Nanomaterial

Abstract

Lipid nanoparticle compositions for delivery of nucleic acids are described. In various embodiments, the lipid nanoparticles comprise an ionizable lipid of formula (I). Also provided are methods of using such lipid nanoparticle compositions to achieve targeted delivery of therapeutic carriers without the need for targeting ligands. # imgabs0 #

Description

[0001] Related application information

[0002] This application is a divisional application of Chinese Patent Application No. 202180016840.5, and claims priority to U.S. Provisional Application No. 62 / 958,876 filed on January 9, 2020, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to the fields of chemistry, biology and medicine. Drug delivery systems and methods of using the same are disclosed herein. More specifically, nanoparticle compositions for delivering nucleic acids to cells are disclosed herein. Background Art

[0004] Hepatocytes help maintain homeostasis and produce many secreted proteins, and are therefore involved in many genetic diseases. Although RNA-carrying nanoparticles have been directed to hepatocytes, systemic delivery to hepatocytes without a targeting ligand remains challenging. Summary of the invention

[0005] Some embodiments described herein relate to compounds of formula (I):

[0006]

[0007] in:

[0008] R 1 It is C9-C 20 Alkyl or C9-C 20 alkenyl;

[0009] X 1 and X 2 Each independently absent or selected from -O-, -NR 2 -and Each R 2 are independently hydrogen or C1-C6 alkyl;

[0010] Each a is independently an integer from 1 to 6;

[0011] X 3 and X 4 each independently absent or selected from: 4 to 8-membered heterocyclyl optionally substituted by 1 or 2 C1-C6 alkyl groups, 5 to 6-membered heteroaryl optionally substituted by 1 or 2 C1-C6 alkyl groups, 5 to 6-membered aryl optionally substituted by 1 or 2 C1-C6 alkyl groups, 4 to 7-membered cycloalkyl optionally substituted by 1 or 2 C1-C6 alkyl groups, -O- and -NR 3 -, where each R 3 is a hydrogen atom or a C1-C6 alkyl group, and wherein X1 -X 2 -X 3 -X 4 does not contain any oxygen-oxygen, oxygen-nitrogen, or nitrogen-nitrogen bonds;

[0012] X 5 Yes - (CH2) b -, wherein b is an integer from 0 to 6;

[0013] X 6 is hydrogen, C1-C6 alkyl, 5- to 6-membered heteroaryl optionally substituted by 1 or 2 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 The nitrogen to which they are bonded are joined together to form a 4- to 7-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic group optionally includes additional heteroatoms selected from oxygen, sulfur and nitrogen;

[0014] Each X 7 are independently hydrogen, hydroxyl or -NR 6 R 7 , where R 6 and R 7 are each independently hydrogen or C1-C6 alkyl; or alternatively, R 6 and R 7 The nitrogen to which they are bonded are joined together to form a 4- to 7-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic group optionally includes additional heteroatoms selected from oxygen, sulfur and nitrogen;

[0015] X 1 , X 2 , X 3 , X 4 and X 5 At least one of them exists;

[0016] A 1 and A 2 Each independently selected from: C5-C 12 Halogenated alkyl, C5-C 12 Alkenyl, C5-C 12 Alkynyl, (C5-C 12 Alkoxy)-(CH2) n2 -, the ring is optionally substituted by one or two halogen, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy groups (C5-C 10 Aryl)-(CH2) n3-, and (C3-C8 cycloalkyl)-(CH2) wherein the ring is optionally substituted by 1 or 2 C1-C6 alkyl groups n4 -; or alternatively, A 1 and A 2 The atoms to which they are bonded are linked together to form a 10 Alkyl-substituted 5- to 6-membered cyclic acetals;

[0017] n1, n2 and n3 are each independently an integer from 1 to 4; and

[0018] n4 is an integer from 0 to 4.

[0019] Some embodiments feature a lipid nanoparticle composition comprising: an ionizable lipid as described herein; a phospholipid; a polyethylene glycol-lipid; cholesterol; and optionally a nucleic acid. Other embodiments relate to a method of delivering a nucleic acid to an individual in need thereof, the method comprising administering a lipid nanoparticle composition to an individual in need thereof.

[0020] These and other embodiments are described in greater detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Reaction schemes for preparing compounds of formula (I) are illustrated.

[0022] Figure 2 The structures of the compounds of Examples 1 to 148 are summarized. Specific implementation plan

[0023] definition

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Unless otherwise indicated, all patents, applications, published applications and other publications cited herein are incorporated by reference in their entirety. Where multiple definitions exist for a term in this article, those in this section shall prevail unless otherwise indicated.

[0025] 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 , X7 , A 1 and A 2 = represents a substituent that can be attached to the indicated atom. Such R, X, and A groups may be collectively referred to herein as "R" groups. R groups may be substituted or unsubstituted. If two "R" groups are referred to as being "attached together," it is understood that the R groups and the atoms to which they are bonded may form a ring structure, such as a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocycle. For example, but not limited to, if NR a R b R a and R b are indicated as "linked together", this means that they are covalently bonded to each other to form a ring:

[0026]

[0027] Furthermore, if, alternatively, two "R" groups are "joined together" with the atoms to which they are attached to form a ring, then the R groups are not limited to the previously defined variables or substituents.

[0028] Whenever a group is described as "optionally substituted", the group may be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when a group is described as "unsubstituted or substituted", if substituted, the substituents may be selected from one or more of the indicated substituents. If substituents are not shown, it means that the indicated "optionally substituted" or "substituted" group may be substituted with one or more groups individually and independently selected from the following: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, acylalkyl, hydroxy, alkoxy, alkoxyalkyl, aminoalkyl, aminoacid, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxyalkyl, acyl, cyano, halogen, thiocarbonyl, O- carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, S-sulfonylamino, N-sulfonylamino, C-carboxyl, O-carboxyl, isocyanato, thiocyanato, isothiocyanato, azido, nitro, silyl, thio, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonylamino, amino, monosubstituted amino and disubstituted amino.

[0029] As used herein, “C a To C b" (wherein "a" and "b" are integers) refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heteroalicyclic group. That is, the ring of an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heteroalicyclic group can contain from "a" to "b" (inclusive) carbon atoms. Thus, for example, "C1 to C4 alkyl" refers to all alkyl groups having 1 to 4 carbon atoms, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-. If "a" and "b" are not specified for an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heteroalicyclic group, the broadest range described in these definitions is assumed.

[0030] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain containing a fully saturated (no double or triple bond) hydrocarbon group. Alkyl can have 1 to 20 carbon atoms (whenever it appears in this article, a numerical range such as "1 to 20" refers to each integer in a given range; for example, "1 to 20 carbon atoms" means that the alkyl can be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but the definition also covers the existence of the term "alkyl" without a specified numerical range). Alkyl can also be a medium-sized alkyl with 1 to 10 carbon atoms. Alkyl can also be a low alkyl with 1 to 6 carbon atoms. The alkyl of a compound can be designated as "C1-C4 alkyl" or similar designation. For example only, "C1-C4 alkyl" means that there are one to four carbon atoms in the alkyl chain, that is, the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and hexyl. The alkyl group may be substituted or unsubstituted.

[0031] As used herein, "alkenyl" refers to a hydrocarbon group containing one or more double bonds in a straight or branched hydrocarbon chain. Examples of alkenyl groups include allenyl, vinylmethyl, and vinyl. Alkenyl groups may be unsubstituted or substituted.

[0032] As used herein, "alkynyl" refers to a hydrocarbon group containing one or more triple bonds in a straight or branched hydrocarbon chain. Examples of alkynyl groups include ethynyl and propynyl. Alkynyl groups may be unsubstituted or substituted.

[0033] As used herein, "cycloalkyl" refers to a fully saturated (no double bond or triple bond) monocyclic hydrocarbon ring system or a polycyclic hydrocarbon ring system. When composed of two or more rings, the rings can be connected together in a fused, bridged or spiral manner. As used herein, the term "fused" refers to two rings with two atoms and a bond in common. As used herein, the term "bridged cycloalkyl" refers to a compound in which a cycloalkyl contains a bond of one or more atoms connecting non-adjacent atoms. As used herein, the term "spiro" refers to two rings with one atom in common, and the two rings are not connected by a bridge. Cycloalkyl can contain 3 to 30 atoms in the ring, 3 to 20 atoms in the ring, 3 to 10 atoms in the ring, 3 to 8 atoms in the ring, or 3 to 6 atoms in the ring. Cycloalkyl can be unsubstituted or substituted. Typical monocyclic alkyls include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Examples of fused cycloalkyls are decahydronaphthyl, dodecahydro-1H-phenalenyl, and tetradecahydroanthracenyl; examples of bridged cycloalkyls are bicyclo[1.1.1]pentyl, bicyclo[2.1.1]heptane, adamantyl, and norbornyl; and examples of spirocycloalkyls include spiro[3.3]heptane and spiro[4.5]decane.

[0034] As used herein, "cycloalkenyl" refers to a monocyclic hydrocarbon ring system or a polycyclic hydrocarbon ring system containing one or more double bonds in at least one ring; however, if there is more than one ring, a completely delocalized π electron system cannot be formed throughout all ring double bonds (otherwise the group would be an "aryl" as defined herein). A cycloalkenyl group may contain 3 to 10 atoms in the ring or 3 to 8 atoms in the ring. When composed of two or more rings, the rings may be connected together in a fused manner. A cycloalkenyl group may be unsubstituted or substituted.

[0035] As used herein, "aryl" refers to a carbocyclic (all-carbon) monocyclic aromatic ring system or a polycyclic aromatic ring system (including fused ring systems in which two carbon rings share a chemical bond) having a completely delocalized π electron system throughout all rings. The number of carbon atoms in an aryl group can vary. For example, an aryl group can be C6-C 14 Aryl, C6-C 10 Aryl or C6 aryl. Examples of aryl include, but are not limited to, benzene, naphthalene and azulene. Aryl may be substituted or unsubstituted.

[0036] As used herein, "heteroaryl" refers to a monocyclic aromatic ring system or a polycyclic aromatic ring system (a ring system with a completely 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 in the heteroaryl can vary. For example, the heteroaryl can contain 4 to 14 atoms in the ring, 5 to 10 atoms in the ring, or 5 to 6 atoms in the ring. In addition, the term "heteroaryl" includes a fused ring system in which two rings (e.g., at least one aromatic ring and at least one heteroaromatic ring, or at least two heteroaromatic 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. The heteroaryl group may be substituted or unsubstituted.

[0037] As used herein, "heterocyclic group" or "heteroalicyclic group" refers to a three-membered, four-membered, five-membered, six-membered, seven-membered, eight-membered, nine-membered, ten-membered, up to 18-membered monocyclic, bicyclic and tricyclic ring system, wherein carbon atoms together with 1 to 5 heteroatoms constitute the ring system. However, the heterocyclic ring may optionally contain one or more unsaturated bonds arranged in a manner that a completely delocalized π-electron system does not appear throughout all rings. Heteroatoms are elements other than carbon, including but not limited to oxygen, sulfur and nitrogen. The heterocyclic ring may further contain one or more carbonyl or thiocarbonyl functional groups so that the definition includes oxo systems and thio systems, such as lactams, lactones, cyclic imides, cyclic thioimides and cyclic carbamates. When composed of two or more rings, the rings may be connected together in a fused or spiral manner, as described herein for "cycloalkyl". In addition, any nitrogen in the heterocyclic group may be quaternized. The heterocyclic group or heteroalicyclic group may be unsubstituted or substituted. Examples of such "heterocyclyl" or "heteroalicyclic" 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-oxathiinane, 1,4-oxathiinene, 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-oxathiinane, tetrahydro-1,4-thiazine, 1,3-thiazinane, 2H-1,2-thiazinane, 1,3-oxathiinane, 1,4-oxathiinane, 1,3 ... Oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, ethylene oxide, piperidine N-oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidinedione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiomorpholine, thiomorpholine sulfoxide, thiomorpholine sulfone and their benzo-fused analogs (e.g., benzimidazolidinone, tetrahydroquinoline and 3,4-methylenedioxyphenyl).

[0038] As used herein, "aralkyl" and "aryl (alkyl)" refer to an aryl group connected as a substituent through a lower alkylene group. The lower alkylene group and aryl group of the aralkyl group can be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

[0039] As used herein, "heteroaralkyl" and "heteroaryl (alkyl)" refer to heteroaryl groups attached as substituents through lower alkylene groups. The lower alkylene and heteroaryl groups of the heteroaralkyl groups may be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furanylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, imidazolylalkyl, and benzo-fused analogs thereof.

[0040] "Heteroalicyclic (alkyl)" and "heterocyclyl (alkyl)" refer to heterocyclyl or heteroalicyclic groups connected as substituents through lower alkylene groups. The lower alkylene and heterocyclyl groups of heteroalicyclic (alkyl) groups 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-thiazin-4-yl (methyl).

[0041] "Lower alkylene" is a straight-chain -CH2-tethered group that forms a bond connecting the molecular fragments via their terminal carbon atoms. Examples include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and butylene (-CH2CH2CH2CH2-). Lower alkylene can be substituted by replacing one or more hydrogens of the lower alkylene with a substituent listed under the definition of "substituted".

[0042] As used herein, "alkoxy" refers to a group of formula -OR, wherein R is an 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 benzyloxy. Alkoxy can be substituted or unsubstituted.

[0043] As used herein, "acyl" refers to hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl), or heterocyclyl(alkyl) attached as a substituent through a carbonyl group. Examples include formyl, acetyl, propionyl, benzoyl, and acryloyl. Acyl groups may be substituted or unsubstituted.

[0044] As used herein, "acylalkyl" refers to an acyl group attached as a substituent through a lower alkylene group. Examples include aryl-C(=O)-(CH2) n - and heteroaryl-C(=O)-(CH2) n -, wherein n is an integer from 1 to 6.

[0045] As used herein, "alkoxyalkyl" refers to an alkoxy group attached as a substituent through a lower alkylene group. Examples include C 1-4 Alkyl-O-(CH2) n -, wherein n is an integer from 1 to 6.

[0046] As used herein, "aminoalkyl" refers to an optionally substituted amino group attached as a substituent through a lower alkylene group. Examples include H2N(CH2) n -, wherein n is an integer from 1 to 6.

[0047] As used herein, "hydroxyalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a hydroxyl group. Exemplary hydroxyalkyl groups include, but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. Hydroxyalkyl groups may be substituted or unsubstituted.

[0048] As used herein, "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a 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 groups may be substituted or unsubstituted.

[0049] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by a 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. The haloalkoxy group may be substituted or unsubstituted.

[0050] "Thio" 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 thio group can be substituted or unsubstituted.

[0051] "Sulfinyl" refers to a "-S(=O)-R" group, wherein R may be the same as defined for sulfenyl. The sulfinyl group may be substituted or unsubstituted.

[0052] "Sulfonyl" refers to a "SO2R" group, where R can be the same as defined for sulfenyl. A sulfonyl group can be substituted or unsubstituted.

[0053] "O-carboxyl" refers to a "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-carboxyl group can be substituted or unsubstituted.

[0054] The terms "ester" and "C-carboxy" refer to a "-C(=O)OR" group, where R may be the same as defined for O-carboxy. Esters and C-carboxy may be substituted or unsubstituted.

[0055] "Thiocarbonyl" refers to a "-C(=S)R" group, where R may be the same as defined for O-carboxyl. The thiocarbonyl group may be substituted or unsubstituted.

[0056] "Trihalomethanesulfonyl" refers to a "X3CSO2-" group where each X is a halogen.

[0057] "Trihalomethanesulfonylamino" refers to "X3CS(O)2N(R A )-" group, wherein 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).

[0058] The term "amino" as used herein refers to a -NH2 group.

[0059] As used herein, the term "hydroxy" refers to an -OH group.

[0060] "Cyano" refers to a "-CN" group.

[0061] The term "azido" as used herein refers to a -N3 group.

[0062] "Isocyanato" refers to a "-NCO" group.

[0063] "Thiocyanato" refers to a "-CNS" group.

[0064] "Isothiocyanato" refers to a "-NCS" group.

[0065] "Carbonyl" refers to a C=O group.

[0066] "S-sulfonylamino" refers to "-SO2N(R A R B )" group, wherein R A and R BIt can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). S-sulfonylamino can be substituted or unsubstituted.

[0067] "N-sulfonylamino" means "RSO2N(R A )-" group, where R and R A It can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). N-sulfonylamino can be substituted or unsubstituted.

[0068] "O-carbamoyl" refers to "-OC(=O)N(R A R B )" group, wherein R A and R B O-carbamoyl may be substituted or unsubstituted.

[0069] "N-carbamoyl" refers to "ROC(=O)N(R A )-" group, where R and R A It may independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). N-carbamoyl may be substituted or unsubstituted.

[0070] "O-thiocarbamoyl" refers to "-OC(=S)-N(R A R B )" group, wherein R A and R B O-thiocarbamoyl may be substituted or unsubstituted.

[0071] "N-thiocarbamoyl" means "ROC(=S)N(R A )-" group, where R and R A It may be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). N-thiocarbamoyl may be substituted or unsubstituted.

[0072] "C-amido" refers to "-C(=O)N(R A R B )" group, wherein 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-amido can be substituted or unsubstituted.

[0073] "N-amido" refers to "RC(=O)N(R A )-" group, where R and R A It may be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). N-acylamino may be substituted or unsubstituted.

[0074] The term "urea" refers to "N(R)-C(=O)-NR A R B " group, wherein R can be hydrogen or alkyl, and R A and R B May be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). Urea may be substituted or unsubstituted.

[0075] An "oxime" group refers to a "-C(=N-OH)R A ”, where R A may independently be alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). The oxime may be substituted or unsubstituted.

[0076] "Acylhydrazone" refers to "-C(=N-NH-acyl)-R A ”, wherein the acyl moiety has the structure of “acyl” as provided herein, and R A It can be independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). The acylhydrazone can be substituted or unsubstituted.

[0077] "Hydrazine" means "-NHNR A R B ”, where R A and R Bmay independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). Hydrazine may be substituted or unsubstituted.

[0078] The term "halogen atom" or "halogen" as used herein means any of the radiation-stable atoms of column 7 of the periodic table, such as fluorine, chlorine, bromine and iodine.

[0079] As used herein, " ------ ” represents a single bond or a double bond unless otherwise indicated.

[0080] Where the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may include one or more halogens that are the same or different. As another example, "C1-C3 alkoxyphenyl" may include one or more alkoxy groups that are the same or different and contain one, two, or three atoms.

[0081] As used herein, unless otherwise indicated, abbreviations for any protecting groups, amino acids, and other compounds are in accordance with their commonly used, recognized abbreviations or the IUPAC-IUB Biochemical Nomenclature Commission (see Biochem. 11:942-944 (1972)).

[0082] As used herein, the term "protecting group" (protecting group / protecting groups) (and the abbreviation "PG") refers to any atom or group of atoms added to a molecule to prevent an existing group in the molecule from undergoing an undesirable chemical reaction. Examples of protecting group moieties are described in TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd edition. John Wiley & Sons, 1999, and . FW McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, which are hereby incorporated by reference for the limited purpose of disclosing suitable protecting groups. Protecting group moieties can be selected in such a way that they are stable to certain reaction conditions and are easily removed at a convenient stage using methods known in the art. A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butyloxycarbonyl (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, tert-butyldimethylsilyl, triisopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl or tert-butyldiphenylsilyl); esters (e.g., benzoate); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., tosylate or mesylate); acyclic ketals (e.g., dimethyl acetals); 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).

[0083] As used herein, the term "leaving group" (and abbreviation "LG") refers to any atom or moiety that can be replaced by other atoms or moieties in a chemical reaction. More specifically, in some embodiments, a "leaving group" refers to an atom or moiety that is replaced in a nucleophilic substitution reaction. In some embodiments, a "leaving group" is any atom or moiety of the conjugate base of a strong acid. Examples of suitable leaving groups include, but are not limited to, tosylate, mesylate, trifluoroacetate, and halogen (e.g., I, Br, and Cl). Non-limiting properties and examples of leaving groups can be found in, for example, Organic Chemistry, 2nd Edition, Francis Carey (1992), pages 328-331; Introduction to Organic Chemistry, 2nd Edition, Andrew Streitwieser and Clayton Heathcock (1981), pages 169-171; and Organic Chemistry, 5th Edition, John McMurry (2000), pages 398 and 408; all of which are incorporated herein by reference for the limited purpose of disclosing properties and examples of leaving groups.

[0084] The term "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant stimulation to the organism to which it is applied and does not eliminate 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 the 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 the compound with an organic acid such as an aliphatic or aromatic carboxylic acid or sulfonic acid, such as 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. Pharmaceutically acceptable salts can also be obtained by reacting the compound with a base to form a salt, such as ammonium salts, alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), salts of organic bases (e.g., dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamines, cyclohexylamine, triethanolamine, ethylenediamine), and salts formed with amino acids such as arginine and lysine.

[0085] Unless otherwise expressly stated, the terms and phrases used in this application and their variations, especially the terms and phrases used in the appended claims and their variations, should be interpreted as open-ended and not restrictive. As the aforementioned example, the term "including" should be understood to mean "including without limitation", "including but not limited to", etc.; the term "including" as used herein is synonymous with "including", "containing" or "characterized by", and is inclusive or open-ended, and does not exclude additional unlisted elements or method steps; the term "having" should be interpreted as "at least having"; the term "including" should be interpreted as "including but not limited to"; the term "example" is used to provide an illustrative example of the item under discussion, rather than an exhaustive or restrictive list thereof; and the use of terms such as "preferably", "preferably", "desirable" or "desirable" and words with similar meanings should not be understood to imply that certain features are critical, necessary or even important for structure or function, but are intended only to highlight alternative or additional features that may or may not be used in a specific embodiment. In addition, the term "including" should be interpreted as synonymous with the phrase "at least having" or "at least including". When used in the context of a method, the term "comprising" means that the method includes at least the 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 features or components, but may also include additional features or components. Similarly, a group of items connected with the conjunction "and" should not be understood as requiring each and every one of these items to be present in the grouping, but should be understood as "and / or", unless the context otherwise indicates. Similarly, a group of items connected with the conjunction "or" should not be understood as requiring mutual exclusion in the group, but should be understood as "and / or", unless the context otherwise indicates.

[0086] With respect to substantially any plural and / or singular terms used herein, those skilled in the art may translate the plural into the singular and / or the singular into the plural, depending on the context and / or application. For the purpose of clarity, various singular / plural permutations are explicitly set forth herein. The indefinite article "a" or "an" does not exclude a plurality. Certain measures are stated in mutually different dependent claims, but this fact does not mean that a combination of these measures cannot be used advantageously. Any reference signs of the present invention should not be interpreted as limiting the scope.

[0087] It is understood that in any compound described herein having one or more chiral centers, if the absolute stereochemistry is not explicitly indicated, each center can independently have the R configuration or the S configuration or a mixture thereof. Thus, the compounds provided herein can be enantiomerically pure, an enantiomerically enriched racemic mixture, or a diastereomerically pure, diastereomerically enriched stereoisomeric mixture. In addition, it is understood that in any compound described herein having one or more double bonds that produce geometric isomers that can be defined as E or Z, each double bond can independently be E or Z or a mixture thereof.

[0088] Likewise, it is to be understood that in any compound described, all tautomeric forms are also intended to be included.

[0089] It is understood that where compounds of the present disclosure have unfilled valences, the valences are filled with hydrogen or isotopes thereof (eg, hydrogen-1 (protium) and hydrogen-2 (deuterium)).

[0090] It should be understood that the compounds described herein can be isotopically labeled. Substitution with isotopes (e.g., deuterium) can provide certain therapeutic advantages, produce higher metabolic stability, for example, increased half-life in vivo or reduced dosage requirements. Each chemical element as represented in the compound structure can include any isotope of the element. For example, in the compound structure, a hydrogen atom can be explicitly disclosed or understood to be present in the compound. At any position of a compound where a hydrogen atom may be present, the hydrogen atom can be any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Therefore, all possible isotopic forms are covered by reference to compounds herein, unless the context clearly specifies otherwise.

[0091] It should be understood that the methods and combinations described herein include crystalline forms (also referred to as polymorphs, which include different crystal packing arrangements of the same elemental composition of the compound), 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, etc. In other embodiments, the compounds described herein exist in non-solvated forms. Solvates contain stoichiometric or non-stoichiometric amounts of solvents and can be formed during crystallization with pharmaceutically acceptable solvents such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. In addition, the compounds provided herein may exist in non-solvated forms as well as solvated forms. Generally, for the purposes of the compounds and methods provided herein, solvated forms are considered to be equivalent to non-solvated forms.

[0092] Where a numerical range is provided, it is understood that the upper and lower limits and every intervening value between the upper and lower limits of the stated range is encompassed within the embodiments.

[0093] As used herein, "RNA" refers to ribonucleic acid that can be naturally or non-naturally present. For example, RNA can include modified and / or non-naturally present components, such as one or more nucleobases, nucleosides, nucleotides or linkers. RNA can include 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 target polypeptide. For example, RNA can be a messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, such as in vivo translation of an mRNA in a mammalian cell, can produce an encoded polypeptide. RNA can be selected from a non-limiting group consisting of small interfering RNA (siRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single guide RNA (sgRNA), cas9mRNA and mixtures thereof.

[0094] The terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to a string of at least three amino acids linked together by peptide bonds. A peptide may refer to a single peptide or a collection of peptides. A peptide may contain natural amino acids, non-natural amino acids (i.e., compounds that do not occur in nature but can be incorporated into a polypeptide chain), and / or amino acid analogs. In addition, one or more amino acids in a peptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization or other modifications, and the like. Modifications may include cyclization of the peptide, incorporation of D-amino acids, and the like.

[0095] As used herein, the terms "treat", "treatment" and "therapeutic use" refer to the elimination, alleviation or improvement of one or more symptoms of a disease or disorder. As used herein, a "therapeutically effective amount" refers to the amount of a therapeutic agent sufficient to mediate the clinically relevant elimination, alleviation or improvement of such symptoms. An effect is clinically relevant if its magnitude is sufficient to affect the health or prognosis of the recipient individual. A therapeutically effective amount may refer to the amount of a therapeutic agent sufficient to delay or minimize the onset of a disease (e.g., delay or minimize the spread of cancer). A therapeutically effective amount may also refer to the amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0096] Compositions as described herein are preferably provided in unit dosage form. As used herein, "unit dosage form" is a composition comprising a compound or composition of an amount suitable for being applied to an animal (preferably a mammalian individual) in a single dose according to good medical practice. However, the preparation of a single or unit dosage form does not mean that the dosage form is applied once a day or once per course of treatment. It is expected that such dosage forms are applied once, twice, three times or more a day, and can be applied as an infusion over a period of time (e.g., about 30 minutes to about 2-6 hours), or as a continuous infusion, and can be given more than once during the course of treatment, although single administration is not particularly excluded. It will be appreciated by those skilled in the art that the preparation does not specifically consider the entire course of treatment, and such a decision is left to the technicians in the field of treatment rather than the technicians in the field of formulation. As used herein, the term "prophylactic agent" refers to an agent that can be used to prevent such a disorder or disease before any symptom of the disorder or disease is detected. "Preventionally effective" amount is the amount of the prophylactic agent sufficient to mediate this protection. A prevention-effective amount can also refer to the amount of the prophylactic agent providing a preventive benefit in preventing a disease.

[0097] As described above, useful compositions can be any of various suitable forms for various routes of administration, such as oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intraarterial, intravenous, intramuscular or other parenteral routes of administration. It will be appreciated by those skilled in the art that oral and nasal compositions include compositions that are administered by inhalation and prepared using available methods. Depending on the specific route of administration desired, a variety of pharmaceutically acceptable carriers well known in the art can be used. Pharmaceutically acceptable carriers include, for example, solid or liquid fillers, diluents, cosolvents, surfactants and encapsulating materials. Optional pharmaceutically active materials may be included that do not substantially interfere with the inhibitory activity of the compound. The amount of the carrier used in conjunction with the compound is sufficient to provide the actual amount of material for administration per unit dose of the compound. Techniques and compositions for preparing 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 Edition, 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).

[0098] 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 (eg, mice and rats), and other laboratory animals.

[0099] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water, and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents.

[0100] 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 .

[0101] The term "oligonucleotide" refers to a short DNA, RNA or DNA / RNA molecule or oligomer containing a relatively small number of nucleotides.

[0102] A. Lipid Nanoparticles

[0103] The effective targeted delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids is a continuous challenge in the medical field. The relative instability and low cell permeability of nucleic acids make the specific delivery of nucleic acids difficult. It has been found that lipid nanoparticles with ionizable lipids as described herein can more effectively deliver nucleic acids to specific tissues in vivo. In one embodiment, lipid nanoparticles can be prepared by mixing nucleic acids with ionizable lipids, PEG-lipids, phospholipids, cholesterol and optional nucleic acids. In some embodiments, lipid nanoparticles do not include targeting ligands. In some embodiments, the disclosed lipid nanoparticles preferentially target T cells relative to hepatocytes in the absence of targeting ligands.

[0104] The size of lipid nanoparticle is different.In one embodiment, lipid nanoparticle can have the average hydrodynamic diameter of about 30 to about 170nm.Lipid nanoparticle can have the average hydrodynamic diameter of about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm, 165nm, 170nm or have the average hydrodynamic diameter of any scope of the endpoint defined by any two of the above-mentioned values.For example, in embodiments, nanoparticle has the average hydrodynamic diameter of 50nm to 100nm.

[0105] 1. Compound

[0106] Some embodiments described herein relate to compounds of formula (I):

[0107]

[0108] In various embodiments, the compound of formula (I) is an ionizable lipid as described elsewhere herein. In various embodiments, R in formula (I) 1 For C9-C 20 Alkyl or C9-C 20 For example, in some embodiments, R in formula (I) 1 It is a C9-C with 2 unsaturated 20 Alkenyl, such as C 17 Alkenyl.

[0109] In various embodiments, X in formula (I) 1 and X 2 Each independently absent or selected from -O-, -NR 2 -and Where R 2 is hydrogen or C1-C6 alkyl, a is an integer from 1 to 6, 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, R 6 and R 7The nitrogen to which they are bonded are joined together to form a 4- to 7-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic group optionally includes additional heteroatoms selected from oxygen, sulfur and nitrogen. 1 Does not exist, X 2 Does not exist, or X 1 and X 2 Neither exists. As discussed elsewhere in this article, X 1 -X 2 -X 3 -X 4 do not contain any oxygen-oxygen, oxygen-nitrogen or nitrogen-nitrogen bonds to each other. 1 and X 2 Can't both be -O-, and can't both be -NR 2 -. Similarly, X 1 and X 2 Cannot be -O- and -NR respectively 2 -, and cannot be -NR 2 -and-O-.

[0110] In various embodiments, X 1 In various embodiments, X 2 In some embodiments, X 1 for For example -(CH2) a -, -CH(OH)- or -(CH2) a-1 In some embodiments, X 2 yes For example -(CH2) a -, -CH(OH)- or -(CH2) a-1 CH(OH)-. In various embodiments, each a is independently 1, 2, 3, 4, 5 or 6. In various embodiments, X 1 Yes - NR 6 R 7 In various embodiments, X 2 Yes - NR 6 R 7 In some embodiments, R 6 is hydrogen or C1-C6 alkyl. 7 is hydrogen or C1-C6 alkyl. In other embodiments, R 6 and R 7 The nitrogen to which they are bonded are linked together to form a 4 to 7 membered heterocyclic group optionally substituted with 1 or 2 C1-C6 alkyl groups. 6 and R 7The 4 to 7 membered heterocyclic groups linked together optionally contain additional heteroatoms selected from oxygen, sulfur and nitrogen.

[0111] In various embodiments, X in formula (I) 3 and X 4 Each independently absent or selected from:

[0112] (1) a 4- to 8-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups;

[0113] (2) a 5- to 6-membered heteroaryl group optionally substituted by 1 or 2 C1-C6 alkyl groups;

[0114] (3) a 5- to 6-membered aryl group optionally substituted by 1 or 2 C1-C6 alkyl groups;

[0115] (4) a 4- to 7-membered cycloalkyl group optionally substituted by 1 or 2 C1-C6 alkyl groups;

[0116] (5) -O-; or

[0117] (6)-NR 3 -, where each R 3 are independently a hydrogen atom or a C1-C6 alkyl group.

[0118] In some embodiments, X 3 Does not exist, X 4 Does not exist, or X 3 and X 4 Neither exists. As discussed elsewhere in this article, X 1 -X 2 -X 3 -X 4 do not contain any oxygen-oxygen, oxygen-nitrogen or nitrogen-nitrogen bonds to each other. 2 and X 3 Can't both be -O-. When X 2 Is -O- or -NR 2 -, then X 3 Cannot be -NR 3 -. Similarly, when X 3 Is -O- or -NR 3 -, then X 2 Cannot be -NR 2 -. Similarly, X 3 and X 4 Can't both be -O-, and can't both be -NR 3 -. Similarly, X 3 and X 4 Cannot be -O- and -NR respectively 3 -, and cannot be -NR 3 -and-O-.

[0119] In various embodiments, X in formula (I) 3 and X 4 Each is independently a 4- to 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 each is independently azetidinyl, methylazetidinyl, pyrrolidinyl, methylpyrrolidinyl, piperidinyl, methylpiperidinyl, piperazinyl, methylpiperazinyl, dimethylpiperazinyl, morpholinyl, diazepanyl, methyldiazepanyl, octahydro-2H-quinolizinyl, azabicyclo[3.2.1]octyl, methyl-azabicyclo[3.2.1]octyl, diazaspiro[3.5]nonyl or methyldiazaspiro[3.5]nonyl.

[0120] In various embodiments, X in formula (I) 3 and X 4 Each independently is a 5- to 6-membered heteroaryl group optionally substituted with 1 or 2 C1-C6 or C1-C3 alkyl groups. For example, in various embodiments, X 3 and X 4 Each is independently pyrrolyl, methylpyrrolyl, imidazolyl, methylimidazolyl, pyridinyl or methylpyridinyl.

[0121] In various embodiments, X in formula (I) 3 and X 4 Each independently is a 5- to 6-membered aryl group optionally substituted with 1 or 2 C1-C6 or C1-C3 alkyl groups. For example, in various embodiments, X 3 and X 4 Each is independently phenyl, methylphenyl, naphthyl or methylnaphthyl.

[0122] In various embodiments, X in formula (I) 3 and X 4 Each independently is a 4 to 7 membered cycloalkyl group optionally substituted with 1 or 2 C1-C6 or C1-C3 alkyl groups. For example, in various embodiments, X 3 and X 4 Each is independently cyclopentyl, methylcyclopentyl, cyclohexyl or methylcyclohexyl.

[0123] In various embodiments, X in formula (I) 3 In various embodiments, X in formula (I) 4 In various embodiments, X 3 Yes - NR 3 -, where R 3is a hydrogen atom or a C1-C6 alkyl group, such as a C1-C3 alkyl group. For example, in various embodiments, X 3 is -N(CH3)-, -N(CH2CH3)- or N(CH2CH2CH3)-. In other embodiments, X 4 Yes - NR 3 -, where R 3 is a hydrogen atom or a C1-C6 alkyl group, such as a C1-C3 alkyl group. For example, in various embodiments, X 4 It is -N(CH3)-, -N(CH2CH3)- or N(CH2CH2CH3)-.

[0124] In various embodiments, X in formula (I) 5 Yes - (CH2) b -, wherein b is an integer from 0 to 6. In some embodiments, b is 0, in which case X 5 In other embodiments, b is 1, 2, 3, 4, 5 or 6.

[0125] In various embodiments, X in formula (I) 6 is hydrogen, C1-C6 alkyl, 5- to 6-membered heteroaryl optionally substituted by 1 or 2 C1-C6 alkyl groups, or -NR 4 R 5 In some embodiments, R 4 and R 5 Each is independently hydrogen or C1-C6 alkyl. Alternatively, in other embodiments, R 4 and R 5 The nitrogen to which they are bound join together to form a 4 to 7 membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups, wherein the 4 to 7 membered heterocyclic group optionally includes additional heteroatoms selected from oxygen, sulfur and nitrogen.

[0126] In various embodiments of Formula (I), X 1 , X 2 , X 3 , X 4 and X 5 At least one of is present. For example, in various embodiments, in formula (I), X 1 , X 2 , X 3 , X 4 and X 5 In other embodiments, in formula (I), X 1 , X 2 , X 3 , X 4 and X 5At least three of are present. For example, in some embodiments, in Formula (I), X 1 , X 2 , X 3 , X 4 and X 5 In other embodiments, in formula (I), all X 1 , X 2 , X 3 , X 4 and X 5 Yes, it exists.

[0127] In some embodiments, X 6 In other embodiments, X 6 is C1-C6 alkyl, such as C1-C3 alkyl (e.g., methyl, ethyl or propyl). 6 is a 5- to 6-membered heteroaryl group optionally substituted with 1 or 2 C1-C6 alkyl groups. For example, in various embodiments, X 6 is pyrrolyl, methylpyrrolyl, imidazolyl, methylimidazolyl, pyridinyl or methylpyridinyl. 6 Yes - NR 4 R 5 For example, in some embodiments, X 6 is -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -N(CH3)2, -N(CH2CH3)2, or -N(CH2CH2CH3)2. Alternatively, in other embodiments, R 4 and R 5 The nitrogen to which they are bonded is linked together to form a 4 to 7 membered heterocyclic group. The 4 to 7 membered heterocyclic group may be optionally substituted with 1 or 2 C1-C6 alkyl groups (e.g., C1-C3 alkyl groups), and / or the 4 to 7 membered heterocyclic group may optionally include 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.

[0128] In various embodiments, each X in Formula (I) 7 is hydrogen. In other embodiments, each X 7 In other embodiments, each X 7 Yes - NR 6 R 7 For embodiments where a is 2 to 6, each X 7Can be the same or different. For example, in various embodiments, X 7 Yes (CH2) a-1 CH(X 7 )-, where a is 2, 3, 4, 5 or 6. 7 Yes - NR 6 R 7 In some embodiments, R 6 and R 7 Each is independently hydrogen or C1-C6 alkyl, such as C1-C3 alkyl. For example, in some embodiments, X 7 is -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -N(CH3)2, -N(CH2CH3)2 or -N(CH2CH2CH3)2. Alternatively, wherein X 7 Yes - NR 6 R 7 In some embodiments, R 6 and R 7 The nitrogen to which they are bonded are linked together to form a 4- to 7-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups. 7 Yes - NR 6 R 7 In other embodiments, R 6 and R 7 The nitrogen to which they are bonded may be linked together to form a 4 to 7 membered heterocyclic group. The 4 to 7 membered heterocyclic group may be optionally substituted with 1 or 2 C1-C6 alkyl groups (e.g., C1-C3 alkyl groups), and / or the 4 to 7 membered heterocyclic group may optionally include 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.

[0129] In various embodiments, A in formula (I) 1 and A 2 Each independently selected from:

[0130] (1) C5-C 12 Haloalkyl;

[0131] (2) C5-C 12 alkenyl;

[0132] (3)C5-C 12 Alkynyl;

[0133] (4)(C5-C 12 Alkoxy)-(CH2)n2 -;

[0134] (5) the ring is optionally substituted by one or two halogens, C1-C6 alkyls, C1-C6 haloalkyls or C1-C6 alkoxys (C5-C6 10 Aryl)-(CH2) n3 -;as well as

[0135] (6) (C3-C8 cycloalkyl)-(CH2) wherein the ring is optionally substituted by 1 or 2 C1-C6 alkyl groups n4 -;

[0136] Or alternatively, A 1 and A 2 The atoms to which they are bonded are linked together to form a 10 Alkyl-substituted 5- to 6-membered cyclic acetals.

[0137] In various embodiments of Formula (I), n1, n2 and n3 are each independently an integer from 1 to 4 (i.e., 1, 2, 3 or 4), and n4 is an integer from 0 to 4 (i.e., 0, 1, 2, 3 or 4). 1 and A 2 Have the same chemical structure.

[0138] In various embodiments of Formula (I), A 1 and A 2 Each independently is C5-C 12 For example, in various embodiments, C5-C 12 Haloalkyl is C5-C 12 Fluorinated alkyl, such as C6 fluoroalkyl, C7 fluoroalkyl, C8 fluoroalkyl, C9 fluoroalkyl, C 10 Fluorinated alkyl, C 11 Fluorinated alkyl or C 12 Fluorinated alkyl. Attached to C5-C 12 The number of halogen atoms in a haloalkyl group may vary within wide limits, depending on the length of the alkyl chain and the degree of halogenation. 12 The haloalkyl group contains 1 to 25 halogen atoms, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 halogen atoms. 12 A haloalkyl group is a C5-C 12 Fluorinated alkyl, such as CF3(CF2) n5 -, wherein n5 is an integer from 0 to 5. For example, in various embodiments, C5-C 12 Fluorinated alkyl is CF3(CF2) n5 (CH2)n6 -, where n5 is an integer from 0 to 5, n6 is an integer from 0 to 11, and n5+n6+1 is equal to C5-C 12 The number of carbons in the fluoroalkyl group.

[0139] In various embodiments of Formula (I), A 1 and A 2 Each independently is C5-C 12 The position of the double bond of the alkenyl group can vary. For example, in various embodiments, C5-C 12 Alkenyl is CH3CH2CH=CH(CH2) n7 -, wherein n7 is an integer from 1 to 8, such as CH3CH2CH=CH(CH2)4-. In some embodiments, C5-C 12 The alkenyl group is branched, for example (CH3)2C=CH(CH2) n8 -CH(CH3)-(CH2) n9 -, wherein n8 and n9 are each independently 1, 2 or 3.

[0140] In various embodiments of Formula (I), A 1 and A 2 Each independently is C5-C 12 The position of the triple bond of the alkynyl group can vary. For example, in various embodiments, C5-C 12 Alkynyl is CH3CH2C≡C(CH2) n10 -, wherein n10 is an integer from 1 to 8, such as CH3CH2C≡C(CH2)4-. In some embodiments, C5-C 12 Alkynyl is branched, 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 2 to 5.

[0141] In various embodiments of Formula (I), A 1 and A 2 Each independently is (C5-C 12 Alkoxy)-(CH2) n2 -. In various embodiments, each n2 is independently an integer from 1 to 4 (i.e., 1, 2, 3, or 4). The position of the oxygen may vary. For example, in various embodiments, (C5-C 12 Alkoxy)-(CH2) n2 - is CH3O(CH2) n13 -(CH2) n2-, wherein n13 is an integer from 1 to 11, such as CH3O(CH2)7-. In other embodiments, (C5-C 12 Alkoxy)-(CH2) n2 - is CH3(CH2) n14 -O-(CH2) n15 -(CH2) n2 -, wherein n14 and n15 are each independently an integer from 1 to 8, and n14+n15 is an integer from 4 to 11, for example CH3(CH2)7-O-(CH2)2-(CH2) n2 -. In some embodiments, C5-C 12 The alkoxy group is branched, 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 from 2 to 9.

[0142] In various embodiments of Formula (I), A 1 and A 2 are each independently optionally substituted by one or two halogen, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy groups (C5-C 10 Aryl)-(CH2) n3 -. In various embodiments, each n3 is independently an integer from 1 to 4 (i.e., 1, 2, 3, or 4). In some embodiments, C5-C 10 Aryl is phenyl. For example, in various embodiments, (C5-C 10 Aryl)-(CH2) n3 - is C6H5-(CH2) ring optionally substituted by one or two halogen, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy n3 -. In an embodiment, the ring is optionally substituted (C5-C 10 Aryl)-(CH2) n3 - is CF3-C6H4-(CH2) n3 -, for example CF3-C6H4-CH2- or CF3-C6H4-(CH2)2-. In another embodiment, the ring is optionally substituted (C5-C 10 Aryl)-(CH2) n3 - is CH3-(CH2) n18 -C6H4-(CH2) n2-, wherein n18 is 1, 2 or 3 and n2 is 1, 2, 3 or 4, for example CH3(CH2)3-C6H4-CH2- or CH3(CH2)3-C6H4-(CH2)2-.

[0143] In various embodiments of Formula (I), A 1 and A 2 Each independently is (C3-C8 cycloalkyl)-(CH2) wherein the ring is optionally substituted with 1 or 2 C1-C6 alkyl groups n4 -. In various embodiments, each n4 is independently an integer from 0 to 4 (i.e., 0, 1, 2, 3, or 4). In some embodiments, the C3-C8 cycloalkyl group is a cyclohexyl group or a cyclopentyl group. For example, in various embodiments, (C3-C8 cycloalkyl)-(CH2) n4 - is C6H ring optionally substituted by 1 or 2 C1-C6 alkyl groups 11 -(CH2) n4 -, such as C6H 11 -(CH2)2-、C6H 11 -(CH2)3- or CH3-C6H 10 -(CH2)3-.

[0144] Alternatively, in other embodiments of Formula (I), A 1 and A 2 The atoms to which they are bonded are linked together to form a 10 Alkyl-substituted 5 to 6-membered cyclic acetal. For example, in an embodiment, A 1 and A 2 The atoms to which they are bonded are linked together to form a 6-membered cyclic acetal, which is a ring substituted with 2 C8 alkyl groups, as shown below: In another embodiment, A 1 and A 2 The atoms to which they are bonded are linked together to form a 5-membered cyclic acetal, which is a ring substituted with 2 C8 alkyl groups, as shown below:

[0145] 2. Ionizable lipids

[0146] In one embodiment, disclosed lipid nanoparticles include ionizable lipids. Ionizable lipids generally include amine-containing groups on the head group. In various embodiments, the ionizable lipid is a compound of formula (I). In some embodiments, based on the total moles of the components of the lipid nanoparticles, the ionizable lipid is present in the lipid nanoparticles with 35 mol%, 45 mol%, 50 mol% or 65 mol%. In another embodiment, based on the total moles of the components of the lipid nanoparticles, the ionizable lipid is present with about 33 mol% to about 36 mol%. In yet another embodiment, based on the total moles of the components of the lipid nanoparticles, the ionizable lipid is present with about 35 mol%.

[0147] Additional embodiments relate to lipid nanoparticle compositions comprising: ionizable lipids; phospholipids; polyethylene glycol-lipids; cholesterol; and optionally nucleic acids. In some embodiments, the ionizable lipids comprise a structure according to any one of formulas (I), (Ia), (II), (IIa), (IIb), and (IIc). In some embodiments, the amount of ionizable lipids is present in a range of about 35 mol % to 65 mol % based on the total moles of the components of the lipid nanoparticles.

[0148] 3. Sterols

[0149] In some embodiments, the disclosed lipid nanoparticles include one or more sterols. In one embodiment, sterol is cholesterol, or a variant or derivative thereof. In some embodiments, cholesterol is modified, for example, oxidized. Unmodified cholesterol can be acted upon by enzymes to form variants in which side chains or rings are oxidized. Cholesterol can be oxidized on a β-ring structure or on a hydrocarbon terminal structure. Exemplary cholesterols considered to be used 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, β-sitosterol, stigmasterol, brassica sterol, campesterol, or a combination thereof. In one embodiment, relative to other cholesterol variants, cholesterol oxidized on the side chain can enhance carrier delivery. In one embodiment, cholesterol is unmodified cholesterol.

[0150] 4. PEG-lipid

[0151] In some embodiments, the disclosed nanoparticle compositions further include one or more PEG or PEG-modified lipids. Such lipids may also be referred to as PEGylated lipids or PEG-lipids. The inclusion of PEGylated lipids can be used to enhance the in vitro stability and in vivo circulation time of lipid nanoparticle colloids. In some embodiments, PEGylation is reversible, so the PEG portion is gradually released in the blood circulation. Exemplary PEG-lipids include, but are not limited to, PEGylated lipids with a length of C6-C 20 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 lipid.

[0152] 5. Phospholipids

[0153] The phospholipid component of nanoparticles can include one or more phospholipids, such as one or more (poly) unsaturated lipids. Phospholipids can be assembled into one or more lipid bilayers. In some embodiments, phospholipids can include a phospholipid portion and one or more fatty acid portions.

[0154] 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, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, docosanoic acid, docosapentaenoic acid and docosahexaenoic acid. Non-natural substances including natural substances with modifications and substitutions including branching, oxidation, cyclization and alkynes are also contemplated. For example, phospholipids can be functionalized or cross-linked with one or more alkynes (for example, one or more double bonds in the alkenyl group are replaced by triple bonds). Under suitable reaction conditions, the alkyne group can undergo copper-catalyzed cycloaddition when exposed to azide. Such reactions can be used to functionalize the lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition, or to conjugate the nanoparticle composition to useful components such as targeting or imaging moieties (eg, dyes).

[0155] 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-heneicosyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glyceryl-3-phosphocholine, 1,2-arachidonoyl-sn-glyceryl-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glyceryl-3-phosphocholine, 1,2-diphytanoyl-sn-glyceryl-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glyceryl-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glyceryl-3-phosphoethanolamine, , 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.

[0156] E. Cargo

[0157] In one embodiment, the disclosed lipid nanoparticle compositions include a therapeutic or prophylactic agent for delivery to an individual. In some embodiments, the therapeutic or prophylactic agent is encapsulated by the lipid nanoparticles. In one embodiment, the lipid nanoparticles are loaded with one or more nucleic acids.

[0158] 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 embodiments, the nucleic acid is siRNA, miRNA, mRNA, expressed DNA, antisense oligonucleotides, or immunostimulatory oligonucleotides.

[0159] Gene editing based on CRISPR (clustered regularly interspaced short palindromic repeats) requires two components: guide RNA and CRISPR-associated endonuclease protein (Cas). The guide RNA guides the Cas nuclease to a specific target DNA sequence. Cas then produces 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 a lipid nanoparticle, the nucleic acid carrier is a guide RNA. In such embodiments, the second lipid nanoparticle may contain a nucleic acid carrier encoding an RNA-guided endonuclease. Two lipid nanoparticles can be administered together. Exemplary RNA-guided endonucleases include, but are not limited to, Cas9, CasX, CasY, Cas13, or Cpf1.

[0160] In one embodiment, the carrier is siRNA. Short interfering RNA (siRNA) is a double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby reducing or even inhibiting gene expression. In one embodiment, siRNA triggers the specific degradation of homologous RNA molecules such as mRNA in the sequence identity region between siRNA and target RNA. For example, WO02 / 44321 discloses siRNA that can sequence-specifically degrade target mRNA when paired with 3' overhanging end bases, and the method for preparing these siRNAs is incorporated herein by reference. Sequence-specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that simulate siRNAs produced by the enzyme dicer (Elbashir et al., (2001) Nature, 411: 494 498) (Ui-Tei et al., (2000) FEBS Lett 479: 79-82).

[0161] In one embodiment, the carrier is messenger RNA (mRNA). Messenger RNA is a single-stranded RNA that can be translated into its encoded protein using the cellular protein synthesis machinery once the carrier reaches the cytoplasm. The translation and stability of mRNA can be affected by modifications of the 5' and 3' UTRs responsible for recruiting RNA binding proteins and microRNAs, which can have an impact on translation activity (Sahin et al., (2014) Nat Rev Drug Disco 13: 759-580) (Kariko et al., (2008) Mol Ther 11: 1833-1840).

[0162] In one embodiment, the lipid nanoparticle contains an inhibitory nucleic acid less than 1.0mg / kg. The nanoparticle can contain an inhibitory nucleic acid of 1.0mg / kg, 0.9mg / kg, 0.8mg / kg, 0.7mg / kg, 0.6mg / kg or 0.5mg / kg. In another embodiment, the lipid nanoparticle contains an inhibitory nucleic acid of 0.5mg / kg. This is an advantage over the prior art, in which nanoparticles require a high dose of nucleic acid (>1mg / kg) to achieve gene silencing, which is not approved for human delivery. Disclosed technology can use an inhibitory nucleic acid of 0.5mg / kg to achieve gene silencing in a lipid nanoparticle that does not include a targeting ligand.

[0163] In some embodiments, nucleic acids, including but not limited to oligonucleotides, are modified or include one or more modified nucleotides to increase stability, half-life, and / or nuclease stability. To limit nuclease sensitivity, natural phosphodiester oligodeoxyribonucleotides, natural phosphodiester oligoribonucleotides, ribonucleotide polymers, and deoxyribonucleotide polymers may include one or more different modifications. Exemplary modifications include but are not limited to phosphorothioate (PS) bonds, 2"-O-methyl (2'OMe), 2' fluoro bases, reverse dT and ddT, phosphorylation of the 3' end of the oligonucleotide, locked nucleic acids, and including phosphoramidite C3 spacers.

[0164] Phosphorothioate bonds replace non-bridging oxygens with sulfur atoms in the phosphate backbone of an oligonucleotide. About 50% of the time (due to the 2 resulting stereoisomers that can be formed), PS modifications make the internucleotide linker more resistant to nuclease degradation. In some embodiments, the nucleic acid includes one or more PS bonds, such as at least 3 PS bonds at the 5' and 3' oligonucleotide ends to inhibit exonuclease degradation. Some nucleic acids include PS bonds throughout the entire oligonucleotide to help reduce endonuclease attack.

[0165] The naturally occurring post-transcriptional modification of RNA, 2'OMe, has been found in tRNA and other small RNAs. In some embodiments, nucleic acids or oligonucleotides are directly synthesized to contain 2'OMe. This modification increases the Tm of the RNA: RNA duplex, but only causes a slight change in RNA: DNA stability. It prevents single-stranded endonuclease attack, but does not prevent exonuclease digestion. In some embodiments, these nucleic acids or oligonucleotides are also end-blocked. The DNA oligonucleotides comprising this modification are generally 5 to 10 times less sensitive to DNases than unmodified DNA. 2'OMe modifications are commonly used in antisense oligonucleotides as a means of increasing stability and binding affinity with target transcripts.

[0166] 2'-Fluoro bases have fluorine-modified ribose sugars that increase binding affinity (Tm) and also confer some relative nuclease resistance compared to native RNA.In some embodiments, the nucleic acid or oligonucleotide includes a 2' fluoro base bound to a PS-modified bond.

[0167] Reverse dT can be incorporated at the 3' end of the oligonucleotide to form a 3'-3' bond, which will inhibit 3' exonuclease degradation and DNA polymerase extension. In addition, placing a reverse 2', 3' dideoxy-dT base (5' reverse ddT) at the 5' end of the oligonucleotide prevents spurious ligation and can prevent some forms of enzymatic degradation.

[0168] Some embodiments provide nucleic acids or oligonucleotides comprising a phosphoramidite C3 spacer. The phosphoramidite C3 spacer can be introduced internally, or a long hydrophilic spacer arm for attaching a fluorophore or other side group is introduced at either end of the oligomer. The C3 spacer can also be used to inhibit degradation by 3' exonucleases.

[0169] In some embodiments, the nucleic acid or oligonucleotide comprises a locked nucleic acid. A locked nucleic acid comprises a modified RNA nucleotide in which the 2'-O and 4'-C atoms of the ribose are linked by a methylene bridge. This additional bridge limits the flexibility normally associated with the ring, essentially locking the structure into a rigid structure. LNAs can be inserted into RNA and DNA oligonucleotides.

[0170] Other types of cargo that can be delivered by the disclosed nanoparticles include, but are not limited to, chemotherapeutic agents, cytotoxic agents, radioactive ions, small molecules, proteins, polynucleotides, and nucleic acids.

[0171] Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cretasone, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel. Doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, folinic acid, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, salaplatin, streptozotocin, tegafluuracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, troxodan, vinblastine, vincristine, vindesine, vinorelbine or a combination thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine, taurine, actinomycin D, lactosylceramide, 15d-PGJ (2) and a combination thereof.

[0172] Some embodiments relate to methods of delivering a nucleic acid to an individual in need thereof, comprising administering to the individual a lipid nanoparticle composition as described herein. In some embodiments, the nucleic acid is siRNA, miRNA, mRNA, expressed DNA, antisense oligonucleotide or immunostimulatory oligonucleotide.

[0173] B. Exemplary lipid nanoparticle formulations

[0174] In one embodiment, the lipid nanoparticle preparation comprises about 30mol% to about 70mol% ionizable lipids, about 5mol% to about 25mol% phospholipids, about 25mol% to about 45mol% cholesterol and about 0mol% to about 5mol% PEG-lipids. In another embodiment, the lipid nanoparticle preparation comprises about 45mol% ionizable lipids, about 9mol% phospholipids, about 44mol% cholesterol and about 2mol% PEG-lipids. In another embodiment, the lipid nanoparticle preparation comprises about 50mol% ionizable lipids, about 9mol% phospholipids, about 38mol% cholesterol and about 3mol% PEG-lipids.

[0175] One embodiment provides a lipid nanoparticle formulation comprising 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, about 1 mol% to about 5 mol% of C 14 PEG 2000 and about 30 mol % to about 47 mol % cholesterol, based on the total moles of these four components.

[0176] Another embodiment provides a lipid nanoparticle formulation comprising 50 mol% of an ionizable lipid of formula (I), 9 mol% of 1-2-distearoyl-sn-glycero-3-phosphocholine and 3 mol% of C 14 PEG 2000 , 38 mol % cholesterol, based on the total moles of these four components.

[0177] Another embodiment provides a lipid nanoparticle formulation wherein the mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):mRNA is about 2:1 to 50:1.

[0178] C. Pharmaceutical composition

[0179] 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 comprising one or more different therapeutic and / or prophylactic agents, including but not limited to one or more different types of nucleic acids or encoding different agents. In some embodiments, the pharmaceutical compositions include one or more pharmaceutically acceptable excipients or auxiliary ingredients, including but not limited to pharmaceutically acceptable carriers.

[0180] Pharmaceutical compositions containing nanoparticles can be formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermal (passive or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts, and can be formulated into dosage forms suitable for each route of administration.

[0181] In some in vivo methods, the nanoparticle compositions disclosed herein are administered to an individual in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dose sufficient to treat, inhibit or alleviate one or more symptoms of the disorder being treated or otherwise provide the desired pharmacological and / or physiological effects. The exact dose will vary according to a variety of factors, such as individual dependent variables (e.g., age, immune system health, etc.), disease, and treatment being performed.

[0182] For the disclosed nanoparticles, as further studies are conducted, information about appropriate dosage levels for treating various conditions in various patients will emerge, and considering the treatment background, age and general health of the recipient, the ordinary technician will be able to determine the appropriate dosage. The selected dosage depends on the desired therapeutic effect, the route of administration and the duration of the desired treatment. For the disclosed nanoparticles, a dosage level of 0.001 mg to 5 mg nucleic acid / kg body weight is usually administered to mammals daily. More specifically, the preferred dosage of the disclosed nanoparticles is 0.01 mg / kg to 0.25 mg / kg. For the disclosed nanoparticles, a dosage level of 0.2 mg to 100 mg of the four components (ionizable lipids, cholesterol, PEG-lipids and phospholipids) / kg body weight is usually administered to mammals. More specifically, the preferred dosage of the disclosed nanoparticles is 0.05 mg / kg to 0.5 mg / kg of the four components / kg body weight.

[0183] In certain embodiments, the lipid nanoparticle composition is administered locally, for example, by direct injection into the site to be treated. Typically, injection causes an increase in the local concentration of the lipid nanoparticle composition that is greater than that achievable by systemic administration. The lipid nanoparticle composition can be combined with a matrix as described above to assist in increasing the local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide beyond the site to be treated.

[0184] 1. Preparations for parenteral administration

[0185] In some embodiments, nanoparticle compositions disclosed herein, including those containing lipid nanoparticles, are administered by parenteral injection in aqueous solution. The formulation can also be in the form of a suspension or emulsion. Generally, a pharmaceutical composition comprising an effective amount of lipid nanoparticles is provided, and optionally includes a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier. Such compositions optionally include one or more of the following: diluent, sterile water, buffered saline with various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives, such as detergents and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium pyrosulfite) and preservatives (e.g., thimerosal, benzyl alcohol) and filler materials (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters (e.g., ethyl oleate). The formulation can be lyophilized and redissolved / resuspended immediately prior to use. The formulation can be sterilized, for example, by filtering through a bacteria-retaining filter, by adding a bactericide to the composition, by irradiating the composition, or by heating the composition.

[0186] 2. Controlled delivery polymer matrix

[0187] Lipid nanoparticles disclosed herein can also be used in the form of controlled release formulations. Can be after implanting polymer devices (rods, cylinders, films, discs) or injections (microparticles), prepared for the controlled release polymer devices of systemic long-term release. Matrix can be microparticles such as microspheres in the form of, wherein the medicament is dispersed in a solid polymer matrix or microcapsules, wherein the core has a material different from the polymer shell, and peptides are dispersed or suspended in the core, which can be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres and microcapsules are used interchangeably. Alternatively, polymers can be cast into nanometer to 4 cm thin plates or films, powders produced by grinding or other standard techniques, or even gels such as hydrogels.

[0188] Non-biodegradable or biodegradable substrates can be used for delivering lipid nanoparticles, although biodegradable substrates are preferred in some embodiments. These can be natural or synthetic polymers, although due to better characterization of degradation and release curves, synthetic polymers are preferred in some embodiments. Select polymers based on the time period of expected release. In some cases, linear release may be the most useful, although pulse release or "overall release" can provide more effective results in other cases. Polymer can be in the form of a hydrogel (usually absorbing up to about 90% by weight of water), and can optionally be cross-linked with multivalent ions or polymers.

[0189] 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 method developed for preparing microspheres for drug delivery, for example, as described in 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).

[0190] The devices can be formulated for local release to treat the area of ​​implantation or injection, which typically delivers a much smaller dose than that used to treat the entire body - or systemic delivery. These can be implanted or injected subcutaneously into muscle, fat, or swallowed.

[0191] D. Methods for making lipid nanoparticles

[0192] Methods for making lipid nanoparticles are known in the art. In one embodiment, the disclosed lipid nanoparticles are made 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 short, a carrier, such as an oligonucleotide or siRNA, is prepared in a buffer. Other lipid nanoparticle components (ionizable lipids, PEG-lipids, cholesterol and DSPC) are prepared in another buffer. A syringe pump introduces the two solutions into a microfluidic device. The two solutions are contacted in a microfluidic device to form lipid nanoparticles that encapsulate the carrier.

[0193] The method of screening disclosed lipid nanoparticles is discussed in International Patent Application No. PCT / US / 2018 / 058171, which is incorporated herein by reference in its entirety. The screening method characterizes the delivery formulation of the vehicle to identify the formulation with the desired tropism, and delivers the functional carrier to the cytoplasm of a specific cell. The screening method uses a reporter with a function that can be detected when delivered to the cell. The function of the reporter in the detection cell indicates that the preparation of the delivery vehicle will deliver a functional carrier to the cell. A chemical composition marker is included in each different delivery vehicle preparation to track the specific chemical composition for each different delivery vehicle preparation. In one embodiment, the chemical composition marker is a nucleic acid barcode. The sequence of the nucleic acid barcode is paired with the chemical component of the delivery vehicle for preparing the nucleic acid barcode, so that when the nucleic acid barcode is sequenced, the chemical composition of the delivery vehicle of the delivery barcode is identified. Representative reporters include but are not limited to siRNA, mRNA, nuclease protein, nuclease mRNA, small molecules, epigenetic modifiers and phenotypic modifiers.

[0194] E. How to use

[0195] Disclosed herein are methods for delivering cargo (e.g., nucleic acids) to specific cells or organs using the disclosed lipid nanoparticles. In some embodiments, the nanoparticles deliver therapeutic or preventive agents to specific cells or organs of individuals in need in the absence of a targeting ligand. In another embodiment, the disclosed lipid nanoparticles can be used to treat or prevent a disease in an individual in need.

[0196] In some embodiments, the disclosed nanoparticles are delivered directly to an individual. In other embodiments, the lipid nanoparticles are contacted with cells ex vivo, and the treated cells are administered to an individual. The 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.

[0197] 1. Methods of delivering cargo to cells

[0198] Provided herein are methods of delivering therapeutic and / or prophylactic nucleic acids to individuals in need thereof.

[0199] In some embodiments, the disclosed lipid nanoparticle compositions target specific types or categories of cells (e.g., cells of a specific organ or system thereof). For example, a nanoparticle composition comprising a target therapeutic agent and / or a preventive agent can be specifically delivered to an individual's immune cells. Exemplary immune cells include, but are not limited to, CD8+, CD4+, or CD8+CD4+ cells. In other embodiments, the lipid nanoparticles can be formulated to be delivered to mammalian liver hepatocytes, liver immune cells, spleen T cells, or lung endothelial cells in the absence of a targeting ligand. Specific delivery of cells of a specific class or type indicates that a higher proportion of lipid nanoparticles are delivered to cells of the target type or class. In some embodiments, specific delivery can result in an increase in the amount of therapeutic agents and / or preventive agents of tissues per 1g of the targeted endpoint by more than 2 times, 5 times, 10 times, 15 times, or 20 times.

[0200] 2. Gene Regulation Methods

[0201] Provided herein are methods for gene regulation using disclosed lipid nanoparticles. In one embodiment, lipid nanoparticles can be used to reduce gene expression in target cells of individuals in need. Lipid nanoparticles can deliver inhibitory nucleic acids to target cells of individuals without targeting ligands. Inhibitory nucleic acids can be siRNAs.

[0202] Another embodiment provides a method of using the disclosed lipid nanoparticles to edit genes in cells of an individual in need thereof.

[0203] In one embodiment, the cell regulated by the targeted gene is an immune cell. The immune cell can be a T cell, such as a CD8+T cell, a CD4+T cell, or a T regulatory 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 regulated by the targeted gene is a hepatocyte.

[0204] 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, TNFR-II, IL-7R, OX-4 , CD4, CD8, CD94, CD10, CD114, CD124, 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.

[0205] Exemplary tumor-associated antigens that can be recognized by T cells and are expected to be targeted 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.

[0206] 3. Individuals to be treated

[0207] In some embodiments, the treated individual is a mammal suffering from cancer, autoimmune disease, infectious disease, organ transplantation, organ failure, protein deficiency, or a combination thereof. In embodiments, the individual is a human. In some embodiments, the methods described herein can cause liver cells to translate certain proteins. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to liver cells.

[0208] Example

[0209] General comments: Unless otherwise noted, all reactions were performed under a nitrogen atmosphere in a flask or vial with magnetic stirring using anhydrous grade solvents. 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+ with a pre-filled Biotage Sfar silica gel column. Thin layer chromatography was performed using Merck silica gel 60 plates, and iodine was used to visualize the compounds. Nuclear magnetic resonance (NMR) spectroscopy was performed using a Varian INOVA 500 MHz spectrometer; chemical shifts were reported in δ parts per million (ppm) high field of tetramethylsilane, with reference to the residual solvent peak of CHCl3 at δ = 7.26 ppm. A HPLC-MS / MS setup was used with a Waters Acquity UPLC BEH C18 column ( Liquid chromatography-mass spectrometry (LCMS) analysis was performed on a Waters Acquity UPLCH-class Plus with a QDa detector (ESI) (1.7 μM, 2.1 mm×50 mm). Compounds were analyzed using one of the following general 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 minutes, then held at 95% B for 2 minutes, then ramped back up to 10% B for 1 minute; flow rate=0.5 mL / min. Method B: Column - XTERRA RP 18 (4.6 x 50 mm), 5μ, (mobile phase: initially 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF]; then within 2.65 minutes, to 2% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN:THF], maintaining this mobile phase composition until 3.75 minutes, and finally returning to the initial conditions, i.e., within 4.90 minutes, 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF], maintaining this mobile phase composition until 5.10 minutes. Flow rate = 1.2 ml / min

[0210] List of abbreviations

[0211] DCM: dichloromethane

[0212] DIPEA: N,N-diisopropylethylamine

[0213] DMAP: 4-(dimethylamino)pyridine

[0214] DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine

[0215] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine

[0216] EDC: N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

[0217] Eq: equivalent

[0218] ESI: electrospray ionization

[0219] LCMS: Liquid chromatography-mass spectrometry

[0220] LNP: lipid nanoparticle

[0221] NMR: Nuclear Magnetic Resonance

[0222] PPTS: Pyridinium p-toluenesulfonate

[0223] RT: retention time

[0224] according to Figure 1 Example lipids were prepared according to the synthetic schemes shown.

[0225] A representative synthetic procedure is illustrated using the preparation of Example 1: 3-((4,4-bis((((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadec-9,12-dienoate

[0226] Step 1: 4,4-Bis(((Z)-oct-5-en-1-yl)oxy)butyronitrile (Intermediate Ia)

[0227]

[0228] The following is a representative of General Procedure A: 4,4-diethoxybutyronitrile (1.5 g, 9.5 mmol, 1 eq) and cis-5-octen-1-ol (3.7 g, 29 mmol, 3 eq) were added to a vial containing pyridinium p-toluenesulfonate (0.12 g, 0.48 mmol, 0.05 eq). The vial was capped tightly and the resulting mixture was heated at 105 ° C for 72 hours. Thereafter, the mixture was cooled to room temperature. The crude material was purified by flash column chromatography (100 g silica, 0% to 100% dichloromethane in hexane for 20 minutes). 4,4-bis(((Z)-oct-5-ene-1-yl)oxy)butyronitrile (1.14 g, 37%) was obtained as a colorless oil. 1H NMR (500MHz, chloroform-d) δ5.43-5.27 (m, 4H), 4.56 (t, J=5.3Hz, 1H), 3.61 (dt, J=9.3, 6.6Hz, 2H), 3.44 (dt, J=9.3, 6.6 Hz, 2H), 2.42 (t, J=7.4Hz, 2H), 2.12-1.91 (m, 9H), 1.66-1.54 (m, 5H), 1.49-1.36 (m, 4H), 0.96 (t, J=7.6Hz, 6H).

[0229] Step 2: 4,4-Bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (Intermediate IIa)

[0230]

[0231] The following is representative of General Procedure B: To a vial containing 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyronitrile (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 capped tightly and the reaction mixture was heated to 110 °C for 18 hours. Thereafter, the mixture was cooled to room temperature. The mixture was diluted with ethyl acetate (20 mL) and the pH was adjusted to ~5 by adding 1 M HCl. The resulting two-phase 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 (400MHz, chloroform-d) δ5.41-5.24 (m, 4H), 4.45 (t, J=5.6Hz, 1H), 3.51 (dt, J=9.0, 6.7Hz, 2H), 3.39 (dt, J=9.0, 6.7Hz, 2H), 2. 17 (t, J=7.6Hz, 2H), 2.08-1.98 (m, 8H), 1.81 (q, J=7.3Hz, 2H), 1.59-1.52 (m, 4H), 1.44-1.32 (m, 4H), 0.94 (t, J=7.5Hz, 6H).

[0232] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester (Intermediate III)

[0233]

[0234] Linoleic acid (7.9 g, 1 equivalent, 28 mmol), DIPEA (5.5 g, 7.4 mL, 1.5 equivalents, 42 mmol) and DMAP (0.69 g, 0.2 equivalents, 5.7 mmol) were added to a mixture of trimethylolmethane (3.0 g, 1 equivalent, 28 mmol) in dichloromethane (100 mL). EDC (8.1 g, 1.5 equivalents, 42 mmol) was finally added and stirred at 23 ° C for 18 hours. Thereafter, the reaction mixture was concentrated and purified by flash column chromatography (200 g silica, 0% to 90% ethyl acetate in hexane for 20 minutes). (9Z, 12Z)-octadecane-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl) propyl ester (2.6 g, 25%) was obtained as a colorless oil. 1 H NMR (500MHz, chloroform-d) δ5.43-5.27 (m, 5H), 4.24 (d, J=6.3Hz, 2H), 3.76 (ddt, J=21.1, 11.1, 5.3Hz, 4H), 2.77 (d, J=6.8Hz, 2 H), 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).

[0235] Step 4: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate (Intermediate IVa).

[0236]

[0237] 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) was added (9Z,12Z)-octadec-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester (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). Finally, EDC (666 mg, 2 eq, 3.47 mmol) was added and stirred at 23° C. for 18 hours. 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). 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate was obtained as a colorless oil (450 mg, 38%). 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, 7.2Hz, 2H), 2.25-2.15 (m, 2H), 2.12-1.97 (m, 12H), 1.94 (ddd, J=8.0, 7 .2, 5.5Hz, 2H), 1.67-1.53 ​​(m, 8H), 1.45-1.26 (m, 17H), 0.96 (t, J=7.6Hz, 5H), 0.92-0.87 (m, 3H).

[0238] Step 5: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 1)

[0239]

[0240] 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)-octadec-9,12-dienoate (Intermediate IVa, 100 mg, 1 eq, 145 μmol) in dichloromethane (1 mL) were 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. Thereafter, DIPEA (74.8 mg, 101 μL, 4 eq, 579 μmol) and 3-(diethylamino)propan-1-ol (7 6.0 mg, 85.9 μL, 4 equivalents, 579 μmol). The resulting mixture was stirred at 23 ° C for another 18 hours. Thereafter, the reaction mixture was diluted with dichloromethane (10 mL), washed with 0.75 M aqueous sodium carbonate solution (3 × 10 mL), water (10 mL) and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was dried over sodium sulfate, concentrated, and the residue was purified by flash column chromatography (10 g silica, 0% to 25% methanol in dichloromethane for 12 minutes). (9Z, 12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-ene-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (81 mg, 66%) was obtained as a light yellow oil. 1 H 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), 2.3 1 (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): found m / z (M+H) = 848.7, RT = 3.73 min.

[0241] Example 2: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(((4-(pyrrolidin-1-yl)butyryl)oxy)methyl)propyl ester

[0242]

[0243] 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)-octadec-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 equivalents, 0.23 mmol) was added and stirred at 23 ° C for 18 hours. Thereafter, the reaction mixture was concentrated and purified by flash column chromatography (10 g silica, 0% to 20% methanol in DCM over 12 column volumes). (9Z, 12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-ene-1-yl)oxy)butanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl ester (74 mg, 78%) was obtained as a colorless oil. 1 H NMR (500MHz, chloroform-d) δ5.42-5.28 (m, 8H), 4.49 (t, J=5.6Hz, 1H), 4.15-4.10 (m, 6H), 3.57 (dt, J=9.3, 6.6Hz, 2H), 3.41 (dt, J=9.3, 6.6Hz, 2H), 2.77 (tdq, J=7.1, 1.4, 0.8Hz, 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): found m / z (M+H) = 830.7, RT = 3.75 min.

[0244] The following intermediates Ib-Ih were prepared according to General Procedure A, but varying the alcohol building block.

[0245]

[0246] Intermediate Ib, 4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butyronitrile: Prepared from rac-citronellol on a 9.5 mmol scale, yield 0.87 g (24%).

[0247]

[0248] Intermediate Ic, 4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butyronitrile: Prepared from 4,4,5,5,5-pentafluoropentan-1-ol on a 9.5 mmol scale, yield 1.8 g (45%).

[0249]

[0250] Intermediate Id, 4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butyronitrile: prepared from 5,5,6,6,6-pentafluorohexan-1-ol on a 9.5 mmol scale, yield 0.70 g (16%).

[0251]

[0252] 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%).

[0253]

[0254] Intermediate If, ​​4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butyronitrile: prepared from 7,7,8,8,8-pentafluorooctan-1-ol on a 9.5 mmol scale, yield 1.6 g (33%). 1 H NMR (500MHz, chloroform-d) δ4.57 (t, J=5.3Hz, 1H), 3.61 (dt, J=9.2, 6.5Hz, 2H), 3.45 (dt, J=9.3, 6 .5Hz, 2H), 2.43 (t, J=7.3Hz, 2H), 2.09-1.91 (m, 6H), 1.65-1.53 ​​(m, 8H), 1.47-1.35 (m, 8H).

[0255]

[0256] Intermediate Ig, 4,4-bis(4-(trifluoromethyl)phenethoxy)butyronitrile: prepared from (4-trifluoromethylphenyl)ethanol on a 9.5 mmol scale, yield 2.2 g (52%).

[0257]

[0258] Intermediate Ih, 4,4-bis(3-cyclohexylpropoxy)butyronitrile: prepared from 3-cyclohexylpropan-1-ol on a 9.5 mmol scale, yield 2.3 g (68%).

[0259] The following intermediates IIb-IIh were prepared according to general procedure B from the corresponding intermediates Ib-IIh.

[0260]

[0261] 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)butyronitrile (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=7.3Hz, 2H), 2.06-1.89 (m, 6H), 1.68 (d, J=1.4Hz, 6H), 1.66-1.52 (m, 8H), 1.42 -1.29 (m, 5H), 1.16 (dddd, J=13.4, 9.8, 7.8, 5.8Hz, 3H), 0.89 (dd, J=6.6, 1.7Hz, 6H).

[0262]

[0263] 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)butyronitrile (Intermediate Ic) on a 4.3 mmol scale, yield 1.6 g (83%). 1H 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).

[0264]

[0265] 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)butyronitrile (Intermediate Id) on a 1.6 mmol scale, yield 0.53 g (73%).

[0266]

[0267] 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)butyronitrile (Intermediate Ie) on a 3.3 mmol scale, yield 1.9 g (91%). 1 HNMR (500MHz, chloroform-d) δ4.55 (t, J=5.5Hz, 1H), 3.65 (dt, J=9.5, 6.1Hz, 2H), 3.50 (dt, J=9.5, 6.1Hz, 2H), 2.43 (td, J=7.2, 3.5Hz, 2H), 2.18 (tt, J=18.5, 7.8Hz, 4H), 2.02-1.83 (m, 6H).

[0268]

[0269] 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)butyronitrile (Intermediate If) on a 3.1 mmol scale, yield 1.6 g (96%). 1 H NMR (500MHz, chloroform-d) δ4.50 (t, J=5.5Hz, 1H), 3.57 (dt, J=9.4, 6.6Hz, 2H), 3.41 (dt, J=9.4, 6.6Hz , 2H), 2.38 (t, J=7.5Hz, 2H), 2.08-1.87 (m, 6H), 1.69-1.51 (m, 8H), 1.41-1.38 (m, J=3.5Hz, 8H).

[0270]

[0271] Intermediate IIg, 4,4-bis(4-(trifluoromethyl)phenethoxy)butanoic acid: Prepared from 4,4-bis(4-(trifluoromethyl)phenethoxy)butyronitrile (Intermediate Ig) on ​​a 5.0 mmol scale, yield 1.7 g (74%). 1 H NMR (500MHz, chloroform-d) δ7.52 (d, J=7.6Hz, 4H) 7.27 (d, J=7.6Hz, 4H), 4.49 (t, J=5.5Hz, 1H), 3.66 (dt, J=9.4, 6.6H z, 2H), 3.53 (dt, J=9.4, 6.6Hz, 2H), 2.84 (t, J=6.6Hz, 4H), 2.31 (t, J=7.3Hz, 2H), 1.88 (td, J=7.4, 5.6Hz, 2H).

[0272]

[0273] Intermediate IIh, 4,4-bis(3-cyclohexylpropoxy)butanoic acid: Prepared from 4,4-bis(3-cyclohexylpropoxy)butyronitrile (Intermediate Ih) on a 6.5 mmol scale, yield 1.9 g (79%). 1 H NMR (500MHz, chloroform-d) δ4.50 (t, J=5.5Hz, 1H), 3.55 (dt, J=9.3, 6.8Hz, 2H), 3.40 (dt, J=9.3, 6.8Hz, 2H), 2.41 (t, J=7.3Hz, 2H), 1.92 (td, J=7.4, 5.4Hz, 2H), 1.76-1.49 (m, 14H), 1.33-1.06 (m, 12H), 0.94-0.73 (m, 4H).

[0274] The following intermediates IVb-IVh were prepared according to general procedure C from the corresponding intermediates IIb-IIh and intermediate III.

[0275]

[0276] Intermediate IVb, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester: prepared from 4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoic acid (Intermediate IIb) on a 1.1 mmol scale with a yield of 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 = 7.5Hz, 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).

[0277]

[0278] Intermediate IVc, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester: prepared from 4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanoic acid (Intermediate IIc) on a 1.4 mmol scale with a yield of 0.40 g (37%). 1 H NMR (500MHz, chloroform-d) δ5.43-5.28(m, 4H), 4.53(t, J=5.5Hz, 1H), 4.23-4.08( m, 4H), 3.68-3.57 (m, 4H), 3.49 (dt, J=9.5, 6.0Hz, 2H), 2.80-2.74 (m, 2H), 2.40 (t, J=7.4Hz, 2H), 2.32 (dd, J=7.9, 7.2Hz, 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).

[0279]

[0280] Intermediate IVd, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester: prepared from 4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanoic acid (Intermediate IId) on a 1.1 mmol scale with a yield of 0.20 g (23%).

[0281]

[0282] Intermediate IVe, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester: 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 with a yield of 0.50 g (37%). 1 H 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, 1H). z, 2H), 2.32 (dd, J=8.0, 7.2Hz, 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).

[0283]

[0284] Intermediate IVf, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester: prepared from 4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoic acid (Intermediate IIf) on a 1.1 mmol scale with a yield of 0.39 g (41%). 1 H NMR (500MHz, chloroform-d) δ5.43-5.28 (m, 4H), 4.49 (t, J=5.5Hz, 1H), 4.23-4.12 (m, 4H), 3 .62 (t, J=5.9Hz, 2H), 3.57 (dt, J=9.3, 6.5Hz, 2H), 3.41 (dt, J=9.3, 6.6Hz, 2H), 2.8 1-2.74 (m, 2H), 2.41 (t, J=7.5Hz, 2H), 2.32 (dd, J=7.9, 7.2Hz, 2H), 2.24-2.16 (m, 2 H), 2.10-1.89 (m, 12H), 1.66-1.57 (m, 9H), 1.46-1.24 (m, 21H), 0.92-0.86 (m, 3H).

[0285]

[0286] Intermediate IVg, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(4-(trifluoromethyl)phenethoxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester: prepared from 4,4-bis(4-(trifluoromethyl)phenethoxy)butanoic acid (Intermediate IIg) on ​​a 1.4 mmol scale with a yield of 0.38 g (35%). 1 H 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=6.7Hz, 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).

[0287]

[0288] Intermediate IVh, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester: prepared from 4,4-bis(3-cyclohexylpropoxy)butanoic acid (Intermediate IIh) on a 1.4 mmol scale with a yield of 0.40 g (41%). 1 H NMR (500MHz, chloroform-d) δ5.43-5.28 (m, 4H), 4.49 (t, J=5.5Hz, 1H), 4.23-4.11 (m, 4H), 3.62 (t, J=5. 8Hz, 2H), 3.55 (dt, J=9.3, 6.8Hz, 2H), 3.39 (dt, J=9.3, 6.8Hz, 2H), 2.77 (ttd, J=7.0, 1.4, 0.7Hz , 2H), 2.41 (t, J=7.5Hz, 2H), 2.32 (dd, J=7.9, 7.2Hz, 2H), 2.25-2.16 (m, 1H), 2.09-2.01 (m, 4H) , 1.93 (ddd, J=7.9, 7.2, 5.5Hz, 2H), 1.74-1.50 (m, 18H), 1.42-1.09 (m, 25H), 0.92-0.83 (m, 7H).

[0289] The following Examples 3 to 11 were prepared according to General Procedure D from Intermediate IVa.

[0290]

[0291] Example 3, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVa and (1-ethylpiperidin-3-yl)methanol on a 0.15 mmol scale, yield 0.082 g (66%). 1 H NMR (500MHz, chloroform-d) δ5.43-5.27 (m, 8H), 4.49 (t, J=5.6Hz, 1H), 4.22-4.12 (m, 6H), 4.09-3.93 (m, 1 H), 3.57 (dt, J=9.3, 6.6Hz, 2H), 3.41 (dt, J=9.4, 6.6Hz, 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): found m / z (M+H) = 860.6, RT = 3.75 min.

[0292]

[0293] Example 4, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(dipropylamino)propoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 876.6, RT = 3.76 minutes.

[0294]

[0295] Example 5, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-methylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 846.7, RT = 3.72 minutes.

[0296]

[0297] Example 6, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpyrrolidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVa and (1-ethylpyrrolidin-3-yl)methanol on a 0.072 mmol scale, yield 0.039 g (64%). LCMS (Method A): found m / z (M+H) = 846.6, RT = 3.72 minutes.

[0298]

[0299] Example 7, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)-2-hydroxypropoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 864.6, RT = 3.66 minutes.

[0300]

[0301] Example 8, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 820.7, RT = 3.77 minutes.

[0302]

[0303] Example 9, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 846.7, RT = 3.76 minutes.

[0304]

[0305] Example 10, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-methylpiperidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 860.6, RT = 3.83 minutes.

[0306]

[0307] Example 11, (9Z, 12Z)-octadec-9, 12-dienoic acid 3-(((3-(azetidin-1-yl)propoxy)carbonyl)oxy)-2-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl ester: 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 (M+H) = 832.5, RT = 3.74 minutes.

[0308] The following Examples 12 and 13 were prepared according to General Procedure E from Intermediate IVa.

[0309]

[0310] Example 12, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(4-methylpiperazin-1-yl)propanoyl)oxy)methyl)propyl ester: prepared from intermediate IVa and 3-(4-methylpiperazin-1-yl)propanoic acid dihydrochloride on a 0.072 mmol scale, yield 0.056 g (91%). 1H 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): found m / z (M+H)=845.6, RT=3.67 min.

[0311]

[0312] Example 13, 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadec-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 (M+H) = 816.6, RT = 3.76 minutes.

[0313] The following Examples 14 to 22 were prepared according to General Procedure D from Intermediate IVb.

[0314]

[0315] Example 14, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: 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), 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): found m / z (M+H) = 904.8, RT = 4.06 min.

[0316]

[0317] Example 15, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVb and (1-ethylpiperidin-3-yl)methanol on a 0.13 mmol scale, yield 0.070 g (57%). LCMS (Method A): found m / z (M+H) = 916.7, RT = 4.06 minutes.

[0318]

[0319] Example 16, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpyrrolidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVb and (1-ethylpyrrolidin-3-yl)methanol on a 0.08 mmol scale, yield 0.040 g (55%). LCMS (Method A): found m / z (M+H) = 902.7, RT = 4.00 min.

[0320]

[0321] Example 17, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: prepared from intermediate IVb and 2-(1-methylpiperidin-3-yl)ethan-1-ol on a 0.08 mmol scale, yield 0.038 g (52%). LCMS (Method A): found m / z (M+H) = 916.8, RT = 3.94 minutes.

[0322]

[0323] Example 18, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((4-morpholinobutoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 932.6, RT = 3.95 minutes.

[0324]

[0325] Example 19, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((((1r,4r)-4-morpholinocyclohexyl)oxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 958.6, RT = 3.95 minutes.

[0326]

[0327] Example 20, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: prepared from intermediate IVb and 4-(4-methylpiperazin-1-yl)butan-1-ol on a 0.08 mmol scale, yield 0.027 g (36%). LCMS (Method A): found m / z (M+H) = 945.7, RT = 3.95 minutes.

[0328]

[0329] Example 21, (9Z,12Z)-octadec-9,12-dienoic acid 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]oct-3-yl)oxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVb and pseudotropine base on a 0.08 mmol scale, yield 0.019 g (26%). LCMS (Method A): found m / z (M+H) = 914.6, RT = 3.88 min.

[0330]

[0331] Example 22, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((2-((dimethylamino)methyl)benzyl)oxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVb and (2-((dimethylamino)methyl)phenyl)methanol on a 0.08 mmol scale, yield 0.030 g (40%). LCMS (Method A): found m / z (M+H) = 938.8, RT = 4.28 min.

[0332] The following Examples 23 to 30 were prepared according to General Procedure E from Intermediate IVb.

[0333]

[0334] Example 23, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl ester: prepared from intermediate IVb and 4-(pyrrolidin-1-yl)butanoic acid hydrochloride on a 0.09 mmol scale, yield 0.052 g (64%). LCMS (Method A): found m / z (M+H) = 886.7, RT = 3.98 minutes.

[0335]

[0336] Example 24, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(4-methylpiperazin-1-yl)propanoyl)oxy)methyl)propyl ester: prepared from intermediate IVb and 3-(4-methylpiperazin-1-yl)propanoic acid dihydrochloride on a 0.09 mmol scale, yield 0.060 g (72%). LCMS (Method A): found m / z (M+H) = 901.8, RT = 4.08 minutes.

[0337]

[0338] Example 25, 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)propyl 1'-ethyl-[1,4'-bipiperidine]-4-carboxylate: prepared from intermediate IVb and 1'-ethyl-[1,4'-bipiperidine]-4-carboxylic acid dihydrochloride on a 0.08 mmol scale, yield 0.021 g (27%). LCMS (Method A): found m / z (M+H) = 969.8, RT = 3.60 min.

[0339]

[0340] Example 26, 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)propyl 1-(pyridin-4-yl)piperidine-4-carboxylate: prepared from intermediate IVb and 1-(pyridin-4-yl)piperidine-4-carboxylic acid on a 0.08 mmol scale, yield 0.020 g (27%). LCMS (Method A): found m / z (M+H) = 935.5, RT = 3.88 min.

[0341]

[0342] Example 27, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propyl ester: prepared from intermediate IVb and 5-(dimethylamino)pentanoyl hydrochloride on a 0.08 mmol scale, yield 0.057 g (81%). LCMS (Method A): found m / z (M+H) = 874.8, RT = 3.95 min.

[0343]

[0344] Example 28, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(dipropylamino)butanoyl)oxy)methyl)propyl ester: prepared from intermediate IVb and 4-(dipropylamino)butanoyl hydrochloride on a 0.08 mmol scale, yield 0.062 g (84%). LCMS (Method A): found m / z (M+H) = 916.8, RT = 4.00 min.

[0345]

[0346] Example 29, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-morpholinopentanoyl)oxy)methyl)propyl ester: prepared from intermediate IVb and 5-morpholinopentanoyl hydrochloride on a 0.08 mmol scale, yield 0.064 g (87%). LCMS (Method A): found m / z (M+H) = 916.8, RT = 3.96 min.

[0347]

[0348] Example 30, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((2-(1-methylpiperidin-4-yl)acetoxy)methyl)propyl ester: prepared from intermediate IVb and 2-(1-methylpiperidin-4-yl)acetic acid on a 0.08 mmol scale, yield 0.040 g (56%). LCMS (Method A): found m / z (M+H) = 886.7, RT = 3.96 minutes.

[0349] The following Examples 31 and 32 were prepared according to General Procedure D from Intermediate IVc.

[0350]

[0351] Example 31, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 948.5, RT = 3.49 min.

[0352]

[0353] Example 32, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((4,4,5,5,5-pentafluoropentyl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVc and (1-ethylpiperidin-3-yl)methanol on a 0.13 mmol scale, yield 0.050 g (41%). LCMS (Method A): found m / z (M+H) = 960.6, RT = 3.52 minutes.

[0354] The following Examples 33 and 34 were prepared according to General Procedure D from Intermediate IVd.

[0355]

[0356] Example 33, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: 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), 2.4 6-2.37 (m, 3H), 2.31 (t, J = 7.6 Hz, 2H), 2.13-1.99 (m, 9H), 1.93 (td, J = 7.5, 5.4 Hz, 2H), 1.86-1.76 (m, 2H), 1.73-1.56 (m, 11H), 1.41-1.24 (m, 12H), 1.01 (t, J = 7.1 Hz, 6H), 0.92-0.83 (m, 3H). LCMS (Method A): found m / z (M+H) = 976.6, RT = 3.52 min.

[0357]

[0358] Example 34, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((5,5,6,6,6-pentafluorohexyl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVd and (1-ethylpiperidin-3-yl)methanol on a 0.12 mmol scale, yield 0.035 g (29%). 1 H NMR (500MHz, chloroform-d) δ5.43-5.29 (m, 4H), 4.51 (t, J=5.5Hz, 1H), 4.21-4.11 (m, 6H), 4.05 (dd, J=10.6, 5.8Hz, 1H), 3.96 (dd, J=10.6, 7.2Hz, 1H), 3.60 (dt, J=9.3, 5.8Hz, 2H), 3.44 (dt, J=9.3, 5.8Hz, 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): found m / z (M+H) = 988.4, RT = 3.52 min.

[0359] The following Examples 35 and 36 were prepared according to General Procedure D from Intermediate IVe.

[0360]

[0361] Example 35, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: prepared from intermediate IVe and 3-(diethylamino)propan-1-ol on a 0.12 mmol scale, yield 0.050 g (36%). LCMS (Method A): found m / z (M+H) = 1148.4, RT = 3.72 min.

[0362]

[0363] Example 36, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: prepared from intermediate IVe and (1-ethylpiperidin-3-yl)methanol on a 0.12 mmol scale, yield 0.072 g (51%). LCMS (Method A): found m / z (M+H) = 1160.4, RT = 3.73 min.

[0364] The following Examples 37 to 45 were prepared according to General Procedure D from Intermediate IVf.

[0365]

[0366] Example 37, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: 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): found m / z (M+H) = 1032.5, RT = 3.69 min.

[0367]

[0368] Example 38, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVf and (1-ethylpiperidin-3-yl)methanol on a 0.11 mmol scale, yield 0.081 g (68%). 1 H NMR (500MHz, chloroform-d) δ 5.43-5.28 (m, 4H), 4.49 (t, J = 5.5Hz, 1H), 4.19 (d, J = 6.0Hz, 2H), 4.17-4.12 (m, 4H), 4.05 ( dd, J=10.6, 5.8Hz, 1H), 3.96 (dd, J=10.7, 7.2Hz, 1H), 3.57 (dt, J=9.3, 6.6Hz, 2H), 3.41 (dt, J=9.3, 6.5Hz, 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): found m / z (M+H) = 1044.6, RT = 3.67 min.

[0369]

[0370] Example 39, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVf and (1-methylpiperidin-4-yl)methanol on a 0.09 mmol scale, yield 0.055 g (63%). LCMS (Method A): found m / z (M+H) = 1030.5, RT = 3.68 minutes.

[0371]

[0372] Example 40, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVf and 3-(piperidin-1-yl)propan-1-ol on a 0.09 mmol scale, yield 0.052 g (58%). LCMS (Method A): found m / z (M+H) = 1044.5, RT = 3.70 min.

[0373]

[0374] Example 41, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((octahydro-2H-quinolizin-1-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVf and (octahydro-2H-quinolizin-1-yl)methanol on a 0.09 mmol scale, yield 0.050 g (55%). LCMS (Method A): found m / z (M+H) = 1070.6, RT = 3.65 minutes.

[0375]

[0376] Example 42, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVf and 3-(4-methylpiperazin-1-yl)propan-1-ol on a 0.09 mmol scale, yield 0.049 g (54%). LCMS (Method A): found m / z (M+H) = 1059.5, RT = 3.66 minutes.

[0377]

[0378] Example 43, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((4-(diethylamino)butoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVf and 4-(diethylamino)butan-1-ol on a 0.09 mmol scale, yield 0.049 g (55%). LCMS (Method A): found m / z (M+H) = 1046.5, RT = 3.62 min.

[0379]

[0380] Example 44, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: prepared from intermediate IVf and 3-(3-(piperidin-1-yl)propoxy)propan-1-ol on a 0.09 mmol scale, yield 0.044 g (46%). LCMS (Method A): found m / z (M+H) = 1102.5, RT = 3.66 min.

[0381]

[0382] Example 45, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((1,3-dimethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVf and (1,3-dimethylpiperidin-3-yl)methanol on a 0.06 mmol scale, yield 0.042 g (70%). LCMS (Method A): found m / z (M+H) = 1044.5, RT = 3.68 minutes.

[0383]

[0384] Example 46, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl ester: Prepared according to General Procedure E from Intermediate IVf and 4-(pyrrolidin-1-yl)butanoic acid hydrochloride on a 0.09 mmol scale, yield 0.079 g (91%). 1 H 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): found m / z (M+H) = 1014.4, RT = 3.68 min.

[0385] The following Examples 47 and 48 were prepared according to General Procedure D from Intermediate IVg.

[0386]

[0387] Example 47, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(4-(trifluoromethyl)phenethoxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVg and 3-(diethylamino)propan-1-ol on a 0.12 mmol scale, yield 0.067 g (56%). LCMS (Method A): found m / z (M+H) = 972.5, RT = 3.58 minutes.

[0388]

[0389] Example 48, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(4-(trifluoromethyl)phenethoxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVg and (1-ethylpiperidin-3-yl)methanol on a 0.12 mmol scale, yield 0.045 g (37%). LCMS (Method A): found m / z (M+H) = 984.5, RT = 3.56 minutes.

[0390] The following Examples 49 and 50 were prepared according to General Procedure D from Intermediate IVh.

[0391]

[0392] Example 49, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVh and 3-(diethylamino)propan-1-ol on a 0.14 mmol scale, yield 0.059 g (48%). LCMS (Method A): found m / z (M+H) = 876.6, RT = 4.08 min.

[0393]

[0394] Example 50, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from intermediate IVh and (1-ethylpiperidin-3-yl)methanol on a 0.14 mmol scale, yield 0.048 g (39%). LCMS (Method A): found m / z (M+H) = 888.7, RT = 4.04 min.

[0395] The following Examples 51 and 52 were prepared according to General Procedure E from Intermediate IVh.

[0396]

[0397] Example 51, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl ester: prepared from intermediate IVh and 4-(pyrrolidin-1-yl)butanoic acid hydrochloride on a 0.14 mmol scale, yield 0.104 g (87%). LCMS (Method A): found m / z (M+H) = 858.8, RT = 4.06 min.

[0398]

[0399] Example 52, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(3-cyclohexylpropoxy)butanoyl)oxy)-2-(((3-(4-methylpiperazin-1-yl)propanoyl)oxy)methyl)propyl ester: prepared from intermediate IVh and 3-(4-methylpiperazin-1-yl)propanoic acid dihydrochloride on a 0.14 mmol scale, yield 0.074 g (61%). LCMS (Method A): found m / z (M+H) = 873.7, RT = 4.17 min.

[0400]

[0401] Example 53, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((1-ethylpyrrolidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: Prepared according to General Procedure D from Intermediate IVb and (1-ethylpyrrolidin-3-yl)methanol on a 0.08 mmol scale, yield 40 mg (55%). LCMS (Method A): Found m / z (M+H) = 902.7, RT = 3.99 min.

[0402]

[0403] Example 54, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((((1r,4r)-4-morpholinocyclohexyl)oxy)carbonyl)oxy)methyl)propyl ester: Prepared according to General Procedure D from Intermediate IVb and trans-4-morpholinocyclohexan-1-ol on a 0.08 mmol scale, yield 36 mg (47%). LCMS (Method A): Found m / z (M+H) = 958.6, RT = 4.56 min.

[0404]

[0405] 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)-octadec-9,12-dienoate: Prepared according to General Procedure D from Intermediate IVb and 4-(4-methylpiperazin-1-yl)butan-1-ol on a 0.08 mmol scale, yield 27 mg (36%). LCMS (Method A): Found m / z (M+H) = 945.7, RT = 3.95 min.

[0406]

[0407] Example 56, (9Z,12Z)-octadec-9,12-dienoic acid 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]oct-3-yl)oxy)carbonyl)oxy)methyl)propyl ester: Prepared according to General Procedure D from Intermediate IVb and pseudotropine base on a 0.08 mmol scale, yield 19 mg (26%). LCMS (Method A): Found m / z (M+H) = 914.8, RT = 3.88 min.

[0408]

[0409] 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)-octadec-9,12-dienoate: Prepared according to General Procedure D from Intermediate IVb and 4-morpholinobutan-1-ol on a 0.08 mmol scale, yield 39 mg (52%). LCMS (Method A): Found m / z (M+H) = 932.7, RT = 4.58 min.

[0410]

[0411] 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)-octadec-9,12-dienoate: Prepared according to General Procedure D from Intermediate IVb and 2-(1-methylpiperidin-3-yl)ethan-1-ol on a 0.08 mmol scale, yield 38 mg (52%). LCMS (Method A): Found m / z (M+H) = 916.8, RT = 3.94 min.

[0412]

[0413] Example 59, 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)propyl 1'-ethyl-[1,4'-bipiperidine]-4-carboxylate: Prepared according to General Procedure E from Intermediate IVb and 1'-ethyl-[1,4'-bipiperidine]-4-carboxylic acid dihydrochloride on a 0.08 mmol scale, yield 21 mg (27%). LCMS (Method A): Found m / z (M+H) = 969.7, RT = 3.41 min.

[0414]

[0415] Example 60, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(dipropylamino)butanoyl)oxy)methyl)propyl ester: Prepared according to General Procedure E from Intermediate IVb and 4-(dipropylamino)butanoic acid hydrochloride on a 0.08 mmol scale, yield 62 mg (84%). LCMS (Method A): Found m / z (M+H) = 916.8, RT = 4.00 min.

[0416]

[0417] Example 61, 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)propyl 1-(pyridin-4-yl)piperidine-4-carboxylate: Prepared according to General Procedure E from Intermediate IVb and 1-(pyridin-4-yl)piperidine-4-carboxylic acid hydrochloride on a 0.08 mmol scale, yield 20 mg (27%). LCMS (Method A): Found m / z (M+H) = 935.7, RT = 3.87 min.

[0418]

[0419] 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)-octadec-9,12-dienoate: Prepared from Intermediate IVb and 2-(1-methylpiperidin-4-yl)acetic acid on a 0.08 mmol scale according to General Procedure E, yield 40 mg (56%). LCMS (Method A): Found m / z (M+H) = 886.7, RT = 3.95 min.

[0420]

[0421] Example 63, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-morpholinopentanoyl)oxy)methyl)propyl ester: Prepared from intermediate IVb and 5-morpholinopentanoyl hydrochloride on a 0.08 mmol scale according to General Procedure E, yield 64 mg (87%). LCMS (Method A): Found m / z (M+H) = 916.9, RT = 4.50 min.

[0422]

[0423] 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)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-morpholinopentanoyl)oxy)methyl)propyl (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-(((5-morpholinopentanoyl)oxy)methyl)propyl ester: 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): Found m / z (M+H) = 874.8, RT = 3.95 min.

[0424]

[0425] Example 65, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-methylpiperidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 860.7, RT = 3.83 min.

[0426]

[0427] Example 66, (9Z,12Z)-octadec-9,12-dienoic acid 3-(((3-(azetidin-1-yl)propoxy)carbonyl)oxy)-2-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl ester: 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 (M+H) = 832.6, RT = 3.74 min.

[0428]

[0429] Example 67, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((3,7-dimethyloct-6-en-1-yl)oxy)butanoyl)oxy)-2-((((2-((dimethylamino)methyl)benzyl)oxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 938.7, RT = 4.28 min.

[0430]

[0431] 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)-octadec-9,12-dienoate: Prepared from Intermediate IVf and 4-(diethylamino)butan-1-ol on a 0.086 mmol scale according to General Procedure D, yield 49 mg (55%). LCMS (Method A): Found m / z (M+H) = 1046.5, RT = 3.63 min.

[0432]

[0433] Example 69, 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((3-(3-(piperidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadec-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 (M+H) = 1102.5, RT = 3.66 min.

[0434]

[0435] Example 70, 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadec-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 (M+H) = 816.6, RT = 3.74 min.

[0436]

[0437] 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)-octadec-9,12-dienoate: Prepared from Intermediate IVa and 3-(dimethylamino)propan-1-ol on a 0.077 mmol scale according to General Procedure D, yield 22 mg (35%). LCMS (Method A): Found m / z (M+H) = 820.7, RT = 3.76 min.

[0438]

[0439] 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)-octadec-9,12-dienoate: Prepared from Intermediate IVa and 3-(pyrrolidin-1-yl)propan-1-ol on a 0.077 mmol scale according to General Procedure D, yield 27 mg (41%). LCMS (Method A): Found m / z (M+H) = 846.7, RT = 3.73 min.

[0440]

[0441] Example 73, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-((((1,3-dimethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: 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 (M+H) = 1044.5, RT = 3.69 min.

[0442]

[0443] 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)-octadec-9,12-dienoate

[0444] Step 1: 2-(Hydroxymethyl)butane-1,4-diol

[0445]

[0446] To a stirred solution of ethane-1,1,2-tricarboxylic acid triethyl ester (5 g, 20.3 mmol) in tert-butanol (80 mL) was added NaBH4 (2.3 g, 60.9 mmol) at 25 ° C under an argon atmosphere. The resulting suspension was heated to reflux, and methanol (3 mL) was added dropwise in three portions over 30 minutes. The resulting solution was heated to reflux for another 3 hours. The reaction mixture was then cooled to 25 ° C and neutralized with 5N HCl (2.5 mL). The precipitate was filtered and the filtrate was evaporated to obtain a crude material, which was purified by combined fast (combiflash) column chromatography, eluted with 10% to 15% MeOH / DCM to obtain 2- (hydroxymethyl) butane-1,4-diol (1.7 g, 69%) as a yellow liquid. 1 H NMR (400MHz, DMSO-d6): δ1.34-1.46 (m, 2H), 1.49-1.63 (m, 1H), 3.27-3.48 (m, 6H), 4.34 (t, J=5.2Hz, 2H), 4.40 (t, J=5.1Hz, 1H).

[0447] Step 2: 2-(2,2-Dimethyl-1,3-dioxan-5-yl)ethan-1-ol

[0448]

[0449] 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) at 25 ° C under an argon atmosphere was added p-toluenesulfonic acid monohydrate (0.36 g, 3.1 mmol). The reaction mixture was stirred at 25 ° C for 16 hours. Thereafter, the reaction was neutralized with triethylamine (5 mL). The solvent was removed under reduced pressure to obtain a crude material, which was purified by combined flash column chromatography, eluted with 15% ethyl acetate-hexane to obtain 2-(2,2-dimethyl-1,3-dioxane-5-yl) ethane-1-ol (1.2 g, 53%) as a light yellow liquid. 1 H NMR (400MHz, CDCl3): δ1.41 (s, 6H), 1.50-1.60 (m, 2H), 1.88-1.98 (m, 1H), 3.6 3 (dd, J=7.9, 11.8Hz, 2H), 3.70 (t, J=6.4Hz, 2H), 3.93 (dd, J=4.5, 11.8Hz, 2H).

[0450] Step 3: 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl (4-nitrophenyl)carbonate

[0451]

[0452] To a solution of 2-(2,2-dimethyl-1,3-dioxane-5-yl)ethane-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 hours. Thereafter, 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 layer was dried over Na2SO4. The solvent was removed under reduced pressure to give the crude material, which was purified by combined flash column chromatography eluting with 30% ethyl acetate-hexanes 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 (400MHz, CDCl3) δ1.29-1.34 (m, 6H), 1.62 (q, J=6.7Hz, 2H), 1.80 (bs, 1H), 3.54 (q, J=8.8Hz, 2H), 3.83 (dd, J=11.4Hz, 4.0Hz, 2H), 4.28 (t, J=6.4Hz, 2H), 7.57 (d, J=9.0Hz, 2H), 8.31 (d, J=9.0Hz, 2H).

[0453] Step 4: 3-(Diethylamino)propyl (2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl)carbonate

[0454]

[0455] To a stirred solution of (4-nitrophenyl)carbonic acid 2-(2,2-dimethyl-1,3-dioxane-5-yl)ethyl ester (150mg, 0.46mmol) in 5mL DCM was added DMAP (5.6mg, 0.04mmol) and pyridine (0.07mL, 0.9mmol) and stirred for 5 minutes at 25°C. Then 3-(diethylamino)-1-propanol (78.7mg, 0.6mmol) was added at 25°C. The reaction mass was stirred for 9 hours at 25°C. The reaction was judged to be complete by TLC (5% MeOH-DCM). The reaction mixture was diluted with water and extracted with DCM (3×15mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material thus obtained was purified by combined flash column chromatography eluting with 10% MeOH-DCM to afford 3-(diethylamino)propyl (2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl)carbonate (98 mg, 67%) as a light yellow liquid. 1 H NMR (400MHz, CDCl3) δ1.03 (t, J=7.0Hz, 3H), 1.40 (s, 3H), 1.63 (q, J=6.7Hz, 1H), 1.80-1.87 (m, 1H), 2.54 (t, J=7.0Hz, 3H), 3.58 (q, J=8.2Hz, 1H), 3.91 (dd, J=11.8Hz, 4.2Hz, 1H), 4.13 (q, J=6.2Hz, 2H).

[0456] Step 5: 3-(Diethylamino)propyl (4-hydroxy-3-(hydroxymethyl)butyl)carbonate

[0457]

[0458] 1N HCl (0.9mL, 0.9mmol) was added to a stirred solution of (2-(2,2-dimethyl-1,3-dioxane-5-yl)ethyl)carbonic acid 3-(diethylamino)propyl ester (92mg, 0.3mmol) in MeOH (1mL) at 25°C. The reaction mass was stirred for 2 hours. The reaction was judged to be complete by TLC (5% MeOH-DCM). The reaction mixture was concentrated and azeotroped twice with toluene to obtain a crude product (4-hydroxy-3-(hydroxymethyl)butyl)carbonic acid 3-(diethylamino)propyl ester (120mg), which was directly used in the next step without purification.

[0459] Step 6: (9Z,12Z)-octadec-9,12-dienoic acid 4-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(hydroxymethyl)butyl ester

[0460]

[0461] To the stirring solution of linoleic acid (0.08mL, 0.26mmol) in 3mL DCM, add EDC (82.9mg, 0.43mmol) and DMAP (7.2mg, 0.06mmol) and stir for 5 minutes at 25 ℃.Add DIPEA (0.147mL, 0.86mmol) and (4-hydroxy-3-(hydroxymethyl) butyl) carbonate 3-(diethylamino) propyl ester (80mg, 0.29mmol) at 25 ℃.Then the reaction mixture was stirred for 16 hours at 25 ℃.The reaction was judged to be complete by LCMS of the crude reaction mixture.The reaction mixture was diluted with water (10mL) and extracted with DCM (15mL×3).The organic layer merged was washed with brine (10mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material thus obtained was purified by combined flash column chromatography eluting with 10% MeOH-DCM to afford (9Z,12Z)-octadeca-9,12-dienoic acid 4-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(hydroxymethyl)butyl ester (42 mg, 27%) as a light yellow liquid. 1 H NMR (400MHz, CDCl3) δ0.87 (d, J=6.6Hz, 3H), 1.00-1.10 (m, 5H), 1.24-1.29 (m, 20H), 1.69-1.83 (m, 4H), 2.02 (t, J=6.6Hz, 5H), 2.30 (t, J=7.3Hz, 2H), 2.50-2.55 (m, 5H), 2.75 (d, J=7.3Hz, 2H), 3.54-3.61 (m, 2H), 4.13-4.23 (m, 5H), 5.34 (t, J=4.5Hz, 4H).

[0462] Step 7: (9Z,12Z)-octadec-9,12-dienoic acid 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)butyl ester (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 of DCM at 25 °C was added EDC (22.3 mg, 0.12 mmol) and DMAP (1.9 mg, 0.015 mmol) and stirred for 5 minutes. Then DIPEA (0.04mL, 0.23mmol) and (9Z, 12Z)-octadec-9,12-dienoic acid 4-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(hydroxymethyl)butyl ester (42mg, 0.08mmol) were added at 25°C. The reaction mixture was stirred at 25°C for 6 hours. The reaction was judged to be complete by LCMS of the crude reaction mixture. The reaction mixture was diluted with NaHCO3 solution (5mL) and extracted with DCM (10mL×3). The combined organic layers were washed with brine (10mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material thus obtained was purified by preparative-HPLC to give 2-(((4,4-bis((((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)butyl (9Z,12Z)-octadec-9,12-dienoate as a light yellow liquid (24 mg, 36%). LCMS (Method B): found m / z (M+H) = 867.7, RT = 2.04 min.

[0463]

[0464] 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)-octadec-9,12-dienoate

[0465] Step 1: (9Z,12Z)-octadec-9,12-dienoic acid 3-(acryloyloxy)-2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl ester (Intermediate V)

[0466]

[0467] To a stirred solution of (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester (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. Thereafter, 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 combined flash column chromatography eluting with 10% ethyl acetate-hexanes to afford 3-(acryloyloxy)-2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadec-9,12-dienoate as a colorless liquid (860 mg, 79%). 1 H NMR (400 MHz, chloroform-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, 2 H), 3.34-3.45 (m, 2H), 3.56 (q, J=7.4Hz, 2H), 4.11-4.18 (m, 4H), 4.22 (d, J=6.0Hz, 2H), 4.47 (t, J=5.6 Hz, 1H), 5.22-5.45 (m, 8H), 5.85 (d, J=11.3Hz, 1H), 6.11 (dd, J=10.5, 17.3Hz, 1H), 6.36-6.45 (m, 1H).

[0468] 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)-octadec-9,12-dienoate (Example 75): The following is representative of General Procedure F. 3-(propanoyloxy)-2-(((4,4-bis((((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)propyl (Intermediate V) (50 mg, 0.067 mmol, 1 eq) and 1-ethylpiperazine (38 mg, 0.34 mmol, 5 eq) were heated to 50 °C without solvent for 16 h. After cooling to room temperature, the crude reaction mixture was stirred at 4 °C for 16 h. Purification by column chromatography eluting with 9:1 DCM / MeOH gave 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)-octadec-9,12-dienoate (26 mg, 44%). LCMS (Method B): found m / z (M+H) = 859.9, RT = 1.67 min.

[0469]

[0470] Example 76, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: 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 (M+H) = 873.9, RT = 1.56 min.

[0471]

[0472] Example 77, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: prepared from intermediate V and 1-(2-methoxyethyl)piperazine according to general procedure F, yield 34 mg (57%). LCMS (Method B): found m / z (M+H) = 889.9, RT = 1.69 min.

[0473]

[0474] Example 78, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(3,5-dimethylpiperazin-1-yl)propanoyl)oxy)methyl)propyl ester: prepared from intermediate V and 2,6-dimethylpiperazine according to general procedure F, yield 38 mg (65%). LCMS (Method B): found m / z (M+H) = 859.9, RT = 1.71 min.

[0475]

[0476] Example 79, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-((2-(diethylamino)ethyl)(ethyl)amino)propanoyl)oxy)methyl)propyl ester: prepared according to General Procedure F from Intermediate V and N1,N1,N2-triethylethane-1,2-diamine, yield 24 mg (48%). LCMS (Method B): Found m / z (M+H) = 889.9, RT = 2.29 minutes.

[0477]

[0478] 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)-octadec-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 (M+H) = 887.9, RT = 1.34 min.

[0479]

[0480] Example 81, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-((pyridin-3-ylmethyl)amino)propanoyl)oxy)methyl)propyl ester: prepared according to General Procedure F from Intermediate V and pyridin-3-ylmethanamine, yield 20 mg (43%). LCMS (Method B): Found m / z (M+H) = 854.0, RT = 1.34 min.

[0481]

[0482] Example 82, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: prepared according to General Procedure F from Intermediate V and N-methyl-2-(pyridin-2-yl)ethan-1-amine, yield 34 mg (49%). LCMS (Method B): Found m / z (M+H) = 882.0, RT = 1.07 min.

[0483]

[0484] Example 83, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((3-(3-(dimethylamino)pyrrolidin-1-yl)propanoyl)oxy)methyl)propyl ester: prepared from intermediate V and 3-(dimethylamino)pyrrolidine according to general procedure F, yield 27 mg (58%). LCMS (Method B): found m / z (M+H) = 859.9, RT = 1.62 minutes.

[0485]

[0486] 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)-octadec-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 (M+H) = 859.9, RT = 1.34 min.

[0487]

[0488] 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)-octadec-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 (M+H) = 873.8, RT = 2.28 min.

[0489]

[0490] Example 86, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: prepared according to General Procedure F from Intermediate V and 2-(pyridin-4-yl)ethan-1-amine, yield 18 mg (39%). LCMS (Method B): Found m / z (M+H) = 867.9, RT = 1.34 minutes.

[0491]

[0492] Example 87, (9Z,12Z)-octadec-9,12-dienoic acid 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 ester: prepared according to General Procedure F from Intermediate V and 2-(pyridin-4-yl)ethan-1-amine, yield 26 mg (43%). LCMS (Method B): Found m / z (M+H) = 867.9, RT = 1.34 minutes.

[0493]

[0494] 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)-octadec-9,12-dienoate

[0495] Step 1: 4,4-Bis(((Z)-non-3-en-1-yl)oxy)butyronitrile

[0496]

[0497] Prepared according to general procedure A using (Z)-non-3-en-1-ol, yield 280 mg (41%). 1 H NMR (400MHz, chloroform-d): δ0.88 (t, J=6.7Hz, 6H), 1.21-1.41 (m, 12H), 1.94 (q, J=7.2Hz, 2H), 2.03 (q, J=7.2Hz, 4H), 2.32 (q, J=7.0Hz , 4H), 2.41 (t, J=7.4Hz, 2H), 3.39-3.50 (m, 2H), 3.56-3.66 (m, 2H), 4.58 (t, J=5.3Hz, 1H), 5.29-5.40 (m, 2H), 5.41-5.53 (m, 2H).

[0498] Step 2: 4,4-Bis(((Z)-non-3-en-1-yl)oxy)butanoic acid

[0499]

[0500] Prepared according to general procedure B from 4,4-bis(((Z)-non-3-en-1-yl)oxy)butanenitrile, yield 230 mg (78%). 1 H NMR (400MHz, DMSO-d6): δ0.85 (t, J=6.6Hz, 6H), 1.13-1.38 (m, 13H), 1.72 (q, J=7.1Hz, 2H), 2.00 (q, J=7.0Hz, 4H), 2.16-2.29 (m, 5H), 3.33-3.42 (m, 2H), 3.43-3.54 (m, 2H), 4.49 (t, J=5.6Hz, 1H), 5.30-5.47 (m, 4H), 12.06 (s, 1H).

[0501] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0502]

[0503] Prepared according to general procedure C from 4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoic acid, yield 340 mg (58%). 1 H NMR (400MHz, chloroform-d): δ0.82-0.92 (m, 9H), 1.21-1.40 (m, 26H), 1.61 (t, J=7.1Hz, 2H), 1.88-2.09 (m, 10H), 2.13-2.23 (m, 2H), 2.30 (q, J=7.0Hz, 6H) , 2.41 (t, J=7.5Hz, 2H), 2.76 (t, J=6.4Hz, 2H), 3.36-3.47 (m, 2H), 3.52-3 .65(m, 4H), 4.13-4.23(m, 4H), 4.52(t, J=5.5Hz, 1H), 5.26-5.51(m, 8H).

[0504] 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)-octadec-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)-octadec-9,12-dienoate and 3-(diethylamino)propan-1-ol according to General Procedure D. Yield 79 mg (83%). LCMS (Method B): Found m / z (M+H) = 876.9, RT = 1.91 min.

[0505]

[0506] 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)-octadec-9,12-dienoate: prepared from 3-((4,4-bis(((Z)-non-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 50 mg (69%). LCMS (Method B): Found m / z (M+H) = 889.0, RT = 1.77 min.

[0507]

[0508] Example 90, 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadecane-9,12-dienoate

[0509] Step 1: 4,4-Bis((4-butylbenzyl)oxy)butanenitrile

[0510]

[0511] Prepared according to general procedure A using (4-butylphenyl)methanol, yield 1.1 g (36%). 1H NMR (400MHz, chloroform-d): δ0.92 (t, J=7.3Hz, 6H), 1.28-1.40 (m, 4H), 1.51-1.65 (m, 4H), 1.97-2.07 (m, 2H), 2.41 (t, J=7.4Hz, 2H), 2.60 (t, J=7.7Hz, 4H), 4.51 (d, J=11.5Hz, 2H), 4.64 (d, J=11.5Hz, 2H), 4.79 (t, J=5.3Hz, 1H), 7.16 (d, J=7.9Hz, 4H), 7.20-7.28 (m, 4H).

[0512] Step 2: 4,4-Bis((4-butylbenzyl)oxy)butyric acid

[0513]

[0514] Prepared according to general procedure B from 4,4-bis((4-butylbenzyl)oxy)butanenitrile, yield 1.1 g (91%). 1 HNMR (400MHz, DMSO-d6): δ0.89 (t, J=7.3Hz, 6H), 1.22-1.36 (m, 4H), 1.47-1.60 (m, 4H), 1.87 (q, J=7.2Hz, 2H), 2.27 (t, J=7.4Hz, 2H), 2.56 (t, J =7.6Hz, 4H), 4.45 (d, J = 11.7Hz, 2H), 4.56 (d, J = 11.7Hz, 2H), 4.72 (t, J = 5.5Hz, 1H), 7.15 (d, J = 7.9Hz, 4H), 7.22 (d, J = 7.6Hz, 4H), 12.09 (s, 1H).

[0515] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0516]

[0517] Prepared according to general procedure C from 4,4-bis((4-butylbenzyl)oxy)butanoic acid, yield 400 mg (64%). 1H 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, 4H). z, 2H), 3.55 (t, J=5.7Hz, 2H), 4.04-4.19 (m, 4H), 4.50 (d, J=11.5Hz, 2H), 4.61 (d, J=11.5Hz, 2H), 4.74 (t, J=5.4Hz, 1H), 5.26-5.42 (m, 4H), 7.15 (d, J=7.9Hz, 4H), 7.22 (d, J=7.8Hz, 4H).

[0518] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 90). Prepared according to General Procedure D from (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 3-(diethylamino)propan-1-ol to give 62 mg (83%). LCMS (Method B): Found m / z (M+H) = 920.9, RT = 1.81 min.

[0519]

[0520] Example 91, 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadec-9,12-dienoate. Prepared according to General Procedure D from 3-((4,4-bis((4-butylbenzyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol, yield 50 mg, 69%. LCMS (Method B): Found m / z (M+H) = 889.0, RT = 1.77 min.

[0521]

[0522] 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)-octadec-9,12-dienoate

[0523] Step 1: 4,4-Bis(((Z)-dec-4-en-1-yl)oxy)butyronitrile

[0524]

[0525] Prepared according to general procedure A using (Z)-dec-4-en-1-ol, yield 750 mg (51%). 1 H NMR (400MHz, DMSO-d6): δ0.86 (t, J=6.8Hz, 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=7.2Hz, 2H), 3.33-3.44 (m, 2H), 3.47-3.60 (m, 2H), 4.51 (t, J=5.4Hz, 1H), 5.28-5.42 (m, 4H).

[0526] Step 2: 4,4-Bis(((Z)-dec-4-en-1-yl)oxy)butanoic acid

[0527]

[0528] Prepared according to general procedure B from 4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanenitrile, yield 300 mg (71%). 1 H NMR (400MHz, DMSO-d6): δ0.85 (t, J=6.7Hz, 6H), 1.19-1.36 (m, 12H), 1.53 (q, J=6.9Hz, 4H), 1.73 (q, J=7.0Hz, 2H), 1.91-2.09 (m, 8H), 2.22 (t, J=7.5Hz, 2H), 3.29-3.40 (m, 2H), 3.44-3.55 (m, 2H), 4.45 (t, J=5.5Hz, 1H), 5.27-5.42 (m, 4H), 12.05 (s, 1H).

[0529] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0530]

[0531] Prepared according to general procedure C from 4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoic acid, 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=7.4Hz, 2H), 2.76(t, J=6.6Hz, 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=5.4Hz, 1H), 5.26-5.44 (m, 8H).

[0532] 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)-octadec-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)-octadec-9,12-dienoate and 3-(diethylamino)-1-propanol according to General Procedure D, yield 40 mg (61%). LCMS (Method B): Found m / z (M+H) = 905.0, RT = 1.90 min.

[0533]

[0534] Example 93, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-dec-4-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 50 mg (68%). LCMS (Method B): Found m / z (M+H) = 917.0, RT = 1.91 min.

[0535]

[0536] Example 94, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-((3-(5,5-dioctyl-1,3-dioxane-2-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadecane-9,12-dienoate

[0537] Step 1: 3-(5,5-dioctyl-1,3-dioxan-2-yl)propionitrile

[0538]

[0539] 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).

[0540] Step 2: 3-(5,5-dioctyl-1,3-dioxan-2-yl)propanoic acid

[0541]

[0542] Prepared according to General Procedure B from 3-(5,5-dioctyl-1,3-dioxan-2-yl)propionitrile, 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).

[0543] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((3-(5-hexyl-5-octyl-1,3-dioxan-2-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0544]

[0545] Prepared according to general procedure C from 3-(5,5-dioctyl-1,3-dioxan-2-yl)propanoic acid, 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=7.5Hz, 2H), 2.76 (t, J=6.3Hz, 2H), 3.34 (d, J=11.0Hz, 2H), 3.60 (t, J=6.0Hz, 2H), 3.74 (d, J=11.1Hz, 2H), 4.17 (t, J=7.1Hz, 4H), 4.42-4.54 (m, 1H), 5.30-5.39 (m, 4H).

[0546] Step 4: 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-((3-(5,5-dioctyl-1,3-dioxan-2-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadec-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)-octadec-9,12-dienoate and 3-(diethylamino)-1-propanol according to General Procedure D, yield 27 mg (73%). LCMS (Method B): Found m / z (M+H) = 892.9, RT = 1.93 min.

[0547]

[0548] 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)-octadec-9,12-dienoate: prepared from 3-((3-(5-hexyl-5-octyl-1,3-dioxan-2-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 27 mg (59%). LCMS (Method B): Found m / z (M+H) = 905.0, RT = 1.94 min.

[0549]

[0550] Example 96, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(2-(octyloxy)ethoxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester

[0551] Step 1: 4,4-Bis(2-(octyloxy)ethoxy)butyronitrile

[0552]

[0553] Prepared according to general procedure A using 2-(octyloxy)ethan-1-ol, yield 340 mg (21%). 1 H NMR (400MHz, chloroform-d): δ0.87 (t, J=6.7Hz, 6H), 1.23-1.37 (m, 20H), 1.53 (d, J=7.7Hz, 2H), 1.58 (d, J=7.8Hz, 2H), 1.93-2.03 (m, 2H), 2.44 (t, J=7.4Hz, 2H), 3.43 (t, J=6.8Hz, 4H), 3.55 (t, J=4.8Hz, 4H), 3.58-3.69 (m, 2H), 3.70-3.80 (m, 2H), 4.70 (t, J=5.4Hz, 1H).

[0554] Step 2: 4,4-Bis(2-(octyloxy)ethoxy)butanoic acid

[0555]

[0556] Prepared according to general procedure B from 4,4-bis(2-(octyloxy)ethoxy)butyronitrile, yield 136 mg (81%). 1 HNMR (400MHz, DMSO-d6): δ0.85 (t, J=6.7Hz, 6H), 1.07-1.36 (m, 20H), 1.47 (t, J=6.9Hz, 4H), 1.68-1.79 (m, 2H), 2.23 (t, J=7.5Hz, 2H), 3.36 (t, J=6.5Hz, 4H), 3.42-3.56 (m, 6H), 3.58-3.63 (m, 2H), 4.55 (t, J=5.6Hz, 1H), 12.04 (s, 1H).

[0557] Step 3, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(2-(octyloxy)ethoxy)butyryl)oxy)-2-(hydroxymethyl)propyl ester

[0558]

[0559] Prepared according to general procedure C from 4,4-bis(2-(octyloxy)ethoxy)butanoic acid, yield 147 mg (53%). 1 HNMR (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=6.5Hz, 2H), 3.43 (t, J=6.8Hz, 4H), 3.55 (t, J=4.8Hz, 4H), 3.56-3.66 (m, 4H ), 3.67-3.77(m, 2H), 4.08-4.23(m, 4H), 4.64(t, J=5.5Hz, 1H), 5.28-5.41(m, 4H).

[0560] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(2-(octyloxy)ethoxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 96). Prepared according to General Procedure D from (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(2-(octyloxy)ethoxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 3-(diethylamino)-1-propanol, yield 36 mg (62%). LCMS (Method B): Found m / z (M+H) = 941.0, RT = 1.80 min.

[0561]

[0562] Example 97, 3-((4,4-bis(2-(octyloxy)ethoxy)butyryl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadec-9,12-dienoate: prepared from 3-((4,4-bis(2-(octyloxy)ethoxy)butyryl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 27 mg (58%). LCMS (Method B): Found m / z (M+H) = 953.0, RT = 1.81 min.

[0563]

[0564] 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)-octadec-9,12-dienoate

[0565] Step 1: 4,4-Bis(((Z)-non-2-en-1-yl)oxy)butyronitrile

[0566]

[0567] Prepared according to general procedure A using (Z)-non-2-en-1-ol, yield 610 mg (38%). 1 H NMR (400MHz, chloroform-d) δ0.87 (t, J=6.7Hz, 6H), 1.19-1.41 (m, 18H), 1.97 (q, J=7.3Hz, 2H), 2.05 (q, J =7.2Hz, 4H), 2.42 (t, J = 7.4Hz, 2H), 4.04-4.21 (m, 4H), 4.65 (t, J = 5.3Hz, 1H), 5.45-5.65 (m, 4H).

[0568] Step 2: 4,4-Bis(((Z)-non-2-en-1-yl)oxy)butanoic acid

[0569]

[0570] Prepared according to general procedure B from 4,4-bis(((Z)-non-2-en-1-yl)oxy)butyronitrile, yield 550 mg, 87%. 1 H NMR (400MHz, DMSO-d6) δ0.85 (t, J=6.4Hz, 6H), 1.09-1.39 (m, 16H), 1.76 (q, J=6.9Hz, 2H), 2.02 (q, J=6.8H z, 4H), 2.22 (t, J=7.4Hz, 2H), 3.95-4.11 (m, 4H), 4.53 (t, J=5.6Hz, 1H), 5.41-5.57 (m, 4H), 12.06 (s, 1H).

[0571] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0572]

[0573] Prepared according to general procedure C from 4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoic acid, yield 390 mg, 71%. 1 H NMR (400MHz, chloroform-d) δ0.80-0.94(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=7.6Hz, 2H), 2.4 1(t, J=7.4Hz, 2H), 2.76 (t, J=6.4Hz, 2H), 3.60 (t, J=5.9Hz, 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).

[0574] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 98). Prepared according to General Procedure D from (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 3-(diethylamino)-1-propanol, yield 70 mg (71%). LCMS (Method B): Found m / z (M+H) = 876.9, RT = 1.81 min.

[0575]

[0576] 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)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-2-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 45 mg (81%). LCMS (Method B): Found m / z (M+H) = 860.9, RT = 1.56 min.

[0577]

[0578] 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)-octadec-9,12-dienoate

[0579] Step 1: 4,4-Bis(((Z)-hex-3-en-1-yl)oxy)butyronitrile

[0580]

[0581] Prepared according to general procedure A using (Z)-hex-3-en-1-ol, yield 350 mg (51%).

[0582] Step 2: 4,4-Bis(((Z)-hex-3-en-1-yl)oxy)butanoic acid

[0583]

[0584] Prepared according to general procedure B from 4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanenitrile, yield 310 mg (82%). 1 H NMR (400MHz, DMSO-d6) δ0.92 (t, J=7.5Hz, 6H), 1.72 (q, J=7.0Hz, 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=5.6Hz, 1H), 5.27-5.38 (m, 2H), 5.36-5.47 (m, 2H), 12.05 (s, 1H).

[0585] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0586]

[0587] Prepared according to general procedure C from 4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoic acid, yield 165 mg (37%). 1H 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=7.5Hz, 2H), 2.76 (t, J=6.4Hz, 2H), 3.42 (q, J=7.4Hz, 2H), 3.52-3.65 (m , 4H), 4.17 (t, J=5.3Hz, 4H), 4.52 (t, J=5.6Hz, 1H), 5.27-5.41 (m, 4H), 5.41-5.50 (m, 2H).

[0588] 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)-octadec-9,12-dienoate (Example 100). Prepared according to General Procedure D from 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and 3-(diethylamino)-1-propanol, yield 29 mg (52%). LCMS (Method B): Found m / z (M+H) = 792.8, RT = 1.49 min.

[0589]

[0590] 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)-octadec-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 29 mg (51%). LCMS (Method B): Found m / z (M+H) = 804.8, RT = 1.50 min.

[0591]

[0592] 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)-octadec-9,12-dienoate

[0593] Step 1: 4,4-Bis(((Z)-hept-3-en-1-yl)oxy)butanenitrile

[0594]

[0595] Prepared according to general procedure A using (Z)-hept-3-en-1-ol, yield 310 mg (41%). 1 H NMR (400MHz, chloroform-d) δ0.90 (t, J=7.4Hz, 6H), 1.30-1.44 (m, 4H), 1.89-1.99 (m, 2H), 2.02 (q, J=7.3Hz, 4H), 2.32 (q, J=7.0Hz, 4H ), 2.41 (t, J=7.4Hz, 2H), 3.40-3.48 (m, 2H), 3.54-3.66 (m, 2H), 4.58 (t, J=5.3Hz, 1H), 5.31-5.42 (m, 2H), 5.42-5.53 (m, 2H).

[0596] Step 2: 4,4-Bis(((Z)-hept-3-en-1-yl)oxy)butanoic acid

[0597]

[0598] Prepared according to general procedure B from 4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanenitrile, yield 320 mg (83%). 1 H NMR (400MHz, DMSO-d6) δ0.86 (t, J=7.3Hz, 6H), 1.21-1.40 (m, 4H), 1.72 (q, J=7.2Hz, 2H), 1.99 (q, J=6.9Hz, 4H), 2 .18-2.28(m, 6H), 3.31-3.42(m, 2H), 3.44-3.55(m, 2H), 4.49(t, J=5.6Hz, 1H), 5.31-5.47(m, 4H), 12.05(s, 1H).

[0599] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0600]

[0601] Prepared according to general procedure C from 4,4-bis(((Z)-hept-3-en-1-yl)oxy)butanoic acid, yield 295 mg, 45%. 1 H NMR (400MHz, chloroform-d) δ0.83-0.93 (m, 9H), 1.22-1.44 (m, 18H), 1.57-1.67 (m, 2H), 1.88-2.09 (m, 10H), 2.19 (q, J=5.9Hz, 2H), 2.25-2.35 (m, 6H), 2 .41 (t, J=7.5Hz, 2H), 2.76 (t, J=6.5Hz, 2H), 3.36-3.47 (m, 2H), 3.52-3. 65 (m, 4H), 4.10-4.23 (m, 4H), 4.52 (t, J=5.5Hz, 1H), 5.26-5.51 (m, 8H).

[0602] 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)-octadec-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)-octadec-9,12-dienoate and 3-(diethylamino)-1-propanol according to General Procedure D, yield 40 mg (72%). LCMS (Method B): Found m / z (M+H) = 820.8, RT = 1.60 min.

[0603]

[0604] 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)-octadec-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 46 mg (83%). LCMS (Method B): Found m / z (M+H) = 832.8, RT = 1.61 min.

[0605]

[0606] Example 104, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(oct-3-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester

[0607] Step 1: 4,4-Bis(oct-3-yn-1-yloxy)butyronitrile

[0608]

[0609] 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).

[0610] Step 2: 4,4-Bis(oct-3-yn-1-yloxy)butanoic acid

[0611]

[0612] Prepared according to general procedure B from 4,4-bis(oct-3-yn-1-yloxy)butanenitrile, yield 310 mg (82%). 1 HNMR (400MHz, DMSO-d6) δ0.86 (t, J=7.0Hz, 6H), 1.26-1.45 (m, 8H), 1.74 (q, J=7.0Hz, 2H), 2.07-2.17 (m, 4H), 2.2 5 (t, J=7.5Hz, 2H), 2.30-2.41 (m, 4H), 3.40-3.51 (m, 2H), 3.52-3.62 (m, 2H), 4.56 (t, J=5.7Hz, 1H), 12.06 (s, 1H).

[0613] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(oct-3-yn-1-yloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0614]

[0615] Prepared according to general procedure C from 4,4-bis(oct-3-yn-1-yloxy)butanoic acid, yield 390 mg (60%). 1HNMR (400MHz, chloroform-d) δ0.83-0.93 (m, 9H), 1.22-1.51 (m, 22H), 1.57-1.65 (m, 2H), 1.90-2.00 (m, 2H), 2.04 (q, J=6.9Hz, 4H), 2.08-2.23 (m, 6H), 2 .31 (t, J=7.6Hz, 2H), 2.36-2.47 (m, 6H), 2.76 (t, J=6.4Hz, 2H), 3.48-3. 71 (m, 6H), 4.08-4.23 (m, 4H), 4.60 (t, J=5.6Hz, 1H), 5.26-5.42 (m, 4H).

[0616] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(oct-3-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 104). Prepared according to General Procedure D from (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(oct-3-yn-1-yloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 3-(diethylamino)-1-propanol, yield 62 mg (67%). LCMS (Method B): Found m / z (M+H) = 844.9, RT = 1.56 min.

[0617]

[0618] 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)-octadec-9,12-dienoate: Prepared from 3-((4,4-bis((((Z)-hex-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 43 mg (51%). LCMS (Method B): Found m / z (M+H) = 856.9, RT = 1.57 min.

[0619]

[0620] 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)-octadec-9,12-dienoate

[0621] Step 1: 4,4-Bis(((Z)-oct-3-en-1-yl)oxy)butyronitrile

[0622]

[0623] Prepared according to general procedure A using (Z)-oct-3-en-1-ol, yield 100 mg (40%). 1 H NMR (400MHz, chloroform-d) δ0.79-0.99 (m, 7H), 1.16-1.39 (m, 9H), 1.90-1.98 (m, 2H), 2.03 (d, J=6.9Hz, 4H), 2.32 (q, J=7.0Hz, 4H) , 2.41 (t, J=7.4Hz, 2H), 3.39-3.49 (m, 2H), 3.56-3.66 (m, 2H), 4.57 (t, J=5.4Hz, 1H), 5.29-5.40 (m, 2H), 5.41-5.52 (m, 2H).

[0624] Step 2: 4,4-Bis(((Z)-oct-3-en-1-yl)oxy)butanoic acid

[0625]

[0626] Prepared according to general procedure B from 4,4-bis(((Z)-oct-3-en-1-yl)oxy)butyronitrile, yield 320 mg, 83%. 1 H NMR (400MHz, DMSO-d6) δ0.83-0.89 (m, 6H), 1.18-1.36 (m, 8H), 1.72 (q, J=7.0Hz, 2H), 2.01 (q, J=6.7Hz, 4H), 2. 15-2.29 (m, 6H), 3.31-3.42 (m, 2H), 3.44-3.54 (m, 2H), 4.49 (t, J=5.5Hz, 1H), 5.30-5.47 (m, 4H), 12.06 (s, 1H).

[0627] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0628]

[0629] Prepared according to general procedure C from 4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoic acid, yield 310 mg, 48%. 1H 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=7.5Hz, 2H), 2.76 (t, J=6.0Hz, 2H), 3.42 (q, J=7.6Hz, 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).

[0630] 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)-octadec-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)-octadec-9,12-dienoate and 3-(diethylamino)-1-propanol according to General Procedure D, yield 47 mg (82%). LCMS (Method B): Found m / z (M+H) = 848.9, RT = 1.72 min.

[0631]

[0632] 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)-octadec-9,12-dienoate: Prepared from 3-((4,4-bis(((Z)-oct-3-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 45 mg (81%). LCMS (Method B): Found m / z (M+H) = 860.9, RT = 1.56 min.

[0633]

[0634] 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)-octadec-9,12-dienoate

[0635] Step 1: 4,4-Bis(((Z)-non-6-en-1-yl)oxy)butyronitrile

[0636]

[0637] 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).

[0638] Step 2: 4,4-Bis(((Z)-non-6-en-1-yl)oxy)butanoic acid

[0639]

[0640] Prepared according to general procedure B from 4,4-bis(((Z)-non-6-en-1-yl)oxy)butyronitrile. Yield 280 mg, 73%. 1 H NMR (400MHz, DMSO-d6) δ0.91 (t, J=7.5Hz, 6H), 1.21-1.34 (m, 13H), 1.40 (t, J=6.7Hz, 2H), 1.43-1.52 (m, 2H), 1.72 (q, J=7. 1Hz, 1H), 1.91-2.07 (m, 8H), 2.21 (t, J=7.4Hz, 1H), 3.32-3.41 (m, 3H), 3.42-3.53 (m, 1H), 4.32 (s, 1H), 5.24-5.39 (m, 4H).

[0641] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0642]

[0643] Prepared according to general procedure C from 4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoic acid, 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=7.6Hz, 3H), 2.40 (t, J=7.5Hz, 3H), 2.76 (t, J=6.4Hz, 3H), 3.34-3.44 (m, 3H ), 3.50-3.65 (m, 6H), 4.06-4.23 (m, 7H), 4.48 (t, J=5.5Hz, 2H), 5.17-5.50 (m, 12H).

[0644] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 108). Prepared according to General Procedure D from (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 3-(diethylamino)-1-propanol, yield 36 mg (69%). LCMS (Method B): Found m / z (M+H) = 876.9, RT = 1.78 min.

[0645]

[0646] 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)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-non-6-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 37 mg (67%). LCMS (Method B): Found m / z (M+H) = 888.9, RT = 1.77 min.

[0647]

[0648] Example 110, 3-((4,4-bis((8-fluorooctyl)oxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadecane-9,12-dienoate

[0649] Step 1: 4,4-Bis((8-bromooctyl)oxy)butyronitrile

[0650]

[0651] Prepared according to the general procedure using 8-bromooctan-1-ol, yield 750 mg (33%). 1 H NMR (400MHz, chloroform-d) δ1.22-1.41 (m, 12H), 1.37-1.48 (m, 4H), 1.56 (t, J=7.3Hz, 4H), 1.78-1.91 (m, 4H), 1.88-1.98 (m, 2H), 2.41 (t, J=7.3Hz, 2H), 3.33-3.47 (m, 6H), 3.51-3.64 (m, 2H), 4.54 (t, J=5.3Hz, 1H).

[0652] Step 2: 4,4-Bis((8-fluorooctyl)oxy)butyronitrile

[0653]

[0654] A solution of tetrabutylammonium fluoride in THF (1M, 4.65mL, 4.65mmol, 3 equivalents) was added to 4,4-bis((8-bromooctyl)oxy)butyronitrile (750mg, 1.55mmol). The mixture was stirred at 80 °C for 24 hours. Thereafter, the reaction mass was poured into ice-cold water, and the organic layer was extracted with ethyl acetate (30mL×3). The organic layer was washed with brine (5mL×3) and then dried over sodium sulfate. The organic layer was evaporated to give a crude product. The crude product was subjected to column chromatography (using ethyl acetate / hexane) to give 4,4-bis((8-fluorooctyl)oxy)butyronitrile (210mg, 37%) as a light 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).

[0655] Step 3: 4,4-Bis((8-fluorooctyl)oxy)butanoic acid

[0656]

[0657] Prepared according to general procedure B from 4,4-bis((8-fluorooctyl)oxy)butanenitrile, yield 200 mg (92%). 1 H NMR (400MHz, DMSO-d6) δ1.21-1.37 (m, 14H), 1.48 (t, J=6.6Hz, 4H), 1.53-1.77 (m, 6H), 2.22 (t, J=7.4H z, 2H), 3.27-3.40 (m, 3H), 3.43-3.54 (m, 2H), 4.36 (t, J=6.1Hz, 2H), 4.41-4.52 (m, 3H), 12.05 (s, 1H).

[0658] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((8-fluorooctyl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl ester

[0659]

[0660] Prepared according to general procedure C from 4,4-bis((8-fluorooctyl)oxy)butanoic acid, yield 160 mg, 37%. 1 H NMR (400MHz, chloroform-d) δ0.88 (t, J=6.9Hz, 3H), 1.21-1.44 (m, 34H), 1.58-1.77 (m, 6H), 1.87-1.97 (m, 2H), 2.04 (q, J=6.8Hz, 4H), 2.13-2.24 (m, 2H), 2.31 (t, J=7.6Hz, 2H) , 2.40 (t, J=7.5Hz, 2H), 2.76 (t, J=6.4Hz, 2H), 3.34-3.44 (m, 2H), 3.50-3.65 (m, 4H ), 4.10-4.23 (m, 4H), 4.36 (t, J=6.1Hz, 2H), 4.44-4.52 (m, 3H), 5.25-5.44 (m, 4H).

[0661] Step 5: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 110). Prepared according to General Procedure D from (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 3-(diethylamino)-1-propanol. Yield 18 mg (21%). LCMS (Method B): Found m / z (M+H) = 888.9, RT = 1.40 min.

[0662]

[0663] Example 111, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared from (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((8-fluorooctyl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 41 mg (48%). LCMS (Method B): Found m / z (M+H) = 900.9, RT = 1.41 min.

[0664]

[0665] 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)-octadec-9,12-dienoate

[0666] Step 1: 2-Methoxycyclohexan-1-one oxime

[0667]

[0668] Sodium acetate (6.41 g, 78.1 mmol) was added to a stirred solution of 2-methoxycyclohexan-1-one (5 g, 39.0 mmol) in MeOH-HO (150 ml, 1: 2), followed by hydroxylamine hydrochloride (5.43 g, 78.1 mmol). The resulting mixture was heated at 70 ° C for 16 hours in a sealed tube. Thereafter, 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, eluted with 3% ethyl acetate / hexane to give 2-methoxycyclohexan-1-one oxime (4.1 g, 88%) as a colorless oil. 1 H NMR (400MHz, DMSO-d6) δ1.17-1.32 (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).

[0669] Step 2: 6,6-Dimethoxyhexanenitrile

[0670]

[0671] To a stirred solution of 2-methoxycyclohexan-1-one oxime (2.0 g, 14.0 mmol) in CCl4 (20 ml) was added thionyl chloride (1.2 ml, 16.8 mmol) at 0 ° C and stirred for 10 minutes. After that, anhydrous MeOH (20 ml) was added to the reaction mixture at 5-10 ° C. After the addition was completed, the reaction mixture was allowed to reach room temperature and left for 2 hours. The mixture was then diluted with ethyl acetate and washed with brine, dried over sodium sulfate and concentrated to give 6,6-dimethoxyhexanenitrile (1.6 g, 81%) as a light 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).

[0672] Step 3: 6,6-Bis(((Z)-oct-3-en-1-yl)oxy)hexanenitrile

[0673]

[0674] Prepared according to general procedure A from 6,6-dimethoxyhexanenitrile and (Z)-oct-3-en-1-ol, yield 280 mg (50%). 1H NMR (400MHz, DMSO-d6) δ0.83-0.89 (m, 6H), 1.21-1.32 (m, 8H), 1.31-1.43 (m, 2H), 1.47-1.61 (m, 4H), 2.01 (q, J=6.7Hz, 4H), 2 .23 (q, J=6.8Hz, 4H), 2.47 (d, J=7.1Hz, 2H), 3.32-3.42 (m, 2H), 3.44-3.54 (m, 2H), 4.47 (t, J=5.6Hz, 1H), 5.30-5.47 (m, 4H).

[0675] Step 4: 6,6-Bis(((Z)-oct-3-en-1-yl)oxy)hexanoic acid

[0676]

[0677] Prepared according to general procedure B from 6,6-dimethoxyhexanenitrile, yield 260 mg (80%). 1 H NMR (400MHz, DMSO-d6) δ0.86 (q, J=3.8, 5.1Hz, 6H), 1.21-1.34 (m, 10H), 1.48 (t, J=7.6Hz, 4H), 2.00 (t, J=6.7Hz, 4H ), 2.14-2.27(m, 6H), 3.30-3.41(m, 2H), 3.42-3.53(m, 2H), 4.44(t, J=5.6Hz, 1H), 5.30-5.46(m, 4H), 11.97(s, 1H).

[0678] Step 5: (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0679]

[0680] Prepared according to general procedure C from 6,6-bis(((Z)-oct-3-en-1-yl)oxy)hexanoic acid, yield 70 mg (55%). 1H NMR (400MHz, chloroform-d) δ0.83-0.95 (m, 9H), 1.17-1.42 (m, 25H), 1.57-1.70 (m, 5H), 2.04 (d, J=7.0Hz, 8H), 2.18 (t, J=6.2Hz, 2H), 2.31 (t, J=6.7Hz, 8H) , 2.76 (t, J=6.5Hz, 2H), 3.41 (q, J=7.5Hz, 2H), 3.50-3.64 (m, 4H), 4.09-4 .20(m, 4H), 4.48(t, J=5.6Hz, 1H), 5.32-5.37(m, 5H), 5.33-5.48(m, 3H).

[0681] Step 6: (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 112). Prepared according to General Procedure D from (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester and 3-(diethylamino)-1-propanol, yield 37 mg (64%). LCMS (Method B): Found m / z (M+H) = 877.0, RT = 1.70 min.

[0682]

[0683] Example 113, 3-((6,6-di(((Z)-oct-3-en-1-yl)oxy)hexanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadec-9,12-dienoate: prepared from 3-((6,6-di((Z)-oct-3-en-1-yl))oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 89 mg (70%). LCMS (Method B): Found m / z (M+H) = 889.0, RT = 1.79 min.

[0684]

[0685] 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)-octadec-9,12-dienoate

[0686] Step 1: 6,6-Bis(((Z)-hex-3-en-1-yl)oxy)hexanenitrile

[0687]

[0688] Prepared according to general procedure A from 6,6-dimethoxyhexanenitrile and (Z)-hex-3-en-1-ol. Yield: 190 mg, 42%. 1 H NMR (400MHz, chloroform-d) δ0.96 (t, J=7.5Hz, 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=5.5Hz, 1H), 5.27-5.39 (m, 2H), 5.46 (q, J=7.6Hz, 2H).

[0689] Step 2: 6,6-Bis(((Z)-hex-3-en-1-yl)oxy)hexanoic acid

[0690]

[0691] Prepared according to general procedure B from 6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanenitrile, yield 170 mg, 82%. 1 H NMR (400MHz, DMSO-d6) δ0.92 (t, J=7.5Hz, 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=5.6Hz, 1H), 5.27-5.38(m, 2H), 5.36-5.47(m, 2H), 11.96(s, 1H).

[0692] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((((Z)-hex-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0693]

[0694] Prepared according to general procedure C from 6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoic acid, yield 60 mg (45%). 1H NMR (400MHz, chloroform-d) δ0.88 (t, J=6.7Hz, 3H), 0.95 (t, J=7.5Hz, 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=6.4Hz, 2H), 3.36-3.47(m, 2H), 3.50-3. 64 (m, 4H), 4.09-4.24 (m, 4H), 4.48 (t, J=5.6Hz, 1H), 5.26-5.51 (m, 8H).

[0695] 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)-octadec-9,12-dienoate (Example 114). Prepared according to General Procedure D from 3-((6,6-bis((((Z)-hex-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and 3-(diethylamino)-1-propanol, yield 36 mg (62%). LCMS (Method B): Found m / z (M+H) = 820.9, RT = 1.70 min.

[0696]

[0697] 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)-octadec-9,12-dienoate: Prepared from 3-((6,6-bis(((Z)-hex-3-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethylmethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 69 mg (72%). LCMS (Method B): Found m / z (M+H) = 833.0, RT = 1.60 min.

[0698]

[0699] 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)-octadec-9,12-dienoate

[0700] Step 1: 6,6-Bis(((Z)-oct-5-en-1-yl)oxy)hexanenitrile

[0701]

[0702] Prepared according to general procedure A from 6,6-dimethoxyhexanenitrile and (Z)-oct-5-en-1-ol, yield 210 mg (47%). 1 H 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).

[0703] Step 2: 6,6-Bis(((Z)-oct-5-en-1-yl)oxy)hexanoic acid

[0704]

[0705] Prepared according to general procedure B from 6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanenitrile, yield 150 mg, 94%. 1 H NMR (400MHz, DMSO-d6) δ0.91 (t, J=7.5Hz, 6H), 1.24-1.41 (m, 6H), 1.42-1.53 ​​(m, 8H), 1.93-2.06 (m, 8H), 2.18 ( t, J=7.3Hz, 2H), 3.31-3.40 (m, 2H), 3.43-3.53 (m, 2H), 4.40 (t, J=5.6Hz, 1H), 5.19-5.45 (m, 4H), 11.97 (s, 1H).

[0706] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0707]

[0708] Prepared according to general procedure C from 6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoic acid, yield 90 mg (45%). 1H 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), 2.27-2.3 7(m, 4H), 2.76(t, J=6.4Hz, 2H), 3.34-3.44(m, 2H), 3.50-3.58(m, 2H), 3.60( t, J=5.6Hz, 2H), 4.09-4.24 (m, 4H), 4.44 (t, J=5.6Hz, 1H), 5.25-5.43 (m, 8H).

[0709] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 116). Prepared according to General Procedure D from (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester and 3-(diethylamino)-1-propanol, yield 24 mg (57%). LCMS (Method B): Found m / z (M+H) = 876.8, RT = 1.70 min.

[0710]

[0711] Example 117, (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: Prepared from (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis(((Z)-oct-5-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 50 mg (50%). LCMS (Method B): Found m / z (M+H) = 889.2, RT = 1.73 minutes.

[0712]

[0713] 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)-octadec-9,12-dienoate

[0714] Step 1: 6,6-Bis(((Z)-dec-4-en-1-yl)oxy)hexanenitrile

[0715]

[0716] Prepared according to general procedure A from 6,6-dimethoxyhexanenitrile and (Z)-dec-4-en-1-ol, yield 150 mg (38%). 1 H 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)

[0717] Step 2: 6,6-Bis(((Z)-dec-4-en-1-yl)oxy)hexanoic acid

[0718]

[0719] Prepared according to general procedure B from 6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanenitrile, yield 150 mg, 95%. 1 H NMR (400MHz, DMSO-d6) δ0.85 (t, J=6.7Hz, 6H), 1.18-1.37 (m, 14H), 1.45-1.56 (m, 8H), 1.91-2.10 (m, 8H), 2.1 8 (t, J=7.4Hz, 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).

[0720] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0721]

[0722] Prepared according to general procedure C from 6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoic acid, yield 87 mg (42%). 1H NMR (400MHz, chloroform-d) δ0.88 (t, J=6.8Hz, 9H), 1.17-1.45 (m, 24H), 1.51-1.71 (m, 10H), 1.96-2.17 (m, 12H), 2.32 (q, J=7.3Hz, 4H ), 2.76 (t, J=6.5Hz, 2H), 3.35-3.45 (m, 2H), 3.51-3.64 (m, 4H), 4.09-4.24 (m, 4H), 4.44 (t, J=5.6Hz, 1H), 5.28-5.44 (m, 8H).

[0723] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester. Prepared according to General Procedure D from (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester and 3-(diethylamino)-1-propanol, yield 32 mg (59%). LCMS (Method B): Found m / z (M+H) = 932.9, RT = 1.70 min.

[0724]

[0725] 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)-octadec-9,12-dienoate: Prepared from 3-((6,6-bis(((Z)-dec-4-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 51 mg (52%). LCMS (Method B): Found m / z (M+H) = 945.2, RT = 1.70 min.

[0726]

[0727] 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)-octadec-9,12-dienoate

[0728] Step 1: 6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanenitrile

[0729]

[0730] Prepared according to general procedure A from 6,6-dimethoxyhexanenitrile and 3,7-dimethyloct-6-en-1-ol, yield 220 mg (42%). 1 H NMR (400MHz, chloroform-d) δ0.88 (d, J=6.4Hz, 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=7.1Hz, 2H), 3.35-3.50 (m, 2H), 3.52-3.66 (m, 2H), 4.44 (t, J=5.5Hz, 1H), 5.08 (t, J=7.3Hz, 2H).

[0731] Step 2: 6,6-Bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoic acid

[0732]

[0733] Prepared according to general procedure B from 6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanenitrile, yield 220 mg (95%). 1 H NMR (400MHz, DMSO-d6) δ0.85 (d, J=5.9Hz, 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=7.3Hz, 1H), 3.44-3.33 (m, 2H), 3.60-3.44 (m, 2H), 4.40 (t, J=5.4Hz, 1H), 5.07 (t, J=7.3Hz, 2H), 11.97 (s, 1H).

[0734] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0735]

[0736] Prepared according to general procedure C from 6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoic acid, yield 90 mg (42%). 1H NMR (400MHz, chloroform-d) δ0.88 (d, J=6.1Hz, 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=7.5Hz, 4H), 2.75 (d, J=6.1Hz, 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).

[0737] 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)-octadec-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)-octadec-9,12-dienoate and 3-(diethylamino)-1-propanol according to General Procedure D, yield 32 mg (59%). LCMS (Method B): Found m / z (M+H) = 933.0, RT = 1.70 min.

[0738]

[0739] 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)-octadec-9,12-dienoate: prepared from 3-((6,6-bis((3,7-dimethyloct-6-en-1-yl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 32 mg (59%). LCMS (Method B): Found m / z (M+H) = 945.2, RT = 1.96 min.

[0740]

[0741] 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)-octadecane-9,12-dienoate

[0742] Step 1: 6,6-Bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanenitrile

[0743]

[0744] Prepared according to general procedure A from 6,6-dimethoxyhexanenitrile and 7,7,8,8,8-pentafluorooctan-1-ol, yield 300 mg (44%). 1 H 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).

[0745] Step 2: 6,6-Bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoic acid

[0746]

[0747] Prepared according to general procedure B from 6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanenitrile, yield 296 mg (95%). 1 H NMR (400MHz, DMSO-d6) δ1.18-1.40(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=5.6Hz, 1H), 11.93(s, 1H).

[0748] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0749]

[0750] Prepared according to general procedure C from 6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoic acid, yield 140 mg (57%). 1H NMR (400MHz, chloroform-d) δ0.87 (d, J=7.3Hz, 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 =7.3Hz, 2H), 3.36-3.44 (m, 2H), 3.49-3.58 (m, 2H), 3.60 (t, J = 5.7Hz, 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).

[0751] 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)-octadec-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)-octadec-9,12-dienoate and 3-(diethylamino)-1-propanol according to General Procedure D, yield 51 mg (61%). LCMS (Method B): Found m / z (M+H) = 1060.7, RT = 1.70 min.

[0752]

[0753] 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)-octadec-9,12-dienoate: Prepared from 3-((6,6-bis((7,7,8,8,8-pentafluorooctyl)oxy)hexanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and (1-ethylpiperidin-3-yl)methanol according to General Procedure D, yield 80 mg (74%). LCMS (Method B): Found m / z (M+H) = 1073.2, RT = 1.81 min.

[0754]

[0755] 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)-octadec-9,12-dienoate

[0756] Step 1: Ethyl 1-(2-hydroxyethyl)piperidine-3-carboxylate

[0757]

[0758] 2-bromoethane-1-ol (1.89 g, 15.28 mmol), dried powder KCO (3.5 g, 25.47 mmol) and KI (0.42 g, 2.55 mmol) were added to a stirred solution of ethyl piperidine-3-carboxylate (2 g, 12.74 mmol) in anhydrous acetone (10 mL). 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 combined flash column chromatography, eluted with 30% ethyl acetate-hexane to obtain 1- (2-hydroxyethyl) piperidine-3-carboxylic acid ethyl ester (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).

[0759] Step 2: Ethyl 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-3-carboxylate

[0760]

[0761] Triethylamine (0.35mL, 2.48mmol) and tert-butyldimethylsilyl chloride (225mg, 1.49mmol) were added to a stirred solution of 1-(2-hydroxyethyl)piperidine-3-ethyl formate (200mg, 0.99mmol) in DCM (2mL) at 0°C. The reaction mixture was stirred for 14 hours at 25°C. The reaction mixture was then quenched with water and extracted with DCM (50mL), washed with saline solution (25mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material thus obtained was purified by combined flash chromatography, eluted with 5% ethyl acetate-hexane, to obtain 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-3-ethyl formate (160mg, 51%) in a sticky solid state. 1H NMR (400MHz, chloroform-d) δ0.04 (s, 6H), 0.87 (s, 9H), 1.23 (t, J=7.1Hz, 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 = 10.9Hz, 1H), 2.48-2.59 (m, 3H), 2.78 (d, J = 11.2Hz , 1H), 3.04 (d, J=13.4Hz, 1H), 3.73 (t, J=6.4Hz, 2H), 4.11 (q, J=7.1Hz, 2H).

[0762] Step 3: (1-(2-((tert-Butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)methanol

[0763]

[0764] 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 hour. Upon completion, the reaction mass was quenched with saturated aqueous Na2SO4 solution (10 mL) at 0°C and filtered. The filtrate was concentrated under reduced pressure to give (1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)methanol (400 mg, 92%) as a colorless oil which was used in the next step without further purification. 1 H NMR (400 MHz, chloroform-d) δ 0.04 (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=11.6Hz, 1H), 2.48 (t, J=6.5Hz, 2H), 2.55-2.72 (m, 1H), 2.82 (d, J=10.8Hz , 1H), 3.53 (dd, J=5.8, 10.6Hz, 1H), 3.57-3.70 (m, 1H), 3.73 (t, J=6.4Hz, 2H).

[0765] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 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 ester

[0766]

[0767] To a stirred solution of 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate (Intermediate IVa) (100 mg, 0.14 mmol) in DCM (1 mL) was 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 hour. 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 another 16 hours. The reaction mixture was diluted with DCM (10 mL), washed with 1M Na2CO3 solution (3 × 10 mL) and washed with water (10 mL) and brine. The combined organic portion was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material thus obtained was purified by combined flash column chromatography, eluted with 2% MeOH-DCM to give (9Z, 12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((((Z)-oct-5-ene-1-yl)oxy)butanoyl)oxy)-2-((((1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester (50 mg, 35%) as a light yellow oil. LCMS (method B): found m / z(M+H)=991.1, RT=1.14 minutes.

[0768] Step 5: (9Z,12Z)-octadec-9,12-dienoic acid 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 ester (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)-octadec-9,12-dienoate (60 mg, 0.061 mmol) in anhydrous THF (1 mL) was added tetra-n-butylammonium fluoride (TBAF) (0.12 mL, 0.12 mmol) (1 M solution in tetrahydrofuran) at 0°C. After the mixture was allowed to warm to room temperature, the resulting solution was stirred for 4 hours. 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), and dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material thus obtained was purified by combined flash column chromatography eluting with 2% MeOH-DCM to afford 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)-octadec-9,12-dienoate as a brown viscous gum (35 mg, 66%). LCMS (Method B): found m / z (M+H) = 876.7, RT = 1.58 min.

[0769]

[0770] 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)-octadec-9,12-dienoate

[0771] Step 1: Ethyl 1-(3-hydroxypropyl)piperidine-3-carboxylate

[0772]

[0773] 3-bromopropan-1-ol (2.12 g, 15.28 mmol), dried powder KCO (3.5 g, 25.47 mmol) and KI (0.42 g, 2.55 mmol) were added to a stirred solution of ethyl piperidine-3-carboxylate (2 g, 12.74 mmol) in anhydrous acetone (10 mL). 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 combined flash column chromatography, eluted with 25% ethyl acetate-hexane to obtain 1- (3-hydroxypropyl) piperidine-3-carboxylic acid ethyl ester (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).

[0774] Step 2: Ethyl 1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidine-3-carboxylate

[0775]

[0776] Triethylamine (0.3mL, 3.31mmol) and tert-butyldimethylsilyl chloride (211mg, 1.39mmol) were added to a stirred solution of 1-(3-hydroxypropyl)piperidine-3-ethyl formate (200mg, 0.93mmol) in DCM (2mL) 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 and extracted with DCM and washed with a saline solution. The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material thus obtained was purified by combined flash column chromatography, eluted with 5% ethyl acetate-hexane, and 1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidine-3-ethyl formate (160mg, 52%) was obtained as a sticky solid. 1H 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).

[0777] Step 3: (1-(3-((tert-Butyldimethylsilyl)oxy)propyl)piperidin-3-yl)methanol

[0778]

[0779] 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 hour. After completion, the reaction mass was quenched with saturated aqueous Na2SO4 solution (10 mL) 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).

[0780] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 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 ester

[0781]

[0782] To a stirred solution of 3-((4,4-di((((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-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 hour, (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. Stir for 16 hours. After completion, the reaction mixture was diluted with DCM (10 mL), washed with 1M sodium carbonate solution (3×10 mL), and washed with water (10 mL) and brine. The combined organic portion was dried over Na2SO4, filtered and concentrated under reduced pressure and purified by combined flash column chromatography, eluted with 2% MeOH-DCM to give (9Z, 12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((((Z)-oct-5-ene-1-yl)oxy)butanoyl)oxy)-2-((((1-(3-((tert-butyldimethylsilyl)oxy)propyl)piperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester (50 mg, 30%) as a light yellow oil. LCMS (Method B): Found m / z(M+H)=1005.1, RT=1.12 minutes.

[0783] 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)-octadec-9,12-dienoate (Example 125). To a solution of (9Z,12Z)-octadec-9,12-dienoic acid 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 ester (85 mg, 0.085 mmol) in anhydrous THF (1.0 mL) was added tetra-n-butylammonium fluoride (TBAF) (0.17 mL, 0.17 mmol) (1 M solution in tetrahydrofuran) at 0°C. After the mixture was warmed to 25°C, the resulting solution was stirred for 4 hours. The resulting solution was diluted with DCM (10 mL) and quenched with water (5 mL). The organic layer was extracted with DCM (2 x 5 mL), brine (5 mL) and dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material thus obtained was purified by combined flash 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)-octadec-9,12-dienoate as a light yellow viscous gum (36 mg, 52%). LCMS (Method B): found m / z (M+H) = 891.0, RT = 1.61 min.

[0784]

[0785] 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)-octadec-9,12-dienoate

[0786] Step 1: 3,3'-(Methylazanediyl)bis(propan-1-ol)

[0787]

[0788] Under argon atmosphere, Na2CO3(260mg, 2.24mmol) and 3-chloropropanol (0.1mL, 1.12mmol) were added to a stirred solution of 3-(methylamino)propan-1-ol (100mg, 1.12mmol) in EtOH (2mL) under argon atmosphere. The reaction mixture was stirred at 80°C for 4 hours. After completion, the reaction mixture was concentrated under reduced pressure. The crude material thus obtained was purified by combined flash column chromatography, eluted with 2% acetone-hexane, and 3,3'-(methylazanediyl)bis(propan-1-ol) (90mg, 55%) was obtained as a colorless oil. 1 H NMR (400MHz, DMSO-d6) δ1.53-1.67 (m, 2H), 1.68-1.78 (m, 1H), 1.84 (t, J=6.2Hz, 1H), 2.26 (s , 2H), 2.52 (d, J=6.7Hz, 3H), 2.90 (t, J=7.5Hz, 1H), 3.39-3.51 (m, 6H), 3.68 (t, J=6.5Hz, 1H).

[0789] Step 2: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-((3-hydroxypropyl)(methyl)amino)propoxy)carbonyl)oxy)methyl)propyl ester (Example 126). Prepared according to General Procedure D from Intermediate IVa and 3,3'-(methylazanediyl)bis(propan-1-ol), yield 18 mg (23%). LCMS (Method B): Found m / z (M+H) = 864.7, RT = 1.55 min.

[0790]

[0791] Example 127, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propyl)carbamoyl)oxy)methyl)propyl ester: prepared according to General Procedure D from Intermediate IVa and N1,N1-diethylpropane-1,3-diamine, yield 32 mg (53%). LCMS (Method B): Found m / z (M+H) = 848.0, RT = 1.55 min.

[0792]

[0793] 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)-octadec-9,12-dienoate

[0794] Step 1: 2-(1-ethylpyrrolidin-2-yl)ethan-1-ol

[0795]

[0796] The following is a representation 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 nitrogen atmosphere. The resulting mixture was stirred at 56°C for 16 hours. The reaction mass was cooled to room temperature and filtered through a celite bed 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).

[0797] (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis((((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-ethylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl ester (Example 128). Prepared according to General Procedure D from Intermediate IVa and 2-(1-ethylpyrrolidin-2-yl)ethan-1-ol, yield 28 mg (52%). LCMS (Method B): Found m / z (M+H) = 860.8, RT = 1.55 min.

[0798]

[0799] Example 129, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1R,3s,5S)-8-ethyl-8-azabicyclo[3.2.1]oct-3-yl)oxy)carbonyl)oxy)methyl)propyl ester

[0800] Step 1: (1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-ol

[0801]

[0802] Prepared according to general procedure G from (1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-ol. Yield: 180 mg, 73%.

[0803] Step 2: (9Z,12Z)-octadec-9,12-dienoic acid 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 ester (Example 129). Prepared according to General Procedure D from Intermediate IVa and (1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-ol, yield 16 mg (51%). LCMS (Method B): Found m / z (M+H) = 872.8, RT = 1.70 min.

[0804]

[0805] Example 130, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((((1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]oct-3-yl)oxy)carbonyl)oxy)methyl)propyl ester

[0806] Step 1: (1R,3R,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-ol

[0807]

[0808] Prepared according to general procedure G from (1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-ol, yield 80 mg (92%).

[0809] Step 2: (9Z,12Z)-octadec-9,12-dienoic acid 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 ester (Example 130). Prepared according to General Procedure D from Intermediate IVa and (1R,3r,5S)-8-ethyl-8-azabicyclo[3.2.1]octan-3-ol, yield 16 mg (51%). LCMS (Method B): Found m / z (M+H) = 872.9, RT = 1.64 min.

[0810]

[0811] Example 131, 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((9Z,12Z)-octadec-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 (M+H) = 913.7, RT = 3.21 min.

[0812]

[0813] Example 132, 3-((4,4-bis((7,7,8,8,8-pentafluorooctyl)oxy)butanoyl)oxy)-2-(((9Z,12Z)-octadec-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 (M+H) = 1097.5, RT = 3.19 min.

[0814]

[0815] Example 133, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((((2-(1-ethylpiperidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl ester: prepared according to General Procedure D from Intermediate IVa and 2-(1-ethylpiperidin-2-yl)ethan-1-ol, yield 38 mg (40%). LCMS (Method A): Found m / z (M+H) = 874.7, RT = 3.98 minutes.

[0816]

[0817] Example 134, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-((2-(1-ethylpiperidin-4-yl)acetoxy)methyl)propyl ester: prepared from intermediate IVa and 2-(1-ethylpiperidin-4-yl)acetic acid according to general procedure E, yield 66 mg (72%). LCMS (Method A): found m / z (M+H) = 844.7, RT = 3.95 minutes.

[0818]

[0819] Example 135, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(oct-2-yn-1-yloxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: 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 in the final step: 91 mg (92%). LCMS (Method A): Found m / z (M+H) = 856.7, RT = 3.63 min.

[0820]

[0821] Example 136, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(oct-2-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: prepared using a procedure analogous to Example 1, substituting oct-2-yn-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in the final step: 80 mg (82%). LCMS (Method A): Found m / z (M+H) = 844.7, RT = 3.66 min.

[0822]

[0823] Example 137, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(non-2-yn-1-yloxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared using a procedure analogous to Example 3, substituting non-2-yn-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in the final step: 86 mg (88%). LCMS (Method A): Found m / z (M+H) = 884.7, RT = 3.73 min.

[0824]

[0825] Example 138, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(non-2-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: prepared using a procedure analogous to Example 1, substituting non-2-yn-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in the final step: 87 mg (90%). LCMS (Method A): Found m / z (M+H) = 872.7, RT = 3.69 min.

[0826]

[0827] Example 139, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(oct-7-yn-1-yloxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: 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 in the final step: 111 mg (84%). LCMS (Method A): Found m / z (M+H) = 856.7, RT = 3.74 min.

[0828]

[0829] Example 140, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(oct-7-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: prepared using a procedure analogous to Example 1, substituting oct-7-yn-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in the final step: 108 mg (83%). LCMS (Method A): Found m / z (M+H) = 844.7, RT = 3.76 min.

[0830]

[0831] Example 141, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(dec-2-yn-1-yloxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared using a procedure analogous to Example 3, substituting dec-2-yn-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in the final step: 122 mg (88%). LCMS (Method A): Found m / z (M+H) = 912.8, RT = 4.13 min.

[0832]

[0833] Example 142, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(dec-2-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: prepared using a procedure analogous to Example 1, substituting dec-2-yn-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in the final step: 115 mg (84%). LCMS (Method A): Found m / z (M+H) = 900.8, RT = 4.04 min.

[0834]

[0835] Example 143, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(dec-3-yn-1-yloxy)butanoyl)oxy)-2-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: 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 in the final step: 104 mg (89%). LCMS (Method A): Found m / z (M+H) = 912.8, RT = 3.97 min.

[0836]

[0837] Example 144, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(dec-3-yn-1-yloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: prepared using a procedure analogous to Example 1, substituting dec-3-yn-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in the final step: 108 mg (94%). LCMS (Method A): Found m / z (M+H) = 900.8, RT = 3.93 min.

[0838]

[0839] Example 145, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((E)-oct-2-en-1-yl)oxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester: prepared using a procedure analogous to Example 3, substituting (E)-oct-2-en-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in the final step: 74 mg (89%). LCMS (Method A): Found m / z (M+H) = 860.7, RT = 3.87 min.

[0840]

[0841] Example 146, (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((E)-oct-2-en-1-yl)oxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester: prepared using a procedure analogous to Example 1, substituting (E)-oct-2-en-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in the final step: 68 mg (83%). LCMS (Method A): Found m / z (M+H) = 848.8, RT = 3.89 min.

[0842]

[0843] 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)-octadec-9,12-dienoate: Prepared using a procedure analogous to Example 3, substituting (E)-non-2-en-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in final step: 68 mg (81%). LCMS (Method A): Found m / z (M+H) = 888.8, RT = 4.01 min.

[0844]

[0845] 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)-octadec-9,12-dienoate: Prepared using a procedure analogous to Example 1, substituting (E)-non-2-en-1-ol for (Z)-oct-5-en-1-ol in General Procedure A. Yield in final step: 34 mg (41%). LCMS (Method A): Found m / z (M+H) = 877.9, RT = 4.07 min.

[0846] Lipid Nanoparticles

[0847] F. LNP formulation

[0848] The lipid nanoparticle components are dissolved in 100% ethanol at a specific lipid component molar ratio. The nucleic acid (NA) cargo is dissolved in 10mM citrate, 100mM NaCl, pH 4.0 to obtain a NA cargo concentration of about 0.22mg / mL. In some embodiments, the NA cargo consists of a functional NA (e.g., siRNA, antisense, expressed DNA, mRNA) and a reporter DNA barcode (as previously described by Sago, 2018PNAS) mixed at a mass ratio of 1:10 to 10:1 functional NA to barcode.

[0849] LNPs are formulated to a mass ratio of total lipids to NA of 11.7. According to the manufacturer's protocol, LNPs are formed by microfluidic mixing of lipid and NA solutions using PrecisionNanosystems NanoAssemblr Spark or Benchtop instruments. A 2:1 ratio of water to organic solvent is maintained during mixing using different flow rates. After mixing, LNPs are collected, diluted in PBS (about 1:1 v / v), and further buffer exchange is performed using dialysis of 20 kDa filters at 4 ° C for 8 to 24 hours in PBS. After this initial dialysis, each individual LNP formulation is characterized by DLS to measure size and polydispersity, and the pKa of LNP subpopulations is measured by TNS determination. LNPs falling within a specific diameter and polydispersity range are merged, and PBS is further dialyzed for 1 to 4 hours at 4 ° C on a 100 kDa dialysis cassette. After the second dialysis, LNPs are sterile filtered using a 0.22 μM filter and stored at 4 ° C for further use.

[0850] G. LNP characterization

[0851] DLS-LNP hydrodynamic diameter and percent polydispersity (PDI%) were measured using high throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt).LNPs were diluted to appropriate concentrations with 1×PBS and analyzed.

[0852] The concentration of NA and encapsulation efficiency-concentration were determined by the Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring uncleaved and cleaved LNPs.

[0853] pKa-prepare a stock solution of 10mM HEPES (Sigma Aldrich), 10mM MES (Sigma Aldrich), 10mM sodium acetate (Sigma) and 140nM sodium chloride (Sigma Aldrich), and use hydrogen chloride and sodium hydroxide to adjust the pH to pH 4-10. Repeat 4 times for each pH, ​​add 140 μL of pH-adjusted buffer to a 96-well plate, then add 5 μL of 2-(p-toluidine)-6-naphthalenesulfonic acid (60 μg / mL). Add 5 μL of LNP to each well. After incubation for 5 minutes under gentle shaking, measure fluorescence using an excitation wavelength of 325nm and an emission wavelength of 435nm (BioTek Synergy H4 Hybrid).

[0854] LNP administration - Male and female mice of about 8-12 weeks of age were used for all studies. Each mouse was temporarily restrained, and the pooled LNPs were administered IV to up to 5 animals per experiment via tail vein injection. Age-matched mice were also used to administer vehicle (1×PBS) via tail vein injection, up to 3 animals per experiment. 72 hours after administration, tissues including liver, spleen, bone marrow and blood were collected for analysis.

[0855] Flow-Liver tissue was mechanically digested and then digested by enzymes using a mixture of proteases, then passed through a 70 μM filter to produce a single cell suspension. Spleen tissue was mechanically digested to produce 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 antibodies. Using BD FACSMelody (Becton Dickinson), all samples were obtained by flow cytometry to generate gates before sorting. Typically, the gating structure is the size of the target → single cell → live cell → cell. T cells are defined as CD45+CD3+, monocytes are defined as CD45+CD11b+, and B cells are defined as CD45+CD19+. In the liver, endothelial cells are defined as CD31+, Kupffer cells are defined as CD45+CD11b+, and hepatocytes are defined as CD31- / CD45-. For siRNA studies, we chose to downregulate target genes, and for mRNA studies, we chose to upregulate target genes. Tissues from mice administered vehicle were used to set gates for sorting. Up to 20,000 cells of each cell subset with the correct phenotype were sorted into 1× PBS. After sorting, cells were pelleted by centrifugation and DNA was extracted using a rapid DNA extraction solution (Lucigen) according to the manufacturer's protocol. DNA was stored at -20°C.

[0856] Barcode sequencing - DNA (genomic and DNA barcodes) was isolated using QuickExtract (Lucigen) and sequenced using Illumina MiniSeq as previously described (Sago et al., PNAS 2018, Sago et al., JACs 2018, Sago, Lokugamage et al., Nano Letters 2018), and DNA barcode counts in FACS-isolated samples were normalized to the frequency of injected input. These data were plotted as "Normalized fold-top input".

[0857] H. confirm

[0858] LNP formulation

[0859] The lipid nanoparticle components are dissolved in 100% ethanol with a specific lipid component molar ratio. The nucleic acid (NA) carrier is dissolved in 10mM citrate, 100mM NaCl, pH 4.0 to obtain a NA carrier concentration of about 0.22mg / mL. In some embodiments, the NA carrier is composed of functional NA (e.g., siRNA, antisense, expressed DNA, mRNA). LNP is formulated into a total lipid and NA mass ratio of 11.7. According to the manufacturer's protocol, LNP is formed by microfluidic mixing of lipid and NA solutions using Precision Nanosystems NanoAssemblr Spark or Benchtop instruments. A 3:1 ratio of water to organic solvent is maintained during mixing using different flow rates. After mixing, LNP is collected, diluted in PBS (about 1:1v / v), and further buffer exchange is performed using dialysis for 20kDa filters at 4°C for 8 to 24 hours in PBS. After this initial dialysis, each individual LNP preparation was characterized by DLS to measure size and polydispersity, and the pKa of LNP subpopulations was measured by TNS assay.After dialysis, the LNPs were sterile filtered using a 0.22 μM sterile filter and stored at 4 °C for further use.

[0860] LNP characterization

[0861] DLS-LNP hydrodynamic diameter and percent polydispersity (PDI%) were measured using high throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt).LNPs were diluted to appropriate concentrations with 1×PBS and analyzed.

[0862] The concentration of NA and encapsulation efficiency-concentration were determined by the Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring uncleaved and cleaved LNPs.

[0863] pKa-prepare a stock solution of 10mM HEPES (Sigma Aldrich), 10mM MES (Sigma Aldrich), 10mM sodium acetate (Sigma) and 140nM sodium chloride (Sigma Aldrich), and use hydrogen chloride and sodium hydroxide to adjust the pH to pH 4-10. Repeat 4 times for each pH, ​​add 140 μL of pH-adjusted buffer to a 96-well plate, then add 5 μL of 2-(p-toluidine)-6-naphthalenesulfonic acid (60 μg / mL). Add 5 μL of LNP to each well. After incubation for 5 minutes under gentle shaking, measure fluorescence using an excitation wavelength of 325nm and an emission wavelength of 435nm (BioTek Synergy H4 Hybrid).

[0864] LNP administration - Male and female mice of about 8-12 weeks of age were used for all studies. Each mouse was temporarily restrained, and the pooled LNPs were administered IV to up to 5 animals per experiment via tail vein injection. Age-matched mice were also used to administer vehicle (1×PBS) via tail vein injection, up to 3 animals per experiment. 72 hours after administration, tissues including liver, spleen, bone marrow and blood were collected for analysis.

[0865] hEPO expression-For human EPO (hEPO) protein expression, mice were temporarily restricted and bled 6 hours after administration (via tail vein). Blood was collected in heparin tubes, processed into plasma, and stored at -80 ° C until ready for use. R&D system ELISA kit (DuoSet; DY286-05) was used to measure hEPO protein according to the manufacturer's instructions. Various exemplary ionizable lipids were formulated into LNP A1 to LNP A17 using the following molar ratios: 45% ionizable lipids / 9% distearoylphosphatidylcholine (DSPC) / 44% cholesterol / 2% PEG lipids or 50% ionizable lipids / 9% distearoylphosphatidylcholine (DSPC) / 38% cholesterol / 3% PEG lipids, wherein the mass ratio of lipid to nucleic acid (human EPO mRNA) was 11.7: 1, 18: 1 or 25: 1. Activity was determined by measuring hEPO expression in plasma 6 hours after administration of nucleic acid at a dose of 0.15 mg / kg by tail vein injection as described. Representative data for LNP encapsulation efficiency, hydrodynamic diameter, polydispersity (PDI) are provided in Table 1, and hEPO in plasma 6 hours after administration of LNP formulations was measured.

[0866] Table 1

[0867]

[0868] 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 not limitation, with the true scope and spirit being indicated by the following claims.

Claims

1. Compounds of formula (I): in: R 1 It is C9-C 20 Alkyl or C9-C 20 alkenyl; X 1 and X 2 Each independently absent or selected from -O-, -NR 2 -and Each R 2 are independently hydrogen or C1-C6 alkyl; Each a is independently an integer from 1 to 6; X 3 and X 4 each independently absent or selected from: 4 to 8-membered heterocyclyl optionally substituted by 1 or 2 C1-C6 alkyl groups, 5 to 6-membered heteroaryl optionally substituted by 1 or 2 C1-C6 alkyl groups, 5 to 6-membered aryl optionally substituted by 1 or 2 C1-C6 alkyl groups, 4 to 7-membered cycloalkyl optionally substituted by 1 or 2 C1-C6 alkyl groups, -O- and -NR 3 -, where each R 3 are independently a hydrogen atom or a C1-C6 alkyl group, and wherein X 1 -X 2 -X 3 -X 4 does not contain any oxygen-oxygen, oxygen-nitrogen, or nitrogen-nitrogen bonds; X 5 Yes - (CH2) b -, wherein b is an integer from 0 to 6; X 6 is hydrogen, C1-C6 alkyl, 5- to 6-membered heteroaryl optionally substituted by 1 or 2 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 The nitrogen to which they are bonded are joined together to form a 4- to 7-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic group optionally includes additional heteroatoms selected from oxygen, sulfur and nitrogen; Each X 7 are independently hydrogen, hydroxyl or -NR 6 R 7 , where R 6 and R 7 are each independently hydrogen or C1-C6 alkyl; or alternatively, R 6 and R 7 The nitrogen to which they are bonded are joined together to form a 4- to 7-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic group optionally includes additional heteroatoms selected from oxygen, sulfur and nitrogen; X 1 , X 2 , X 3 , X 4 and X 5 At least one of them exists; A 1 and A 2 Each independently selected from: C5-C 12 Halogenated alkyl, C5-C 12 Alkenyl, ring optionally substituted by one or two halogen, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy (C5-C 10 Aryl)-(CH2) n3 -, and (C3-C8 cycloalkyl)-(CH2) wherein the ring is optionally substituted by 1 or 2 C1-C6 alkyl groups n4 -; n1 and n3 are each independently an integer from 1 to 4; and n4 is an integer from 0 to 4.

2. The compound of claim 1, wherein A 1 and A 2 Each independently is C5-C 12 Alkenyl, or wherein A 1 and A 2 Each independently is C5-C 12 Halogenated alkyl.

3. The compound as claimed in claim 1, wherein A 1 and A 2 Each independently is a ring optionally substituted by one or two halogen, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy (C5-C 10 Aryl)-(CH2) n3 –, or A 1 and A 2 Each independently is (C3-C8 cycloalkyl)-(CH2) wherein the ring is optionally substituted with 1 or 2 C1-C6 alkyl groups n4 –.

4. The compound of claim 1, wherein: R 1 yes X 1 is –O–; X 2 does not exist; X 3 and X 4 Each does not exist; X 5 Yes – (CH2) b –, where b is an integer from 0 to 6; X 6 Yes – NR 4 R 5 , where R 4 and R 5 The nitrogen to which they are bonded are joined together to form a 4- to 7-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic group optionally includes additional heteroatoms selected from oxygen, sulfur and nitrogen; A 1 and A 2 Each independently is C5-C 12 alkenyl; and n1 is an integer from 1 to 4.

5. The compound according to any one of claims 1 to 3, wherein R 1 It is C9-C with 2 degrees of unsaturation 20 alkenyl, optionally wherein R 1 for 6. The compound according to any one of claims 1 to 3, wherein X 1 does not exist, and X 2 yes Where X 7 Optionally: hydrogen, hydroxy and a is 1, or -NR 6 R 7 and a is 1; X 1 is -O-, and X 2 yes Where X 7 Optionally: hydrogen, hydroxy and a is 1, or -NR 6 R 7 and a is 1; or X 1 yes And X 2 yes Where X 7 Optionally: hydrogen, hydroxy and a is 1, or -NR 6 R 7 And a is 1.

7. A compound as claimed in any one of claims 1 to 6, wherein X 3 Does not exist, or X 3 is a 4- to 8-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups.

8. A compound as claimed in any one of claims 1 to 7, wherein X 4 Does not exist.

9. The compound according to any one of claims 1 to 8, wherein b is an integer from 1 to 6; and X 6 is hydrogen or NR 4 R 5 , optionally wherein R 4 and R 5 The nitrogen to which they are bonded are linked together to form a 4- to 7-membered heterocyclic group optionally substituted by 1 or 2 C1-C6 alkyl groups.

10. The compound of claim 1, wherein the compound is selected from the group consisting of:

11. The compound of claim 1, wherein the compound is selected from the group consisting of:

12. The compound according to claim 1, having the following structure:

13. A lipid nanoparticle composition comprising: An ionizable lipid comprising a compound as described in any one of claims 1 to 12, optionally wherein the ionizable lipid is present in an amount ranging from about 35 mol% to 65 mol% based on the total moles; A phospholipid, optionally wherein the phospholipid is DSPC or DMPC; PEG-lipid; Cholesterol; and optionally Nucleic acid.

14. The lipid nanoparticle composition of claim 13, wherein the nucleic acid is a siRNA, a miRNA, an mRNA, an expressed DNA, an antisense oligonucleotide, or an immunostimulatory oligonucleotide.

15. The lipid nanoparticle composition of claim 13, wherein the nucleic acid is a guide RNA.

16. The lipid nanoparticle composition of any one of claims 13 to 15, for use in a method of delivering a nucleic acid to an individual in need thereof, the method comprising administering the lipid nanoparticle composition to the individual.

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