 2.74 (m, 2H), 2.41 (t, J = 7.5 Hz, 2H), 2.35 - 2.29 (m, 2H), 2.25 - 2.15 (m, 2H), 2.10 - 1.89 (m, 10H), 1.74 - 1.66 (m, 6H), 1.66 - 1.51 (m, 12H), 1.44 - 1.23 (m, 18H), 1.22 - 1.11 (m, 2H), 0.96 - 0.84 (m, 9H).

[0226] [ka]

[0227] Intermediate IVc, 3-((4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate: Prepared from 4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanoic acid (Intermediate IIc) on a 1.4 mmol scale. Yield 0.40 g (37%). 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 4.53 (t, J = 5.5 Hz, 1H), 4.23 - 4.08 (m, 4H), 3.68 - 3.57 (m, 4H), 3.49 (dt, J = 9.5, 6.0 Hz, 2H), 2.80 - 2.74 (m, 2H), 2.40 (t, J = 7.4 Hz, 2H), 2.32 (dd, J = 7.9, 7.2 Hz, 2H), 2.23 - 2.00 (m, 11H), 1.99 - 1.81 (m, 6H), 1.68 - 1.55 (m, 4H), 1.40 - 1.24 (m, 11H), 0.91 - 0.86 (m, 3H).

[0228] [ka]

[0229] Intermediate IVd, 3-((4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanoic acid (Intermediate IId) on a 1.1 mmol scale. Yield 0.20 g (23%).

[0230] [ka]

[0231] Intermediate IVe, 3-((4,4-bis((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 4,4-bis((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)butanoic acid (Intermediate IIe) on a 1.4 mmol scale. Yield 0.50 g (37%). 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 4.54 (t, J = 5.5 Hz, 1H), 4.24 - 4.08 (m, 4H), 3.68 - 3.60 (m, 4H), 3.51 (dt, J = 9.5, 6.1 Hz, 2H), 2.80 - 2.74 (m, 2H), 2.41 (t, J = 7.4 Hz, 2H), 2.32 (dd, J = 8.0, 7.2 Hz, 2H), 2.26 - 2.11 (m, 5H), 2.10 - 2.01 (m, 5H), 1.99 - 1.84 (m, 6H), 1.65 - 1.59 (m, 2H), 1.57 (s, 2H), 1.40 - 1.23 (m, 12H), 0.92 - 0.86 (m, 3H).

[0232] [ka]

[0233] Intermediate IVf, 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoic acid (Intermediate IIf) on a 1.1 mmol scale. Yield 0.39 g (41%). 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 4.49 (t, J = 5.5 Hz, 1H), 4.23 - 4.12 (m, 4H), 3.62 (t, J = 5.9 Hz, 2H), 3.57 (dt, J = 9.3, 6.5 Hz, 2H), 3.41 (dt, J = 9.3, 6.6 Hz, 2H), 2.81 - 2.74 (m, 2H), 2.41 (t, J = 7.5 Hz, 2H), 2.32 (dd, J = 7.9, 7.2 Hz, 2H), 2.24 - 2.16 (m, 2H), 2.10 - 1.89 (m, 12H), 1.66 - 1.57 (m, 9H), 1.46 - 1.24 (m, 21H), 0.92 - 0.86 (m, 3H).

[0234] [ka]

[0235] Intermediate IVg, 3-((4,4-bis(4-(trifluoromethyl)phenetoxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate: Prepared from 4,4-bis(4-(trifluoromethyl)phenetoxy)butanoic acid (Intermediate IIg) on ​​a 1.4 mmol scale. Yield 0.38 g (35%). 1H NMR (500 MHz, chloroform-d) δ 7.54 - 7.50 (m, 4H), 7.29 - 7.26 (m, 4H), 5.43 - 5.28 (m, 4H), 4.48 (t, J = 5.5 Hz, 1H), 4.22 - 4.06 (m, 5H), 3.69 - 3.62 (m, 2H), 3.60 (t, J = 5.7 Hz, 2H), 3.57 - 3.47 (m, 2H), 2.85 (t, J = 6.7 Hz, 4H), 2.80 - 2.74 (m, 2H), 2.38 - 2.23 (m, 4H), 2.23 - 2.12 (m, 2H), 2.10 - 2.01 (m, 4H), 1.95 - 1.85 (m, 2H), 1.65 - 1.56 (m, 4H), 1.41 - 1.23 (m, 11H), 0.92 - 0.86 (m, 3H).

[0236] [ka]

[0237] Intermediate IVh, 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 4,4-bis(3-cyclohexylpropoxy)butanoic acid (Intermediate IIh) on a 1.4 mmol scale. Yield 0.40 g (41%). 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 4.49 (t, J = 5.5 Hz, 1H), 4.23 - 4.11 (m, 4H), 3.62 (t, J = 5.8 Hz, 2H), 3.55 (dt, J = 9.3, 6.8 Hz, 2H), 3.39 (dt, J = 9.3, 6.8 Hz, 2H), 2.77 (ttd, J = 7.0, 1.4, 0.7 Hz, 2H), 2.41 (t, J = 7.5 Hz, 2H), 2.32 (dd, J = 7.9, 7.2 Hz, 2H), 2.25 - 2.16 (m, 1H), 2.09 - 2.01 (m, 4H), 1.93 (ddd, J = 7.9, 7.2, 5.5 Hz, 2H), 1.74 - 1.50 (m, 18H), 1.42 - 1.09 (m, 25H), 0.92 - 0.83 (m, 7H).

[0238] The following Examples 3-11 were prepared according to general procedure D from intermediate IVa.

[0239] [ka]

[0240] [Example 3] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVa and (1-ethylpiperidin-3-yl)methanol on a 0.15 mmol scale. Yield 0.082 g (66%). 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.27 (m, 8H), 4.49 (t, J = 5.6 Hz, 1H), 4.22 - 4.12 (m, 6H), 4.09 - 3.93 (m, 1H), 3.57 (dt, J = 9.3, 6.6 Hz, 2H), 3.41 (dt, J = 9.4, 6.6 Hz, 2H), 2.80 - 2.74 (m, 2H), 2.47 - 2.36 (m, 4H), 2.31 (dd, J = 8.0, 7.2 Hz, 2H), 2.12 - 1.96 (m, 12H), 1.96 - 1.85 (m, 3H), 1.82 - 1.67 (m, 3H), 1.67 - 1.51 (m, 10H), 1.48 - 1.24 (m, 21H), 1.08 (t, J = 7.2 Hz, 3H), 0.96 (t, J = 7.6 Hz, 6H), 0.92 - 0.85 (m, 3H). LCMS (Method A): Observed value of (M+H) m / z = 860.6, RT = 3.75 min.

[0241] [ka]

[0242] [Example 4] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(dipropylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from Intermediate IVa and 3-(dipropylamino)propan-1-ol on a 0.072 mmol scale. Yield 0.057 g (90%). LCMS (Method A): Found m / z for (M+H) = 876.6, RT = 3.76 min.

[0243] [ka]

[0244] [Example 5] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-methylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from Intermediate IVa and 2-(1-methylpyrrolidin-2-yl)ethan-1-ol on a 0.072 mmol scale. Yield 0.042 g (69%). LCMS (Method A): Found m / z for (M+H) = 846.7, RT = 3.72 min.

[0245] [ka]

[0246] [Example 6] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-ethylpyrrolidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.072 mmol scale from Intermediate IVa and (1-ethylpyrrolidin-3-yl)methanol. Yield 0.039 g (64%). LCMS (Method A): Found m / z for (M+H) = 846.6, RT = 3.72 min.

[0247] [ka]

[0248] [Example 7] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)-2-hydroxypropoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from Intermediate IVa and 3-(diethylamino)propane-1,2-diol on a 0.072 mmol scale. Yield 0.002 g (3%). LCMS (Method A): Found m / z for (M+H) = 864.6, RT = 3.66 min.

[0249] [ka]

[0250] [Example 8] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from Intermediate IVa and 3-(dimethylamino)propan-1-ol on a 0.077 mmol scale. Yield 0.022 g (35%). LCMS (Method A): Found m / z for (M+H) = 820.7, RT = 3.77 min.

[0251] [ka]

[0252] [Example 9] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from Intermediate IVa and 3-(pyrrolidin-1-yl)propan-1-ol on a 0.077 mmol scale. Yield 0.027 g (41%). LCMS (Method A): Found m / z for (M+H) = 846.7, RT = 3.76 min.

[0253] [ka]

[0254] [Example 10] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-methylpiperidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from Intermediate IVa and 2-(1-methylpiperidin-2-yl)ethan-1-ol on a 0.087 mmol scale. Yield 0.039 g (52%). LCMS (Method A): Found m / z for (M+H) = 860.6, RT = 3.83 min.

[0255] [ka]

[0256] [Example 11] 3-(((3-(Azetidin-1-yl)propoxy)carbonyl)oxy)-2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from Intermediate IVa and 3-(azetidin-1-yl)propan-1-ol on a 0.087 mmol scale. Yield 0.041 g (57%). LCMS (Method A): Found m / z for (M+H) = 832.5, RT = 3.74 min.

[0257] The following Examples 12 and 13 were prepared from intermediate IVa according to general procedure E.

[0258] [ka]

[0259] [Example 12] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(4-methylpiperazin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVa and 3-(4-methylpiperazin-1-yl)propanoic acid dihydrochloride on a 0.072 mmol scale. Yield 0.056 g (91%). 1 H NMR (500 MHz, chloroform-d) δ 5.42 - 5.27 (m, 8H), 4.49 (t, J = 5.6 Hz, 1H), 4.21 - 4.10 (m, 6H), 3.64 - 3.53 (m, 2H), 3.41 (dt, J = 9.3, 6.6 Hz, 2H), 2.80 - 2.74 (m, 2H), 2.72 - 2.65 (m, 2H), 2.62 - 2.46 (m, 6H), 2.46 - 2.35 (m, 7H), 2.35 - 2.23 (m, 4H), 2.12 - 1.96 (m, 13H), 1.96 - 1.89 (m, 2H), 1.67 - 1.52 (m, 9H), 1.48 - 1.23 (m, 15H), 0.95 (t, J = 7.6 Hz, 6H), 0.92 - 0.86 (m, 3H). LCMS (Method A): Actual value of (M+H) m / z = 845.6, RT = 3.67 minutes.

[0260] [ka]

[0261] [Example 13] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1,3-dimethylpyrrolidine-3-carboxylate: Prepared from intermediate IVa and 1,3-dimethylpyrrolidine-3-carboxylic acid on a 0.077 mmol scale. Yield 0.007 g (11%). LCMS (Method A): Found m / z for (M+H) = 816.6, RT = 3.76 min.

[0262] The following examples 14-22 were prepared according to general procedure D from intermediate IVb.

[0263] [ka]

[0264] [Example 14] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVb and 3-(diethylamino)propan-1-ol on a 0.13 mmol scale. Yield 0.056 g (46%). 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 5.13 - 5.06 (m, 2H), 4.48 (t, J = 5.5 Hz, 1H), 4.22 - 4.10 (m, 8H), 3.66 - 3.55 (m, 2H), 3.50 - 3.38 (m, 2H), 2.80 - 2.74 (m, 2H), 2.51 (q, J = 7.1 Hz, 6H), 2.48 - 2.37 (m, 3H), 2.31 (dd, J = 8.0, 7.2 Hz, 2H), 2.05 (dd, J = 7.2, 1.1 Hz, 3H), 2.02 - 1.89 (m, 3H), 1.81 (p, J = 6.7 Hz, 2H), 1.73 - 1.50 (m, 23H), 1.45 - 1.23 (m, 17H), 1.22 - 1.11 (m, 2H), 1.01 (t, J = 7.1 Hz, 6H), 0.95 - 0.82 (m, 9H). LCMS (Method A): Observed value of (M+H) m / z = 904.8, RT = 4.06 min.

[0265] [ka]

[0266] [Example 15] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.13 mmol scale from Intermediate IVb and (1-ethylpiperidin-3-yl)methanol. Yield 0.070 g (57%). LCMS (Method A): Found m / z for (M+H) = 916.7, RT = 4.06 min.

[0267] [ka]

[0268] [Example 16] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpyrrolidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and (1-ethylpyrrolidin-3-yl)methanol. Yield 0.040 g (55%). LCMS (Method A): Found m / z for (M+H) = 902.7, RT = 4.00 min.

[0269] [ka]

[0270] [Example 17] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-methylpiperidin-3-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and 2-(1-methylpiperidin-3-yl)ethan-1-ol. Yield 0.038 g (52%). LCMS (Method A): Found m / z for (M+H) = 916.8, RT = 3.94 min.

[0271] [ka]

[0272] [Example 18] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((4-morpholinobutoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVb and 4-morpholinobutan-1-ol on a 0.08 mmol scale. Yield 0.039 g (52%). LCMS (Method A): Found m / z for (M+H) = 932.6, RT = 3.95 min.

[0273] [ka]

[0274] [Example 19] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((((1r,4r)-4-morpholinocyclohexyl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVb and trans-4-morpholinocyclohexan-1-ol on a 0.08 mmol scale. Yield 0.036 g (47%). LCMS (Method A): Found m / z for (M+H) = 958.6, RT = 3.95 min.

[0275] [ka]

[0276] [Example 20] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((4-(4-methylpiperazin-1-yl)butoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and 4-(4-methylpiperazin-1-yl)butan-1-ol. Yield 0.027 g (36%). LCMS (Method A): Found m / z for (M+H) = 945.7, RT = 3.95 min.

[0277] [ka]

[0278] [Example 21] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((((1R,3s,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVb and pseudotropine on a 0.08 mmol scale. Yield 0.019 g (26%). LCMS (Method A): Found m / z for (M+H) = 914.6, RT = 3.88 min.

[0279] [ka]

[0280] [Example 22] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((((2-((dimethylamino)methyl)benzyl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from Intermediate IVb and (2-((dimethylamino)methyl)phenyl)methanol. Yield 0.030 g (40%). LCMS (Method A): Found m / z for (M+H) = 938.8, RT = 4.28 min.

[0281] The following Examples 23-30 were prepared according to general procedure E from intermediate IVb.

[0282] [ka]

[0283] [Example 23] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.09 mmol scale from intermediate IVb and 4-(pyrrolidin-1-yl)butanoic acid hydrochloride. Yield 0.052 g (64%). LCMS (Method A): Found m / z for (M+H) = 886.7, RT = 3.98 min.

[0284] [ka]

[0285] [Example 24] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(4-methylpiperazin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.09 mmol scale from Intermediate IVb and 3-(4-methylpiperazin-1-yl)propanoic acid dihydrochloride. Yield 0.060 g (72%). LCMS (Method A): Found m / z for (M+H) = 901.8, RT = 4.08 min.

[0286] [ka]

[0287] [Example 25] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1'-ethyl-[1,4'-bipiperidine]-4-carboxylate: Prepared on a 0.08 mmol scale from intermediate IVb and 1'-ethyl-[1,4'-bipiperidine]-4-carboxylic acid dihydrochloride. Yield 0.021 g (27%). LCMS (Method A): Found m / z for (M+H) = 969.8, RT = 3.60 min.

[0288] [ka]

[0289] [Example 26] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-(pyridin-4-yl)piperidine-4-carboxylate: Prepared on a 0.08 mmol scale from intermediate IVb and 1-(pyridin-4-yl)piperidine-4-carboxylic acid. Yield 0.020 g (27%). LCMS (Method A): Found m / z for (M+H) = 935.5, RT = 3.88 min.

[0290] [ka]

[0291] [Example 27] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVb and 5-(dimethylamino)pentanoic acid hydrochloride on a 0.08 mmol scale. Yield 0.057 g (81%). LCMS (Method A): Found m / z for (M+H) = 874.8, RT = 3.95 min.

[0292] [ka]

[0293] [Example 28] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(dipropylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVb and 4-(dipropylamino)butanoic acid hydrochloride on a 0.08 mmol scale. Yield 0.062 g (84%). LCMS (Method A): Found m / z for (M+H) = 916.8, RT = 4.00 min.

[0294] [ka]

[0295] [Example 29] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-morpholinopentanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVb and 5-morpholinopentanoic acid hydrochloride on a 0.08 mmol scale. Yield 0.064 g (87%). LCMS (Method A): Found m / z for (M+H) = 916.8, RT = 3.96 min.

[0296] [ka]

[0297] [Example 30] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((2-(1-methylpiperidin-4-yl)acetoxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from Intermediate IVb and 2-(1-methylpiperidin-4-yl)acetic acid. Yield 0.040 g (56%). LCMS (Method A): Found m / z for (M+H) = 886.7, RT = 3.96 min.

[0298] The following Examples 31 and 32 were prepared according to general procedure D from intermediate IVc.

[0299] [ka]

[0300] [Example 31] 3-((4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from Intermediate IVc and 3-(diethylamino)propan-1-ol on a 0.13 mmol scale. Yield 0.042 g (35%). LCMS (Method A): Found m / z for (M+H) = 948.5, RT = 3.49 min.

[0301] [ka]

[0302] [Example 32] 3-((4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.13 mmol scale from Intermediate IVc and (1-ethylpiperidin-3-yl)methanol. Yield 0.050 g (41%). LCMS (Method A): Found m / z for (M+H) = 960.6, RT = 3.52 min.

[0303] The following Examples 33 and 34 were prepared according to general procedure D from intermediate IVd.

[0304] [ka]

[0305] [Example 33] 3-((4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVd and 3-(diethylamino)propan-1-ol on a 0.12 mmol scale. Yield 0.044 g (37%). 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.29 (m, 4H), 4.50 (t, J = 5.5 Hz, 1H), 4.22 - 4.09 (m, 8H), 3.60 (dt, J = 9.2, 5.8 Hz, 2H), 3.44 (dt, J = 9.3, 5.8 Hz, 2H), 2.77 (t, J = 6.7 Hz, 2H), 2.51 (q, J = 7.2 Hz, 6H), 2.46 - 2.37 (m, 3H), 2.31 (t, J = 7.6 Hz, 2H), 2.13 - 1.99 (m, 9H), 1.93 (td, J = 7.5, LCMS (Method A): Actual value of (M+H) m / z = 976.6, RT = 3.52 minutes.

[0306] [ka]

[0307] [Example 34] 3-((4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVd and (1-ethylpiperidin-3-yl)methanol on a 0.12 mmol scale. Yield 0.035 g (29%). 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.29 (m, 4H), 4.51 (t, J = 5.5 Hz, 1H), 4.21 - 4.11 (m, 6H), 4.05 (dd, J = 10.6, 5.8 Hz, 1H), 3.96 (dd, J = 10.6, 7.2 Hz, 1H), 3.60 (dt, J = 9.3, 5.8 Hz, 2H), 3.44 (dt, J = 9.3, 5.8 Hz, 2H), 2.89 (dd, J = 35.3, 11.1 Hz, 2H), 2.77 (t, J = 6.7 Hz, 2H), 2.47 - 2.36 (m, 5H), 2.31 (t, J = 7.6 Hz, 2H), 2.13 - 1.83 (m, 9H), 1.78 - 1.55 (m, 15H), 1.42 - 1.23 (m, 17H), 1.08 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H). LCMS (Method A): Observed value of (M+H) m / z = 988.4, RT = 3.52 min.

[0308] The following Examples 35 and 36 were prepared according to general procedure D from intermediate IVe.

[0309] [ka]

[0310] [Example 35] 3-((4,4-bis((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.12 mmol scale from Intermediate IVe and 3-(diethylamino)propan-1-ol. Yield 0.050 g (36%). LCMS (Method A): Found m / z for (M+H) = 1148.4, RT = 3.72 min.

[0311] [ka]

[0312] [Example 36] 3-((4,4-bis((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.12 mmol scale from Intermediate IVe and (1-ethylpiperidin-3-yl)methanol. Yield 0.072 g (51%). LCMS (Method A): Found m / z for (M+H) = 1160.4, RT = 3.73 min.

[0313] The following examples 37-45 were prepared from intermediate IVf according to general procedure D.

[0314] [ka]

[0315] [Example 37] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVf and 3-(diethylamino)propan-1-ol on a 0.11 mmol scale. Yield 0.084 g (71%). 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 4.48 (t, J = 5.5 Hz, 1H), 4.22 - 4.10 (m, 8H), 3.57 (dt, J = 9.3, 6.5 Hz, 2H), 3.41 (dt, J = 9.3, 6.5 Hz, 2H), 2.80 - 2.74 (m, 2H), 2.51 (q, J = 7.2 Hz, 6H), 2.45 - 2.36 (m, 3H), 2.31 (dd, J = 8.0, 7.2 Hz, 2H), 2.09 - 1.88 (m, 10H), 1.86 - 1.76 (m, 2H), 1.73 - 1.49 (m, 12H), 1.46 - 1.23 (m, 20H), 1.01 (t, J = 7.1 Hz, 6H), 0.92 - 0.86 (m, 3H). LCMS (Method A): Actual value of (M+H) m / z = 1032.5, RT = 3.69 minutes.

[0316] [ka]

[0317] [Example 38] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVf and (1-ethylpiperidin-3-yl)methanol on a 0.11 mmol scale. Yield 0.081 g (68%). 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 4.49 (t, J = 5.5 Hz, 1H), 4.19 (d, J = 6.0 Hz, 2H), 4.17 - 4.12 (m, 4H), 4.05 (dd, J = 10.6, 5.8 Hz, 1H), 3.96 (dd, J = 10.7, 7.2 Hz, 1H), 3.57 (dt, J = 9.3, 6.6 Hz, 2H), 3.41 (dt, J = 9.3, 6.5 Hz, 2H), 2.90 (dd, J = 35.1, 11.3 Hz, 2H), 2.80 - 2.74 (m, 2H), 2.47 - 2.36 (m, 5H), 2.31 (dd, J = 8.0, 7.2 Hz, 2H), 2.11 - 1.79 (m, 11H), 1.79 - 1.66 (m, 3H), 1.66 - 1.49 (m, 12H), 1.49 - 1.23 (m, 24H), 1.08 (t, J = 7.2 Hz, 3H), 0.94 - 0.86 (m, 2H). LCMS (Method A): Observed value of (M+H) m / z = 1044.6, RT = 3.67 min.

[0318] [ka]

[0319] [Example 39] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.09 mmol scale from intermediate IVf and (1-methylpiperidin-4-yl)methanol. Yield 0.055 g (63%). LCMS (Method A): Found m / z for (M+H) = 1030.5, RT = 3.68 min.

[0320] [ka]

[0321] [Example 40] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((3-(piperidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.09 mmol scale from intermediate IVf and 3-(piperidin-1-yl)propan-1-ol. Yield 0.052 g (58%). LCMS (Method A): Found m / z for (M+H) = 1044.5, RT = 3.70 min.

[0322] [ka]

[0323] [Example 41] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((((octahydro-2H-quinolizin-1-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.09 mmol scale from intermediate IVf and (octahydro-2H-quinolizin-1-yl)methanol. Yield 0.050 g (55%). LCMS (Method A): Found m / z for (M+H) = 1070.6, RT = 3.65 min.

[0324] [ka]

[0325] [Example 42] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.09 mmol scale from intermediate IVf and 3-(4-methylpiperazin-1-yl)propan-1-ol. Yield 0.049 g (54%). LCMS (Method A): Found m / z for (M+H) = 1059.5, RT = 3.66 min.

[0326] [ka]

[0327] [Example 43] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((4-(diethylamino)butoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.09 mmol scale from intermediate IVf and 4-(diethylamino)butan-1-ol. Yield 0.049 g (55%). LCMS (Method A): Found m / z for (M+H) = 1046.5, RT = 3.62 min.

[0328] [ka]

[0329] [Example 44] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((3-(3-(piperidin-1-yl)propoxy)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.09 mmol scale from intermediate IVf and 3-(3-(piperidin-1-yl)propoxy)propan-1-ol. Yield 0.044 g (46%). LCMS (Method A): Found m / z for (M+H) = 1102.5, RT = 3.66 min.

[0330] [ka]

[0331] [Example 45] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((((1,3-dimethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.06 mmol scale from intermediate IVf and (1,3-dimethylpiperidin-3-yl)methanol. Yield 0.042 g (70%). LCMS (Method A): Found m / z for (M+H) = 1044.5, RT = 3.68 min.

[0332] [ka]

[0333] [Example 46] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure E from intermediate IVf and 4-(pyrrolidin-1-yl)butanoic acid hydrochloride on a 0.9 mmol scale. Yield 0.079 g (91%). 1H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 4.51 - 4.46 (m, 1H), 4.22 - 4.10 (m, 6H), 3.57 (dtd, J = 9.3, 6.6, 1.3 Hz, 2H), 3.41 (dt, J = 9.3, 6.5 Hz, 2H), 2.80 - 2.74 (m, 2H), 2.55 - 2.27 (m, 12H), 2.11 - 1.72 (m, 15H), 1.69 - 1.52 (m, 16H), 1.47 - 1.23 (m, 18H), 0.92 - 0.86 (m, 3H). LCMS (Method A): (M+H) found m / z = 1014.4, RT = 3.68 min.

[0334] The following Examples 47 and 48 were prepared from intermediate IVg according to general procedure D.

[0335] [ka]

[0336] [Example 47] 3-((4,4-bis(4-(trifluoromethyl)phenethoxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.12 mmol scale from Intermediate IVg and 3-(diethylamino)propan-1-ol. Yield 0.067 g (56%). LCMS (Method A): Found m / z for (M+H) = 972.5, RT = 3.58 min.

[0337] [ka]

[0338] [Example 48] 3-((4,4-bis(4-(trifluoromethyl)phenethoxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.12 mmol scale from Intermediate IVg and (1-ethylpiperidin-3-yl)methanol. Yield 0.045 g (37%). LCMS (Method A): Found m / z for (M+H) = 984.5, RT = 3.56 min.

[0339] The following Examples 49 and 50 were prepared from intermediate IVh according to general procedure D.

[0340] [ka]

[0341] [Example 49] 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.14 mmol scale from intermediate IVh and 3-(diethylamino)propan-1-ol. Yield 0.059 g (48%). LCMS (Method A): Found m / z for (M+H) = 876.6, RT = 4.08 min.

[0342] [ka]

[0343] [Example 50] 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.14 mmol scale from intermediate IVh and (1-ethylpiperidin-3-yl)methanol. Yield 0.048 g (39%). LCMS (Method A): Found m / z for (M+H) = 888.7, RT = 4.04 min.

[0344] The following Examples 51 and 52 were prepared according to general procedure E from intermediate IVh.

[0345] [ka]

[0346] [Example 51] 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.14 mmol scale from intermediate IVh and 4-(pyrrolidin-1-yl)butanoic acid hydrochloride. Yield 0.104 g (87%). LCMS (Method A): Found m / z for (M+H) = 858.8, RT = 4.06 min.

[0347] [ka]

[0348] [Example 52] 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-(((3-(4-methylpiperazin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.14 mmol scale from intermediate IVh and 3-(4-methylpiperazin-1-yl)propanoic acid dihydrochloride. Yield 0.074 g (61%). LCMS (Method A): Found m / z for (M+H) = 873.7, RT = 4.17 min.

[0349] [ka]

[0350] [Example 53] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpyrrolidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from Intermediate IVb and (1-ethylpyrrolidin-3-yl)methanol according to general procedure D. Yield 40 mg (55%). LCMS (Method A): Found m / z for (M+H) = 902.7, RT = 3.99 min.

[0351] [ka]

[0352] [Example 54] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((((1r,4r)-4-morpholinocyclohexyl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and trans-4-morpholinocyclohexan-1-ol according to general procedure D. Yield 36 mg (47%). LCMS (Method A): Found m / z for (M+H) = 958.6, RT = 4.56 min.

[0353] [ka]

[0354] [Example 55] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((4-(4-methylpiperazin-1-yl)butoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and 4-(4-methylpiperazin-1-yl)butan-1-ol according to general procedure D. Yield 27 mg (36%). LCMS (Method A): Found m / z for (M+H) = 945.7, RT = 3.95 min.

[0355] [ka]

[0356] [Example 56] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((((1r,3s,5s)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure D from intermediate IVb and pseudotropine on a 0.08 mmol scale. Yield 19 mg (26%). LCMS (Method A): Found m / z for (M+H) = 914.8, RT = 3.88 min.

[0357] [ka]

[0358] [Example 57] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((4-morpholinobutoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and 4-morpholinobutan-1-ol according to general procedure D. Yield 39 mg (52%). LCMS (Method A): Found m / z for (M+H) = 932.7, RT = 4.58 min.

[0359] [ka]

[0360] [Example 58] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-methylpiperidin-3-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and 2-(1-methylpiperidin-3-yl)ethan-1-ol according to general procedure D. Yield 38 mg (52%). LCMS (Method A): Found m / z for (M+H) = 916.8, RT = 3.94 min.

[0361] [ka]

[0362] [Example 59] 3-((4,4-Bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1'-ethyl-[1,4'-bipiperidine]-4-carboxylate: Prepared on a 0.08 mmol scale from intermediate IVb and 1'-ethyl-[1,4'-bipiperidine]-4-carboxylic acid dihydrochloride according to general procedure E. Yield 21 mg (27%). LCMS (Method A): Found m / z for (M+H) = 969.7, RT = 3.41 min.

[0363] [ka]

[0364] [Example 60] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(dipropylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and 4-(dipropylamino)butanoic acid hydrochloride according to general procedure E. Yield 62 mg (84%). LCMS (Method A): Found m / z for (M+H) = 916.8, RT = 4.00 min.

[0365] [ka]

[0366] [Example 61] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-(pyridin-4-yl)piperidine-4-carboxylate: Prepared on a 0.08 mmol scale from intermediate IVb and 1-(pyridin-4-yl)piperidine-4-carboxylic acid hydrochloride according to general procedure E. Yield 20 mg (27%). LCMS (Method A): Found m / z for (M+H) = 935.7, RT = 3.87 min.

[0367] [ka]

[0368] [Example 62] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((2-(1-methylpiperidin-4-yl)acetoxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and 2-(1-methylpiperidin-4-yl)acetic acid according to general procedure E. Yield 40 mg (56%). LCMS (Method A): Found m / z for (M+H) = 886.7, RT = 3.95 min.

[0369] [ka]

[0370] [Example 63] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-morpholinopentanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared on a 0.08 mmol scale from intermediate IVb and 5-morpholinopentanoic acid hydrochloride according to general procedure E. Yield 64 mg (87%). LCMS (Method A): Found m / z for (M+H) = 916.9, RT = 4.50 min.

[0371] [ka]

[0372] [Example 64] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate: Example 63, 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-morpholinopentanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure E from intermediate IVb and 5-(dimethylamino)pentanoic acid hydrochloride on a 0.08 mmol scale. Yield 57 mg (81%). LCMS (Method A): (M+H) found m / z = 874.8, RT = 3.95 min.

[0373] [ka]

[0374] [Example 65] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-methylpiperidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure D from intermediate IVa and 2-(1-methylpiperidin-2-yl)ethan-1-ol on a 0.087 mmol scale. Yield 39 mg (52%). LCMS (Method A): Found m / z for (M+H) = 860.7, RT = 3.83 min.

[0375] [ka]

[0376] [Example 66] 3-(((3-(Azetidin-1-yl)propoxy)carbonyl)oxy)-2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure D from intermediate IVa and 3-(azetidin-1-yl)propan-1-ol on a 0.087 mmol scale. Yield 41 mg (57%). LCMS (Method A): Found m / z for (M+H) = 832.6, RT = 3.74 min.

[0377] [ka]

[0378] [Example 67] 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((((2-((dimethylamino)methyl)benzyl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure D from intermediate IVb and (2-((dimethylamino)methyl)phenyl)methanol on a 0.087 mmol scale. Yield 30 mg (40%). LCMS (Method A): Found m / z for (M+H) = 938.7, RT = 4.28 min.

[0379] [ka]

[0380] [Example 68] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((4-(diethylamino)butoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure D from intermediate IVf and 4-(diethylamino)butan-1-ol on a 0.086 mmol scale. Yield 49 mg (55%). LCMS (Method A): Found m / z for (M+H) = 1046.5, RT = 3.63 min.

[0381] [ka]

[0382] [Example 69] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((3-(3-(piperidin-1-yl)propoxy)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure D from intermediate IVf and 3-(3-(piperidin-1-yl)propoxy)propan-1-ol on a 0.086 mmol scale. Yield 44 mg (46%). LCMS (Method A): Found m / z for (M+H) = 1102.5, RT = 3.66 min.

[0383] [ka]

[0384] [Example 70] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1,3-dimethylpyrrolidine-3-carboxylate: Prepared according to general procedure E from intermediate IVa and 1,3-dimethylpyrrolidine-3-carboxylic acid on a 0.077 mmol scale. Yield 7 mg (11%). LCMS (Method A): Found m / z for (M+H) = 816.6, RT = 3.74 min.

[0385] [ka]

[0386] [Example 71] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure D from intermediate IVa and 3-(dimethylamino)propan-1-ol on a 0.077 mmol scale. Yield 22 mg (35%). LCMS (Method A): Found m / z for (M+H) = 820.7, RT = 3.76 min.

[0387] [ka]

[0388] [Example 72] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure D from intermediate IVa and 3-(pyrrolidin-1-yl)propan-1-ol on a 0.077 mmol scale. Yield 27 mg (41%). LCMS (Method A): Found m / z for (M+H) = 846.7, RT = 3.73 min.

[0389] [ka]

[0390] [Example 73] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((1,3-dimethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared according to general procedure D from intermediate IVf and (1,3-dimethylpiperidin-3-yl)methanol on a 0.057 mmol scale. Yield 42 mg (70%). LCMS (Method A): Found m / z for (M+H) = 1044.5, RT = 3.69 min.

[0391] [ka]

[0392] [Example 74] 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate

[0393] Step 1: 2-(hydroxymethyl)butane-1,4-diol

[0394] [ka]

[0395] To a stirred solution of triethylethane-1,1,2-tricarboxylate (5 g, 20.3 mmol) in tert-butanol (80 mL) at 25 °C under an argon atmosphere was added NaBH (2.3 g, 60.9 mmol). The resulting suspension was heated to reflux, and methanol (3 mL) was added dropwise in three portions within 30 min. The resulting solution was heated to reflux for another 3 h. The reaction mixture was then cooled to 25 °C and neutralized with 5 N HCl (2.5 mL). The precipitate was filtered, and the filtrate was evaporated to give the crude material, which was purified by CombiFlash column chromatography, eluting with 10–15% MeOH in DCM, to give 2-(hydroxymethyl)butane-1,4-diol (1.7 g, 69%) as a yellow liquid. 1 H NMR (400 MHz, DMSO-d6): δ1.34-1.46 (m, 2H), 1.49-1.63 (m, 1H), 3.27-3.48 (m, 6H), 4.34 (t, J = 5.2 Hz, 2H), 4.40 (t, J = 5.1 Hz, 1H).

[0396] Step 2: 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethan-1-ol

[0397] [ka]

[0398] To a stirred solution of 2-(hydroxymethyl)butane-1,4-diol (1.7 g, 14.1 mmol) and 2,2-dimethoxypropane (4.3 mL, 35.3 mmol) in THF (10 mL) was added p-toluenesulfonic acid monohydrate (0.36 g, 3.1 mmol) at 25 °C under an argon atmosphere. The reaction mixture was stirred at 25 °C for 16 h. After this time, the reaction was neutralized with triethylamine (5 mL). The solvent was removed under reduced pressure to give the crude material, which was purified by CombiFlash column chromatography eluting with 15% ethyl acetate-hexane to give 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethan-1-ol (1.2 g, 53%) as a pale yellow liquid. 1 H NMR (400 MHz, CDCl3): δ1.41 (s, 6H), 1.50-1.60 (m, 2H), 1.88-1.98 (m, 1H), 3.63 (dd, J = 7.9, 11.8 Hz, 2H), 3.70 (t, J = 6.4 Hz, 2H), 3.93 (dd, J = 4.5, 11.8 Hz, 2H).

[0399] Step 3: 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl (4-nitrophenyl) carbonate

[0400] [ka]

[0401] To a solution of 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethan-1-ol (400 mg, 2.5 mmol) in DCM (10 mL) was added pyridine (0.4 mL, 5.0 mmol), DMAP (30.5 mg, 0.25 mmol), and finally 4-nitrophenyl chloroformate (604 mg, 2.9 mmol). The reaction was stirred at 25 °C for 7 h. After this time, the reaction mixture was diluted with water (20 mL) and DCM (30 mL). The organic layer was separated, and the aqueous layer was extracted with DCM (10 mL × 2). The combined organic layers were dried over Na2SO4. The solvent was removed under reduced pressure to give a crude mass, which was purified by Combi-Flash column chromatography eluting with 30% ethyl acetate-hexane to give 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl (4-nitrophenyl)carbonate (0.302 g, 37%) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 1.29-1.34 (m, 6H), 1.62 (q, J = 6.7 Hz, 2H), 1.80 (bs, 1H), 3.54 (q, J = 8.8 Hz, 2H), 3.83 (dd, J = 11.4 Hz, 4.0 Hz, 2H), 4.28 (t, J = 6.4 Hz, 2H), 7.57 (d, J = 9.0 Hz, 2H), 8.31 (d, J = 9.0 Hz, 2H).

[0402] Step 4: 3-(Diethylamino)propyl (2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl)carbonate

[0403] [ka]

[0404] To a stirred solution of 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl (4-nitrophenyl)carbonate (150 mg, 0.46 mmol) in 5 mL of DCM, DMAP (5.6 mg, 0.04 mmol) and pyridine (0.07 mL, 0.9 mmol) were added at 25° C. and stirred for 5 minutes. 3-(diethylamino)-1-propanol (78.7 mg, 0.6 mmol) was then added at 25° C. The reaction mass was stirred at 25° C. for 9 hours. Completion of the reaction was determined by TLC (5% MeOH-DCM). The reaction mixture was diluted with water and extracted with DCM (3×15 mL). The combined organic layers were washed with brine, dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by Combiflash column chromatography eluting with 10% MeOH-DCM to give 3-(diethylamino)propyl (2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl)carbonate (98 mg, 67%) as a pale yellow liquid. 1 H NMR (400 MHz, CDCl3) δ 1.03 (t, J = 7.0 Hz, 3H), 1.40 (s, 3H), 1.63 (q, J = 6.7 Hz, 1H), 1.80-1.87 (m, 1H), 2.54 (t, J = 7.0 Hz, 3H), 3.58 (q, J = 8.2 Hz, 1H), 3.91 (dd, J = 11.8 Hz, 4.2 Hz, 1H), 4.13 (q, J = 6.2 Hz, 2H).

[0405] Step 5: 3-(Diethylamino)propyl (4-hydroxy-3-(hydroxymethyl)butyl)carbonate

[0406] [ka]

[0407] To a stirred solution of 3-(diethylamino)propyl (2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl)carbonate (92 mg, 0.3 mmol) in MeOH (1 mL) was added 1N HCl (0.9 mL, 0.9 mmol) at 25° C. The reaction mass was stirred for 2 h. Completion of the reaction was judged by TLC (5% MeOH-DCM). The reaction mixture was concentrated and azeotroped twice with toluene to give crude product 3-(diethylamino)propyl (4-hydroxy-3-(hydroxymethyl)butyl)carbonate (120 mg), which was used directly for the next step without purification.

[0408] Step 6: 4-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(hydroxymethyl)butyl (9Z,12Z)-octadeca-9,12-dienoate

[0409] [ka]

[0410] To a stirred solution of linoleic acid (0.08 mL, 0.26 mmol) in 3 mL of DCM, EDC (82.9 mg, 0.43 mmol) and DMAP (7.2 mg, 0.06 mmol) were added at 25 °C and stirred for 5 minutes. DIPEA (0.147 mL, 0.86 mmol) and 3-(diethylamino)propyl (4-hydroxy-3-(hydroxymethyl)butyl)carbonate (80 mg, 0.29 mmol) were added at 25 °C. The reaction mixture was then stirred at 25 °C for 16 hours. Completion of the reaction was determined by LCMS of the crude reaction mixture. The reaction mixture was diluted with water (10 mL) and extracted with DCM (15 mL × 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 10% MeOH-DCM to give 4-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(hydroxymethyl)butyl (9Z,12Z)-octadeca-9,12-dienoate (42 mg, 27%) as a pale yellow liquid. 1 H NMR (400 MHz, CDCl3) δ 0.87 (d, J = 6.6 Hz, 3H), 1.00-1.10 (m, 5H), 1.24-1.29 (m, 20H), 1.69-1.83 (m, 4H), 2.02 (t, J = 6.6 Hz, 5H), 2.30 (t, J = 7.3 Hz, 2H), 2.50-2.55 (m, 5H), 2.75 (d, J = 7.3 Hz, 2H), 3.54-3.61 (m, 2H), 4.13-4.23 (m, 5H), 5.34 (t, J = 4.5 Hz, 4H).

[0411] Step 7: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate (Example 74): To a stirred solution of 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (Intermediate IIa) (24.1 mg, 0.07 mmol) in 2 mL DCM was added EDC (22.3 mg, 0.12 mmol) and DMAP (1.9 mg, 0.015 mmol) at 25° C. and stirred for 5 minutes. Then, DIPEA (0.04 mL, 0.23 mmol) and 4-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(hydroxymethyl)butyl (9Z,12Z)-octadeca-9,12-dienoate (42 mg, 0.08 mmol) were added at 25 °C. The reaction mixture was stirred at 25 °C for 16 h. Completion of the reaction was judged by LCMS of the crude reaction mixture. The reaction mixture was diluted with NaHCO3 solution (5 mL) and extracted with DCM (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by Prep-HPLC to give -(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate (24 mg, 36%) as a pale yellow liquid. LCMS (Method B): (M+H) found m / z = 867.7, RT = 2.04 min.

[0412] [ka]

[0413] [Example 75] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(4-ethylpiperazin-1-yl)propanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0414] Step 1: 3-(acryloyloxy)-2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Intermediate V)

[0415] [ka]

[0416] To a stirred solution of 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Intermediate IVa) (1.0 g, 1.4 mmol) in DCM (20 mL) was added DMAP (18.0 mg, 0.1 mmol) and DIPEA (0.50 mL, 2.8 mmol). The resulting mixture was cooled in an ice bath, and then acryloyl chloride (0.2 mL, 2.9 mmol) was added dropwise over 10 minutes. The mixture was stirred at room temperature for 1 hour. After this time, the reaction mixture was extracted with DCM (20 mL × 2). The combined organic layers were washed with water (20 mL × 2) and brine (20 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by Combiflash column chromatography eluting with 10% ethyl acetate-hexane to give 3-(acryloyloxy)-2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (860 mg, 79%) as a colorless liquid. 1H NMR (400 MHz, クロロホルム-d) δ 0.84 - 0.91 (m, 3H), 0.94 (t, J = 7.5 Hz, 6H), 1.17 - 1.47 (m, 20H), 1.53 - 1.65 (m, 4H), 1.86 - 1.95 (m, 2H), 1.97 - 2.09 (m, 12H), 2.30 (t, J = 7.4 Hz, 2H), 2.34 - 2.47 (m, 3H), 2.76 (t, J = 6.4 Hz, 2H), 3.34 - 3.45 (m, 2H), 3.56 (q, J = 7.4 Hz, 2H), 4.11 - 4.18 (m, 4H), 4.22 (d, J = 6.0 Hz, 2H), 4.47 (t, J = 5.6 Hz, 1H), 5.22 - 5.45 (m, 8H), 5.85 (d, J = 11.3 Hz, 1H), 6.11 (dd, J = 10.5, 17.3 Hz, 1H), 6.36-6.45 (m, 1H).

[0417] Step 2: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(4-ethylpiperazin-1-yl)propanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (Example 75): The following is representative of general procedure F. A mixture of 3-(acryloyloxy)-2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (Intermediate V) (50 mg, 0.067 mmol, 1 Eq) and 1-ethylpiperazine (38 mg, 0.34 mmol, 5 Eq) was heated to 50° C. for 16 hours in the absence of solvent. After cooling to room temperature, the crude reaction mixture was purified by column chromatography eluting with 9:1 DCM / MeOH to give 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(4-ethylpiperazin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (26 mg, 44%). LCMS (Method B): Found m / z for (M+H) = 859.9, RT = 1.67 min.

[0418] [ka]

[0419] [Example 76] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(4-ethyl-1,4-diazepan-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and 1-ethyl-1,4-diazepane according to general procedure F. Yield 16 mg (46%). LCMS (Method B): Found m / z for (M+H) = 873.9, RT = 1.56 min.

[0420] [ka]

[0421] [Example 77] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(4-(2-methoxyethyl)piperazin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and 1-(2-methoxyethyl)piperazine according to general procedure F. Yield 34 mg (57%). LCMS (Method B): Found m / z for (M+H) = 889.9, RT = 1.69 min.

[0422] [ka]

[0423] [Example 78] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(3,5-dimethylpiperazin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and 2,6-dimethylpiperazine according to general procedure F. Yield 38 mg (65%). LCMS (Method B): Found m / z for (M+H) = 859.9, RT = 1.71 min.

[0424] [ka]

[0425] [Example 79] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-((2-(diethylamino)ethyl)(ethyl)amino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and N1,N1,N2-triethylethane-1,2-diamine according to general procedure F. Yield 24 mg (48%). LCMS (Method B): Found m / z for (M+H) = 889.9, RT = 2.29 min.

[0426] [ka]

[0427] [Example 80] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-((1-isopropylpiperidin-4-yl)amino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and 1-isopropylpiperidin-4-amine according to general procedure F. Yield 18 mg (37%). LCMS (Method B): Found m / z for (M+H) = 887.9, RT = 1.34 min.

[0428] [ka]

[0429] [Example 81] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-((pyridin-3-ylmethyl)amino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and pyridin-3-ylmethanamine according to general procedure F. Yield 20 mg (43%). LCMS (Method B): Found m / z for (M+H) = 854.0, RT = 1.34 min.

[0430] [ka]

[0431] [Example 82] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(methyl(2-(pyridin-2-yl)ethyl)amino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and N-methyl-2-(pyridin-2-yl)ethan-1-amine according to general procedure F. Yield 34 mg (49%). LCMS (Method B): Found m / z for (M+H) = 882.0, RT = 1.07 min.

[0432] [ka]

[0433] [Example 83] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(3-(dimethylamino)pyrrolidin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and 3-(dimethylamino)pyrrolidine according to general procedure F. Yield 27 mg (58%). LCMS (Method B): Found m / z for (M+H) = 859.9, RT = 1.62 min.

[0434] [ka]

[0435] [Example 84] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-((2-(pyrrolidin-1-yl)ethyl)amino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and 2-(pyrrolidin-1-yl)ethan-1-amine according to general procedure F. Yield 26 mg (56%). LCMS (Method B): Found m / z for (M+H) = 859.9, RT = 1.34 min.

[0436] [ka]

[0437] [Example 85] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-((3-morpholinopropyl)amino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and 2-(1-methylpyrrolidin-2-yl)ethan-1-amine according to general procedure F. Yield 17 mg (35%). LCMS (Method B): Found m / z for (M+H) = 873.8, RT = 2.28 min.

[0438] [ka]

[0439] [Example 86] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-((2-(pyridin-4-yl)ethyl)amino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and 2-(pyridin-4-yl)ethan-1-amine according to general procedure F. Yield 18 mg (39%). LCMS (Method B): Found m / z for (M+H) = 867.9, RT = 1.34 min.

[0440] [ka]

[0441] [Example 87] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-((2-(1-methylpyrrolidin-2-yl)ethyl)amino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate V and 2-(pyridin-4-yl)ethan-1-amine according to general procedure F. Yield 26 mg (43%). LCMS (Method B): Found m / z for (M+H) = 867.9, RT = 1.34 min.

[0442] [ka]

[0443] [Example 88] 3-((4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0444] Step 1: 4,4-bis(((Z)-non-3-en-1-yl)oxy)butanenitrile

[0445] [ka]

[0446] Prepared according to general procedure A using (Z)-non-3-en-1-ol. Yield 280 mg (41%). 1H NMR (400 MHz, chloroform-d): δ 0.88 (t, J = 6.7 Hz, 6H), 1.21-1.41 (m, 12H), 1.94 (q, J = 7.2 Hz, 2H), 2.03 (q, J = 7.2 Hz, 4H), 2.32 (q, J = 7.0 Hz, 4H), 2.41 (t, J = 7.4 Hz, 2H), 3.39-3.50 (m, 2H), 3.56-3.66 (m, 2H), 4.58 (t, J = 5.3 Hz, 1H), 5.29-5.40 (m, 2H), 5.41-5.53 (m, 2H).

[0447] Step 2: 4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoic acid

[0448] [ka]

[0449] Prepared from 4,4-bis(((Z)-non-3-en-1-yl)oxy)butanenitrile according to general procedure B. Yield 230 mg (78%). 1 H NMR (400 MHz, DMSO-d6): δ 0.85 (t, J = 6.6 Hz, 6H), 1.13-1.38 (m, 13H), 1.72 (q, J = 7.1 Hz, 2H), 2.00 (q, J = 7.0 Hz, 4H), 2.16-2.29 (m, 5H), 3.33-3.42 (m, 2H), 3.43-3.54 (m, 2H), 4.49 (t, J = 5.6 Hz, 1H), 5.30-5.47 (m, 4H), 12.06 (s, 1H).

[0450] Step 3: 3-((4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0451] [ka]

[0452] Prepared from 4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoic acid according to general procedure C. Yield 340 mg (58%). 1 H NMR (400 MHz, chloroform-d): δ 0.82-0.92 (m, 9H), 1.21-1.40 (m, 26H), 1.61 (t, J = 7.1 Hz, 2H), 1.88 - 2.09 (m, 10H), 2.13 - 2.23 (m, 2H), 2.30 (q, J = 7.0 Hz, 6H), 2.41 (t, J = 7.5 Hz, 2H), 2.76 (t, J = 6.4 Hz, 2H), 3.36 - 3.47 (m, 2H), 3.52 - 3.65 (m, 4H), 4.13 - 4.23 (m, 4H), 4.52 (t, J = 5.5 Hz, 1H), 5.26 - 5.51 (m, 8H).

[0453] Step 4: 3-((4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 88). Prepared from 3-((4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)propan-1-ol according to general procedure D. Yield 79 mg (83%). LCMS (Method B): Found m / z for (M+H) = 876.9, RT = 1.91 min.

[0454] [ka]

[0455] [Example 89] 3-((4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 50 mg (69%). LCMS (Method B): Found m / z for (M+H) = 889.0, RT = 1.77 min.

[0456] [ka]

[0457] [Example 90] 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0458] Step 1: 4,4-bis((4-butylbenzyl)oxy)butanenitrile

[0459] [ka]

[0460] Prepared according to general procedure A using (4-butylphenyl)methanol. Yield 1.1 g (36%). 1H NMR (400 MHz, chloroform-d): δ 0.92 (t, J = 7.3 Hz, 6H), 1.28 - 1.40 (m, 4H), 1.51 - 1.65 (m, 4H), 1.97 - 2.07 (m, 2H), 2.41 (t, J = 7.4 Hz, 2H), 2.60 (t, J = 7.7 Hz, 4H), 4.51 (d, J = 11.5 Hz, 2H), 4.64 (d, J = 11.5 Hz, 2H), 4.79 (t, J = 5.3 Hz, 1H), 7.16 (d, J = 7.9 Hz, 4H), 7.20 - 7.28 (m, 4H).

[0461] Step 2: 4,4-bis((4-butylbenzyl)oxy)butanoic acid

[0462] [ka]

[0463] Prepared from 4,4-bis((4-butylbenzyl)oxy)butanenitrile according to general procedure B. Yield 1.1 g (91%). 1 H NMR (400 MHz, DMSO-d6): δ 0.89 (t, J = 7.3 Hz, 6H), 1.22 - 1.36 (m, 4H), 1.47 - 1.60 (m, 4H), 1.87 (q, J = 7.2 Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H), 2.56 (t, J = 7.6 Hz, 4H), 4.45 (d, J = 11.7 Hz, 2H), 4.56 (d, J = 11.7 Hz, 2H), 4.72 (t, J = 5.5 Hz, 1H), 7.15 (d, J = 7.9 Hz, 4H), 7.22 (d, J = 7.6 Hz, 4H), 12.09 (s, 1H).

[0464] Step 3: 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0465] [ka]

[0466] Prepared from 4,4-bis((4-butylbenzyl)oxy)butanoic acid according to general procedure C. Yield 400 mg (64%). 1 H NMR (400 MHz, chloroform-d): δ 0.83 - 0.96 (m, 9H), 1.21 - 1.42 (m, 17H), 1.52 - 1.66 (m, 7H), 1.99 - 2.19 (m, 8H), 2.30 (t, J = 7.6 Hz, 2H), 2.44 (t, J = 7.4 Hz, 2H), 2.59 (t, J = 7.7 Hz, 4H), 2.76 (t, J = 6.4 Hz, 2H), 3.55 (t, J = 5.7 Hz, 2H), 4.04 - 4.19 (m, 4H), 4.50 (d, J = 11.5 Hz, 2H), 4.61 (d, J = 11.5 Hz, 2H), 4.74 (t, J = 5.4 Hz, 1H), 5.26 - 5.42 (m, 4H), 7.15 (d, J = 7.9 Hz, 4H), 7.22 (d, J = 7.8 Hz, 4H).

[0467] Step 4: 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 90). Prepared from 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)propan-1-ol according to general procedure D. Yield 62 mg (83%). LCMS (Method B): Found m / z for (M+H) = 920.9, RT = 1.81 min.

[0468] [ka]

[0469] [Example 91] 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Prepared according to general procedure D from 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol. Yield 50 mg, 69%. LCMS (Method B): (M+H) found m / z = 889.0, RT = 1.77 min.

[0470] [ka]

[0471] [Example 92] 3-((4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0472] Step 1: 4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanenitrile

[0473] [ka]

[0474] Prepared according to general procedure A using (Z)-dec-4-en-1-ol. Yield 750 mg (51%). 1 H NMR (400 MHz, DMSO-d6): δ 0.86 (t, J = 6.8 Hz, 6H), 1.18 - 1.37 (m, 12H), 1.48 - 1.60 (m, 4H), 1.74 - 1.86 (m, 2H), 1.94 - 2.10 (m, 8H), 2.46 (t, J = 7.2 Hz, 2H), 3.33 - 3.44 (m, 2H), 3.47 - 3.60 (m, 2H), 4.51 (t, J = 5.4 Hz, 1H), 5.28 - 5.42 (m, 4H).

[0475] Step 2: 4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoic acid

[0476] [ka]

[0477] Prepared from 4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanenitrile according to general procedure B. Yield 300 mg (71%). 1H NMR (400 MHz, DMSO-d6): δ 0.85 (t, J = 6.7 Hz, 6H), 1.19 - 1.36 (m, 12H), 1.53 (q, J = 6.9 Hz, 4H), 1.73 (q, J = 7.0 Hz, 2H), 1.91 - 2.09 (m, 8H), 2.22 (t, J = 7.5 Hz, 2H), 3.29 - 3.40 (m, 2H), 3.44 - 3.55 (m, 2H), 4.45 (t, J = 5.5 Hz, 1H), 5.27 - 5.42 (m, 4H), 12.05 (s, 1H).

[0478] Step 3: 3-((4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0479] [ka]

[0480] Prepared from 4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoic acid according to general procedure C. Yield 270 mg (47%). 1 H NMR (400 MHz, chloroform-d): δ 0.83 - 0.92 (m, 9H), 1.07 - 1.15 (m, 2H), 1.20 - 1.41 (m, 23H), 1.47 - 1.76 (m, 9H), 1.88 - 2.13 (m, 10H), 2.17 - 2.23 (m, 2H), 2.31 (t, J = 7.5 Hz, 2H), 2.41 (t, J = 7.4 Hz, 2H), 2.76 (t, J = 6.6 Hz, 2H), 3.35 - 3.50 (m, 3H), 3.52 - 3.65 (m, 4H), 3.97 - 4.06 (m, 1H), 4.10 - 4.22 (m, 4H), 4.48 (t, J = 5.4 Hz, 1H), 5.26 - 5.44 (m, 8H).

[0481] Step 4: 3-((4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 92). Prepared from 3-((4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 40 mg (61%). LCMS (Method B): Found m / z for (M+H) = 905.0, RT = 1.90 min.

[0482] [ka]

[0483] [Example 93] 3-((4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 50 mg (68%). LCMS (Method B): Found m / z for (M+H) = 917.0, RT = 1.91 min.

[0484] [ka]

[0485] [Example 94] 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((3-(5,5-dioctyl-1,3-dioxan-2-yl)propanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0486] Step 1: 3-(5,5-dioctyl-1,3-dioxan-2-yl)propanenitrile

[0487] [ka]

[0488] Prepared according to general procedure A using 2,2-dioctylpropane-1,3-diol. Yield 500 mg (70%). 1 H NMR (400 MHz, chloroform-d): δ 0.83-0.91 (m, 6H), 0.92-1.02 (m, 2H), 1.05-1.35 (m, 24H), 1.53-1.63 (m, 2H), 1.89-1.99 (m, 2H), 2.47 (t, J = 7.4 Hz, 2H), 3.37 (d, J = 11.1 Hz, 2H), 3.76 (d, J = 11.1 Hz, 2H), 4.55 (t, J = 4.6 Hz, 1H).

[0489] Step 2: 3-(5,5-dioctyl-1,3-dioxan-2-yl)propanoic acid

[0490] [ka]

[0491] Prepared from 3-(5,5-dioctyl-1,3-dioxan-2-yl)propanenitrile according to general procedure B. Yield 400 mg (70%). 1H NMR (400 MHz, DMSO-d6): δ 0.78-0.97 (m, 8H), 1.02-1.35 (m, 24H), 1.47-1.57 (m, 2H), 1.67-1.76 (m, 2H), 2.26 (t, J = 7.5 Hz, 2H), 3.33 (d, J = 10.3 Hz, 2H), 3.67 (d, J = 10.9 Hz, 2H), 4.44 (t, J = 4.8 Hz, 1H), 12.05 (s, 1H).

[0492] Step 3: 3-((3-(5-hexyl-5-octyl-1,3-dioxan-2-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0493] [ka]

[0494] Prepared from 3-(5,5-dioctyl-1,3-dioxan-2-yl)propanoic acid according to general procedure C. Yield 80 mg (40%). 1 H NMR (400 MHz, chloroform-d): δ 0.79-1.01 (m, 11H), 1.02-1.45 (m, 38H), 1.56-1.65 (m, 4H), 1.89-1.99 (m, 2H), 1.99-2.09 (m, 4H), 2.12-2.26 (m, 2H), 2.31 (t, J = 7.6 Hz, 2H), 2.47 (t, J = 7.5 Hz, 2H), 2.76 (t, J = 6.3 Hz, 2H), 3.34 (d, J = 11.0 Hz, 2H), 3.60 (t, J = 6.0 Hz, 2H), 3.74 (d, J = 11.1 Hz, 2H), 4.17 (t, J = 7.1 Hz, 4H), 4.42-4.54 (m, 1H), 5.30-5.39 (m, 4H).

[0495] Step 4: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((3-(5,5-dioctyl-1,3-dioxan-2-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 94). Prepared from 3-((3-(5-hexyl-5-octyl-1,3-dioxan-2-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 27 mg (73%). LCMS (Method B): Found m / z for (M+H) = 892.9, RT = 1.93 min.

[0496] [ka]

[0497] [Example 95] 3-((3-(5,5-Dioctyl-1,3-dioxan-2-yl)propanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((3-(5-hexyl-5-octyl-1,3-dioxan-2-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 27 mg (59%). LCMS (Method B): Found m / z for (M+H) = 905.0, RT = 1.94 min.

[0498] [ka]

[0499] [Example 96] 3-((4,4-bis(2-(octyloxy)ethoxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0500] Step 1: 4,4-bis(2-(octyloxy)ethoxy)butanenitrile

[0501] [ka]

[0502] Prepared according to general procedure A using 2-(octyloxy)ethan-1-ol. Yield 340 mg (21%). 1 H NMR (400 MHz, chloroform-d): δ 0.87 (t, J = 6.7 Hz, 6H), 1.23 - 1.37 (m, 20H), 1.53 (d, J = 7.7 Hz, 2H), 1.58 (d, J = 7.8 Hz, 2H), 1.93 - 2.03 (m, 2H), 2.44 (t, J = 7.4 Hz, 2H), 3.43 (t, J = 6.8 Hz, 4H), 3.55 (t, J = 4.8 Hz, 4H), 3.58 - 3.69 (m, 2H), 3.70 - 3.80 (m, 2H), 4.70 (t, J = 5.4 Hz, 1H).

[0503] Step 2: 4,4-bis(2-(octyloxy)ethoxy)butanoic acid

[0504] [ka]

[0505] Prepared from 4,4-bis(2-(octyloxy)ethoxy)butanenitrile according to general procedure B. Yield 136 mg (81%). 1H NMR (400 MHz, DMSO-d6): δ 0.85 (t, J = 6.7 Hz, 6H), 1.07 - 1.36 (m, 20H), 1.47 (t, J = 6.9 Hz, 4H), 1.68 - 1.79 (m, 2H), 2.23 (t, J = 7.5 Hz, 2H), 3.36 (t, J = 6.5 Hz, 4H), 3.42 - 3.56 (m, 6H), 3.58-3.63 (m, 2H), 4.55 (t, J = 5.6 Hz, 1H), 12.04 (s, 1H).

[0506] Step 3, 3-((4,4-bis(2-(octyloxy)ethoxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0507] [ka]

[0508] Prepared from 4,4-bis(2-(octyloxy)ethoxy)butanoic acid according to general procedure C. Yield 147 mg (53%). 1 H NMR (400 MHz, chloroform-d): δ 0.83-0.92 (m, 9H), 1.13 - 1.41 (m, 37H), 1.55 - 1.65 (m, 3H), 1.92-2.09 (m, 6H), 2.13-2.22 (m, 1H), 2.27 - 2.36 (m, 3H), 2.42 (t, J = 7.4 Hz, 2H), 2.76 (t, J = 6.5 Hz, 2H), 3.43 (t, J = 6.8 Hz, 4H), 3.55 (t, J = 4.8 Hz, 4H), 3.56 - 3.66 (m, 4H), 3.67 - 3.77 (m, 2H), 4.08 - 4.23 (m, 4H), 4.64 (t, J = 5.5 Hz, 1H), 5.28 - 5.41 (m, 4H).

[0509] Step 4: 3-((4,4-bis(2-(octyloxy)ethoxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 96). Prepared from 3-((4,4-bis(2-(octyloxy)ethoxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 36 mg (62%). LCMS (Method B): Found m / z for (M+H) = 941.0, RT = 1.80 min.

[0510] [ka]

[0511] [Example 97] 3-((4,4-bis(2-(octyloxy)ethoxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis(2-(octyloxy)ethoxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 27 mg (58%). LCMS (Method B): Found m / z for (M+H) = 953.0, RT = 1.81 min.

[0512] [ka]

[0513] [Example 98] 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0514] Step 1: 4,4-bis(((Z)-non-2-en-1-yl)oxy)butanenitrile

[0515] [ka]

[0516] Prepared according to general procedure A using (Z)-non-2-en-1-ol. Yield 610 mg (38%). 1 H NMR (400 MHz, chloroform-d) δ 0.87 (t, J = 6.7 Hz, 6H), 1.19 - 1.41 (m, 18H), 1.97 (q, J = 7.3 Hz, 2H), 2.05 (q, J = 7.2 Hz, 4H), 2.42 (t, J = 7.4 Hz, 2H), 4.04 - 4.21 (m, 4H), 4.65 (t, J = 5.3 Hz, 1H), 5.45 - 5.65 (m, 4H).

[0517] Step 2: 4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoic acid

[0518] [ka]

[0519] Prepared from 4,4-bis(((Z)-non-2-en-1-yl)oxy)butanenitrile according to general procedure B. Yield 550 mg, 87%. 1H NMR (400 MHz, DMSO-d6) δ 0.85 (t, J = 6.4 Hz, 6H), 1.09 - 1.39 (m, 16H), 1.76 (q, J = 6.9 Hz, 2H), 2.02 (q, J = 6.8 Hz, 4H), 2.22 (t, J = 7.4 Hz, 2H), 3.95 - 4.11 (m, 4H), 4.53 (t, J = 5.6 Hz, 1H), 5.41 - 5.57 (m, 4H), 12.06 (s, 1H).

[0520] Step 3: 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0521] [ka]

[0522] Prepared from 4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoic acid according to general procedure C. Yield 390 mg, 71%. 1 H NMR (400 MHz, chloroform-d) δ 0.80 - 0.94 (m, 9H), 1.19 - 1.40 (m, 29H), 1.56 - 1.67 (m, 2H), 1.90 - 2.10 (m, 10H), 2.12 - 2.27 (m, 2H), 2.31 (t, J = 7.6 Hz, 2H), 2.41 (t, J = 7.4 Hz, 2H), 2.76 (t, J = 6.4 Hz, 2H), 3.60 (t, J = 5.9 Hz, 2H), 4.01 - 4.23 (m, 8H), 4.58 (t, J = 5.5Hz, 1H), 5.26 - 5.43 (m, 4H), 5.44 - 5.62 (m, 4H).

[0523] Step 4: 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 98). Prepared from 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 70 mg (71%). LCMS (Method B): Found m / z for (M+H) = 876.9, RT = 1.81 min.

[0524] [ka]

[0525] [Example 99] 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 45 mg (81%). LCMS (Method B): Found m / z for (M+H) = 860.9, RT = 1.56 min.

[0526] [ka]

[0527] [Example 100] 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0528] Step 1: 4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanenitrile

[0529] [ka]

[0530] Prepared according to general procedure A using (Z)-hex-3-en-1-ol. Yield 350 mg (51%).

[0531] Step 2: 4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoic acid

[0532] [ka]

[0533] Prepared from 4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanenitrile according to general procedure B. Yield 310 mg (82%). 1 H NMR (400 MHz, DMSO-d6) δ 0.92 (t, J = 7.5 Hz, 6H), 1.72 (q, J = 7.0 Hz, 2H), 1.95 - 2.07 (m, 4H), 2.17 - 2.27 (m, 6H), 3.31 - 3.42 (m, 2H), 3.44 - 3.55 (m, 2H), 4.49 (t, J = 5.6 Hz, 1H), 5.27 - 5.38 (m, 2H), 5.36 - 5.47 (m, 2H), 12.05 (s, 1H).

[0534] Step 3: 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0535] [ka]

[0536] Prepared from 4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoic acid according to general procedure C. Yield 165 mg (37%). 1 H NMR (400 MHz, chloroform-d) δ 0.88 (t, J = 6.7 Hz, 3H), 0.95 (t, J = 7.5 Hz, 6H), 1.30 (s, 16H), 1.56 - 1.66 (m, 2H), 1.88 - 1.98 (m, 2H), 2.04 (q, J = 7.2 Hz, 8H), 2.18 (t, J = 6.8 Hz, 2H), 2.31 (t, J = 7.3 Hz, 6H), 2.41 (t, J = 7.5 Hz, 2H), 2.76 (t, J = 6.4 Hz, 2H), 3.42 (q, J = 7.4 Hz, 2H), 3.52 - 3.65 (m, 4H), 4.17 (t, J = 5.3 Hz, 4H), 4.52 (t, J = 5.6 Hz, 1H), 5.27 - 5.41 (m, 4H), 5.41 - 5.50 (m, 2H).

[0537] Step 4: 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 100). Prepared from 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 29 mg (52%). LCMS (Method B): Found m / z for (M+H) = 792.8, RT = 1.49 min.

[0538] [ka]

[0539] [Example 101] 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 29 mg (51%). LCMS (Method B): Found m / z for (M+H) = 804.8, RT = 1.50 min.

[0540] [ka]

[0541] [Example 102] 3-((4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0542] Step 1: 4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanenitrile

[0543] [ka]

[0544] Prepared according to general procedure A using (Z)-hept-3-en-1-ol. Yield 310 mg (41%). 1 H NMR (400 MHz, chloroform-d) δ 0.90 (t, J = 7.4 Hz, 6H), 1.30 - 1.44 (m, 4H), 1.89 - 1.99 (m, 2H), 2.02 (q, J = 7.3 Hz, 4H), 2.32 (q, J = 7.0 Hz, 4H), 2.41 (t, J = 7.4 Hz, 2H), 3.40 - 3.48 (m, 2H), 3.54 - 3.66 (m, 2H), 4.58 (t, J = 5.3 Hz, 1H), 5.31 - 5.42 (m, 2H), 5.42 - 5.53 (m, 2H).

[0545] Step 2: 4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanoic acid

[0546] [ka]

[0547] Prepared from 4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanenitrile according to general procedure B. Yield 320 mg (83%). 1H NMR (400 MHz, DMSO-d6) δ 0.86 (t, J = 7.3 Hz, 6H), 1.21 - 1.40 (m, 4H), 1.72 (q, J = 7.2 Hz, 2H), 1.99 (q, J = 6.9 Hz, 4H), 2.18 - 2.28 (m, 6H), 3.31 - 3.42 (m, 2H), 3.44 - 3.55 (m, 2H), 4.49 (t, J = 5.6 Hz, 1H), 5.31 - 5.47 (m, 4H), 12.05 (s, 1H).

[0548] Step 3: 3-((4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0549] [ka]

[0550] Prepared from 4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanoic acid according to general procedure C. Yield 295 mg, 45%. 1 H NMR (400 MHz, chloroform-d) δ 0.83 - 0.93 (m, 9H), 1.22 - 1.44 (m, 18H), 1.57 - 1.67 (m, 2H), 1.88 - 2.09 (m, 10H), 2.19 (q, J = 5.9 Hz, 2H), 2.25 - 2.35 (m, 6H), 2.41 (t, J = 7.5 Hz, 2H), 2.76 (t, J = 6.5 Hz, 2H), 3.36 - 3.47 (m, 2H), 3.52 - 3.65 (m, 4H), 4.10 - 4.23 (m, 4H), 4.52 (t, J = 5.5 Hz, 1H), 5.26 - 5.51 (m, 8H).

[0551] Step 4: 3-((4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 102). Prepared from 3-((4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 40 mg (72%). LCMS (Method B): Found m / z for (M+H) = 820.8, RT = 1.60 min.

[0552] [ka]

[0553] [Example 103] 3-((4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 46 mg (83%). LCMS (Method B): Found m / z for (M+H) = 832.8, RT = 1.61 min.

[0554] [ka]

[0555] [Example 104] 3-((4,4-bis(oct-3-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0556] Step 1: 4,4-bis(oct-3-yn-1-yloxy)butanenitrile

[0557] [ka]

[0558] Prepared according to general procedure A using oct-3-yn-1-ol. Yield 410 mg (28%). 1 H NMR (400 MHz, chloroform-d) δ 0.89 (t, J = 7.1 Hz, 6H), 1.31 - 1.51 (m, 8H), 1.92 - 2.02 (m, 2H), 2.09 - 2.19 (m, 4H), 2.38 - 2.49 (m, 6H), 3.50 - 3.61 (m, 2H), 3.64 - 3.74 (m, 2H), 4.67 (t, J = 5.4 Hz, 1H).

[0559] Step 2: 4,4-bis(oct-3-yn-1-yloxy)butanoic acid

[0560] [ka]

[0561] Prepared from 4,4-bis(oct-3-yn-1-yloxy)butanenitrile according to general procedure B. Yield 310 mg (82%). 1H NMR (400 MHz, DMSO-d6) δ 0.86 (t, J = 7.0 Hz, 6H), 1.26 - 1.45 (m, 8H), 1.74 (q, J = 7.0 Hz, 2H), 2.07 - 2.17 (m, 4H), 2.25 (t, J = 7.5 Hz, 2H), 2.30 - 2.41 (m, 4H), 3.40 - 3.51 (m, 2H), 3.52 - 3.62 (m, 2H), 4.56 (t, J = 5.7 Hz, 1H), 12.06 (s, 1H).

[0562] Step 3: 3-((4,4-bis(oct-3-yn-1-yloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0563] [ka]

[0564] Prepared from 4,4-bis(oct-3-yn-1-yloxy)butanoic acid according to general procedure C. Yield 390 mg (60%). 1 H NMR (400 MHz, chloroform-d) δ 0.83 - 0.93 (m, 9H), 1.22 - 1.51 (m, 22H), 1.57 - 1.65 (m, 2H), 1.90 - 2.00 (m, 2H), 2.04 (q, J = 6.9 Hz, 4H), 2.08 - 2.23 (m, 6H), 2.31 (t, J = 7.6 Hz, 2H), 2.36 - 2.47 (m, 6H), 2.76 (t, J = 6.4 Hz, 2H), 3.48 - 3.71 (m, 6H), 4.08 - 4.23 (m, 4H), 4.60 (t, J = 5.6 Hz, 1H), 5.26 - 5.42 (m, 4H).

[0565] Step 4: 3-((4,4-bis(oct-3-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 104). Prepared from 3-((4,4-bis(oct-3-yn-1-yloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 62 mg (67%). LCMS (Method B): Found m / z for (M+H) = 844.9, RT = 1.56 min.

[0566] [ka]

[0567] [Example 105] 3-((4,4-Bis(oct-3-yn-1-yloxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 43 mg (51%). LCMS (Method B): Found m / z for (M+H) = 856.9, RT = 1.57 min.

[0568] [ka]

[0569] [Example 106] 3-((4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0570] Step 1: 4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanenitrile

[0571] [ka]

[0572] Prepared according to general procedure A using (Z)-oct-3-en-1-ol. Yield 100 mg (40%). 1 H NMR (400 MHz, chloroform-d) δ 0.79 - 0.99 (m, 7H), 1.16 - 1.39 (m, 9H), 1.90 - 1.98 (m, 2H), 2.03 (d, J = 6.9 Hz, 4H), 2.32 (q, J = 7.0 Hz, 4H), 2.41 (t, J = 7.4 Hz, 2H), 3.39 - 3.49 (m, 2H), 3.56 - 3.66 (m, 2H), 4.57 (t, J = 5.4 Hz, 1H), 5.29 - 5.40 (m, 2H), 5.41 - 5.52 (m, 2H).

[0573] Step 2: 4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoic acid

[0574] [ka]

[0575] Prepared from 4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanenitrile according to general procedure B. Yield 320 mg, 83%. 1H NMR (400 MHz, DMSO-d6) δ 0.83 - 0.89 (m, 6H), 1.18 - 1.36 (m, 8H), 1.72 (q, J = 7.0 Hz, 2H), 2.01 (q, J = 6.7 Hz, 4H), 2.15 - 2.29 (m, 6H), 3.31 - 3.42 (m, 2H), 3.44 - 3.54 (m, 2H), 4.49 (t, J = 5.5 Hz, 1H), 5.30 - 5.47 (m, 4H), 12.06 (s, 1H).

[0576] Step 3: 3-((4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0577] [ka]

[0578] Prepared from 4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoic acid according to general procedure C. Yield 310 mg, 48%. 1 H NMR (400 MHz, chloroform-d) δ 0.84 - 0.93 (m, 9H), 1.20 - 1.42 (m, 25H), 1.55 - 1.69 (m, 2H), 1.89 - 1.98 (m, 2H), 1.98 - 2.08 (m, 7H), 2.13 - 2.22 (m, 2H), 2.31 (t, J = 7.2 Hz, 5H), 2.41 (t, J = 7.5 Hz, 2H), 2.76 (t, J = 6.0 Hz, 2H), 3.42 (q, J = 7.6 Hz, 2H), 3.52 - 3.65 (m, 4H), 4.09 - 4.28 (m, 4H), 4.48 - 4.56 (m, 1H), 5.27 - 5.41 (m, 5H), 5.39 - 5.53 (m, 2H).

[0579] Step 4: 3-((4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 106). Prepared from 3-((4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 47 mg (82%). LCMS (Method B): Found m / z for (M+H) = 848.9, RT = 1.72 min.

[0580] [ka]

[0581] [Example 107] 3-((4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 45 mg (81%). LCMS (Method B): Found m / z for (M+H) = 860.9, RT = 1.56 min.

[0582] [ka]

[0583] [Example 108] 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0584] Step 1: 4,4-bis(((Z)-nona-6-en-1-yl)oxy)butanenitrile

[0585] [ka]

[0586] Prepared according to general procedure A using (Z)-non-6-en-1-ol. Yield 610 mg (37%). 1 H NMR (400 MHz, chloroform-d) δ 0.94 (t, J = 7.5 Hz, 6H), 1.29 - 1.43 (m, 8H), 1.53 - 1.63 (m, 4H), 1.89 - 1.97 (m, 2H), 1.96 - 2.11 (m, 8H), 2.41 (t, J = 7.4 Hz, 2H), 3.36 - 3.47 (m, 2H), 3.53 - 3.65 (m, 2H), 4.54 (t, J = 6.9 Hz, 1H), 5.18 - 5.52 (m, 4H).

[0587] Step 2: 4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoic acid

[0588] [ka]

[0589] Prepared from 4,4-bis(((Z)-non-6-en-1-yl)oxy)butanenitrile according to general procedure B. Yield 280 mg, 73%. 1H NMR (400 MHz, DMSO-d6) δ 0.91 (t, J = 7.5 Hz, 6H), 1.21 - 1.34 (m, 13H), 1.40 (t, J = 6.7 Hz, 2H), 1.43 - 1.52 (m, 2H), 1.72 (q, J = 7.1 Hz, 1H), 1.91 - 2.07 (m, 8H), 2.21 (t, J = 7.4 Hz, 1H), 3.32 - 3.41 (m, 3H), 3.42 - 3.53 (m, 1H), 4.32 (s, 1H), 5.24 - 5.39 (m, 4H).

[0590] Step 3: 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0591] [ka]

[0592] Prepared from 4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoic acid according to general procedure C. Yield 318 mg, 49%. 1 H NMR (400 MHz, chloroform-d) δ 0.86 - 0.90 (m, 4H), 0.94 (t, J = 7.5 Hz, 9H), 1.30 - 1.41 (m, 15H), 1.56 - 1.67 (m, 2H), 1.92 (q, J = 7.3 Hz, 3H), 2.01 - 2.04 (m, 5H), 2.19 (q, J = 6.5 Hz, 3H), 2.31 (t, J = 7.6 Hz, 3H), 2.40 (t, J = 7.5 Hz, 3H), 2.76 (t, J = 6.4 Hz, 3H), 3.34 - 3.44 (m, 3H), 3.50 - 3.65 (m, 6H), 4.06 - 4.23 (m, 7H), 4.48 (t, J = 5.5 Hz, 2H), 5.17 - 5.50 (m, 12H).

[0593] Step 4: 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 108). Prepared from 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 36 mg (69%). LCMS (Method B): Found m / z for (M+H) = 876.9, RT = 1.78 min.

[0594] [ka]

[0595] [Example 109] 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 37 mg (67%). LCMS (Method B): Found m / z for (M+H) = 888.9, RT = 1.77 min.

[0596] [ka]

[0597] [Example 110] 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0598] Step 1: 4,4-bis((8-bromooctyl)oxy)butanenitrile

[0599] [ka]

[0600] Prepared according to general procedure A using 8-bromootan-1-ol. Yield 750 mg (33%). 1 H NMR (400 MHz, chloroform-d) δ 1.22 - 1.41 (m, 12H), 1.37 - 1.48 (m, 4H), 1.56 (t, J = 7.3 Hz, 4H), 1.78 - 1.91 (m, 4H), 1.88 - 1.98 (m, 2H), 2.41 (t, J = 7.3 Hz, 2H), 3.33 - 3.47 (m, 6H), 3.51 - 3.64 (m, 2H), 4.54 (t, J = 5.3 Hz, 1H).

[0601] Step 2: 4,4-bis((8-fluorooctyl)oxy)butanenitrile

[0602] [ka]

[0603] A solution of tetrabutylammonium fluoride in THF (1 M, 4.65 mL, 4.65 mmol, 3 Eq) was added to 4,4-bis((8-bromooctyl)oxy)butanenitrile (750 mg, 1.55 mmol). The mixture was stirred at 80° C. for 24 hours. After this time, the reaction mass was rinsed with ice-cold water, and then the organic layer was extracted with ethyl acetate (30 mL×3). The organic layer was washed with brine (5 mL×3) and then dried over sodium sulfate. The organic layer was evaporated to give the crude product. The crude product was subjected to column chromatography (using ethyl acetate / hexane) to give 4,4-bis((8-fluorooctyl)oxy)butanenitrile (210 mg, 37%) as a pale yellow gum. 1 H NMR (400 MHz, chloroform-d) δ 1.30 - 1.45 (m, 18H), 1.56 - 1.77 (m, 6H), 1.88 - 1.98 (m, 2H), 2.41 (t, J = 7.3 Hz, 2H), 3.32 - 3.47 (m, 2H), 3.54 - 3.64 (m, 2H), 4.37 (t, J = 6.2 Hz, 2H), 4.48 (t, J = 6.1 Hz, 2H), 4.54 (t, J = 5.3 Hz, 1H).

[0604] Step 3: 4,4-bis((8-fluorooctyl)oxy)butanoic acid

[0605] [ka]

[0606] Prepared from 4,4-bis((8-fluorooctyl)oxy)butanenitrile according to general procedure B. Yield 200 mg (92%). 1H NMR (400 MHz, DMSO-d6) δ 1.21 - 1.37 (m, 14H), 1.48 (t, J = 6.6 Hz, 4H), 1.53 - 1.77 (m, 6H), 2.22 (t, J = 7.4 Hz, 2H), 3.27 - 3.40 (m, 3H), 3.43 - 3.54 (m, 2H), 4.36 (t, J = 6.1 Hz, 2H), 4.41 - 4.52 (m, 3H), 12.05 (s, 1H).

[0607] Step 4: 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0608] [ka]

[0609] Prepared from 4,4-bis((8-fluorooctyl)oxy)butanoic acid according to general procedure C. Yield 160 mg, 37%. 1 H NMR (400 MHz, chloroform-d) δ 0.88 (t, J = 6.9 Hz, 3H), 1.21 - 1.44 (m, 34H), 1.58 - 1.77 (m, 6H), 1.87 - 1.97 (m, 2H), 2.04 (q, J = 6.8 Hz, 4H), 2.13 - 2.24 (m, 2H), 2.31 (t, J = 7.6 Hz, 2H), 2.40 (t, J = 7.5 Hz, 2H), 2.76 (t, J = 6.4 Hz, 2H), 3.34 - 3.44 (m, 2H), 3.50 - 3.65 (m, 4H), 4.10 - 4.23 (m, 4H), 4.36 (t, J = 6.1 Hz, 2H), 4.44 - 4.52 (m, 3H), 5.25 - 5.44 (m, 4H).

[0610] Step 5: 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 110). Prepared from 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 18 mg (21%). LCMS (Method B): Found m / z for (M+H) = 888.9, RT = 1.40 min.

[0611] [ka]

[0612] [Example 111] 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 41 mg (48%). LCMS (Method B): Found m / z for (M+H) = 900.9, RT = 1.41 min.

[0613] [ka]

[0614] [Example 112] 3-((6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0615] Step 1: 2-Methoxycyclohexan-1-one oxime

[0616] [ka]

[0617] To a stirred solution of 2-methoxycyclohexan-1-one (5 g, 39.0 mmol) in MeOH-HO (150 mL, 1:2) was added sodium acetate (6.41 g, 78.1 mmol), followed by hydroxylamine hydrochloride (5.43 g, 78.1 mmol). The resulting mixture was heated in a sealed tube at 70 °C for 16 h. After that time, the MeOH was evaporated, and the mixture was diluted with water, extracted with ethyl acetate (3 × 50 mL), washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude product thus obtained was purified by flash column chromatography eluting with 3% ethyl acetate in hexane to give 2-methoxycyclohexan-1-one oxime (4.1 g, 88%) as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) δ 1.17-1.32 (m, 1H), 1.40-1.56 (m, 2H), 1.59-1.82 (m, 3H), 1.92-1.99 (m, 1H), 2.93-3.01 (m, 1H), 3.11 (s, 3H), 3.63 (s, 1H), 10.61 (s, 1H).

[0618] Step 2: 6,6-Dimethoxyhexanenitrile

[0619] [ka]

[0620] To a stirred solution of 2-methoxycyclohexan-1-one oxime (2.0 g, 14.0 mmol) in CCl4 (20 mL) was added dropwise thionyl chloride (1.2 mL, 16.8 mmol) at 0 °C and stirred for 10 min. After that time, dry MeOH (20 mL) was added to the reaction mixture at 5-10 °C. After the addition was complete, the reaction mixture was allowed to reach room temperature and stand for 2 h. The mixture was then diluted with ethyl acetate, washed with brine, dried over sodium sulfate, and concentrated to give 6,6-dimethoxyhexanenitrile (1.6 g, 81%) as a pale yellow oil. 1 H NMR (400 MHz, chloroform-d) δ 1.44–1.56 (m, 2H), 1.57–1.75 (m, 4H), 2.34 (t, J = 7.0 Hz, 2H), 3.31 (s, 6H), 4.34 (t, J = 5.5 Hz, 1H).

[0621] Step 3: 6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanenitrile

[0622] [ka]

[0623] Prepared from 6,6-dimethoxyhexanenitrile and (Z)-oct-3-en-1-ol according to general procedure A. Yield 280 mg (50%). 1 H NMR (400 MHz, DMSO-d6) δ 0.83 - 0.89 (m, 6H), 1.21 - 1.32 (m, 8H), 1.31 - 1.43 (m, 2H), 1.47 - 1.61 (m, 4H), 2.01 (q, J = 6.7 Hz, 4H), 2.23 (q, J = 6.8 Hz, 4H), 2.47 (d, J = 7.1 Hz, 2H), 3.32 - 3.42 (m, 2H), 3.44 - 3.54 (m, 2H), 4.47 (t, J = 5.6 Hz, 1H), 5.30 - 5.47 (m, 4H).

[0624] Step 4: 6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoic acid

[0625] [ka]

[0626] Prepared from 6,6-dimethoxyhexanenitrile according to general procedure B. Yield 260 mg (80%). 1 H NMR (400 MHz, DMSO-d6) δ 0.86 (q, J = 3.8, 5.1 Hz, 6H), 1.21 - 1.34 (m, 10H), 1.48 (t, J = 7.6 Hz, 4H), 2.00 (t, J = 6.7 Hz, 4H), 2.14 - 2.27 (m, 6H), 3.30 - 3.41 (m, 2H), 3.42 - 3.53 (m, 2H), 4.44 (t, J = 5.6 Hz, 1H), 5.30 - 5.46 (m, 4H), 11.97 (s, 1H).

[0627] Step 5: 3-((6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0628] [ka]

[0629] Prepared from 6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoic acid according to general procedure C. Yield 70 mg (55%). 1H NMR (400 MHz, chloroform-d) δ 0.83 - 0.95 (m, 9H), 1.17 - 1.42 (m, 25H), 1.57 - 1.70 (m, 5H), 2.04 (d, J = 7.0 Hz, 8H), 2.18 (t, J = 6.2 Hz, 2H), 2.31 (t, J = 6.7 Hz, 8H), 2.76 (t, J = 6.5 Hz, 2H), 3.41 (q, J = 7.5 Hz, 2H), 3.50 - 3.64 (m, 4H), 4.09 - 4.20 (m, 4H), 4.48 (t, J = 5.6 Hz, 1H), 5.32 - 5.37 (m, 5H), 5.33 - 5.48 (m, 3H).

[0630] Step 6: 3-((6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 112). Prepared from 3-((6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 37 mg (64%). LCMS (Method B): Found m / z for (M+H) = 877.0, RT = 1.70 min.

[0631] [ka]

[0632] [Example 113] 3-((6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 89 mg (70%). LCMS (Method B): Found m / z for (M+H) = 889.0, RT = 1.79 min.

[0633] [ka]

[0634] [Example 114] 3-((6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0635] Step 1: 6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanenitrile

[0636] [ka]

[0637] Prepared from 6,6-dimethoxyhexanenitrile and (Z)-hex-3-en-1-ol according to general procedure A. Yield 190 mg, 42%. 1H NMR (400 MHz, chloroform-d) δ 0.96 (t, J = 7.5 Hz, 6H), 1.58 - 1.74 (m, 6H), 1.99 - 2.11 (m, 4H), 2.20 - 2.38 (m, 6H), 3.36 - 3.47 (m, 2H), 3.52 - 3.62 (m, 2H), 4.48 (t, J = 5.5 Hz, 1H), 5.27 - 5.39 (m, 2H), 5.46 (q, J = 7.6 Hz, 2H).

[0638] Step 2: 6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoic acid

[0639] [ka]

[0640] Prepared from 6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanenitrile according to general procedure B. Yield 170 mg, 82%. 1 H NMR (400 MHz, DMSO-d6) δ 0.92 (t, J = 7.5 Hz, 6H), 1.21 - 1.32 (m, 2H), 1.42 - 1.55 (m, 4H), 1.95 - 2.07 (m, 4H), 2.14 - 2.27 (m, 6H), 3.31 - 3.42 (m, 2H), 3.43 - 3.54 (m, 2H), 4.45 (t, J = 5.6 Hz, 1H), 5.27 - 5.38 (m, 2H), 5.36 - 5.47 (m, 2H), 11.96 (s, 1H).

[0641] Step 3: 3-((6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0642] [ka]

[0643] Prepared from 6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoic acid according to general procedure C. Yield 60 mg (45%). 1 H NMR (400 MHz, chloroform-d) δ 0.88 (t, J = 6.7 Hz, 3H), 0.95 (t, J = 7.5 Hz, 6H), 1.18 - 1.44 (m, 15H), 1.56 - 1.70 (m, 7H), 1.99 - 2.11 (m, 8H), 2.13 - 2.23 (m, 2H), 2.23 - 2.37 (m, 8H), 2.76 (t, J = 6.4 Hz, 2H), 3.36-3.47 (m, 2H), 3.50 - 3.64 (m, 4H), 4.09 - 4.24 (m, 4H), 4.48 (t, J = 5.6 Hz, 1H), 5.26 - 5.51 (m, 8H).

[0644] Step 4: 3-((6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 114). Prepared from 3-((6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 36 mg (62%). LCMS (Method B): Found m / z for (M+H) = 820.9, RT = 1.70 min.

[0645] [ka]

[0646] [Example 115] 3-((6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 69 mg (72%). LCMS (Method B): Found m / z for (M+H) = 833.0, RT = 1.60 min.

[0647] [ka]

[0648] [Example 116] 3-((6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0649] Step 1: 6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanenitrile

[0650] [ka]

[0651] Prepared from 6,6-dimethoxyhexanenitrile and (Z)-oct-5-en-1-ol according to general procedure A. Yield 210 mg (47%). 1H NMR (400 MHz, chloroform-d) δ 0.94 (t, J = 7.5 Hz, 6H), 1.34 - 1.52 (m, 4H), 1.47 - 1.74 (m, 10H), 1.95 - 2.10 (m, 8H), 2.33 (t, J = 7.1 Hz, 2H), 3.34 - 3.45 (m, 2H), 3.51 - 3.61 (m, 2H), 4.44 (t, J = 5.5 Hz, 1H), 5.25 - 5.42 (m, 4H).

[0652] Step 2: 6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoic acid

[0653] [ka]

[0654] Prepared from 6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanenitrile according to general procedure B. Yield 150 mg, 94%. 1 H NMR (400 MHz, DMSO-d6) δ 0.91 (t, J = 7.5 Hz, 6H), 1.24 - 1.41 (m, 6H), 1.42 - 1.53 (m, 8H), 1.93 - 2.06 (m, 8H), 2.18 (t, J = 7.3 Hz, 2H), 3.31 - 3.40 (m, 2H), 3.43 - 3.53 (m, 2H), 4.40 (t, J = 5.6 Hz, 1H), 5.19 - 5.45 (m, 4H), 11.97 (s, 1H).

[0655] Step 3: 3-((6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0656] [ka]

[0657] Prepared from 6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoic acid according to general procedure C. Yield 90 mg (45%). 1 H NMR (400 MHz, chloroform-d) δ 0.88 (t, J = 6.8 Hz, 3H), 0.94 (t, J = 7.5 Hz, 6H), 1.20 - 1.47 (m, 23H), 1.50 - 1.70 (m, 9H), 1.94 - 2.11 (m, 12H), 2.10 - 2.23 (m, 2H), 2.27 - 2.37 (m, 4H), 2.76 (t, J = 6.4 Hz, 2H), 3.34 - 3.44 (m, 2H), 3.50 - 3.58 (m, 2H), 3.60 (t, J = 5.6 Hz, 2H), 4.09 - 4.24 (m, 4H), 4.44 (t, J = 5.6 Hz, 1H), 5.25 - 5.43 (m, 8H).

[0658] Step 4: 3-((6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 116). Prepared from 3-((6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 24 mg (57%). LCMS (Method B): Found m / z for (M+H) = 876.8, RT = 1.70 min.

[0659] [ka]

[0660] [Example 117] 3-((6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 50 mg (50%). LCMS (Method B): Found m / z for (M+H) = 889.2, RT = 1.73 min.

[0661] [ka]

[0662] [Example 118] 3-((6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0663] Step 1: 6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanenitrile

[0664] [ka]

[0665] Prepared from 6,6-dimethoxyhexanenitrile and (Z)-dec-4-en-1-ol according to general procedure A. Yield 150 mg (38%). 1H NMR (400 MHz, chloroform-d) δ 0.88 (t, J = 6.7 Hz, 6H), 1.17 - 1.41 (m, 12H), 1.45 - 1.57 (m, 2H), 1.59 - 1.74 (m, 8H), 1.87 - 2.20 (m, 8H), 2.33 (t, J = 7.1 Hz, 2H), 3.35 - 3.45 (m, 2H), 3.50 - 3.70 (m, 2H), 4.44 (t, J = 5.5 Hz, 1H), 5.28 - 5.45 (m, 4H)

[0666] Step 2: 6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoic acid

[0667] [ka]

[0668] Prepared from 6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanenitrile according to general procedure B. Yield 150 mg, 95%. 1 H NMR (400 MHz, DMSO-d6) δ 0.85 (t, J = 6.7 Hz, 6H), 1.18 - 1.37 (m, 14H), 1.45 - 1.56 (m, 8H), 1.91 - 2.10 (m, 8H), 2.18 (t, J = 7.4 Hz, 2H), 3.32 - 3.41 (m, 2H), 3.43 - 3.53 (m, 2H), 4.37 - 4.44 (m, 1H), 5.25 - 5.46 (m, 4H), 11.96 (s, 1H).

[0669] Step 3: 3-((6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0670] [ka]

[0671] Prepared from 6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoic acid according to general procedure C. Yield 87 mg (42%). 1 H NMR (400 MHz, chloroform-d) δ 0.88 (t, J = 6.8 Hz, 9H), 1.17 - 1.45 (m, 24H), 1.51 - 1.71 (m, 10H), 1.96 - 2.17 (m, 12H), 2.32 (q, J = 7.3 Hz, 4H), 2.76 (t, J = 6.5 Hz, 2H), 3.35 - 3.45 (m, 2H), 3.51 - 3.64 (m, 4H), 4.09 - 4.24 (m, 4H), 4.44 (t, J = 5.6 Hz, 1H), 5.28 - 5.44 (m, 8H).

[0672] Step 4: 3-((6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Prepared from 3-((6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 32 mg (59%). LCMS (Method B): Found m / z for (M+H) = 932.9, RT = 1.70 min.

[0673] [ka]

[0674] [Example 119] 3-((6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 51 mg (52%). LCMS (Method B): Found m / z for (M+H) = 945.2, RT = 1.70 min.

[0675] [ka]

[0676] [Example 120] 3-((6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0677] Step 1: 6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanenitrile

[0678] [ka]

[0679] Prepared from 6,6-dimethoxyhexanenitrile and 3,7-dimethyloct-6-en-1-ol according to general procedure A. Yield 220 mg (42%). 1H NMR (400 MHz, chloroform-d) δ 0.88 (d, J = 6.4 Hz, 6H), 1.11 - 1.22 (m, 3H), 1.25 - 1.43 (m, 6H), 1.45 - 1.77 (m, 19H), 1.87 - 2.04 (m, 4H), 2.33 (t, J = 7.1 Hz, 2H), 3.35 - 3.50 (m, 2H), 3.52 - 3.66 (m, 2H), 4.44 (t, J = 5.5 Hz, 1H), 5.08 (t, J = 7.3 Hz, 2H).

[0680] Step 2: 6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoic acid

[0681] [ka]

[0682] Prepared from 6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanenitrile according to general procedure B. Yield 220 mg (95%). 1 H NMR (400 MHz, DMSO-d6) δ 0.85 (d, J = 5.9 Hz, 6H), 1.13 - 1.03 (m, 2H), 1.40 - 1.17 (m, 8H), 1.58 - 1.44 (m, 12H), 1.70 - 1.59 (m, 6H), 2.03 - 1.87 (m, 5H), 2.18 (t, J = 7.3 Hz, 1H), 3.44 - 3.33 (m, 2H), 3.60 - 3.44 (m, 2H), 4.40 (t, J = 5.4 Hz, 1H), 5.07 (t, J = 7.3 Hz, 2H), 11.97 (s, 1H).

[0683] Step 3: 3-((6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0684] [ka]

[0685] Prepared from 6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoic acid according to general procedure C. Yield 90 mg (42%). 1 H NMR (400 MHz, chloroform-d) δ 0.88 (d, J = 6.1 Hz, 10H), 1.18 - 1.45 (m, 15H), 1.51 - 1.57 (m, 22H), 1.67 (s, 5H), 1.83 - 2.09 (m, 8H), 2.16 - 2.23 (m, 2H), 2.32 (q, J = 7.5 Hz, 4H), 2.75 (d, J = 6.1 Hz, 2H), 3.32 - 3.69 (m, 7H), 4.10 - 4.21 (m, 4H), 4.40 - 4.48 (m, 1H), 5.03 - 5.16 (m, 2H), 5.28 - 5.42 (m, 4H).

[0686] Step 4: 3-((6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 120). Prepared from 3-((6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 32 mg (59%). LCMS (Method B): Found m / z for (M+H) = 933.0, RT = 1.70 min.

[0687] [ka]

[0688] [Example 121] 3-((6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 32 mg (59%). LCMS (Method B): Found m / z for (M+H) = 945.2, RT = 1.96 min.

[0689] [ka]

[0690] [Example 122] 3-((6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0691] Step 1: 6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanenitrile

[0692] [ka]

[0693] Prepared from 6,6-dimethoxyhexanenitrile and 7,7,8,8,8-pentafluorooctan-1-ol according to general procedure A. Yield 300 mg (44%). 1H NMR (400 MHz, chloroform-d) δ 1.35 - 1.45 (m, 8H), 1.45 - 1.56 (m, 2H), 1.54 - 1.74 (m, 12H), 1.91 - 2.09 (m, 4H), 2.34 (t, J = 7.0 Hz, 2H), 3.34 - 3.45 (m, 2H), 3.51 - 3.61 (m, 2H), 4.44 (t, J = 5.5 Hz, 1H)

[0694] Step 2: 6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoic acid

[0695] [ka]

[0696] Prepared from 6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanenitrile according to general procedure B. Yield 296 mg (95%). 1 H NMR (400 MHz, DMSO-d6) δ 1.18 - 1.40 (m, 10H), 1.38 - 1.55 (m, 12H), 2.04 - 2.23 (m, 6H), 3.29 - 3.38 (m, 2H), 3.40 - 3.51 (m, 2H), 4.39 (t, J = 5.6 Hz, 1H), 11.93 (s, 1H).

[0697] Step 3: 3-((6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0698] [ka]

[0699] Prepared from 6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoic acid according to general procedure C. Yield 140 mg (57%). 1 H NMR (400 MHz, chloroform-d) δ 0.87 (d, J = 7.3 Hz, 3H), 1.22 - 1.46 (m, 27H), 1.56 - 1.70 (m, 9H), 1.88 - 2.10 (m, 9H), 2.11 - 2.25 (m, 2H), 2.26 - 2.37 (m, 4H), 2.76 (t, J = 7.3 Hz, 2H), 3.36 - 3.44 (m, 2H), 3.49 - 3.58 (m, 2H), 3.60 (t, J = 5.7 Hz, 2H), 3.91 - 4.10 (m, 1H), 4.09 - 4.26 (m, 4H), 4.36 - 4.53 (m, 1H), 5.22 - 5.52 (m, 4H).

[0700] Step 4: 3-((6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 122). Prepared from 3-((6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate and 3-(diethylamino)-1-propanol according to general procedure D. Yield 51 mg (61%). LCMS (Method B): Found m / z for (M+H) = 1060.7, RT = 1.70 min.

[0701] [ka]

[0702] [Example 123] 3-((6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate: Prepared from 3-((6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to general procedure D. Yield 80 mg (74%). LCMS (Method B): Found m / z for (M+H) = 1073.2, RT = 1.81 min.

[0703] [ka]

[0704] [Example 124] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(2-hydroxyethyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0705] Step 1: Ethyl 1-(2-hydroxyethyl)piperidine-3-carboxylate

[0706] [ka]

[0707] To a stirred solution of ethyl piperidine-3-carboxylate (2 g, 12.74 mmol) in anhydrous acetone (10 mL) was added 2-bromoethan-1-ol (1.89 g, 15.28 mmol), dry powdered K2CO3 (3.5 g, 25.47 mmol), and KI (0.42 g, 2.55 mmol). The reaction mixture was stirred at 25 °C for 18 hours. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 30% ethyl acetate-hexane to give ethyl 1-(2-hydroxyethyl)piperidine-3-carboxylate (2.2 g, 85%) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ 1.17 - 1.29 (m, 3H), 1.46 - 1.62 (m, 2H), 1.60 - 1.76 (m, 1H), 1.81 - 1.95 (m, 1H), 2.14 - 2.19 (m, 1H), 2.38 (t, J = 10.4 Hz, 1H), 2.42 - 2.59 (m, 4H), 2.62 - 2.71 (m, 1H), 2.80 - 2.89 (m, 1H), 3.52 - 3.63 (m, 2H), 4.10 (q, J = 7.1 Hz, 2H).

[0708] Step 2: Ethyl 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-3-carboxylate

[0709] [ka]

[0710] To a stirred solution of ethyl 1-(2-hydroxyethyl)piperidine-3-carboxylate (200 mg, 0.99 mmol) in DCM (2 mL) was added triethylamine (0.35 mL, 2.48 mmol) and tert-butyldimethylsilyl chloride (225 mg, 1.49 mmol) at 0° C. The reaction mixture was stirred at 25° C. for 14 hours. The reaction mixture was then quenched with water, extracted with DCM (50 mL), and washed with brine solution (25 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by Combiflash chromatography eluting with 5% ethyl acetate-hexane to give ethyl 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-3-carboxylate (160 mg, 51%) as a sticky solid. 1 H NMR (400 MHz, chloroform-d) δ 0.04 (s, 6H), 0.87 (s, 9H), 1.23 (t, J = 7.1 Hz, 3H), 1.32 - 1.48 (m, 1H), 1.47 - 1.64 (m, 1H), 1.63 - 1.77 (m, 1H), 1.87 - 1.98 (m, 1H), 2.00 - 2.13 (m, 1H), 2.22 (t, J = 10.9 Hz, 1H), 2.48 - 2.59 (m, 3H), 2.78 (d, J = 11.2 Hz, 1H), 3.04 (d, J = 13.4 Hz, 1H), 3.73 (t, J = 6.4 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H).

[0711] Step 3: (1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)methanol

[0712] [ka]

[0713] To a stirred solution of ethyl 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-3-carboxylate (500 mg, 1.58 mmol) in THF (5 mL) was added lithium aluminum hydride (1 M in THF) (3.2 mL, 3.17 mmol) at 0° C. and the reaction mixture was stirred at 25° C. for 1 h. Upon completion, the reaction mass was quenched with saturated aqueous NaSO (10 mL) at 0° C. and filtered. The filtrate was concentrated under reduced pressure to afford (1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)methanol (400 mg, 92%) as a colorless oil, which was carried on to the next step without further purification. 1 H NMR (400 MHz, chloroform-d) δ 0.04 (s, 6H), 0.87 (s, 9H), 1.07 - 1.22 (m, 1H), 1.49 - 1.62 (m, 1H), 1.62 - 1.72 (m, 1H), 1.72 - 1.85 (m, 3H), 2.11 (d, J = 10.5 Hz, 1H), 2.23 (d, J = 11.6 Hz, 1H), 2.48 (t, J = 6.5 Hz, 2H), 2.55 - 2.72 (m, 1H), 2.82 (d, J = 10.8 Hz, 1H), 3.53 (dd, J = 5.8, 10.6 Hz, 1H), 3.57 - 3.70 (m, 1H), 3.73 (t, J = 6.4 Hz, 2H).

[0714] Step 4: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0715] [ka]

[0716] To a stirred solution of 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (Intermediate IVa) (100 mg, 0.14 mmol) in DCM (1 mL) were added pyridine (0.03 mL, 0.29 mmol), N,N-dimethylpyridin-4-amine (5.3 mg, 0.04 mmol), and 4-nitrophenyl chloroformate (72.36 mg, 0.29 mmol) and stirred at 25° C. for 1 h. Then, (1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)methanol (158 mg, 0.58 mmol) and DIPEA (0.1 mL, 0.58 mmol) were added and stirred for an additional 16 h. The reaction mixture was diluted with DCM (10 mL) and washed with 1 M NaCO solution (3 × 10 mL), water (10 mL), and brine. The combined organic portions were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 2% MeOH-DCM to give 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (50 mg, 35%) as a pale yellow oil. LCMS (Method B): (M+H) found m / z = 991.1, RT = 1.14 min.

[0717] Step 5: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(2-hydroxyethyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 124). To a stirred solution of 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (60 mg, 0.061 mmol) in anhydrous THF (1 mL) at 0° C. was added tetra-n-butylammonium fluoride (TBAF) (0.12 mL, 0.12 mmol) (1 M solution in tetrahydrofuran). The resulting solution was stirred for 4 h after allowing the mixture to warm to room temperature. The resulting solution was diluted with DCM (5 mL) and quenched with water (5 mL). The organic layer was extracted with DCM (2 × 5 mL), brine (5 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 2% MeOH-DCM to give 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(2-hydroxyethyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (35 mg, 66%) as a brown sticky gum. LCMS (Method B): (M+H) found m / z = 876.7, RT = 1.58 min.

[0718] [ka]

[0719] [Example 125] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(3-hydroxypropyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0720] Step 1: Ethyl 1-(3-hydroxypropyl)piperidine-3-carboxylate

[0721] [ka]

[0722] To a stirred solution of ethyl piperidine-3-carboxylate (2 g, 12.74 mmol) in anhydrous acetone (10 mL) was added 3-bromopropan-1-ol (2.12 g, 15.28 mmol), dry powdered K2CO3 (3.5 g, 25.47 mmol), and KI (0.42 g, 2.55 mmol). The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 25% ethyl acetate-hexane to give ethyl 1-(3-hydroxypropyl)piperidine-3-carboxylate (2.0 g, 73%) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ 1.23 (t, J = 7.1 Hz, 3H), 1.37 - 1.59 (m, 2H), 1.62 - 1.79 (m, 3H), 1.88 - 1.95 (m, 1H), 1.96 - 2.08 (m, 1H), 2.11 - 2.22 (m, 2H), 2.47 - 2.56 (m, 1H), 2.58 (t, J = 5.7 Hz, 2H), 2.84 (d, J = 11.3 Hz, 1H), 3.06 (d, J = 9.7 Hz, 1H), 3.76 (t, J = 5.2 Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H).

[0723] Step 2: Ethyl 1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidine-3-carboxylate

[0724] [ka]

[0725] To a stirred solution of ethyl 1-(3-hydroxypropyl)piperidine-3-carboxylate (200 mg, 0.93 mmol) in DCM (2 mL) was added triethylamine (0.3 mL, 3.31 mmol) and tert-butyldimethylsilyl chloride (211 mg, 1.39 mmol) at 0° C. The reaction mixture was slowly warmed to 25° C. and stirred for 14 hours. The reaction mixture was then quenched with water, extracted with DCM, and washed with brine solution. The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 5% ethyl acetate-hexane to give ethyl 1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidine-3-carboxylate (160 mg, 52%) as a sticky solid. 1 H NMR (400 MHz, chloroform-d) δ 0.03 (s, 6H), 0.87 (s, 9H), 1.24 (t, J = 7.1 Hz, 3H), 1.34 - 1.63 (m, 2H), 1.63 - 1.75 (m, 3H), 1.87 - 2.01 (m, 2H), 2.11 (t, J = 10.7 Hz, 1H), 2.35 - 2.44 (m, 2H), 2.47 - 2.59 (m, 1H), 2.75 (d, J = 11.2 Hz, 1H), 2.96 (d, J = 10.1 Hz, 1H), 3.63 (t, J = 6.3 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H).

[0726] Step 3: (1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidin-3-yl)methanol

[0727] [ka]

[0728] To a stirred solution of 1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidine-3-carboxylate (100 mg, 0.30 mmol) in THF (2 mL) was added lithium aluminum hydride (1 M in THF) (0.6 mL, 0.60 mmol) at 0° C. and the reaction mass was stirred at 0° C. for 1 h. Upon completion, the reaction mass was quenched with saturated aqueous NaSO at 0° C. and filtered. The filtrate was evaporated to give (1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidin-3-yl)methanol (80 mg, 92%) as a colorless oil, which was used in the next step without further purification. 1 H NMR (400 MHz, chloroform-d) δ 0.03 (s, 6H), 0.88 (s, 9H), 1.10 - 1.23 (m, 1H), 1.49 - 1.87 (m, 7H), 1.95 - 2.24 (m, 2H), 2.32 - 2.41 (m, 2H), 2.50 - 2.67 (m, 1H), 2.77 (d, J = 11.0 Hz, 1H), 3.54 (dd, J = 5.7, 10.5 Hz, 1H), 3.59 - 3.71 (m, 3H).

[0729] Step 4: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0730] [ka]

[0731] To a stirred solution of 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (Intermediate IVa) (50 mg, 0.07 mmol) in DCM (1.0 mL) was added pyridine (0.01 mL, 0.14 mmol), N,N-dimethylpyridin-4-amine (2.65 mg, 0.02 mmol), and 4-nitrophenyl chloroformate (36.18 mg, 0.14 mmol) and stirred at room temperature for 1 hour. After 1 h, (1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidin-3-yl (83.06 mg, 0.28 mmol) and DIPEA (0.05 mL, 0.28 mmol) were added and stirred at 25 °C for 16 h. Upon completion, the reaction mixture was diluted with DCM (10 mL), washed with 1 M sodium carbonate solution (3 x 10 mL), water (10 mL) and brine. The combined organic portions were dried over NaSO, filtered and concentrated under reduced pressure to give 2% Purification by Combiflash column chromatography eluting with MeOH-DCM afforded 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (50 mg, 30%) as a pale yellow oil. LCMS (Method B): Found m / z for (M+H) = 1005.1, RT = 1.12 min.

[0732] Step 5: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(3-hydroxypropyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 125). To a solution of 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (85 mg, 0.085 mmol) in anhydrous THF (1.0 mL) at 0° C. was added tetra-n-butylammonium fluoride (TBAF) (0.17 mL, 0.17 mmol) (1 M solution in tetrahydrofuran). The resulting solution was stirred for 4 h after warming the mixture to 25° C. The resulting solution was diluted with DCM (10 mL) and quenched with water (5 mL). The organic layer was extracted with DCM (2 × 5 mL), brine (5 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 1.5% MeOH-DCM to afford 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-(3-hydroxypropyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (36 mg, 52%) as a pale yellow sticky gum. LCMS (Method B): Found m / z for (M+H) = 891.0, RT = 1.61 min.

[0733] [ka]

[0734] [Example 126] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-((3-hydroxypropyl)(methyl)amino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate.

[0735] Step 1: 3,3'-(methylazanediyl)bis(propan-1-ol)

[0736] [ka]

[0737] To a stirred solution of 3-(methylamino)propan-1-ol (100 mg, 1.12 mmol) in EtOH (2 mL) was added NaCO (260 mg, 2.24 mmol) and 3-chloropropanol (0.1 mL, 1.12 mmol) under an argon atmosphere. The reaction mixture was refluxed at 80 °C for 4 h. Upon completion, the reaction mixture was concentrated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 2% acetone-hexane to give 3,3'-(methylazanediyl)bis(propan-1-ol) (90 mg, 55%) as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) δ 1.53 - 1.67 (m, 2H), 1.68 - 1.78 (m, 1H), 1.84 (t, J = 6.2 Hz, 1H), 2.26 (s, 2H), 2.52 (d, J = 6.7 Hz, 3H), 2.90 (t, J = 7.5 Hz, 1H), 3.39 - 3.51 (m, 6H), 3.68 (t, J = 6.5 Hz, 1H).

[0738] Step 2: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-((3-hydroxypropyl)(methyl)amino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 126). Prepared from Intermediate IVa and 3,3'-(methylazanediyl)bis(propan-1-ol) according to general procedure D. Yield 18 mg (23%). LCMS (Method B): Found m / z for (M+H) = 864.7, RT = 1.55 min.

[0739] [ka]

[0740] [Example 127] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propyl)carbamoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVa and N1,N1-diethylpropane-1,3-diamine according to general procedure D. Yield 32 mg (53%). LCMS (Method B): Found m / z for (M+H) = 848.0, RT = 1.55 min.

[0741] [ka]

[0742] [Example 128] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-ethylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0743] Step 1: 2-(1-ethylpyrrolidin-2-yl)ethan-1-ol

[0744] [ka]

[0745] The following is representative of general procedure G. To a stirred solution of 2-(pyrrolidin-2-yl)ethan-1-ol (20 mg, 0.21 mmol) in acetonitrile (1 mL) was added KCO (72 mg, 0.65 mmol) and EtI (0.01 mL, 0.21 mmol) under a nitrogen atmosphere. The resulting mixture was stirred at 56 °C for 16 h. The reaction mass was cooled to room temperature, filtered through a bed of Celite, and the filtrate was evaporated under reduced pressure to give 2-(1-ethylpyrrolidin-2-yl)ethan-1-ol (18 mg, 58%) as a colorless liquid. 1 H NMR (400 MHz, chloroform-d) δ 1.08 (t, J = 7.2 Hz, 3H), 1.37 - 1.48 (m, 1H), 1.65 - 1.96 (m, 5H), 1.92 - 2.05 (m, 1H), 2.04 - 2.18 (m, 2H), 2.72 - 2.83 (m, 1H), 2.87 - 3.04 (m, 1H), 3.10 - 3.20 (m, 1H), 3.62 - 3.72 (m, 1H), 3.89 - 4.05 (m, 1H).

[0746] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-ethylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 128). Prepared from intermediate IVa and 2-(1-ethylpyrrolidin-2-yl)ethan-1-ol according to general procedure D. Yield 28 mg (52%). LCMS (Method B): Found m / z for (M+H) = 860.8, RT = 1.55 min.

[0747] [ka]

[0748] [Example 129] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((((1R,3s,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl)oxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0749] Step 1: (1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-ol

[0750] [ka]

[0751] Prepared from (1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-ol according to general procedure G. Yield 180 mg, 73%.

[0752] Step 2: -((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1R,3s,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 129). Prepared from intermediate IVa and (1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-ol according to general procedure D. Yield 16 mg (51%). LCMS (Method B): Found m / z for (M+H) = 872.8, RT = 1.70 min.

[0753] [ka]

[0754] [Example 130] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((((1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl)oxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate

[0755] Step 1: (1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-ol

[0756] [ka]

[0757] Prepared from (1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-ol according to general procedure G. Yield 80 mg (92%).

[0758] Step 2: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-yl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Example 130). Prepared from intermediate IVa and (1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-ol according to general procedure D. Yield 16 mg (51%). LCMS (Method B): Found m / z for (M+H) = 872.9, RT = 1.64 min.

[0759] [ka]

[0760] [Example 131] 3-((4,4-Bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1'-ethyl-[1,4'-bipiperidine]-4-carboxylate: Prepared from intermediate IVa and 1'-ethyl-[1,4'-bipiperidine]-4-carboxylic acid dihydrochloride according to general procedure E. Yield 138 mg (52%). LCMS (Method A): Found m / z for (M+H) = 913.7, RT = 3.21 min.

[0761] [ka]

[0762] [Example 132] 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1'-ethyl-[1,4'-bipiperidine]-4-carboxylate: Prepared from intermediate IVf and 1'-ethyl-[1,4'-bipiperidine]-4-carboxylic acid dihydrochloride according to general procedure E. Yield 195 mg (78%). LCMS (Method A): Found m / z for (M+H) = 1097.5, RT = 3.19 min.

[0763] [ka]

[0764] [Example 133] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-ethylpiperidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVa and 2-(1-ethylpiperidin-2-yl)ethan-1-ol according to general procedure D. Yield 38 mg (40%). LCMS (Method A): Found m / z for (M+H) = 874.7, RT = 3.98 min.

[0765] [ka]

[0766] [Example 134] 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((2-(1-ethylpiperidin-4-yl)acetoxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared from intermediate IVa and 2-(1-ethylpiperidin-4-yl)acetic acid according to general procedure E. 66 mg (72%). LCMS (Method A): Found m / z for (M+H) = 844.7, RT = 3.95 min.

[0767] [ka]

[0768] [Example 135] 3-((4,4-bis(oct-2-yn-1-yloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 3, substituting oct-2-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 91 mg (92%). LCMS (Method A): Found m / z for (M+H) = 856.7, RT = 3.63 min.

[0769] [ka]

[0770] [Example 136] 3-((4,4-bis(oct-2-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 1, substituting oct-2-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 80 mg (82%). LCMS (Method A): Found m / z for (M+H) = 844.7, RT = 3.66 min.

[0771] [ka]

[0772] [Example 137] 3-((4,4-bis(non-2-yn-1-yloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 3, substituting non-2-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 86 mg (88%). LCMS (Method A): Found m / z for (M+H) = 884.7, RT = 3.73 min.

[0773] [ka]

[0774] [Example 138] 3-((4,4-bis(non-2-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 1, substituting non-2-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 87 mg (90%). LCMS (Method A): Found m / z for (M+H) = 872.7, RT = 3.69 min.

[0775] [ka]

[0776] [Example 139] 3-((4,4-bis(oct-7-yn-1-yloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 3, substituting oct-7-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 111 mg (84%). LCMS (Method A): Found m / z for (M+H) = 856.7, RT = 3.74 min.

[0777] [ka]

[0778] [Example 140] 3-((4,4-bis(oct-7-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 1, substituting oct-7-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 108 mg (83%). LCMS (Method A): Found m / z for (M+H) = 844.7, RT = 3.76 min.

[0779] [ka]

[0780] [Example 141] 3-((4,4-bis(dec-2-yn-1-yloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 3, substituting dec-2-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 122 mg (88%). LCMS (Method A): Found m / z for (M+H) = 912.8, RT = 4.13 min.

[0781] [ka]

[0782] [Example 142] 3-((4,4-bis(dec-2-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadec-9,12-dienoate: Prepared using a procedure similar to Example 1, substituting dec-2-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 115 mg (84%). LCMS (Method A): Found m / z for (M+H) = 900.8, RT = 4.04 min.

[0783] [ka]

[0784] [Example 143] 3-((4,4-bis(dec-3-yn-1-yloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 3, substituting dec-3-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 104 mg (89%). LCMS (Method A): Found m / z for (M+H) = 912.8, RT = 3.97 min.

[0785] [ka]

[0786] [Example 144] 3-((4,4-bis(dec-3-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadec-9,12-dienoate: Prepared using a procedure similar to Example 1, substituting dec-3-yn-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 108 mg (94%). LCMS (Method A): Found m / z for (M+H) = 900.8, RT = 3.93 min.

[0787] [ka]

[0788] [Example 145] 3-((4,4-bis(((E)-oct-2-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 3, substituting (E)-oct-2-en-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 74 mg (89%). LCMS (Method A): Found m / z for (M+H) = 860.7, RT = 3.87 min.

[0789] [ka]

[0790] [Example 146] 3-((4,4-bis(((E)-oct-2-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 1, substituting (E)-oct-2-en-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 68 mg (83%). LCMS (Method A): Found m / z for (M+H) = 848.8, RT = 3.89 min.

[0791] [ka]

[0792] [Example 147] 3-((4,4-bis(((E)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 3, substituting (E)-non-2-en-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 68 mg (81%). LCMS (Method A): Found m / z for (M+H) = 888.8, RT = 4.01 min.

[0793] [ka]

[0794] [Example 148] 3-((4,4-bis(((E)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate: Prepared using a procedure similar to Example 1, substituting (E)-non-2-en-1-ol for (Z)-oct-5-en-1-ol in general procedure A. Yield for final step: 34 mg (41%). LCMS (Method A): Found m / z for (M+H) = 877.9, RT = 4.07 min.

[0795] lipid nanoparticles F. LNP Formulation Lipid nanoparticle components were dissolved in 100% ethanol at the designated lipid component molar ratio. Nucleic acid (NA) cargo was dissolved in 10 mM citrate, 100 mM NaCl, pH 4.0, resulting in a NA cargo concentration of approximately 0.22 mg / mL. In some embodiments, the NA cargo consists of both functional NAs (e.g., siRNA, antisense, expressed DNA, mRNA) and reporter DNA barcodes (previously described by Sago, 2018 PNAS) mixed at a mass ratio of functional NA to barcode of 1:10 to 10:1.

[0796] LNPs are formulated at a total lipid to NA mass ratio of 11.7. LNPs are formed by microfluidic mixing of lipid and NA solutions using a Precision Nanosystems NanoAssemblr Spark or Benchtop Instrument according to the manufacturer's protocol. A 2:1 ratio of aqueous to organic solvents is maintained during mixing using differential pressure flow. After mixing, LNPs are collected, diluted with PBS (approximately 1:1 v / v), and further buffer exchanged using dialysis against a 20 kDa filter in PBS at 4°C for 8-24 hours. After this initial dialysis, each individual LNP formulation is characterized via DLS to measure size and polydispersity, and the pKa of LNP subpopulations is measured via a TNS assay. LNPs falling within specific size and polydispersity ranges are pooled and further dialyzed against PBS in a 100 kDa dialysis cassette at 4°C for 1-4 hours. After the second dialysis, LNPs are filter-sterilized using a 0.22 μM filter and stored at 4°C for further use.

[0797] G. LNP Characterization DLS - LNP hydrodynamic diameter and percent polydispersity (PDI%) are measured using high-throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt). LNPs were diluted to appropriate concentrations in 1x PBS and analyzed.

[0798] Concentration and encapsulation efficiency - The concentration of NAs is determined by Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. The encapsulation efficiency is determined by measuring the ratio of undissolved LNPs to dissolved LNPs.

[0799] Prepare a stock solution of 10 mM HEPES (Sigma Aldrich), 10 mM MES (Sigma Aldrich), 10 mM sodium acetate (Sigma), and 140 nM sodium chloride (Sigma Aldrich) and adjust the pH to a range of pH 4-10 using hydrogen chloride and sodium hydroxide. Using four replicate samples for each pH, ​​add 140 μL of pH-adjusted buffer to a 96-well plate, followed by 5 μL of 2-(p-toluidino)-6-naphthalenesulfonic acid (60 μg / mL). Add 5 μL of LNP to each well. After a 5-minute incubation under gentle shaking, measure fluorescence using an excitation wavelength of 325 nm and an emission wavelength of 435 nm (BioTek Synergy H4 Hybrid).

[0800] LNP Administration - All studies use male and female mice approximately 8-12 weeks of age. Each mouse is temporarily restrained, and pooled LNP is administered IV via tail vein injection in up to five animals per experiment. Age-matched mice are also administered vehicle (1x PBS) via tail vein injection in up to three animals per experiment. 72 hours after administration, tissues including liver, spleen, bone marrow, and blood are harvested for analysis.

[0801] Flow - Liver tissue was mechanically digested, then enzymatically digested using a mixture of proteinases and then passed through a 70 μM filter to generate a single-cell suspension. Spleen tissue was mechanically digested to generate a single-cell suspension. All tissues were treated with ACK buffer to lyse red blood cells and then stained with fluorescently labeled antibodies for flow cytometry and fluorescence-activated cell sorting (FACS). All antibodies were commercially available. All samples were acquired by flow cytometry using a BD FACSMelody (Becton Dickinson) to create gates before sorting. The overall gating structure was size → single cells → live cells → cells of interest. T cells were defined as CD45+CD3+, monocytes as CD45+CD11b+, and B cells as CD45+CD19+. In the liver, endothelial cells were defined as CD31+, Kupffer cells as CD45+CD11b+, and hepatocytes as CD31- / CD45-. For siRNA studies, downregulation of the target gene is gated, while for mRNA studies, upregulation of the target gene is gated. Tissue from vehicle-treated mice is used to set the sorting gate. Up to 20,000 cells for each cell subset with the correct phenotype are sorted and added to 1x PBS. After sorting, cells are pelleted via centrifugation, and DNA is extracted using Quick Extract DNA Extraction Solution (Lucigen) according to the manufacturer's protocol. DNA is stored at -20°C.

[0802] Barcode sequencing - DNA (genomic and DNA barcode) was isolated using QuickExtract (Lucigen) and sequenced using an Illumina MiniSeq as previously described (Sago et al. PNAS 2018, Sago et al. JACs 2018, Sago, Lokugamage et al. Nano Letters 2018). DNA barcode counts in FACS-isolated samples were normalized to the frequency of injected input. These data were plotted as "Normalized Fold Above Input."

[0803] H. Confirm LNP formulation Lipid nanoparticle components are dissolved in 100% ethanol at the designated lipid component molar ratio. Nucleic acid (NA) cargo is dissolved in 10 mM citrate, 100 mM NaCl, pH 4.0, resulting in a concentration of approximately 0.22 mg / mL of NA cargo. In some embodiments, the NA cargo consists of functional NA (e.g., siRNA, antisense, expressed DNA, mRNA). LNPs are formulated at a total lipid to NA mass ratio of 11.7. LNPs are formed by microfluidic mixing of the lipid and NA solutions using a Precision Nanosystems NanoAssemblr Spark or Benchtop Instrument according to the manufacturer's protocol. A 3:1 ratio of aqueous to organic solvent is maintained during mixing using differential flow rates. After mixing, the LNPs are collected and diluted with PBS (approximately 1:1 v / v), and further buffer exchange is performed using dialysis against a 20 kDa filter against PBS at 4 °C for 8-24 hours. After this initial dialysis, each individual LNP formulation is characterized via DLS to measure size and polydispersity, and via a TNS assay to measure the pKa of LNP subpopulations. After dialysis, the LNPs are filter-sterilized using a 0.22 micron sterile filter and stored at 4°C for further use.

[0804] LNP characterization DLS - LNP hydrodynamic diameter and percent polydispersity (PDI%) are measured using high-throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt). LNPs are diluted to appropriate concentrations in 1x PBS and analyzed.

[0805] Concentration and encapsulation efficiency - The concentration of NAs is determined by Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. The encapsulation efficiency is determined by measuring the ratio of undissolved LNPs to dissolved LNPs.

[0806] Prepare a stock solution of 10 mM HEPES (Sigma Aldrich), 10 mM MES (Sigma Aldrich), 10 mM sodium acetate (Sigma), and 140 nM sodium chloride (Sigma Aldrich) and adjust the pH to a range of pH 4-10 using hydrogen chloride and sodium hydroxide. Using four replicate samples for each pH, ​​add 140 μL of pH-adjusted buffer to a 96-well plate, followed by 5 μL of 2-(p-toluidino)-6-naphthalenesulfonic acid (60 μg / mL). Add 5 μL of LNP to each well. After a 5-minute incubation under gentle shaking, measure fluorescence using an excitation wavelength of 325 nm and an emission wavelength of 435 nm (BioTek Synergy H4 Hybrid).

[0807] LNP Administration - All studies use male and female mice approximately 8-12 weeks of age. Each mouse is temporarily restrained, and pooled LNP is administered IV via tail vein injection in up to five animals per experiment. Age-matched mice are also administered vehicle (1x PBS) via tail vein injection in up to three animals per experiment. 72 hours after administration, tissues including liver, spleen, bone marrow, and blood are harvested for analysis.

[0808] hEPO Expression - For human EPO (hEPO) protein expression, mice were briefly restrained and bled (via the tail vein) 6 hours after administration. Blood was collected into heparin tubes, processed into plasma, and stored at -80°C until ready for use. hEPO protein was measured using an R&D Systems ELISA kit (DuoSet; DY286-05) according to the manufacturer's instructions, using appropriate dilutions of plasma. Various exemplary ionizable lipids were formulated into LNPs A1-A17 using the following molar ratios: 45% ionizable lipid / 9% distearoylphosphatidylcholine (DSPC) / 44% cholesterol / 2% PEG lipid or 50% ionizable lipid / 9% distearoylphosphatidylcholine (DSPC) / 38% cholesterol / 3% PEG lipid, with lipid-to-nucleic acid (human EPO mRNA) mass ratios of either 11.7:1, 18:1, or 25:1. Activity was determined by measuring hEPO expression in plasma 6 hours after a 0.15 mg / kg nucleic acid dose was administered via tail vein injection as described. Representative data for LNP encapsulation efficiency, hydrodynamic diameter, polydispersity index (PDI), and plasma hEPO measurements 6 hours after administration of the LNP formulations are provided in Table 1.

[0809] [Table 1]

[0810] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A compound of formula (I): 【Chemistry 1】 R １ teeth, 【Transformation 3】 and X １ is -O-, X ２ is absent, X ３ and X ４ are absent, X ５ Ha-(CH ２ ) ｂ - where b is an integer between 1 and 6; X ６ Ha-NR ４ R ５ where R ４ and R ５ together with the nitrogen to which they are attached, form one or two C １ ~C ６ forming a 4- to 7-membered heterocyclyl optionally substituted with an alkyl group, wherein said heterocyclyl optionally contains an additional heteroatom selected from oxygen, sulfur, and nitrogen; A １ and A ２ are each independently C ５ ~C １２ is alkenyl, The compound, wherein n1 is an integer between 1 and 4.

2. A compound selected from the group consisting of: 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 【Chemistry 9-4】 【Chemistry 9-5】 【Chemistry 9-6】 【Chemistry 9-7】 【Chemistry 9-8】 【Chemistry 9-9】 [Chemistry 9-10] 【Chemistry 9-11】 【Chemistry 9-12】 【Chemistry 9-13】 [Chemistry 9-14] 【Chemistry 9-15】 【Chemistry 9-16】 【Chemistry 9-17】 【Chemistry 9-18】 【Chemistry 9-19】 【Chemistry 9-20】 【Chemistry 9-21】 【Chemistry 9-22】 [Chemistry 9-23]

3. A compound selected from the group consisting of: 【Chemistry 10】 【change】 【change】 【change】 【change】 【change】 【change】

4. A compound selected from the group consisting of: 【Chemistry 11】 【change】 【change】

5. 3. The compound of claim 2, wherein: 【Chemistry 12】

6. 10. An ionizable lipid comprising the compound of any one of claims 1 to 5, wherein the amount of ionizable lipid is present in the range of 35 to 65 mole percent based on the total moles; phospholipids; polyethylene glycol-lipid; and cholesterol A lipid nanoparticle composition comprising:

7. The lipid nanoparticle composition of claim 6, wherein the phospholipid is DSPC or DMPC.

8. The lipid nanoparticle composition of claim 6 or 7, further comprising a nucleic acid.

9. The lipid nanoparticle composition of claim 8, wherein the nucleic acid is an siRNA, miRNA, mRNA, expressed DNA, an antisense oligonucleotide, or an immunostimulatory oligonucleotide.

10. The lipid nanoparticle composition of claim 8, wherein the nucleic acid is a guide RNA.

11. The lipid nanoparticle composition according to any one of claims 6 to 10, for delivering a nucleic acid to a subject.

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