Nanomaterial

Abstract

Lipid nanoparticle compositions for nucleic acid delivery are described.Lipid nanoparticles can contain conformationally constrained ionizable lipids as part of the composition.These compositions can allow cargo delivery without the need for targeting ligands.

Description

[Technical field]

[0001] Related Application Information This application claims the benefit of U.S. Provisional Patent Application No. 62 / 944,735, filed December 6, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

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

[0003] explanation T cells, B cells, macrophages, and other immune cells help regulate homeostasis and immunity, making them important targets for RNA therapy. Although RNA-carrying nanoparticles have been targeted to immune cells in vivo using protein- and aptamer-based targeting ligands, systemic delivery to immune cells without targeting ligands remains a challenge. Summary of the Invention [Means for solving the problem]

[0004] Some embodiments described herein include a compound represented by formula (I):

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

[0006] [Chemical formula] selected from, where R 2 is C1-C6 alkyl, and X 1 and X 2 are both not -O- or NR 2 ; a is an integer between 1 and 6; X 3 and X 4 are each independently absent, or are a 4- to 7-membered heterocyclyl optionally substituted with one or two C1-C6 alkyl groups, a 5- to 6-membered heteroaryl optionally substituted with one or two C1-C6 alkyl groups, and -NR 3 selected from the group consisting of -, where each R 3 is a hydrogen atom or C1-C6 alkyl; X 5 is -(CH2) b -, where b is an integer between 0 and 6; X 6 is hydrogen, C1-C6 alkyl, a 5- to 6-membered heteroaryl optionally substituted with one or two C1-C6 alkyl groups, or -NR 4 R 5 where R 4 and R 5 are each independently hydrogen or C1-C6 alkyl; alternatively, R 4 and R 5 together with the nitrogen to which they are attached form a 4- to 7-membered heterocyclyl optionally substituted with one or two C1-C6 alkyl groups, where the heterocyclyl optionally contains an additional heteroatom selected from oxygen, sulfur and nitrogen; X 7 is hydrogen or -NR 6 R 7 where R 6and R 7 are each independently hydrogen or C1-C6 alkyl; alternatively, R 6 and R 7 together with the nitrogen to which they are attached form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C1-C6 alkyl groups, where the heterocyclyl optionally contains an additional heteroatom selected from oxygen, sulfur, and nitrogen; X 1 、X 2 、X 3 、X 4 、and X 5 at least one of which is present; provided that X 1 or X 2 is -O-, then neither X 3 nor X 4 is

[0007]

Chemical formula

[0008]

Chemical formula

[0009]

Chemical formula

[0010]

Chemical formula

[0011]

Chemical formula

[0012] One embodiment features a lipid nanoparticle composition comprising an ionizable lipid with a constrained conformation: phospholipid; polyethylene glycol-lipid; cholesterol; and optionally a nucleic acid. Further embodiments are directed to a method of delivering a nucleic acid to a subject in need thereof, the method comprising administering the lipid nanoparticle composition to a subject in need thereof.

[0013] These and other embodiments are described in more detail below.

Brief Description of the Drawings

[0014]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 1H

Figure 1I

Figure 1J

Figure 1K

Figure 1L

Figure 1M

Figure 1N

Figure 1O

Figure 1P

Figure 1Q

Figure 1R

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0015] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications referenced herein are hereby incorporated by reference in their entirety, unless otherwise indicated. In the case of multiple definitions for terms herein, the definitions in this section shall control, unless otherwise indicated.

[0016] As used herein, any "R" group or "X" group, e.g., but not limited to, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 、R 13 、R 14 、R 15 、R 16 、X 1 、X 2 、X 3 、X 4 、X 5 、X 6 、X 7 、X 8 、X 9、 X 10 、X 11 、X 12 、X 13 、X 14 、X 15 、X 16 and X 17 represent substituents that can be attached to the indicated atom. Such R groups and X groups can be referred to herein as "R" groups generally. The R groups may be either substituted or unsubstituted. When two "R" groups are described as being "together", the R groups, and the atoms to which they are attached, can form a cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocycle. For example, but not limited to, the R a R b groups of the NR a and Rb When they are instructed to be "combined together", they covalently bond to each other to form a ring:

[0017]

Chemical formula

[0018] Whenever a group is described as being "optionally substituted", the group may be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when a group is described as being "unsubstituted or substituted", and the group is substituted, the substituents can be selected from one or more of the indicated substituents. When no substituents are indicated, the indicated "optionally substituted" or "substituted" group may be substituted with one or more groups individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, acylalkyl, hydroxy, alkoxy, alkoxyalkyl, aminoalkyl, amino acid, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxyalkyl, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, isocyanate, thiocyanate, isothiocyanate, azide, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, amino, monosubstituted amino group and disubstituted amino group.

[0019] As used herein, "a" and "b" are integers, "C a ~Cb "a" to "b" refers to the number of carbon atoms in each group of alkyl, alkenyl or alkynyl, or the number of carbon atoms in the ring of each group of cycloalkyl, cycloalkenyl, aryl, heteroaryl or heteroaricyclic. That is, the rings of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl or heteroaricyclic can contain carbon atoms, including from "a" to "b". Therefore, for example, the "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbons, that is, CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-. When "a" and "b" are not specified for each group of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl or heteroaricyclic, the broadest range described in these definitions is assumed.

[0020] As used herein, "alkyl" refers to a straight-chain or branched hydrocarbon chain containing a fully saturated (having no double or triple bonds) hydrocarbon group. An alkyl group can have from 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as "1 to 20" refers to each integer within the given range; for example, "from 1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also covers the presence of the term "alkyl" when no numerical range is specified). The alkyl group can also be a medium-sized alkyl having 1 to 10 carbon atoms. The alkyl group can also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group of a compound can be designated as "C1-C4 alkyl" or a similar symbol. By way of mere illustration, "C1-C4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl. The alkyl group can be either substituted or unsubstituted.

[0021] As used herein, "alkenyl" refers to an alkyl group containing one or more double bonds in a straight-chain or branched hydrocarbon chain. Examples of alkenyl groups include arylenyl, vinylmethyl, and ethenyl. The alkenyl group can be either unsubstituted or substituted.

[0022] As used herein, "alkynyl" refers to an alkyl group containing one or more triple bonds in a straight-chain or branched hydrocarbon chain. Examples of alkynyl include ethynyl and propynyl. The alkynyl group can be either unsubstituted or substituted.

[0023] As used herein, "cycloalkyl" refers to a completely saturated (having no double or triple bonds) monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings can be joined together in each of the fused, bridged, or spiro fashions. As used herein, the term "fused" refers to two rings that commonly share two atoms and one bond. As used herein, the term "bridged cycloalkyl" refers to a compound in which the cycloalkyl contains a linkage of one or more atoms that connect non-adjacent atoms. As used herein, the term "spiro" refers to two rings that have a common single atom, but the two rings are not linked by a bridge. The cycloalkyl group 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. The cycloalkyl group can be unsubstituted or substituted. Typical monocycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of fused cycloalkyl groups are decahydronaphthalenyl, dodecahydro-1H-phenalenyl, and tetradecahydroanthracenyl; examples of bridged cycloalkyl groups are bicyclo[1.1.1]pentyl, bicyclo[2.1.1]heptane, adamantanyl, and norbornanyl; examples of spirocycloalkyl groups include spiro[3.3]heptane and spiro[4.5]decane.

[0024] As used herein, "cycloalkenyl" refers to a monocyclic or polycyclic hydrocarbon ring system containing one or more double bonds in at least one ring; provided that when there are two or more, the double bonds cannot form a completely delocalized π - electron system over the entire ring (otherwise, the group is "aryl" as defined herein). The cycloalkenyl group can contain 3 to 10 atoms in the ring, or can contain 3 to 8 atoms in the ring. When composed of two or more rings, these rings can be connected together in a fused manner. The cycloalkenyl group may be unsubstituted or substituted.

[0025] As used herein, "aryl" refers to a carbocyclic (all carbon) monocyclic or polycyclic aromatic ring system (including fused ring systems in which two carbocyclic rings share a chemical bond) having a completely delocalized π - electron system over the entire ring. The number of carbon atoms in the aryl group can vary. For example, the aryl group can be C6 - C 14 an aryl group, or C6 - C 10 an aryl group, or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene, and azulene. The aryl group may be substituted or unsubstituted.

[0026] As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic ring system (a ring system having a fully delocalized π-electron system) containing one, two, three or more heteroatoms, i.e., elements other than carbon, including but not limited to nitrogen, oxygen and sulfur. The number of atoms in the ring of a heteroaryl group can vary. For example, a heteroaryl group can contain 4 to 14 atoms in the ring, 5 to 10 atoms in the ring, or 5 to 6 atoms in the ring. Further, the term "heteroaryl" includes fused ring systems, in which case two rings, for example, at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings, share at least one chemical bond. Examples of heteroaryl rings include, but are not limited to, those described herein and the following: furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, and triazine. A heteroaryl group may be substituted or unsubstituted.

[0027] As used herein, "heterocyclyl" or "heteroaricyclic" refers to monocyclic, bicyclic, and tricyclic ring systems of 3, 4, 5, 6, 7, 8, 9, 10, up to 18 members, where in this case, carbon atoms together with 1 to 5 heteroatoms constitute the ring system. The heterocycle can optionally contain one or more unsaturated bonds, provided that the unsaturated bonds are not in a position such that a fully delocalized π - electron system exists throughout the entire ring. Heteroatoms are elements other than carbon, including but not limited to oxygen, sulfur, and nitrogen. The heterocycle can further contain one or more carbonyl or thiocarbonyl functional groups, and as a result, its definition includes oxo - and thio - based compounds such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, these rings can be joined together in a fused or spiro fashion as described herein with respect to "cycloalkyl". Additionally, any nitrogen within the heterocyclyl may be quaternized. The heterocyclyl group or heteroaricyclic group may be unsubstituted or substituted.Examples of such "heterocyclyl" or "heteroaricyclic" groups include, but are not limited to, those described herein and the following: 1,3-dioxine, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3,4-oxadiazol-2(3H)-one, 1,2,3-oxadiazol-5(2H)-one, 1,3-oxathiolane, 1,3-dithiol, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 1,3-thiazinane, 2H-1,2-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, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiomorpholine, thiomorpholine sulfoxide, thiomorpholine sulfone, and their benzofused ring analogs (e.g., benzimidazolidinone, tetrahydroquinoline, and 3,4-methylenedioxyphenyl).

[0028] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl group connected as a substituent via a lower alkylene group. The lower alkylene group and aryl group of the aralkyl may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

[0029] As used herein, "heteroalkyl" and "heteroaryl(alkyl)" refer to a heteroaryl group connected as a substituent via a lower alkylene group. The lower alkylene group and heteroaryl group of heteroalkyl may be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, imidazolylalkyl, and their benzo-fused ring analogs.

[0030] "Heteroaracyclic(alkyl)" and "heterocyclic(alkyl)" refer to a heterocyclic group or heteroaracyclic group connected as a substituent via a lower alkylene group. The lower alkylene and heterocyclic of heteroaracyclic(alkyl) may be substituted or unsubstituted. Examples include, but are not limited to, tetrahydro-2H-pyran-4-yl(methyl), piperidin-4-yl(ethyl), piperidin-4-yl(propyl), tetrahydro-2H-thiopyran-4-yl(methyl), and 1,3-thiazinan-4-yl(methyl).

[0031] The "lower alkylene group" is a straight-chain -CH2- tether group that forms a bond connecting a molecular fragment via its terminal carbon atom. Examples include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and butylene (-CH2CH2CH2CH2-). The lower alkylene group can be substituted by replacing one or more hydrogens of the lower alkylene group with substituents listed under the definition of "substituted".

[0032] As used herein, "alkoxy" refers to the formula -OR (wherein R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) as defined herein). A non-limiting list of alkoxys is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), cyclopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, cyclobutoxy, phenoxy and benzyloxy. Alkoxy may be substituted or unsubstituted.

[0033] As used herein, "acyl" refers to hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) connected as a substituent via a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl and acrylyl. Acyl may be substituted or unsubstituted.

[0034] As used herein, "acylalkyl" refers to acyl connected as a substituent via a lower alkylene group. Examples include aryl-C(=O)-(CH2) n - and heteroaryl-C(=O)-(CH2) n - are included (wherein n is an integer in the range of 1 to 6).

[0035] As used herein, "alkoxyalkyl" refers to an alkoxy group connected as a substituent via a lower alkylene group. Examples include C 1~4 alkyl-O-(CH2) n - are included (wherein n is an integer in the range of 1 to 6).

[0036] As used herein, "aminoalkyl" refers to an optionally substituted amino group connected as a substituent via a lower alkylene group. Examples include H2N(CH2) n - (wherein n is an integer in the range of 1 to 6).

[0037] As used herein, "hydroxyalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a hydroxy group. Exemplary hydroxyalkyl groups include, but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. Hydroxyalkyl may be substituted or unsubstituted.

[0038] As used herein, "haloalkyl" refers to an alkyl group in which one or more of the 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 may be substituted or unsubstituted.

[0039] As used herein, "haloalkoxy" refers to an alkoxy group in which one or more of the 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-fluoroalkoxy, chloro-difluoroalkoxy, and 2-fluoroisobutoxy. Haloalkoxy may be substituted or unsubstituted.

[0040] The "sulfenyl" group refers to the "-SR" group (wherein R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)). Sulfenyl may be substituted or unsubstituted.

[0041] The "sulfinyl" group refers to the "-S(=O)-R" group (wherein R can be the same as those defined for sulfenyl). Sulfinyl may be substituted or unsubstituted.

[0042] The "sulfonyl" group refers to the "SO2R" group (wherein R can be the same as those defined for sulfenyl). Sulfonyl may be substituted or unsubstituted.

[0043] The "O-carboxy" group refers to the "RC(=O)O-" group (wherein R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) as defined herein). O-carboxy may be substituted or unsubstituted.

[0044] The terms "ester" and "C-carboxy" refer to the "-C(=O)OR" group (wherein R can be the same as those defined for O-carboxy). Ester and C-carboxy may be substituted or unsubstituted.

[0045] The "thiocarbonyl" group refers to the "-C(=S)R" group (wherein R can be the same as those defined for O-carboxy). Thiocarbonyl may be substituted or unsubstituted.

[0046] The term "trihalomethanesulfonyl" refers to the group "X3CSO2-" (where each X is a halogen).

[0047] The term "trihalomethanesulfonamide" refers to the group "X3CS(O)2N(R A )-" (where each X is a halogen and R A is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)).

[0048] As used herein, the term "amino" refers to the -NH2 group.

[0049] As used herein, the term "hydroxy" refers to the -OH group.

[0050] The term "cyano" refers to the group "-CN".

[0051] As used herein, the term "azide" refers to the -N3 group.

[0052] The term "isocyanate" refers to the group "-NCO".

[0053] The term "thiocyanate" refers to the group "-CNS".

[0054] The term "isothiocyanate" refers to the group "-NCS".

[0055] The term "carbonyl" refers to the C=O group.

[0056] The term "S-sulfonamide" refers to the group "-SO2N(R A R B )" (where R A and R Bcan be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). The S-sulfonamide may be substituted or unsubstituted.

[0057] The "N-sulfonamide" group refers to the group "RSO2N(R A )-" (wherein R and R A can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)). The N-sulfonamide may be substituted or unsubstituted.

[0058] The "O-carbamyl" group refers to the group "-OC(=O)N(R A R B )" (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)). The O-carbamyl may be substituted or unsubstituted.

[0059] The "N-carbamyl" group refers to the group "ROC(=O)N(R A )-" (wherein R and R A can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)). The N-carbamyl may be substituted or unsubstituted.

[0060] The "O-thiocarbamyl" group refers to the "-OC(=S)-N(R A R B )" group (wherein R A and R B are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)). O-thiocarbamyl may be substituted or unsubstituted.

[0061] The "N-thiocarbamyl" group refers to the "ROC(=S)N(R A )-" group (wherein R and R A are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)). N-thiocarbamyl may be substituted or unsubstituted.

[0062] The "C-amide" group refers to the "-C(=O)N(R A R B )" group (wherein R A and R B are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)). C-amide may be substituted or unsubstituted.

[0063] The "N-amide" group refers to the "RC(=O)N(R A )-" group (wherein R and R Acan be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). The N-amide may be substituted or unsubstituted.

[0064] The term "urea" group refers to the group "N(R)-C(=O)-NR A R B " (wherein R can be hydrogen or alkyl, and 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)). The urea may be substituted or unsubstituted.

[0065] The term "oxime" group refers to "-C(=N-OH)R A " (wherein R A can be independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)). The oxime may be substituted or unsubstituted.

[0066] The term "acylhydrazone" refers to "-C(=N-NH-acyl)-R A ." (wherein the acyl moiety has the structure provided herein for "acyl" and R A can be independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)). The acylhydrazone may be substituted or unsubstituted.

[0067] "Hydrazine" refers to "-NHNR A R B " (wherein R A and R B can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl)). Hydrazine may be substituted or unsubstituted.

[0068] As used herein, the term "halogen atom" or "halogen" means any one of the radiation-stable atoms in column 7 of the periodic table of elements such as fluorine, chlorine, bromine and iodine.

[0069] As used herein,

[0070]

Chem.

[0071] When the number of substituents is not specified (e.g., haloalkyl), one or more substituents can be present. For example, "haloalkyl" can contain one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" can contain one or more of the same or different alkoxy groups containing 1, 2, or 3 atoms.

[0072] As used herein, the abbreviations for all protecting groups, amino acids and other compounds follow their general usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. 11:942-944 (1972)) unless otherwise indicated.

[0073] As used herein, the terms "protecting group" and "protecting groups" (and the abbreviation "PG") refer to any atom or group of atoms that is added to a molecule to prevent an existing group in the molecule from undergoing an unwanted chemical reaction. Examples of protecting group moieties are described in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3. Ed. John Wiley & Sons, 1999, and in J.F.W. McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, both of which are incorporated herein by reference for the limited purpose of disclosing suitable protecting groups. The protecting group moiety can be selected such that the protecting group is stable to a particular set of reaction conditions and can be readily removed at a convenient stage using methodologies known in the art.A non-limiting list of protecting groups includes the following: benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., t-butoxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ether (e.g., methoxymethyl ether); substituted ethyl ether; substituted benzyl ether; tetrahydropyranyl ether; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl or t-butyldiphenylsilyl); ester (e.g., benzoic acid ester); carbonate (e.g., methoxymethylcarbonate); sulfonate (e.g., tosylate or mesylate); acyclic ketal (e.g., dimethyl acetal); cyclic ketal (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetal; cyclic acetal (e.g., those described herein); acyclic hemiacetal; cyclic hemiacetal; cyclic dithioketal (e.g., 1,3-dithiane or 1,3-dithiolane); orthoester (e.g., those described herein) and triarylmethyl group (e.g., trityl; monomethoxytrityl (MMTr); 4,4'-dimethoxytrityl (DMTr); 4,4',4''-trimethoxytrityl (TMTr); and those described herein).

[0074] As used herein, the term "leaving group" (and the abbreviation "LG") refers to any atom or moiety that can be replaced by another atom or moiety in a chemical reaction. More specifically, in some embodiments, a "leaving group" refers to an atom or moiety that is replaced in a nucleophilic substitution reaction. In some embodiments, a "leaving group" is any atom or moiety that is the conjugate base of a strong acid. Examples of suitable leaving groups include, but are not limited to, tosylate, mesylate, trifluoroacetate, and halogens (e.g., I, Br, and Cl). For non-limiting characteristics and examples of leaving groups, see, for example, Organic Chemistry, 2d ed., Francis Carey (1992), pages 328-331; Introduction to Organic Chemistry, 2d ed., Andrew Streitwieser and Clayton Heathcock (1981), pages 169-171; and Organic Chemistry, 5th ed., John McMurry (2000), pages 398 and 408; all of which are hereby incorporated by reference herein for the limited purpose of disclosing characteristics and examples of leaving groups.

[0075] The term "pharmaceutically acceptable salt" refers to salts of the compound that do not cause significant irritation to the organism to which it is administered and do not inactivate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutically acceptable salts can be obtained by reacting the compound with inorganic acids such as hydrohalic acids (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid. Pharmaceutically acceptable salts can also be obtained by reacting the compound with organic acids such as aliphatic or aromatic carboxylic acids or sulfonic acids, e.g., 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 salts such as ammonium salts, alkali metal salts such as sodium salts or potassium salts, alkaline earth metal salts such as calcium salts or magnesium salts, salts of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamines, cyclohexylamine, triethanolamine, ethylenediamine, and salts with amino acids such as arginine and lysine.

[0076] The terms and phrases used in this application and their variants should be construed as open-ended rather than limiting, unless otherwise clearly stated in the appended claims. By way of example, the term "including" should be read to mean "including, without limitation", "including but not limited to", etc.; the term "comprising" as used herein is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional unrecited elements or method steps; the term "having" should be construed to mean "having at least"; the term "includes" should be construed to mean "includes but is not limited to"; the term "example" is used to provide illustrative instances of matters under discussion, not an exhaustive or limiting enumeration; the use of terms such as "preferably", "preferred", "desired", or "desirable", and words of similar import, should not be understood to imply that a particular characteristic is critical, essential, or even important for a structure or function, but rather is simply intended to highlight alternative or additional characteristics that may or may not be utilized in a particular embodiment. Additionally, the term "comprising" should be construed as synonymous with the phrase "having at least" or "including at least".When used in the context of a method, the term "comprising" means that the method includes at least the recited steps, but may also include additional steps. When used in the context of a compound, composition or device, the term "comprising" means that the compound, composition or device includes at least the recited features or components, but may also include additional features or components. Similarly, a group of items joined by the conjunction "and" should not be read as requiring that each one of those items be present in the grouping, but rather, unless context dictates otherwise, should be read as "and / or". Similarly, a group of items joined by the conjunction "or" should not be read as requiring that the items within the group be mutually exclusive, but rather, unless context dictates otherwise, should be read as "and / or".

[0077] Regarding substantially any use of plural and / or singular terms herein, one of ordinary skill in the art can, where appropriate in the context and / or application, change from the plural to the singular and / or from the singular to the plural. Various singular / plural permutations may be explicitly set forth herein for clarity. The indefinite articles "a" or "an" do not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. No reference signs in the claims should be construed as limiting the scope.

[0078] In any of the compounds described herein that have one or more chiral centers, if the absolute stereochemistry is not explicitly indicated, it is understood that each center can independently be of the R-configuration, the S-configuration, or a mixture thereof. Thus, the compounds provided herein can be enantiomerically pure, enantiomerically enriched, racemic mixtures, diastereomerically pure, diastereomerically enriched, or mixtures of stereoisomers. Additionally, in any of the compounds described herein that have one or more double bonds that can be defined as E or Z, it is understood that each double bond can independently be E, Z, or a mixture thereof.

[0079] Similarly, it is understood that all tautomeric forms are intended to be included in any of the compounds described.

[0080] It should be understood that if a compound disclosed herein has a vacant valence, that valence will be filled with hydrogen or an isotope thereof, such as hydrogen-1 (protium) and hydrogen-2 (deuterium).

[0081] It is understood that the compounds described herein can be isotopically labeled. Substitution with isotopes such as deuterium can provide certain therapeutic advantages resulting from greater metabolic stability, such as an improvement in in vivo half-life or a reduction in the required dosage. Each chemical element represented in the compound structure can include any isotope of said element. For example, in the compound structure, a hydrogen atom may or may be understood to be explicitly disclosed as being present in the compound. At any position in the compound where a hydrogen atom can be present, the hydrogen atom can be any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Thus, references to compounds herein include all isotopic forms thereof that are possible, unless the context indicates otherwise.

[0082] The methods and combinations described herein are understood to include crystalline forms (also known as polymorphs, which include different crystal packing arrangements of compounds of the same elemental composition), amorphous phases, salts, solvates, and hydrates. In some embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In other embodiments, the compounds described herein exist in unsolvated forms. Solvates can contain either stoichiometric or non-stoichiometric amounts of solvent and can be formed during the crystallization process using pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Additionally, the compounds provided herein can exist in unsolvated and solvated forms. Generally, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0083] When ranges of values are provided, it is understood that the upper and lower limits, as well as intervening values between the upper and lower limits of the range, are included within the embodiments.

[0084] As used herein, "RNA" refers to ribonucleic acid, whether naturally occurring or non-naturally occurring. For example, RNA can include modified and / or non-naturally occurring 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 polypeptide of interest. For example, RNA can be messenger RNA (mRNA). Translation of mRNA encoding a specific polypeptide, for example, in vivo translation of mRNA within mammalian cells, can produce the encoded polypeptide. RNA can be selected from the non-limiting group consisting of small interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single guide RNA (sgRNA), cas9 mRNA, and mixtures thereof.

[0085] The terms "polypeptide", "peptide", and "protein" can be used interchangeably to refer to a string of at least three amino acids linked together by peptide bonds. Peptide can refer to an individual peptide or a collection of peptides. Peptides can contain natural amino acids, 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. Also, one or more of the amino acids in a peptide can be modified 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. Modifications can include cyclization of the peptide, incorporation of D-amino acids, and the like.

[0086] As used herein, the terms "treating," "treatment," "treat," and "therapeutic use" refer to the elimination, reduction, or amelioration of one or more symptoms of a disease or disorder. As used herein, "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to mediate a clinically significant elimination, reduction, or amelioration of such symptoms. An effect is clinically significant if its magnitude is sufficient to affect the health or prognosis of the recipient subject. A therapeutically effective amount can refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, e.g., sufficient to delay or minimize the spread of cancer. A therapeutically effective amount can also refer to an amount of a therapeutic agent that provides a therapeutic effect in the treatment or management of a disease.

[0087] The compositions described herein are preferably provided in unit dosage form. As used herein, "unit dosage form" refers to a composition containing an amount of a compound or composition suitable for administration in a single dose to an animal, preferably a mammalian subject, in accordance with good medical practice. However, the preparation of a single or unit dosage form does not implicitly mean that the dosage form is administered once daily or once per course of treatment. Single administration is not particularly excluded, but such dosage forms are contemplated to be administered once, twice, three times, or more times per day, and may be administered as an infusion over a period of time (e.g., from 30 minutes to about 2 - 6 hours), as a continuous infusion, and may be given multiple times during the course of treatment. One of ordinary skill in the art will recognize that a formulation is not particularly contemplated for the entire course of treatment, and such determination is left to the skilled artisan in the art of treatment rather than the formulation. As used herein, the term "preventive agent" refers to an agent that can be used to prevent a disorder or disease before any symptoms related to the disorder or disease are detected. "Preventively effective" amount refers to an amount of a preventive agent sufficient to mediate such prevention. A preventively effective amount can also refer to an amount of a preventive agent that provides a preventive effect in the prevention of a disease.

[0088] The above useful compositions can be in any of a variety of forms suitable for various routes of administration, for example, oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intraarterial, intravenous, intramuscular, or other parental routes of administration. One of ordinary skill in the art will understand that oral and nasal compositions include compositions that are administered by inhalation and made using available methodologies. Depending on the particular 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, hydrotropies, surfactants, and encapsulating materials. Pharmaceutically active materials, as required, can be included that do not substantially interfere with the inhibitory activity of the compounds. The amount of carrier used in conjunction with the compound is sufficient per unit dose of the compound to provide a practical quantity of material for administration. Techniques and compositions for manufacturing dosage forms useful in the methods described herein are described in the following references, all of which are incorporated herein by reference: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

[0089] As used herein, the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and refer to mammals, including but not limited to humans, rodents such as mice and rats, and other laboratory animals.

[0090] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents.

[0091] As used herein, the term "conformationally restricted lipid" refers to a lipid whose molecular structure is mainly in one construct such as adamantane, the shape of which resembles an "armchair".

[0092] 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 、およびC 18 PEG 2000 are included.

[0093] The term "oligonucleotide" refers to a short DNA, RNA, or DNA / RNA molecule or oligomer containing a relatively small number of nucleotides.

[0094] A. Lipid nanoparticles The effective targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids remains an ongoing challenge in the field of medicine. The delivery of nucleic acids is particularly difficult due to the relative instability and low cell permeability of nucleic acids. It has been discovered that nucleic acids can be more effectively delivered to specific tissues in the body by lipid nanoparticles having constrained lipids. In one embodiment, the lipid nanoparticles can be formulated by mixing nucleic acids with ionizable lipids with constrained conformations, PEG-lipids, phospholipids, cholesterol, and optionally nucleic acids. In some embodiments, the lipid nanoparticles do not contain targeting ligands. In some embodiments, the disclosed lipid nanoparticles preferentially target T cells over hepatocytes in the absence of targeting ligands.

[0095] The size of the lipid nanoparticles varies. In one embodiment, the lipid nanoparticles can have an average hydrodynamic diameter ranging from about 30 to about 170 nm. The lipid nanoparticles can have an average hydrodynamic diameter that is any range defined by about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, or any two of the foregoing values. For example, in one embodiment, the nanoparticles have an average hydrodynamic diameter ranging from between 50 nm and 100 nm.

[0096] 1. Compound Some embodiments relate to a compound of formula (I):

[0097]

Chemical formula

[0098]

Chemical formula

[0099]

Chemical formula

[0100] In other embodiments,

[0101]

Chemical formula

[0102]

Chemical formula

[0103]

Chem.

[0104]

Chem.

[0105] In some embodiments, the compounds of formula (I) are:

[0106]

Chem.

[0107]

Chem.

[0108]

Chem.

[0109]

Chem.

[0110]

Chem.

[0111]

Chem.

[0112]

Chem.

[0113]

Chem.

[0114]

Chem.

[0115] A further embodiment relates to a compound of formula (II):

[0116]

Chem.

[0117]

Chem.

[0118]

Chemical formula

[0119]

Chemical formula

[0120] In some embodiments, R 9 is

[0121]

Chemical formula

[0122]

Chemical formula

[0123] [Chemical formula] and X 13 is

[0124] [Chemical formula] is. In some embodiments, c, d, and e are each 1. In some embodiments, R 9 is

[0125] [Chemical formula] is. In some embodiments, R 8 is hydrogen. In some embodiments, R 9 and

[0126] [Chemical formula] are each

[0127] [Chemical formula] is.

[0128] Further embodiments relate to compounds of formula (IIa), (IIb), or (IIc):

[0129] [Chemical formula]

[0130] In some embodiments, the compound of formula (II) is:

[0131] [Chemical formula]

[0132] [Chemical formula]

[0133] [Chemistry]

[0134] [Chemistry]

[0135] [Chemistry]

[0136] [Chemistry]

[0137] [Chemistry]

[0138] [Chemistry] is selected from the group consisting of.

[0139] 2. Ionizable lipid In one embodiment, the disclosed lipid nanoparticles comprise an ionizable lipid. Ionizable lipids typically contain an amine-containing group on the head group. In one embodiment, the ionizable lipid is a conformationally constrained ionizable lipid as described elsewhere herein. In some embodiments, the conformationally constrained lipid is present in the lipid nanoparticles at 35, 45, 50, or 65 mole percent relative to the total moles of the components of the lipid nanoparticles. In another embodiment, the conformationally constrained lipid is present at about 33 mol% to about 36 mol% relative to the total moles of the components of the lipid nanoparticles. In yet another embodiment, the conformationally constrained lipid is present at about 35 mol% relative to the total moles of the components of the lipid nanoparticles.

[0140] Further embodiments relate to lipid nanoparticle compositions comprising conformationally restricted ionizable lipids; phospholipids; polyethylene glycol-lipids; cholesterol; and optionally nucleic acids. In some embodiments, the conformationally restricted ionizable lipid comprises a structure according to any one of formulas (I), (Ia), (II), (IIa), (IIb), and (IIc). In some embodiments, the amount of the conformationally restricted ionizable lipid is present in the range of about 35 to 65 mole percent relative to the total moles of the components of the lipid nanoparticles.

[0141] 3. Sterol In some embodiments, the disclosed lipid nanoparticles comprise one or more sterols. In one embodiment, the sterol is cholesterol, or a variant or derivative thereof. In some embodiments, cholesterol is modified, for example, oxidized. Unmodified cholesterol can be acted upon by enzymes to form variants with oxidized side chains or oxidized rings. Cholesterol may be oxidized on the beta-ring structure or on the hydrocarbon tail structure. Exemplary cholesterols contemplated for use in the disclosed lipid nanoparticles include, but are not limited to, 25-hydroxy cholesterol (25-OH), 20α-hydroxy cholesterol (20α-OH), 27-hydroxy cholesterol, 6-keto-5α-hydroxy cholesterol, 7-ketocholesterol, 7β-hydroxy cholesterol, 7α-hydroxy cholesterol, 7β-25-dihydroxy cholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof. In one embodiment, side-chain oxidized cholesterol can enhance cargo delivery compared to other cholesterol variants. In one embodiment, cholesterol is unmodified cholesterol.

[0142] 4. PEG-lipid In some embodiments, the disclosed nanoparticle compositions also include one or more PEGs or PEG-modified lipids. Such lipids can alternatively be referred to as PEGylated lipids or PEG-lipids. The inclusion of PEGylated lipids can be used to enhance the stability of lipid nanoparticle colloids in vitro and the circulation time in vivo. In some embodiments, the PEGylation is reversible in that the PEG moiety is gradually released within the bloodstream. Exemplary PEG-lipids include, but are not limited to, PEG conjugated to saturated or unsaturated alkyl chains having a length of C6~C 20 including PEG conjugated to saturated or unsaturated alkyl chains having a length of C6~C. 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 each lipid of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG, or PEG-DSPE. 5. Phospholipids

[0143] The phospholipid component of the nanoparticles can include one or more phospholipids such as one or more (poly)unsaturated lipids. The phospholipids can be assembled into one or more lipid bilayers. In some embodiments, the phospholipids can include a phospholipid moiety and one or more fatty acid moieties.

[0144] In some embodiments, the lipid moiety includes, but is not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin. In some embodiments, the fatty acid moiety includes, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Also contemplated are non-natural species, including natural species, having modifications and substitutions such as branching, oxidation, cyclization, and alkynes. For example, the lipid may be functionalized or cross-linked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, an alkyne group may undergo copper-catalyzed cycloaddition when exposed to an azide. Such reactions can be useful in functionalizing the lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cell recognition, or in conjugating the nanoparticle composition to useful components such as targeting or imaging moieties (e.g., dyes).

[0145] Exemplary phospholipids include, but are not limited to, the following: 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-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 lysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE). In a preferred embodiment, the phospholipid is DSPC. In another embodiment, the phospholipid is DMPC.

[0146] E. Cargo In one embodiment, the disclosed lipid nanoparticle composition comprises a therapeutic or prophylactic agent for a subject. In some embodiments, the therapeutic or prophylactic agent is encapsulated by the lipid nanoparticles. In one embodiment, the lipid nanoparticles carry one or more nucleic acids.

[0147] 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.

[0148] CRISPR (clustered regularly interspaced short palindromic repeats)-based gene editing requires two components: a guide RNA and a CRISPR-associated endonuclease protein (Cas). The guide RNA directs the Cas nuclease to a specific target DNA sequence. The Cas then creates a double-strand break in the DNA at that site. In one embodiment, the disclosed lipid nanoparticles can be used to deliver the components necessary for CRISPR-based gene editing. In one lipid nanoparticle, the nucleic acid cargo is the guide RNA. In such an embodiment, a second lipid nanoparticle can contain a nucleic acid cargo that encodes an RNA-guided endonuclease. The two types of lipid nanoparticles can be administered together. Exemplary RNA-guided endonucleases include, but are not limited to, Cas9, CasX, CasY, Cas13, or Cpf1.

[0149] In one embodiment, the cargo is siRNA. Short interfering RNA (siRNA) is a double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby reducing or even suppressing gene expression. In one example, siRNA induces the specific degradation of homologous RNA molecules, such as mRNA, within the region of sequence identity between both the siRNA and the target RNA. For example, WO 02 / 44321 discloses siRNAs capable of sequence-specific degradation of target mRNA when base-paired with a 3' overhang, and the methods for making these siRNAs are incorporated herein by reference. Sequence-specific gene silencing can be performed in mammalian cells using synthetic, short double-stranded RNAs that mimic siRNAs generated by the enzyme Dicer (Elbashir, et al. (2001) Nature, 411:494 - 498)(Ui-Tei, et al. (2000) FEBS Lett 479:79-82).

[0150] In one embodiment, the lipid nanoparticles contain less than 1.0 mg / kg of the inhibitory nucleic acid. The nanoparticles can contain 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 mg / kg of the inhibitory nucleic acid. In another embodiment, the lipid nanoparticles contain 0.5 mg / kg of the inhibitory nucleic acid. This is an advantage over current technology. In current technology, nanoparticles require high doses of nucleic acid (>1 mg / kg) to achieve gene silencing, and those doses are not approved for human delivery. The disclosed technology can achieve gene silencing using 0.5 mg / kg of the inhibitory nucleic acid in lipid nanoparticles that do not contain targeting ligands.

[0151] In some embodiments, the nucleic acid is modified or includes another modified nucleotide to improve stability, half-life, and nuclease sensitivity, including but not limited to oligonucleotides. To limit nuclease sensitivity, natural phosphodiester oligodeoxyribonucleotides, natural phosphodiester oligoribonucleotides, ribonucleotide polymers, and deoxyribonucleotide polymers can include a different modification. Exemplary modifications include, but are not limited to, phosphorothioate (PS) linkages, 2'-O-methyl (2'OMe), 2'-fluoro bases, inverted dT and ddT, phosphorylation of the 3' end of the oligonucleotide, locked nucleic acids, and phosphoramidite C3 spacers.

[0152] Phosphorothioate bonds use a sulfur atom instead of a non-bridging oxygen in the phosphate backbone of oligonucleotides. Approximately 50% of the time (due to the two stereoisomers that can result), PS modification makes the internucleotide linkages more resistant to nuclease degradation. In some embodiments, the nucleic acid contains one or more PS bonds, e.g., at least three PS bonds at the 5' and 3' oligonucleotide termini, so as to suppress exonuclease degradation. Some nucleic acids contain PS bonds throughout the oligonucleotide to also help reduce attack by endonucleases.

[0153] 2’OMe, a naturally occurring post-transcriptional modification of RNA, is found in tRNA and other small RNAs. In some embodiments, the nucleic acid or oligonucleotide is directly synthesized to contain 2’OMe. This modification improves the Tm of RNA:RNA duplexes, but only causes minor changes in RNA:DNA stability. This modification prevents attack by single-strand endonucleases, but does not prevent exonuclease digestion. In some embodiments, such nucleic acids or oligonucleotides are also end-blocked. DNA oligonucleotides containing this modification are typically 1 / 5 to 1 / 10 as susceptible to DNase as unmodified DNA. 2’OMe modification is commonly used in antisense oligonucleotides as a means to improve stability and binding affinity to target transcripts.

[0154] 2’-Fluoro bases have fluorine-modified riboses, which improve binding affinity (Tm) and also confer some relative nuclease resistance compared to native RNA. In some embodiments, the nucleic acid or oligonucleotide contains 2’-fluoro bases in combination with PS-modified bonds.

[0155] Inverted dT can be incorporated at the 3'-end of the oligonucleotide, resulting in a 3'-3' linkage that inhibits degradation by 3'-exonuclease and elongation by DNA polymerase. Additionally, by placing an inverted 2',3'-dideoxy-dT base (5'-inverted ddT) at the 5'-end of the oligonucleotide, spurious ligation can be prevented and some forms of enzymatic degradation can be defended against.

[0156] Some embodiments provide a nucleic acid or oligonucleotide comprising a phosphoramidite C3 spacer. The phosphoramidite C3 spacer can also be incorporated internally or incorporated at either end of the oligo to introduce a long hydrophilic spacer arm for attachment of a fluorophore or other pendant group. The C3 spacer can also be used to inhibit degradation by 3'-exonuclease.

[0157] In some embodiments, the nucleic acid or oligonucleotide comprises a locked nucleic acid. Locked nucleic acids include modified RNA nucleotides in which the 2'-O atom and 4'-C atom of the ribose are linked through a methylene bridge. This additional bridge typically restricts the flexibility normally associated with the ring and essentially locks the structure into a rigid conformation. LNA can be inserted into both RNA and DNA oligonucleotides.

[0158] Other types of cargo that can be delivered via the disclosed nanoparticles include, but are not limited to, chemotherapeutic agents, cytotoxic drugs, radioactive ions, small molecules, proteins, polynucleotides, and nucleic acids.

[0159] Representative chemotherapeutic agents include, but are not limited to, the following: amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. Representative apoptosis promoters include, but are not limited to, fludarabine taurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

[0160] Some embodiments relate to a method of delivering a nucleic acid to a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle composition described herein. In some embodiments, the nucleic acid is siRNA, miRNA, an antisense oligonucleotide, or an immunostimulatory oligonucleotide.

[0161] B. Exemplary Lipid Nanoparticle Formulations In one embodiment, the lipid nanoparticle formulation comprises about 30 mol% to about 70 mol% of an ionizable lipid with a constrained conformation, about 5 mol% to about 25 mol% of a phospholipid, about 25 mol% to about 45 mol% of cholesterol, and about 0 mol% to about 5 mol% of a PEG-lipid. In another embodiment, the lipid nanoparticle formulation comprises about 35 mol% of an ionizable lipid with a constrained conformation, about 16 mol% of a phospholipid, about 46.5 mol% of cholesterol, and about 2.5 mol% of a PEG-lipid. In another embodiment, the lipid nanoparticle formulation comprises about 50 mol% of an ionizable lipid with a constrained conformation, about 10 mol% of a phospholipid, about 38.5 mol% of cholesterol, and about 1.5 mol% of a PEG-lipid.

[0162] One embodiment provides a lipid nanoparticle formulation comprising, based on the total moles of the following four components, about 33 mol% to about 36 mol% of an ionizable lipid with a constrained conformation having an adamantane tail, about 15 mol% to about 17 mol% of 1,2-distearoyl-sn-glycero-3-phosphocholine, C 14 PEG 2000 about 2 mol% to about 3 mol%, and about 45 mol% to about 47 mol% of cholesterol.

[0163] Another embodiment provides a lipid nanoparticle formulation comprising, based on the total moles of the following four components, 35 mol% of an ionizable lipid with a constrained conformation having an adamantane tail, 16 mol% of 1,2-distearoyl-sn-glycero-3-phosphocholine, C 14 PEG 2000 2.5 mol%, and 46 mol% of cholesterol.

[0164] Another embodiment provides a lipid nanoparticle formulation in which the mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):siRNA is between about 2:1 and 50:1.

[0165] In yet another embodiment, the lipid nanoparticle formulation comprises 3-[(1-adamantanyl)acetoxy]-2-{[3-(diethylamino)propoxycarbonyloxy]methyl}propyl (9Z,12Z)-9,12-octadecadienoate, DSPC, polyethylene glycol-lipid, cholesterol, and a suppressive nucleic acid.

[0166] One embodiment provides a lipid nanoparticle composition containing 3-[(1-adamantanyl)acetoxy]-2-{[3-(diethylamino)propoxycarbonyloxy]methyl}propyl (9Z,12Z)-9,12-octadecadienoate, DSPC, polyethylene glycol-lipid, cholesterol, and an sgRNA specific to a gene. Another embodiment provides a lipid nanoparticle containing 3-[(1-adamantanyl)acetoxy]-2-{[3-(diethylamino)propoxycarbonyloxy]methyl}propyl (9Z,12Z)-9,12-octadecadienoate, DSPC, polyethylene glycol-lipid, cholesterol, and an mRNA encoding an RNA-guided DNA endonuclease.

[0167] C. Pharmaceutical Composition A pharmaceutical composition comprising the disclosed lipid nanoparticles is provided. The lipid nanoparticle composition can be formulated, in whole or in part, as a pharmaceutical composition. The pharmaceutical composition can comprise one or more nanoparticle compositions. For example, the pharmaceutical composition can comprise one or more different nanoparticle compositions containing one or more different therapeutic and / or prophylactic agents, including but not limited to, one or more nucleic acids of different types, or can encode different agents. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients or adjuncts, including but not limited to, a pharmaceutically acceptable carrier.

[0168] The pharmaceutical composition containing nanoparticles can be formulated for administration by various routes of administration, such as parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal or sublingual), or by using a bioerodible insert, and can be formulated in a dosage form suitable for each route of administration.

[0169] In some in vivo approaches, the nanoparticle compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein, the terms "effective amount" or "therapeutically effective amount" mean a dosage sufficient to treat, suppress or alleviate one or more symptoms of the disorder being treated, or to provide the desired pharmacological and / or physiological effect. The exact dosage will vary depending on various factors such as the variable factors depending on the subject (e.g., age, immune system health, etc.), the disease, and the treatment being performed.

[0170] As further research is carried out on the disclosed nanoparticles, information regarding appropriate dosage levels for treating various conditions of various patients will become apparent, and those skilled in the art will be able to confirm the appropriate dosage considering the recipient's treatment status, age and general health. The dosage selected depends on the desired therapeutic effect, the route of administration, and the duration of the desired treatment. For the disclosed nanoparticles, generally, a dosage level of 0.001 mg to 5 mg of nucleic acid per kg of body weight per day is administered to mammals. More specifically, the preferred dosage of the disclosed nanoparticles is 0.01 mg / kg to 0.25 mg / kg. For the disclosed nanoparticles, generally, a dosage level of 0.2 mg to 100 mg of the four components (ionizable lipid, cholesterol, PEG-lipid, and phospholipid) per kg of body weight is administered to mammals. More specifically, the preferred dosage of the disclosed nanoparticles is 0.05 mg / kg to 0.5 mg / 4 components 1 kg / body weight 1 kg.

[0171] In certain embodiments, the lipid nanoparticle composition is administered locally, for example, by direct injection into the site to be treated. Typically, injection results in an increase in the local concentration of the lipid nanoparticle composition that is greater than can be obtained by systemic administration. The lipid nanoparticle composition can be combined with the matrix described above to help create an increase in the local concentration of the polypeptide composition by reducing the passive diffusion of the polypeptide out of the site to be treated.

[0172] 1. Formulations for parenteral administration In some embodiments, the nanoparticle compositions disclosed herein are administered in an aqueous solution by parenteral injection, including those containing lipid nanoparticles. The formulations can also be in the form of a suspension or an emulsion. Generally, the pharmaceutical composition is provided in a form that includes an effective amount of lipid nanoparticles and, optionally, a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions can optionally include one or more of the following: diluents, sterile water, various buffer contents (e.g., Tris-HCl, acetate, phosphate), buffered saline with a buffered pH and ionic strength; as well as surfactants and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimerosal, benzyl alcohol), and additives such as bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and organic esters for injection such as ethyl oleate. The formulations can be lyophilized and re-dissolved / resuspended immediately before use. The formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0173] 2. Controlled release polymer matrix The lipid nanoparticles disclosed herein can also be administered in a controlled release formulation. A controlled release polymeric device can be made for systemic long-term release after implantation of the polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, in which case the drug is dispersed within a solid polymeric matrix or microcapsule, where the core is made of a material different from the polymeric shell and the peptide is dispersed or suspended in a core which can be essentially liquid or solid. Unless otherwise specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer can be cast as a thin slab or film ranging from a few nanometers to 4 centimeters, a powder produced by milling or other standard techniques, or even a gel such as a hydrogel.

[0174] Either a non-biodegradable or biodegradable matrix can be used for the delivery of lipid nanoparticles, although in some embodiments, a biodegradable matrix is preferred. These can be natural polymers or synthetic polymers, although in some embodiments synthetic polymers are preferred due to their better characterized degradation and release profiles. The polymer is selected based on the period over which release is desired. Linear release may sometimes be the most useful, or pulsed release or "bulk release" may result in more effective outcomes. The polymer can be in the form of a hydrogel (typically capable of absorbing up to about 90 wt% water) and can be crosslinked with polyvalent ions or polymers as needed.

[0175] The matrix can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. The biodegradable microspheres can be prepared using, for example, any of the methods developed for making microspheres for drug delivery as described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0176] The devices can be formulated for local delivery to treat the area of implantation or injection - which will typically deliver a much lower dosage than that for systemic treatment - or for systemic delivery. These can be implanted subcutaneously into muscle, fat or injected, or can be swallowed.

[0177] D. Method for manufacturing lipid nanoparticles Methods for manufacturing lipid nanoparticles are known in the art. In one embodiment, the disclosed lipid nanoparticles are manufactured using microfluidics. For exemplary methods of forming lipid nanoparticles using microfluidics, see Leung, A.K.K, et al., J Phys Chem, 116:18440-18450 (2012), Chen, D., et al., J Am Chem Soc, 134:6947-6951 (2012), and Belliveau, N.M., et al., Molecular Therapy- Nucleic Acids, 1: e37 (2012). Briefly, cargos such as oligonucleotides and siRNA are prepared in a certain buffer. Other lipid nanoparticle components (ionizable lipids, PEG-lipids, cholesterol, and DSPC) are prepared in a separate buffer. The two solutions are introduced into the microfluidic device by a syringe pump. The two solutions contact within the microfluidic device to form lipid nanoparticles encapsulating the cargo.

[0178] The method of screening the disclosed lipid nanoparticles is discussed in International Patent Application PCT / US / 2018 / 058171, which is incorporated by reference in its entirety. By the screening method, a vehicle delivery formulation is characterized, and a formulation that has a desired tropism and delivers a functional cargo to the cytoplasm of a specific cell is identified. In the screening method, a reporter having a function that can be detected when delivered to a cell is used. The detection of the function of the reporter within the cell indicates that formulating the delivery vehicle will result in the delivery of the functional cargo to the cell. A chemical composition identifier is included in each of the formulations of different delivery vehicles to continuously track the chemical composition unique to each of the formulations of different delivery vehicles. In one embodiment, the chemical composition identifier is a nucleic acid barcode. By pairing the sequence of the nucleic acid barcode with the chemical components used to formulate the delivery vehicle in which it is loaded, the chemical composition of the delivery vehicle that delivered the barcode is identified when the nucleic acid barcode is sequenced. Representative reporters include, but are not limited to, siRNA, mRNA, nuclease proteins, nuclease mRNA, small molecules, epigenetic modifiers, and phenotypic modifiers.

[0179] E. Method of Use Methods of using the disclosed lipid nanoparticles to deliver a cargo, such as a nucleic acid, to a specific cell or organ are disclosed herein. In some embodiments, the nanoparticles deliver a therapeutic or prophylactic agent to a specific cell or organ in a subject in need thereof in the absence of a targeting ligand. In another embodiment, the disclosed lipid nanoparticles are useful for treating or preventing a disease in a subject in need thereof.

[0180] In some embodiments, the disclosed nanoparticles are delivered directly to the subject. In other embodiments, the lipid nanoparticles are contacted with cells ex vivo, and the treated cells are administered to the subject. The cells can be autologous cells, such as, but not limited to, immune cells, including 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.

[0181] 1. Method for delivering cargo to cells Provided herein is a method for delivering a therapeutic and / or prophylactic nucleic acid to a subject in need thereof.

[0182] In some embodiments, the disclosed lipid nanoparticle compositions target a particular type or class of cells (e.g., cells of a particular organ or its system). For example, a nanoparticle composition comprising a therapeutic agent and / or prophylactic agent of interest can be specifically delivered to immune cells of a subject. 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 immune cells, spleen T cells, or lung endothelial cells in the absence of a targeting ligand. Specific delivery to a particular class or type of cells indicates that a higher percentage of the lipid nanoparticles are delivered to the target type or class of cells. In some embodiments, specific delivery can result in an increase of 2-fold, 5-fold, 10-fold, 15-fold, or more than 20-fold in the amount of therapeutic and / or prophylactic agent per gram of the target delivery tissue.

[0183] 2. Method for gene regulation Methods of using the disclosed lipid nanoparticles for gene regulation are provided herein. In one embodiment, the lipid nanoparticles can be used to reduce gene expression in target cells of a subject in need thereof. The lipid nanoparticles can deliver inhibitory nucleic acids to the target cells of the subject without a targeting ligand. The inhibitory nucleic acid can be siRNA.

[0184] Another embodiment provides a method of using the disclosed lipid nanoparticles to edit a gene in a cell of a subject in need thereof.

[0185] In one embodiment, the cells targeted for gene regulation are immune cells. The immune cells can be T cells such as CD8+ T cells, CD4+ T cells, or regulatory T cells. Other exemplary immune cells for gene editing include, but are not limited to, macrophages, dendritic cells, B cells or natural killer cells.

[0186] 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-40, CD137, CD137L, CD30L, CD40L, FasL, TRAIL, CD257, LIGHT, TRAIL-R1, TRAILR2, TRAIL-R4, TWEAK-R, TNFR, BCMA, B7DC, BTLA, B7-H1, B7-H2, B7-H3, ICOS, VEGFR2, NKG2D, JAG1, GITR, CD4, CCR2, GATA-3, MTORC1, MTORC2, RAPTOR, GATOR, FOXP3, NFAT, IL2R, and IL7.

[0187] Exemplary tumor-associated antigens that can be recognized by T cells and for which targeting is contemplated 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.

[0188] 3. Subject to be Treated In some embodiments, the subject to be treated is a mammal experiencing cancer, an autoimmune disease, an infectious disease, an organ transplant, organ failure, or a combination thereof. In some embodiments, the methods described herein can cause T cells to present specific antigens for the treatment of cancer or an autoimmune disease. In some embodiments, the methods described herein can be used for T cell priming. In some embodiments, the methods described herein can be used to deliver DNA or mRNA that causes T cells to present MHC-peptide complexes. In some embodiments, the methods described herein can be used to deliver one or more of DNA, siRNA, or mRNA to T cells to avoid anergy.

Examples

[0189] General Notes: All reactions were carried out with magnetic stirring in a flask or vial under a nitrogen atmosphere using anhydrous-grade solvents, unless otherwise specified. Anhydrous solvents were purchased from Sigma-Aldrich and used as received. Flash column chromatography was performed using a Biotage Selekt or Teledyne-Isco Combiflash Nextgen300+ equipped with prepacked Biotage Sfar silica gel cartridges. Thin-layer chromatography was performed using Merck silica gel 60 plates, and compounds were visualized using iodine. Nuclear magnetic resonance (NMR) spectroscopy was performed using a Varian INOVA 500 MHz spectrometer; chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane with reference to the residual solvent peak of CHCl3 at δ = 7.26 ppm. Liquid chromatography–mass spectrometry (LCMS) was performed using a Waters Acquity UPLC H-class Plus equipped with a QDa detector (ESI) and a Waters Acquity UPLC BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 50 mm). Unless otherwise noted, compounds were analyzed using the following general LCMS method. 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 hold at 95% B for 2 minutes, then ramp back to 10% B over 1 minute; flow rate = 0.5 mL / min.

[0190] List of Abbreviations AOP: (7-Azabenzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate DCM: Dichloromethane DIPEA: N,N-Diisopropylethylamine DMAP: 4-(Dimethylamino)pyridine DMPC: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine DSPC: 1,2-Distearoyl-sn-glycero-3-phosphocholine EDC: N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride Eq: Equivalent ESI: Electrospray ionization LCMS: Liquid chromatography - mass spectrometry LNP: Lipid nanoparticles NMR: Nuclear magnetic resonance RT: Retention time

[0191] [Example 1] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (1)

[0192] [Chemical formula]

[0193] Step 1: 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0194] [Chemical formula]

[0195] A mixture of trimethylolmethane (3.0 g, 1 Eq, 28 mmol) dissolved in dichloromethane (100 mL) was added to linoleic acid (7.9 g, 1 Eq, 28 mmol), DIPEA (5.5 g, 7.4 mL, 1.5 Eq, 42 mmol), and DMAP (0.69 g, 0.2 Eq, 5.7 mmol). Finally, EDC (8.1 g, 1.5 Eq, 42 mmol) was added and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (200 g silica, 0 - 90% ethyl acetate in hexane over 20 min). 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (2.6 g, 25%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.27 (m, 5H), 4.24 (d, J = 6.3 Hz, 2H), 3.76 (ddt, J = 21.1, 11.1, 5.3 Hz, 4H), 2.77 (d, J = 6.8 Hz, 2H), 2.61 - 2.55 (m, 2H), 2.36 - 2.29 (m, 2H), 2.10 - 1.98 (m, 6H), 1.66 - 1.58 (m, 2H), 1.41 - 1.21 (m, 12H), 0.92 - 0.85 (m, 3H).

[0196] Step 2: 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0197]

Chemical Structure

[0198] (9Z,12Z)-Octadeca-9,12-dienoic acid, 3-hydroxy-2-(hydroxymethyl)propyl ester (2.6 g, 1 Eq, 7.1 mmol) was dissolved in dichloromethane (30 mL), and 2-(adamantan-1-yl)acetic acid (1.4 g, 1 Eq, 7.1 mmol), DIPEA (1.8 g, 2 Eq, 14 mmol) and DMAP (0.17 g, 0.2 Eq, 1.4 mmol) were added. Finally, EDC (2.0 g, 1.5 Eq, 11 mmol) was added, and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (100 g silica, 0 - 40% ethyl acetate in hexane over 20 min). 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (1.86 g, 48%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.41 - 5.28 (m, 4H), 4.23 - 4.10 (m, 5H), 3.62 (t, J = 6.0 Hz, 2H), 2.76 (tt, J = 6.6, 0.9 Hz, 2H), 2.35 - 2.27 (m, 3H), 2.19 (septet, J = 5.9 Hz, 1H), 2.10 - 1.99 (m, 6H), 1.97 (p, J = 2.9 Hz, 3H), 1.74 - 1.57 (m, 17H), 1.39 - 1.23 (m, 10H), 0.92 - 0.85 (m, 3H).

[0199] Step 3: 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1'-ethyl-[1,4'-bipiperidine]-4-carboxylate (1)

[0200]

Chemical Structure

[0201] A solution of 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (50 mg, 1 Eq, 92 μmol) in dichloromethane (30 mL) was added to 1’-ethyl-[1,4’-bipiperidine]-4-carboxylic acid dihydrochloride (29 mg, 1 Eq, 92 μmol), DIPEA (53 mg, 72 μL, 4.5 Eq, 0.41 mmol), and DMAP (2.2 mg, 0.2 Eq, 18 μmol). Finally, EDC (35 mg, 2 Eq, 0.18 mmol) was added and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was purified directly by flash column chromatography (silica 10 g, 0 - 25% methanol in dichloromethane over 12 min). 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (61 mg, 87%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.41 - 5.28 (m, 4H), 4.19 - 4.06 (m, 7H), 3.72 - 3.54 (m, 6H), 3.17 - 3.04 (m, 4H), 2.80 - 2.73 (m, 2H), 2.10 - 2.00 (m, 6H), 1.65 - 1.51 (m, 24H), 1.51 - 1.42 (m, 11H), 1.38 - 1.24 (m, 11H), 0.92 - 0.86 (m, 3H). LCMS: calculated m / z (M+H) = 767.6, found 767.7, RT = 3.15 min.

[0202] Examples 2 - 28 below were prepared using the same procedure as in Example 1, but changing the carboxylic acid building block used in the final step.

[0203] [Example 2] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-methylpiperidine-4-carboxylate (2)

[0204] [Chemical formula]

[0205] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-methylpiperidine-4-carboxylic acid hydrochloride on a 0.18 mmol scale. 109 mg (89% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 670.5, found 670.5, RT = 3.75 min.

[0206] [Example 3] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((3-(4-methylpiperazin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (3)

[0207] [Chemical formula]

[0208] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-(4-methylpiperazin-1-yl)propane dihydrochloride on a 0.18 mmol scale. 54 mg (42% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 699.5, found 699.5, RT = 3.63 min.

[0209] [Example 4] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (4)

[0210]

Chem.

[0211] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 4-(dimethylamino)butanoic acid hydrochloride on a 0.18 mmol scale. 105 mg (87% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 658.5, found 658.3, RT = 3.60 min.

[0212] [Example 5] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((dimethylglycyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (5)

[0213]

Chem.

[0214] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 2-(dimethylamino)acetic acid on a 0.18 mmol scale with N,N-dimethylformamide as the solvent instead of dichloromethane. 6.4 mg (6% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 630.5, found 630.4, RT = 3.76 min.

[0215] [Example 6] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((3-(ethyl(methyl)amino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (6)

[0216]

Chemical Structure

[0217] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-[ethyl(methyl)amino]propanoic acid hydrochloride on a 0.18 mmol scale. 30 mg of the product (25% yield) was isolated. LCMS: calculated m / z (M+H) = 658.5, found 658.7, RT = 3.68 min.

[0218] [Example 7] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((3-(pyrrolidin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (7)

[0219]

Chemical Structure

[0220] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-(pyrrolidin-1-yl)propanoic acid hydrochloride on a 0.18 mmol scale. 18 mg of the product (15% yield) was isolated. LCMS: calculated m / z (M+H) = 670.5, found 670.6, RT = 3.46 min.

[0221] [Example 8] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-isopropylpiperidine-4-carboxylate (8)

[0222] [Chemical formula]

[0223] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-(propan-2-yl)piperidine-4-carboxylate hydrochloride on a 0.18 mmol scale. 88 mg (68% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 698.5, found 698.4, RT = 3.48 min.

[0224] [Example 9] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((3-(piperidin-1-yl)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (9)

[0225] [Chemical formula]

[0226] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-(piperidin-1-yl)propanoate hydrochloride on a 0.18 mmol scale. 97 mg (77% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 684.5, found 684.3, RT = 3.46 min.

[0227] [Example 10] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((4-(piperidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (10)

[0228] [Chemical formula]

[0229] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 4-(piperidin-1-yl)butanoic acid hydrochloride on a 0.18 mmol scale. 97 mg (76% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 698.5, found 698.4, RT = 3.46 min.

[0230] [Example 11] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-propylpiperidine-4-carboxylate (11)

[0231] [Chemical formula]

[0232] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-propylpiperidine-4-carboxylic acid hydrochloride on a 0.18 mmol scale. 111 mg (87% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 698.5, found 698.4, RT = 3.48 min.

[0233] [Example 12] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((3-(dimethylamino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (12)

[0234] [Chemical formula]

[0235] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-(dimethylamino)propanoic acid hydrochloride on a 0.18 mmol scale. 63 mg (54% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 644.5, found 644.7, RT = 3.62 min.

[0236] [Example 13] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(((N-methyl-N-propylglycyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (13)

[0237] [Chemical formula]

[0238] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 2-[methyl(propyl)amino]acetic acid hydrochloride on a 0.18 mmol scale. 25 mg (20% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 658.5, found 658.5, RT = 3.77 min.

[0239] [Example 14] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((diethylglycyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (14)

[0240]

Chem.

[0241] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 2-(diethylamino)acetic acid on a 0.18 mmol scale. 34 mg (28% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 658.5, found 658.8, RT = 3.82 min.

[0242] [Example 15] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((3-(diethylamino)propanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (15)

[0243]

Chem.

[0244] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-(diethylamino)propanoic acid hydrochloride on a 0.18 mmol scale. 96 mg (78% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 672.5, found 672.4, RT = 3.64 min.

[0245] [Example 16] 3-(2-(1H-Imidazol-1-yl)acetoxy)-2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (16)

[0246] [Chemical formula]

[0247] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1H-imidazole-1-acetic acid on a 0.18 mmol scale. 19 mg (16% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 653.5, found 653.5, RT = 3.92 min.

[0248] [Example 17] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((2-(4-methylpiperazin-1-yl)acetoxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (17)

[0249] [Chemical formula]

[0250] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 2-(4-methylpiperazin-1-yl)acetic acid dihydrochloride on a 0.09 mmol scale. 47 mg (75% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 685.5, found 685.6, RT = 3.90 min.

[0251] [Example 18] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (18)

[0252] [Chemical Structure]<

[0253] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 4-(pyrrolidin-1-yl)butanoic acid hydrochloride on a 0.09 mmol scale. 40 mg (64% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 684.5, found 684.5, RT = 3.72 min.

[0254] [Example 19] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((4-(dipropylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (19)

[0255] [Chemical Structure]<

[0256] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 4-(dipropylamino)butanoic acid hydrochloride on a 0.09 mmol scale. 59 mg (90% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 714.6, found 714.6, RT = 3.66 min.

[0257] [Example 20] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl quinuclidine-4-carboxylate (20)

[0258] [Chemical Structure]

[0259] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using quinuclidine-4-carboxylic acid hydrochloride on a 0.09 mmol scale. 8.0 mg (13% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 682.5, found 682.4, RT = 3.66 min.

[0260] [Example 21] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-methylpiperidine-3-carboxylate (21)

[0261] [Chemical Structure]

[0262] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-methylpiperidine-3-carboxylic acid on a 0.09 mmol scale. 55 mg (89% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 670.5, found 670.4, RT = 3.58 min.

[0263] [Example 22] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1,3-dimethylpyrrolidine-3-carboxylate (22)

[0264]

Chemical Structure

[0265] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1,3-dimethylpyrrolidine-3-carboxylic acid on a 0.09 mmol scale. 21 mg of the product (34% yield) was isolated. LCMS: calculated m / z (M+H) = 670.5, found 670.5, RT = 3.54 min.

[0266] [Example 23] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((2-(1-methylpiperidin-4-yl)acetoxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (23)

[0267]

Chemical Structure

[0268] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 2-(1-methylpiperidin-4-yl)acetic acid on a 0.09 mmol scale. 37 mg of the product (59% yield) was isolated. LCMS: calculated m / z (M+H) = 684.5, found 684.5, RT = 3.53 min.

[0269] [Example 24] 3-(2-((3S,5S,7S)-Adamantan-1-yl)acetoxy)-2-(((Nα,Nα-dimethyl-L-histidyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (24)

[0270]

Chem.

[0271] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using Nα,Nα-dimethyl-L-histidine on a 0.09 mmol scale. 2.7 mg (4% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 710.5, found 710.4, RT = 3.70 min.

[0272] [Example 25] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-(pyridin-4-yl)piperidine-4-carboxylate (25)

[0273]

Chem.

[0274] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-(pyridin-4-yl)piperidine-4-carboxylic acid on a 0.09 mmol scale. 37 mg (55% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 733.5, found 733.4, RT = 3.76 min.

[0275] [Example 26] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((5-morpholinopentanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (26)

[0276] [Chemical formula]

[0277] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 5-morpholinopentanoic acid hydrochloride on a 0.09 mmol scale. 64 mg (98% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 714.5, found 714.5, RT = 3.77 min.

[0278] [Example 27] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (27)

[0279] [Chemical formula]

[0280] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 5-(dimethylamino)pentanoic acid on a 0.09 mmol scale. 31 mg (50% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 672.5, found 672.5, RT = 3.61 min.

[0281] [Example 28] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-((2-(pyridin-4-yloxy)acetoxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (28)

[0282]

Chem.

[0283] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 2-(pyridin-4-yloxy)acetic acid on a 0.09 mmol scale. 2.7 mg (4% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 680.4, found 680.3, RT = 4.01 min.

[0284] [Example 29] 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-((((1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carbonyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (29)

[0285]

Chem.

[0286] Step 1: 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate

[0287]

Chem.

[0288] (9Z,12Z)-Octadeca-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester (1.0 g, 1 Eq, 2.7 mmol) was dissolved in dichloromethane (30 mL), and 1’-ethyl-[1,4’-bipiperidine]-4-carboxylic acid dihydrochloride (0.85 g, 1 Eq, 2.7 mmol), DIPEA (1.2 g, 1.7 mL, 3.5 Eq, 9.5 mmol), and DMAP (66 mg, 0.2 Eq, 0.54 mmol) were added. Finally, EDC (0.78 g, 1.5 Eq, 4.1 mmol) was added, and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (100 g silica, 0–40% methanol in dichloromethane over 30 min). 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (540 mg, 34%) was obtained as a colorless oil. LCMS: calculated m / z (M+H) = 591.5, found 591.6, RT = 2.67 min.

[0289] Step 2: 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-((((1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carbonyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (29)

[0290]

Chemical formula

[0291] A solution of 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (59 mg, 1 Eq, 0.10 mmol) in dichloromethane (1 mL) was added to (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylic acid (28 mg, 1 Eq, 0.10 mmol), DIPEA (39 mg, 3 Eq, 0.30 mmol), and DMAP (2.4 mg, 0.2 Eq, 20 μmol). Finally, EDC (38 mg, 2 Eq, 0.20 mmol) was added and the mixture was stirred at 23 °C for 18 h. Purification was performed directly by flash column chromatography (10 g silica, 0 - 30% methanol in dichloromethane over 12 min). 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-((((1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carbonyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (31 mg, 36%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.42 - 5.29 (m, 4H), 4.16 - 4.09 (m, 6H), 3.85 (t, J = 6.9 Hz, 2H), 3.67 - 3.51 (m, 2H), 3.35 - 2.94 (m, 7H), 2.83 - 2.74 (m, 5H), 2.69 - 2.46 (m, 2H), 2.39 (p, J = 6.0 Hz, 1H), 2.31 (t, J = 7.6 Hz, 2H), 2.26 - 2.16 (m, 2H), 2.05 (q, J = 7.0 Hz, 4H), 1.97 (d, J = 13.0 Hz, 2H), 1.85 - 1.39 (m, 11H), 1.39 - 1.08 (m, 28H), 1.08 - 0.80 (m, 14H). LCMS: calculated m / z (M+H) = 853.7, found 853.7, RT = 3.54 min.

[0292] Examples 30 to 37 below were prepared using the same procedure as in Example 29, but the carboxylic acid building block used in the final step was changed.

[0293] [Example 30] 3-(2-((1r,3r)-Adamantan-2-yl)acetoxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (30)

[0294] [Chemical formula]

[0295] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate using 2-(adamantan-2-yl)acetic acid on a 0.10 mmol scale. 32 mg (42% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 767.6, found 767.6, RT = 3.17 min.

[0296] [Example 31] 3-((3-((3r,5r,7r)-Adamantan-1-yl)propanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (31)

[0297] [Chemical formula]

[0298] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate using 3-(adamantan-1-yl)propanoic acid on a 0.10 mmol scale. 34 mg (44% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 781.6, found 781.7, RT = 3.24 min.

[0299] [Example 32] 3-((3,5-Di-tert-butylbenzoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (32)

[0300] [Chem.]

[0301] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate using 3,5-di-tert-butylbenzoic acid on a 0.10 mmol scale. 21 mg (26% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 807.6, found 807.6, RT = 3.29 min.

[0302] [Example 33] 3-((2,3-Diphenylpropanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (33)

[0303] [Chem.]

[0304] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate using 2,3-diphenylpropanoic acid on a 0.10 mmol scale. 39 mg (49% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 799.6, found 799.4, RT = 3.06 min.

[0305] [Example 34] 3-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-2-((((1R,2S,3s,4R,5S)-tricyclo[3.2.1.0,2,4]octane-3-carbonyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (34)

[0306] [Chem.]

[0307] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate using rac-(1R,2S,3R,4R,5S)-tricyclo[3.2.1.0,2,4]octane-3-carboxylic acid on a 0.10 mmol scale. 20 mg (28% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 725.5, found 725.4, RT = 3.04 min.

[0308] [Example 35] 3-(2-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)acetoxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (35)

[0309] [Chem.]

[0310] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate using 2-(3,5-dimethyladamantan-1-yl)acetic acid on a 0.10 mmol scale. 43 mg (54% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 795.6, found 795.8, RT = 3.32 min.

[0311] [Example 36] 3-((Bicyclo[3.3.1]nonane-3-carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (36)

[0312] [Chemical formula]

[0313] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate using bicyclo[3.3.1]nonane-3-carboxylic acid on a 0.10 mmol scale. 37 mg (50% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 741.6, found 741.5, RT = 3.12 min.

[0314] [Example 37] 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-((((3as,6as)-octahydro-2,5-methanopentalen-3a-carbonyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate (37)

[0315] [Chemical formula]

[0316] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1’-ethyl-[1,4’-bipiperidine]-4-carboxylate using 3-noradamantanecarboxylic acid on a 0.10 mmol scale. 45 mg (61% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 739.6, found 739.3, RT = 3.10 min.

[0317] [Example 38] 3-((3-((3r,5r,7r)-adamantan-1-yl)propanoyl)oxy)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (38)

[0318] [Chemical formula]

[0319] Step 1: 3-((4-(dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0320] [Chemical formula]

[0321] 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (1000 mg, 1 Eq, 2.713 mmol) was added to a solution dissolved in dichloromethane (24 mL), along with 4-(dimethylamino)butanoic acid (355.9 mg, 1 Eq, 2.713 mmol), DIPEA (1.753 g, 2.35 mL, 5 Eq, 13.57 mmol), and DMAP (66.30 mg, 0.2 Eq, 542.7 μmol). Finally, EDC (1.040 g, 2 Eq, 5.427 mmol) was added, and the mixture was stirred at 23 °C for 18 hours. After this time, the reaction mixture was concentrated and purified by flash column chromatography (100 g of silica, 0 - 30% methanol in dichloromethane over 20 minutes). 3-((4-(Dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (0.447 g, 34%) was obtained as a pale yellow oil. LCMS: calculated m / z (M+H) = 482.4, measured 482.4, RT = 3.15 min.

[0322] Step 2: 3-((3-((3r,5r,7r)-Adamantan-1-yl)propanoyl)oxy)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (38)

[0323]

Chemical Structure

[0324] 3-((4-(Dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (0.050 g, 1 Eq, 0.10 mmol) was added to a solution dissolved in dichloromethane (1 mL), along with 3-((3r,5r,7r)-adamantan-1-yl)propanoic acid (22 mg, 1 Eq, 0.10 mmol), DIPEA (67 mg, 90 μL, 5 Eq, 0.52 mmol), and DMAP (2.5 mg, 0.2 Eq, 21 μmol). Finally, EDC (40 mg, 2 Eq, 0.21 mmol) was added and the mixture was stirred at 23 °C for 18 hours. After this time, the reaction mixture was concentrated and purified by flash column chromatography (10 g of silica, 0 - 20% methanol in dichloromethane over 12 minutes). 3-((3-((3r,5r,7r)-adamantan-1-yl)propanoyl)oxy)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (28 mg, 41%) was obtained as a pale yellow oil. 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.29 (m, 4H), 4.24 - 4.12 (m, 4H), 3.62 (t, J = 5.6 Hz, 2H), 2.77 (t, J = 6.7 Hz, 2H), 2.36 - 2.25 (m, 4H), 2.24 - 2.16 (m, 3H), 2.05 (q, J = 6.8 Hz, 4H), 1.98 - 1.94 (m, 3H), 1.71 (d, J = 12.3 Hz, 3H), 1.64 - 1.59 (m, 6H), 1.57 (s, 3H), 1.48 - 1.40 (m, 12H), 1.38 - 1.26 (m, 16H), 0.89 (t, J = 6.9 Hz, 3H). LCMS: calculated m / z (M+H) = 672.5, found 672.4, RT = 3.84 min.

[0325] Examples 39 - 46 below were prepared using the same procedure as Example 38, but with a change in the carboxylic acid building block used in the final step.

[0326] [Example 39] 3-((4-(Dimethylamino)butanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,3R,5S)-adamantane-1-carboxylate (39)

[0327] [Chemical formula]

[0328] Prepared from 3-((4-(dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-adamantanecarboxylic acid on a 0.10 mmol scale. 22 mg (34% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 644.5, found 644.4, RT = 3.55 min.

[0329] [Example 40] 3-(2-((1R,3S,5r,7r)-adamantan-2-yl)acetoxy)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (40)

[0330] [Chemical formula]

[0331] Prepared from 3-((4-(dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 2-(adamantan-2-yl)acetic acid on a 0.10 mmol scale. 27 mg (40% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 658.5, found 658.5, RT = 3.60 min.

[0332] [Example 41] 3-((4-(Dimethylamino)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate (41)

[0333] [Chemical formula]

[0334] Prepared from 3-((4-(dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3,5-di-tert-butylbenzoic acid on a 0.10 mmol scale. 31 mg (43% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 698.5, found 698.6, RT = 3.47 min.

[0335] [Example 42] 3-(2-((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)acetoxy)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (42)

[0336] [Chemical formula]

[0337] Prepared from 3-((4-(dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 2-(3,5-dimethyladamantan-1-yl)acetic acid on a 0.10 mmol scale. 33 mg (46% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 686.5, found 686.4, RT = 3.68 min.

[0338] [Example 43] 3-((4-(Dimethylamino)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (2R,3as,5S,6as)-hexahydro-2,5-methanopentalen-3a(1H)-carboxylate (43)

[0339] [Chemistry]

[0340] Prepared from 3-((4-(dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-noradamantane carboxylic acid on a 0.10 mmol scale. 30 mg (46% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 630.5, found 630.4, RT = 3.54 min.

[0341] [Example 44] 3-((4-(Dimethylamino)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl bicyclo[3.3.1]nonane-3-carboxylate (44)

[0342] [Chemistry]

[0343] Prepared from 3-((4-(dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using bicyclo[3.3.1]nonane-3-carboxylic acid on a 0.10 mmol scale. 27 mg (41% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 632.5, found 632.8, RT = 3.57 min.

[0344] [Example 45] 3-((4-(Dimethylamino)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1R,2S,3s,4R,5S)-tricyclo[3.2.1.0,2,4]octane-3-carboxylate (45)

[0345]

Chem.

[0346] Prepared from 3-((4-(dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using rac-(1R,2S,3R,4R,5S)-tricyclo[3.2.1.0,2,4]octane-3-carboxylic acid on a 0.10 mmol scale. 28 mg (44% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 616.4, found 616.4, RT = 3.50 min.

[0347] [Example 46] 3-((4-(Dimethylamino)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate (46)

[0348]

Chem.

[0349] Prepared from 3-((4-(dimethylamino)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using trans,trans-4’-pentylbicyclohexyl-4-carboxylic acid on a 0.10 mmol scale. 41 mg (54% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 744.6, found 744.8, RT = 3.75 min.

[0350] [Example 47] 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate (47)

[0351] [Chemical formula]

[0352] Step 1: 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate

[0353] [Chemical formula]

[0354] A solution of 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (800 mg, 1 Eq, 2.17 mmol) in dichloromethane (5 mL) was added with 3,5-di-tert-butylbenzoic acid (509 mg, 1 Eq, 2.17 mmol), DIPEA (561 mg, 0.76 mL, 2 Eq, 4.34 mmol), and DMAP (53.0 mg, 0.2 Eq, 434 μmol). Finally, EDC (624 mg, 1.5 Eq, 3.26 mmol) was added and the mixture was stirred at 23 °C for 18 hours. After this time, the reaction mixture was concentrated and purified by flash column chromatography (silica 50 g, 0 - 30% ethyl acetate in hexane over 20 minutes). 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate (630 mg, 49.6%) was obtained as a colorless oil. 11H NMR (500 MHz, chloroform-d) δ 7.88 (d, J = 1.9 Hz, 2H), 7.65 (t, J = 1.9 Hz, 1H), 5.43 - 5.30 (m, 4H), 4.45 (dd, J = 6.0, 1.5 Hz, 2H), 4.33 - 4.22 (m, 2H), 3.70 (d, J = 5.5 Hz, 2H), 2.80 - 2.74 (m, 2H), 2.49 (s, 1H), 2.39 - 2.30 (m, 3H), 2.08 - 2.01 (m, 5H), 1.62 (qd, J = 7.5, 3.1 Hz, 2H), 1.41 - 1.21 (m, 31H), 0.92 - 0.86 (m, 3H).

[0355] Step 2: 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate (47)

[0356] [Chemical formula]

[0357] 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate (50 mg, 1 Eq, 85 μmol) was dissolved in dichloromethane (1 mL), and to the solution were added 4-(pyrrolidin-1-yl)butanoic acid hydrochloride (17 mg, 1 Eq, 85 μmol), DIPEA (33 mg, 45 μL, 3 Eq, 0.26 mmol), and N,N-dimethylpyridin-4-amine (2.1 mg, 0.2 Eq, 17 μmol). Finally, EDC (33 mg, 2 Eq, 0.17 mmol) was added, and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was purified directly by flash column chromatography (silica 10 g, 0 - 25% methanol in dichloromethane over 12 min). 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate (44 mg, 71%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 7.90 - 7.84 (m, 2H), 7.65 (t, J = 1.9 Hz, 1H), 5.42 - 5.28 (m, 4H), 4.43 - 4.36 (m, 2H), 4.23 (dd, J = 6.0, 3.2 Hz, 4H), 2.86 (s, 4H), 2.77 (t, J = 6.7 Hz, 2H), 2.57 (septet, J = 6.1 Hz, 1H), 2.46 (t, J = 6.9 Hz, 2H), 2.38 - 2.28 (m, 2H), 2.11 - 1.93 (m, 8H), 1.60 (q, J = 7.1 Hz, 4H), 1.40 - 1.22 (m, 34H), 0.89 (t, J = 6.9 Hz, 3H). LCMS: calculated m / z (M+H) = 724.5, found 724.6, RT = 3.57 min.

[0358] Examples 48 and 49 below were prepared using the same procedure as in Example 47, but the carboxylic acid building block used in the final step was changed.

[0359] [Example 48] 3-((3,5-Di-tert-butylbenzoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-(pyridin-4-yl)piperidine-4-carboxylate (48)

[0360] [Chemical formula]

[0361] Prepared from 3-hydroxy-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate using 1-(pyridin-4-yl)piperidine-4-carboxylic acid on a 0.085 mmol scale. 14 mg (21% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 773.5, found 773.7, RT = 3.61 min.

[0362] [Example 49] 3-((Nα,Nα-dimethyl-L-histidyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate (49)

[0363] [Chemical formula]

[0364] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate using Nα,Nα-dimethyl-L-histidine on a 0.085 mmol scale, AOP instead of EDC as the coupling agent, and N,N-dimethylformamide instead of dichloromethane as the solvent. 13 mg (20% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 750.5, found 750.6, RT = 3.59 min.

[0365] [Example 50] 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate (50)

[0366] [Chemical formula]

[0367] Step 1: 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate

[0368] [Chemical formula]

[0369] 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (800 mg, 1 Eq, 2.17 mmol) was added to a solution of (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylic acid (609 mg, 1 Eq, 2.17 mmol), DIPEA (561 mg, 0.76 mL, 2 Eq, 4.34 mmol), and DMAP (53.0 mg, 0.2 Eq, 434 μmol) in dichloromethane (6 mL). Finally, EDC (624 mg, 1.5 Eq, 3.26 mmol) was added and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (silica 50 g, 0 - 30% ethyl acetate in hexane over 20 min). 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate (686 mg, 50.1%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.42 - 5.27 (m, 4H), 4.22 - 4.08 (m, 5H), 3.63 - 3.56 (m, 2H), 2.77 (dddt, J = 7.8, 6.9, 1.4, 0.8 Hz, 2H), 2.35 - 2.28 (m, 2H), 2.27 - 2.14 (m, 2H), 2.08 - 2.01 (m, 4H), 2.01 - 1.94 (m, 2H), 1.81 - 1.56 (m, 8H), 1.44 - 1.16 (m, 27H), 1.16 - 0.92 (m, 6H), 0.92 - 0.80 (m, 6H).

[0370] Step 2: 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate (50)

[0371] [Chem.]

[0372] 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate (50 mg, 1 Eq, 79 μmol) was added to a solution dissolved in dichloromethane (1 mL), along with 4-(pyrrolidin-1-yl)butanoic acid hydrochloride (15 mg, 1 Eq, 79 μmol), DIPEA (31 mg, 41 μL, 3 Eq, 0.24 mmol), and DMAP (1.9 mg, 0.2 Eq, 16 μmol). Finally, EDC (30 mg, 2 Eq, 0.16 mmol) was added, and the mixture was stirred at 23 °C for 18 hours. After this time, the reaction mixture was purified directly by flash column chromatography (10 g of silica, 0 - 25% methanol in dichloromethane over 12 minutes). 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate (41 mg, 67%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.29 (m, 4H), 4.14 - 4.09 (m, 6H), 2.77 (t, J = 6.7 Hz, 7H), 2.45 - 2.36 (m, 2H), 2.31 (q, J = 7.5 Hz, 2H), 2.21 (tt, J = 11.9, 3.5 Hz, 1H), 2.05 (q, J = 6.9 Hz, 4H), 2.01 - 1.83 (m, 7H), 1.82 - 1.51 (m, 9H), 1.44 - 1.18 (m, 25H), 1.17 - 0.92 (m, 9H), 0.88 (q, J = 6.8 Hz, 7H). LCMS: calculated m / z (M+H) = 770.6, found 770.7, RT = 4.08 min.

[0373] Examples 51 and 52 below were prepared using the same procedure as Example 50, but the carboxylic acid building block used in the final step was changed.

[0374] [Example 51] 3-(((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)-2-((((1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carbonyl)oxy)methyl)propyl 1-(pyridin-4-yl)piperidine-4-carboxylate (51)

[0375] [Chemical formula]

[0376] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate using 1-(pyridin-4-yl)piperidine-4-carboxylic acid on a 0.079 mmol scale. 10 mg (15% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 819.6, found 819.7, RT = 4.01 min.

[0377] [Example 52] 3-((Nα,Nα-dimethyl-L-histidyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1S,1’s,4R,4’S)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate (52)

[0378] [Chemical formula]

[0379] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate using Nα,Nα-dimethyl-L-histidine on a 0.079 mmol scale, using AOP instead of EDC as the coupling agent and N,N-dimethylformamide instead of dichloromethane as the solvent. 2.7 mg (4% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 796.6, found 796.6, RT = 4.30 min.

[0380] [Example 53] 3-(2-((1S,2R,5R)-adamantan-2-yl)acetoxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (53)

[0381] [Chemical Structure]

[0382] Step 1: 3-(2-((1S,2R,5R)-adamantan-2-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0383] [Chemical Structure]

[0384] A solution of 2-(adamantan-2-yl)acetic acid (213 mg, 1 Eq, 1.10 mmol) in dichloromethane (35 mL) was added to 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (405 mg, 1 Eq, 1.10 mmol), DIPEA (426 mg, 572 μL, 3 Eq, 3.30 mmol), and DMAP (13.4 mg, 0.1 Eq, 110 μmol). Finally, EDC (316 mg, 1.5 Eq, 1.65 mmol) was added and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (100 g silica, 0 - 40% ethyl acetate in hexane over 20 min). 3-(2-((1r,5R,7S)-adamantan-2-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (287 mg, 48.0%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.28 (m, 4H), 4.23 - 4.12 (m, 4H), 3.64 - 3.58 (m, 2H), 2.77 (dddt, J = 8.4, 7.0, 1.5, 0.8 Hz, 2H), 2.48 (d, J = 7.6 Hz, 2H), 2.36 - 2.29 (m, 2H), 2.26 - 2.15 (m, 3H), 2.09 - 2.01 (m, 4H), 1.91 - 1.75 (m, 8H), 1.75 - 1.66 (m, 5H), 1.65 - 1.50 (m, 4H), 1.40 - 1.24 (m, 13H), 0.92 - 0.86 (m, 3H).

[0385] Step 2: 3-(2-((1S,2R,5R)-adamantan-2-yl)acetoxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (53)

[0386] [Chemical formula]

[0387] A mixture of 4-(pyrrolidin-1-yl)butanoic acid (17 mg, 1 Eq, 0.11 mmol) dissolved in dichloromethane (1 mL) was added to 3-(2-((1R,2r,3S,5r)-adamantan-2-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (0.060 g, 1 Eq, 0.11 mmol), DIPEA (43 mg, 57 μL, 3 Eq, 0.33 mmol), and DMAP (2.7 mg, 0.2 Eq, 22 μmol). Finally, EDC (32 mg, 1.5 Eq, 0.17 mmol) was added and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (silica 10 g, 0 - 10% methanol in dichloromethane over 10 min). 3-(2-((1S,2R,5R)-adamantan-2-yl)acetoxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (75 mg, 99%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.29 (m, 4H), 4.25 - 4.09 (m, 6H), 3.61 (d, J = 5.6 Hz, 1H), 2.77 (t, J = 6.7 Hz, 2H), 2.48 (t, J = 7.8 Hz, 3H), 2.43 - 2.36 (m, 2H), 2.32 (dt, J = 9.1, 7.5 Hz, 2H), 2.25 - 2.16 (m, 2H), 2.05 (q, J = 7.0 Hz, 4H), 1.92 - 1.75 (m, 12H), 1.75 - 1.51 (m, 11H), 1.40 - 1.22 (m, 17H), 0.89 (t, J = 6.9 Hz, 3H). LCMS: calculated m / z (M+H) = 684.5, found 684.6, RT = 3.49 min.

[0388] Examples 54 and 55 below were prepared using the same procedure as Example 53, but the carboxylic acid building block used in the final step was changed.

[0389] [Example 54] 3-(2-((1S,2R,5R)-Adamantan-2-yl)acetoxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 1-(pyridin-4-yl)piperidine-4-carboxylate (54)

[0390] [Chemical formula]

[0391] Prepared from 3-(2-((1R,2r,3S,5r)-adamantan-2-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-(pyridin-4-yl)piperidine-4-carboxylic acid on a 0.11 mmol scale. 40 mg (49% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 733.5, found 733.7, RT = 3.54 min.

[0392] [Example 55] 3-(2-((1S,2R,5R)-Adamantan-2-yl)acetoxy)-2-((((Nα,Nα-dimethyl-L-histidyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (55)

[0393] [Chemical formula]

[0394] Prepared from 3-(2-((1R,2r,3S,5r)-adamantan-2-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using Nα,Nα-dimethyl-L-histidine on a 0.11 mmol scale, using AOP instead of EDC as the coupling agent and N,N-dimethylformamide instead of dichloromethane as the solvent. 6 mg (8% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 710.5, found 710.6, RT = 3.48 min.

[0395] [Example 56] 3-((3-((3r,5r,7r)-adamantan-1-yl)propanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (56)

[0396] [Chemical formula]

[0397] Step 1: 3-((3-((3r,5r,7r)-adamantan-1-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0398] [Chemical formula]

[0399] A mixture of 3-(adamantan-1-yl)propanoic acid (396 mg, 1 Eq, 1.90 mmol) dissolved in dichloromethane (20 mL) was added with 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (700 mg, 1 Eq, 1.90 mmol), DIPEA (736 mg, 989 μL, 3 Eq, 5.70 mmol), and DMAP (23 mg, 0.1 Eq, 190 μmol). Finally, EDC (546 mg, 1.5 Eq, 2.85 mmol) was added and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (50 g silica, 0 - 40% ethyl acetate in hexane over 16 min). 3-((3-((1s,3R,5S)-adamantan-1-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (0.305 g, 28.7%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.41 - 5.28 (m, 4H), 4.22 - 4.10 (m, 4H), 3.61 (t, J = 5.9 Hz, 2H), 2.76 (td, J = 6.8, 1.1 Hz, 2H), 2.35 - 2.24 (m, 6H), 2.23 - 2.14 (m, 1H), 2.04 (dt, J = 8.1, 6.2 Hz, 4H), 1.96 - 1.93 (m, 4H), 1.75 - 1.67 (m, 4H), 1.65 - 1.55 (m, 5H), 1.48 - 1.37 (m, 10H), 1.37 - 1.22 (m, 9H), 0.92 - 0.85 (m, 3H).

[0400] Step 2: 3-((3-((3r,5r,7r)-adamantan-1-yl)propanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (56)

[0401]

Chemical Structure

[0402] To a mixture of 4-(pyrrolidin-1-yl)butanoic acid (18 mg, 1 Eq, 0.12 mmol) dissolved in dichloromethane (20 mL), 3-((3-((3r,5r,7r)-adamantan-1-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (0.065 g, 1 Eq, 0.12 mmol), DIPEA (45 mg, 61 μL, 3 Eq, 0.35 mmol), and DMAP (2.8 mg, 0.2 Eq, 23 μmol) were added. Finally, EDC (33 mg, 1.5 Eq, 0.17 mmol) was added and the mixture was stirred at 23 °C for 18 h. After this time, the reaction mixture was concentrated and purified by flash column chromatography (10 g silica, 0 - 10% methanol in dichloromethane over 10 min). 3-((3-((3r,5r,7r)-adamantan-1-yl)propanoyl)oxy)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (70 mg, 87%) was obtained as a colorless oil. 1 H NMR (500 MHz, chloroform-d) δ 5.43 - 5.29 (m, 4H), 4.24 - 4.08 (m, 6H), 2.77 (t, J = 6.7 Hz, 2H), 2.51 (d, J = 18.9 Hz, 5H), 2.43 - 2.24 (m, 7H), 2.09 - 2.01 (m, 4H), 1.97 - 1.94 (m, 3H), 1.90 - 1.75 (m, 4H), 1.75 - 1.54 (m, 12H), 1.50 - 1.22 (m, 21H), 0.89 (t, J = 6.9 Hz, 3H). LCMS: calculated m / z (M+H) = 698.5, found 698.7, RT = 3.56 min.

[0403] The following Examples 57 and 58 were prepared using the same procedure as in Example 56, but with a change in the carboxylic acid building block used in the final step.

[0404] [Example 57] 3-((3-((3r,5r,7r)-Adamantan-1-yl)propanoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dieno yl)oxy)methyl)propyl 1-(pyridin-4-yl)piperidine-4-carboxylate (57)

[0405] [Chemical Structure]

[0406] Prepared from 3-((3-((3r,5r,7r)-adamantan-1-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-(pyridin-4-yl)piperidine-4-carboxylic acid on a 0.11 mmol scale. 44 mg (50% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 747.5, found 747.7, RT = 3.61 min.

[0407] [Example 58] 3-((3-((3S,5S,7S)-adamantan-1-yl)propanoyl)oxy)-2-((((Nα,Nα-dimethyl-L-histidyl))oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (58)

[0408] [Chemical Structure]

[0409] Prepared from 3-((3-((3r,5r,7r)-adamantan-1-yl)propanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using Nα,Nα-dimethyl-L-histidine on a 0.11 mmol scale, AOP as a coupling agent instead of EDC, and N,N-dimethylformamide as a solvent instead of dichloromethane. 12 mg (14% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 724.5, found 724.6, RT = 3.56 min.

[0410] [Example 59] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((((2-(4-methylpiperazin-1-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (59)

[0411] [Chemical Structure]

[0412] A solution of 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (100 mg, 1 Eq, 184 μmol) in dichloromethane (1 mL) was added pyridine (29.0 mg, 30 μL, 2 Eq, 367 μmol), DMAP (6.73 mg, 0.3 Eq, 55.1 μmol), and 4-nitrophenyl chloroformate (74.0 mg, 2 Eq, 367 μmol). The resulting mixture was stirred at 23 °C for 1 hour. After this time, DIPEA (94.9 mg, 0.13 mL, 4 Eq, 734 μmol) and 2-(4-methylpiperazin-1-yl)ethan-1-ol (106 mg, 4 Eq, 734 μmol) were added thereto. The resulting mixture was stirred at 23 °C for an additional 18 hours. After this time, the reaction mixture was diluted with dichloromethane (10 mL) and washed with 0.75 M aqueous sodium carbonate (3 × 10 mL), water (10 mL), and aqueous saturated sodium chloride (10 mL). The obtained organic layer was dried over sodium sulfate, concentrated, and the residue was purified by flash column chromatography (silica 10 g, 0 - 25% methanol in dichloromethane over 12 minutes). 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((((2-(4-methylpiperazin-1-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (50 mg, 38%) was obtained as a pale yellow oil. 11H NMR (500 MHz, chloroform-d) δ 5.42 - 5.29 (m, 4H), 4.25 (td, J = 5.9, 1.2 Hz, 2H), 4.21 (dd, J = 6.1, 1.1 Hz, 2H), 4.15 (dd, J = 6.1, 1.2 Hz, 2H), 4.13 (dd, J = 5.9, 1.2 Hz, 2H), 2.77 (t, J = 6.7 Hz, 2H), 2.67 (td, J = 5.9, 1.2 Hz, 2H), 2.48 - 2.38 (m, 1H), 2.33 - 2.28 (m, 5H), 2.10 - 2.02 (m, 6H), 1.97 (s, 4H), 1.70 (d, J = 12.5 Hz, 6H), 1.65 - 1.57 (m, 14H), 1.40 - 1.25 (m, 15H), 0.89 (t, J = 7.1 Hz, 3H). LCMS: calculated m / z (M+H) = 715.5, found 715.5, RT = 3.62 min.

[0413] Examples 60 - 82 below were prepared using the same procedure as in Example 59, but with changes to the alcohol reactant (all intermediates described in the procedure of the previous example or prepared as shown below) and the amino alcohol building block used in the final step.

[0414] [Example 60] 3-(2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)-2-(((((1-ethylpyrrolidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (60)

[0415] [Chemical Structure]

[0416] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using (1-ethylpyrrolidin-3-yl)methanol on a 0.18 mmol scale. 30 mg (23% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 700.5, found 700.7, RT = 3.71 min.

[0417] [Example 61] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(((((1-isopropylpiperidin-4-yl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (61)

[0418] [Chemical Structure]

[0419] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-isopropylpiperidin-4-ol on a 0.18 mmol scale. 67 mg (51% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 714.5, found 714.6, RT = 3.64 min.

[0420] [Example 62] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (62)

[0421] [Chemical Structure]

[0422] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-(4-methylpiperazin-1-yl)propan-1-ol on a 0.18 mmol scale. 59 mg (44% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 729.5, found 729.4, RT = 3.69 min.

[0423] [Example 63] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(((((1-ethylpiperidin-3-yl)oxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (63)

[0424] [Chemical formula]

[0425] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 1-ethylpiperidin-3-ol on a 0.18 mmol scale. 50 mg (39% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 700.5, found 700.8, RT = 3.58 min.

[0426] [Example 64] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((((2-(1-methylpyrrolidin-2-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (64)

[0427] [Chemical formula]

[0428] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 2-(1-methylpyrrolidin-2-yl)ethan-1-ol on a 0.18 mmol scale. 44 mg (34% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 700.5, found 700.2, RT = 3.63 min.

[0429] [Example 65] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-((((4-(dimethylamino)butoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (65)

[0430] [Chemical formula]

[0431] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 4-(dimethylamino)butan-1-ol on a 0.18 mmol scale. 37 mg (29% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 688.5, found 688.3, RT = 3.62 min.

[0432] [Example 66] 13-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2,5-dimethyl-10-oxo-9,11-dioxa-2,5-diazatetradecan-14-yl (9Z,12Z)-octadeca-9,12-dienoate (66)

[0433] [Chemical formula]

[0434] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-((2-(dimethylamino)ethyl)(methyl)amino)propan-1-ol on a 0.18 mmol scale. 32 mg (24% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 731.5, found 731.5, RT = 3.59 min.

[0435] [Example 67] 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate (67)

[0436] [Chemical Structure]

[0437] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 3,5-di-tert-butylbenzoate using 3-(diethylamino)-1-propanol on a 0.17 mmol scale. 95 mg (75% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 742.6, found 742.7, RT = 3.60 min.

[0438] [Example 68] 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate (68)

[0439] [Chemical Structure]

[0440] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate using 3-(diethylamino)-1-propanol on a 0.16 mmol scale. 98 mg (78% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 788.6, found 788.7, RT = 4.05 min.

[0441] [Example 69] 3-(2-((1r,3r)-adamantan-2-yl)acetoxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (69)

[0442] [Chem.]

[0443] Prepared from 3-(2-((1S,2R,5R)-adamantan-2-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-(diethylamino)-1-propanol on a 0.09 mmol scale. 53 mg (82% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 702.5, found 702.6, RT = 3.50 min.

[0444] [Example 70] 3-((3-((1r,3s)-adamantan-1-yl)propanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (70)

[0445] [Chem.]

[0446] Prepared from 3-(2-((1S,2R,5R)-adamantan-2-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-(diethylamino)-1-propanol on a 0.09 mmol scale. 50 mg (78% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 716.5, found 716.6, RT = 3.59 min.

[0447] [Example 71] 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (71)

[0448] [Chemical formula]

[0449] Prepared from 3-(2-((3r,5r,7r)-adamantan-1-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using (1-ethylpiperidin-3-yl)methanol on a 0.70 mmol scale. 163 mg (33% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 714.5, found 714.5, RT = 3.50 min.

[0450] [Example 72] 3-(2-((1r,3r)-adamantan-2-yl)acetoxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (72)

[0451] [Chemical formula]

[0452] Prepared from 3-(2-((1S,2R,5R)-adamantan-2-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using (1-ethylpiperidin-3-yl)methanol on a 0.59 mmol scale. 238 mg (56% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 714.5, found 714.5, RT = 3.46 min.

[0453] [Example 73] 3-((3-((1r,3s)-adamantan-1-yl)propanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (73)

[0454] [Chemical Structure]

[0455] Prepared from 3-(2-((1S,2R,5R)-adamantan-2-yl)acetoxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using (1-ethylpiperidin-3-yl)methanol on a 0.36 mmol scale. 207 mg (79% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 728.5, found 728.7, RT = 3.52 min.

[0456] [Example 74] 3-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate (74)

[0457] [Chemical Structure]

[0458] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-pentyl-[1,1’-bi(cyclohexane)]-4-carboxylate using (1-ethylpiperidin-3-yl)methanol on a 0.32 mmol scale. 186 mg (73% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 800.6, found 800.8, RT = 4.24 min.

[0459] [Example 75] 2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl bis(2-((3r,5r,7r)-adamantan-1-yl)acetate) (75)

[0460] [Chemical formula]

[0461] Step 1: 2-(hydroxymethyl)propane-1,3-diyl bis(2-((3r,5r,7r)-adamantan-1-yl)acetate)

[0462] [Chemical formula]

[0463] A solution of trimethylolmethane (5.00 g, 1 Eq, 47.1 mmol) in dichloromethane (125 mL) and tetrahydrofuran (125 mL) was added with 1-adamantaneacetic acid (9.15 g, 1 Eq, 47.1 mmol), DIPEA (9.13 g, 12.3 mL, 1.5 Eq, 70.7 mmol), and DMAP (576 mg, 0.1 Eq, 4.71 mmol). Finally, EDC (9.94 g, 1.1 Eq, 51.8 mmol) was added and stirred at 23 °C for 18 h. It was concentrated by rotary evaporation, then 5% aqueous citric acid (250 mL) was added and extracted with ethyl acetate (200 mL × 2). The combined organics were washed with 5% citric acid, water, and brine. Dehydrated with sodium sulfate and concentrated. The crude residue was purified by flash column chromatography (200 g silica, 0 - 90% ethyl acetate in hexane over 25 min). 2-(Hydroxymethyl)propane-1,3-diylbis(2-((3r,5r,7r)-adamantan-1-yl)acetate) (5.1 g, 24%) was obtained as a colorless oil.

[0464] Step 2: 2-(((((1-Ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propane-1,3-diylbis(2-((3r,5r,7r)-adamantan-1-yl)acetate) (75)

[0465] Prepared from 2-(Hydroxymethyl)propane-1,3-diylbis(2-((3r,5r,7r)-adamantan-1-yl)acetate) using 3-(diethylamino)-1-propanol on a 0.11 mmol scale. The product 31 mg (46% yield) was isolated. LCMS: calculated m / z (M+H) = 616.4, found 616.6, RT = 2.94 min.

[0466] [Example 76] 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’r,4R,4’R)-4’-ethyl-[1,1’-bi(cyclohexane)]-4-carboxylate (76)

[0467]

Chem.

[0468] Step 1: 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’r,4R,4’R)-4’-ethyl-[1,1’-bi(cyclohexane)]-4-carboxylate

[0469]

Chem.

[0470] Prepared from 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using trans,trans-4’-ethyl-[1,1’-bi(cyclohexane)]-4-carboxylic acid on a 0.53 mmol scale. 161 mg (51% yield) was isolated.

[0471] Step 2: 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’r,4R,4’R)-4’-ethyl-[1,1’-bi(cyclohexane)]-4-carboxylate (76)

[0472] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’r,4R,4’R)-4’-ethyl-[1,1’-bi(cyclohexane)]-4-carboxylate using 3-(diethylamino)-1-propanol on a 0.27 mmol scale. 178 mg (87% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 746.6, found 746.7, RT = 3.92 min.

[0473] [Example 77] 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-butyl-[1,1’-bi(cyclohexane)]-4-carboxylate (77)

[0474] [Chemical Structure]

[0475] Step 1: 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-butyl-[1,1’-bi(cyclohexane)]-4-carboxylate

[0476] [Chemical Structure]

[0477] Prepared from 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using trans,trans-4’-butyl-[1,1’-bi(cyclohexane)]-4-carboxylic acid on a 0.54 mmol scale. 159 mg (48% yield) was isolated.

[0478] Step 2: 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-butyl-[1,1’-bi(cyclohexane)]-4-carboxylate (77)

[0479] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl (1r,1’s,4R,4’R)-4’-butyl-[1,1’-bi(cyclohexane)]-4-carboxylate using 3-(diethylamino)-1-propanol on a 0.26 mmol scale. 153 mg (77% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 774.6, found 774.7, RT = 4.02 min.

[0480] [Example 78] 3-((5-Cyclohexylpentanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (78)

[0481] [Chemical formula]

[0482] Step 1: 3-((5-Cyclohexylpentanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate

[0483] [Chemical formula]

[0484] Prepared from 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 5-cyclohexylpentanoic acid on a 0.54 mmol scale. 140 mg (48% yield) was isolated.

[0485] Step 2: 3-((5-Cyclohexylpentanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (78)

[0486] Prepared from 3-((5-Cyclohexylpentanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 3-(diethylamino)-1-propanol on a 0.26 mmol scale. 155 mg (86% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 692.5, found 692.7, RT = 3.48 min.

[0487] [Example 79] 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)methyl)propyl 4-propylcyclohexane-1-carboxylate (79)

[0488] [Chemical Structure]

[0489] Step 1: 3-Hydroxy-2-((((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)methyl)propyl 4-propylcyclohexane-1-carboxylate

[0490] [Chemical Structure]

[0491] Prepared from 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 4-propylcyclohexane-1-carboxylic acid on a 0.54 mmol scale. 142 mg (50% yield) was isolated.

[0492] Step 2: 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-propylcyclohexane-1-carboxylate (79)

[0493] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-propylcyclohexane-1-carboxylate using 3-(diethylamino)-1-propanol on a 0.27 mmol scale. 108 mg (58% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 678.5, found 678.7, RT = 3.38 min.

[0494] [Example 80] 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-butylcyclohexane-1-carboxylate (80)

[0495] [Chemical formula]

[0496] Step 1: 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-butylcyclohexane-1-carboxylate

[0497] [Chemical formula]

[0498] Prepared from 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 4-butylcyclohexane-1-carboxylic acid on a 0.54 mmol scale. 142 mg (49% yield) was isolated.

[0499] Step 2: 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-butylcyclohexane-1-carboxylate (80)

[0500] Prepared from 3-hydroxy-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-butylcyclohexane-1-carboxylate using 3-(diethylamino)-1-propanol on a 0.27 mmol scale. 111 mg (60% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 692.5, found 692.7, RT = 3.48 min.

[0501] [Example 81] 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-(tert-butyl)cyclohexane-1-carboxylate (81)

[0502] [Chemical formula]

[0503] Step 1: 3-Hydroxy-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-(tert-butyl)cyclohexane-1-carboxylate

[0504] Prepared from 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 4-tert-butylcyclohexane-1-carboxylic acid on a 0.54 mmol scale. 151 mg (52% yield) was isolated.

[0505] Step 2: 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-(tert-butyl)cyclohexane-1-carboxylate (81)

[0506] Prepared from 3-hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-(tert-butyl)cyclohexane-1-carboxylate using 3-(diethylamino)-1-propanol on a 0.28 mmol scale. 123 mg (63% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 692.5, found 692.7, RT = 3.43 min.

[0507] [Example 82] 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-pentylcyclohexane-1-carboxylate (82)

[0508] [Chemical formula]

[0509] Step 1: 3-Hydroxy-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-pentylcyclohexane-1-carboxylate

[0510] [Chemical formula]

[0511] Prepared from 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate using 4-pentylcyclohexane-1-carboxylic acid on a 0.54 mmol scale. 139 mg (47% yield) was isolated.

[0512] Step 2: 3-(((3-(Diethylamino)propoxy)carbonyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-pentylcyclohexane-1-carboxylate (82)

[0513] Prepared from 3-hydroxy-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl 4-pentylcyclohexane-1-carboxylate using 3-(diethylamino)-1-propanol on a 0.25 mmol scale. 106 mg (59% yield) of the product was isolated. LCMS: calculated m / z (M+H) = 706.6, found 706.72, RT = 3.55 min.

[0514] [Example 83] 2-((2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)methyl)-4-((4-(pyrrolidin-1-yl)butanoyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate (83)

[0515] [Chemical formula]

[0516] Step 1: 2-(Hydroxymethyl)butane-1,4-diol

[0517] [Chemical formula]

[0518] Triethyl ethane-1,1,2-tricarboxylate (5 g, 20.3 mmol) was added to a stirred solution of tert-butanol (80 mL) at 25 °C under an argon atmosphere, and NaBH4 (2.3 g, 60.9 mmol) was added. The resulting suspension was heated to reflux, and methanol (3 mL) was added dropwise in three portions within 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 off, and the filtrate was evaporated to give the crude material, which was purified by combiflash column chromatography eluting with 10–15% MeOH in DCM to give 2-(hydroxymethyl)butane-1,4-diol (1.7 g, 69%) as a yellow liquid. 1 H NMR (400 MHz, DMSO-d6): δ1.34-1.46 (m, 2H), 1.49-1.63 (m, 1H), 3.27-3.48 (m, 6H), 4.34 (t, J = 5.2 Hz, 2H), 4.40 (t, J = 5.1 Hz, 1H).

[0519] Step 2: 2-(2,2-Dimethyl-1,3-dioxan-5-yl)ethan-1-ol

[0520] [Chemical formula]

[0521] 2-(Hydroxymethyl)butane-1,4-diol (1.7 g, 14.1 mmol) and 2,2-dimethoxypropane (4.3 mL, 35.3 mmol) were added to a stirred solution of THF (10 mL) under an argon atmosphere at 25 °C, and p-toluenesulfonic acid monohydrate (0.36 g, 3.1 mmol) was added. The reaction mixture was stirred at 25 °C for 16 h. After this time, the reaction was neutralized with triethylamine (5 mL). The solvent was removed under reduced pressure to give the crude material, which was purified by combiflash column chromatography eluting with 15% ethyl acetate - hexane to give 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethan-1-ol (1.2 g, 53%) as a pale yellow liquid. 1 H NMR (400 MHz, CDCl3): δ1.41 (s, 6H), 1.50 - 1.60 (m, 2H), 1.88 - 1.98 (m, 1H), 3.63 (dd, J = 7.9, 11.8 Hz, 2H), 3.70 (t, J = 6.4 Hz, 2H), 3.93 (dd, J = 4.5, 11.8 Hz, 2H).

[0522] Step 3: 2-(2,2-Dimethyl-1,3-dioxan-5-yl)ethyl 4-(pyrrolidin-1-yl)butanoate

[0523]

Chemical Structure

[0524] 4-(Pyrrolidin-1-yl)butanoic acid (255 mg, 1.6 mmol) was added to a stirred solution in DCM (5 mL), and EDC (355 mg, 1.8 mmol) and DMAP (31 mg, 0.2 mmol) were added at 25 °C and stirred for 5 minutes. After this time, triethylamine (0.6 mL, 4.96 mmol) and 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethan-1-ol (307 mg, 1.92 mmol) were added at 25 °C. The reaction mass was stirred at 25 °C for 16 hours. The reaction mixture was diluted with water and extracted with DCM (3 × 15 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material thus obtained was purified by combiflash column chromatography eluting with 2% MeOH-DCM to give 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl 4-(pyrrolidin-1-yl)butanoate (140 mg, 38%) as a pale yellow liquid. LCMS: column - YMC Triart C18 (33 × 2.1 mm, 3 μ), (mobile phase: 98% [water with 0.05% HCOOH] and 2% [CH3CN] held for 0.75 minutes, then to 90% [water with 0.05% HCOOH] and 10% [CH3CN] in 1.0 minute, to 2% [water with 0.05% HCOOH] and 98% [CH3CN] in 2.0 minutes, this mobile phase composition was held for 2.25 minutes and finally returned to the initial conditions in 3.0 minutes). Flow rate = 1.5 ml / min, 25 °C = 1.22 minutes, calculated m / z [M+H] = 300.2, found 300.5.

[0525] Step 4: 4-Hydroxy-3-(hydroxymethyl)butyl 4-(pyrrolidin-1-yl)butanoate

[0526]

Chemical Structure

[0527] A stirred solution of 2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl 4-(pyrrolidin-1-yl)butanoate (140 mg, 0.3 mmol) in MeOH (1 mL) was added 1N HCl (0.9 mL, 0.9 mmol) at 25 °C. The reaction was stirred for 4 hours. After this time, the reaction mixture was concentrated and azeotroped twice with toluene to give the crude product (120 mg), which was used directly in the next step without purification. LCMS: Column - YMC Triart C18 (33×2.1 mm, 3 μ), (Mobile phase: 98% [water with 0.05% HCOOH] and 2% [CH3CN] held for 0.75 min, then to 90% [water with 0.05% HCOOH] and 10% [CH3CN] in 1.0 min, further to 2% [water with 0.05% HCOOH] and 98% [CH3CN] in 2.0 min, this mobile phase composition was held for 2.25 min and finally returned to the initial conditions in 3.0 min). Flow rate = 1.5 ml / min, 25 °C = 0.50 min, calculated m / z [M+H] = 260.2, measured 260.2.

[0528] Step 5: 2-(Hydroxymethyl)-4-((4-(pyrrolidin-1-yl)butanoyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate

[0529]

Chemical formula

[0530] A stirred solution of linoleic acid (0.32 mL, 1.0 mmol) in DCM (4 mL) was added with DIPEA (0.5 mL, 2.8 mmol), EDC (333 mg, 1.8 mmol) and DMAP (14 mg, 0.16 mmol) at 0 °C and stirred for 5 minutes. After this time, 4-hydroxy-3-(hydroxymethyl)butyl 4-(pyrrolidin-1-yl)butanoate (crude product from step 4, 115 mg) was added at 0 °C. The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was diluted with water and extracted with DCM (3 × 15 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by combiflash column chromatography eluting with 2% MeOH-DCM to give 2-(hydroxymethyl)-4-((4-(pyrrolidin-1-yl)butanoyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate (150 mg, 24%) as a pale yellow liquid. LCMS: column - YMC Triart C18 (33 × 2.1 mm, 3 μ) (mobile phase: 98% [water with 0.05% HCOOH] and 2% [CH3CN] held for 0.75 minutes, then to 90% [water with 0.05% HCOOH] and 10% [CH3CN] in 1.0 minute, further to 2% [water with 0.05% HCOOH] and 98% [CH3CN] in 2.0 minutes, this mobile phase composition was held for 2.25 minutes and finally returned to the initial conditions in 3.0 minutes). Flow rate = 1.5 ml / min, 25 °C = 1.74 minutes, calculated m / z [M+H] = 522.41, measured 522.7.

[0531] Step 6: 2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-4-((4-(pyrrolidin-1-yl)butanoyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate (83)

[0532] A stirred solution of 1-adamantaneacetic acid (87.74 mg, 0.43 mmol) in DCM (2 mL) was added with EDC (165.35 mg, 0.86 mmol), DIPEA (0.15 mL, 0.86 mmol) and DMAP (3.5 mg, 0.02 mmol) at 0 °C and stirred for 5 minutes. Then, 2-(hydroxymethyl)-4-((4-(pyrrolidin-1-yl)butanoyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate (210 mg, 0.40 mmol) was added at 0 °C. The reaction mixture was stirred at 25 °C for 16 hours. After this time, the reaction mixture was diluted with water and extracted with DCM (3 × 15 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by Prep-HPLC to give 2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-4-((4-(pyrrolidin-1-yl))butanoyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate (22 mg, 8%) as a yellow sticky liquid. Prep-HPLC method: Waters automatic purification apparatus. Column name: Xterra RP18 (50 × 20 mm, 5 μm), operated at 50 °C and a flow rate of 16 mL / min. Mobile phase: A = water with 0.1% formic acid; B = 70:30:: acetonitrile:THF + 0.1% formic acid; Gradient profile: The initial composition of the mobile phase of 70% A and 30% B was gradually increased to 30% A and 70% B in 14 minutes, then increased to 100% B in 15 minutes and continued at this composition for 17 minutes for column washing, then returned to the initial composition at 18 minutes and held until 20 minutes.LCMS: Column - XTERRA RP 18 (4.6×50 mm), 5 μm, (Mobile phase: Initially 80% [Water with 0.1% HCOOH] and 20% [(70:30) ACN:THF with 0.1% HCOOH]; Hold this initial condition for 0.75 minutes, then to 65% [Water with 0.1% HCOOH] and 35% [(70:30) ACN:THF with 0.1% HCOOH] in 3.0 minutes, then to 2% [Water with 0.1% HCOOH] and 98% [(70:30) ACN:THF with 0.1% HCOOH] in 6.0 minutes, hold this mobile phase composition until 9.0 minutes, and finally return to the initial condition, i.e., 80% [Water with 0.1% HCOOH] and 20% [(70:30) ACN:THF] at 11.00 minutes, hold this mobile phase composition until 12.10 minutes. Flow rate = 1.2 ml / min, RT = 5.19 min, Calculated m / z [M + H] = 698.5, Measured 699.2.

[0533] [Example 84] 2 - ((2 - ((3r,5r,7r)-Adamantan - 1 - yl)acetoxy)methyl)-4 - ((3-(4 - methylpiperazin - 1 - yl)propanoyl)oxy)butyl (9Z,12Z)-octadeca - 9,12 - dienoate (84)

[0534] [Chemical formula]

[0535] In Step 3, 4-(pyrrolidin-1-yl)butanoic acid was replaced with 3-(4-methylpiperazin-1-yl)propanoic acid and prepared by the same procedure as exemplified in Example 83. Yield of the final step: 38 mg, 14%. LCMS: Column - XTERRA RP 18 (4.6×50 mm), 5 μ, (Mobile phase: initially 95% [water with 0.1% HCOOH] and 5% [(70:30) ACN:THF with 0.1% HCOOH]; then to 70% [water with 0.1% HCOOH] and 30% [(70:30) ACN:THF with 0.1% HCOOH] in 0.75 min, then to 2% [water with 0.1% HCOOH] and 98% [(70:30) ACN:THF with 0.1% HCOOH] in 3.0 min, this mobile phase composition was held for 4.90 min, and finally returned to the initial conditions, i.e.; 95% [water with 0.1% HCOOH] and 5% [(70:30) ACN:THF with 0.1% HCOOH] at 5.10 min. Flow rate = 1.2 mL / min, RT = 2.57 min, calculated m / z [M+H] = 713.5, MS measured value 713.7.

[0536] [Example 85] 2-((2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)methyl)-4-((4-(dipropylamino)butanoyl)oxy)butyl (9Z,12Z)-octadeca-9,12-dienoate

[0537] [Chemical formula]

[0538] In Step 3, 4-(pyrrolidin-1-yl)butanoic acid was replaced with 4-(dipropylamino)butanoic acid and prepared by the same procedure as exemplified in Example 83. Yield of the final step: 21 mg, 7%. LCMS: Column - XTERRA RP 18 (4.6×50 mm), 5 μ, (Mobile phase: initially 80% [water with 0.1% HCOOH] and 20% [(70:30) ACN:THF with 0.1% HCOOH]; hold this initial condition for 0.75 min, then to 65% [water with 0.1% HCOOH] and 35% [(70:30) ACN:THF with 0.1% HCOOH] in 3.0 min, then to 2% [water with 0.1% HCOOH] and 98% [(70:30) ACN:THF with 0.1% HCOOH] in 6.0 min, hold this mobile phase composition for up to 9.0 min, and finally return to the initial condition, i.e., 80% [water with 0.1% HCOOH] and 20% [(70:30) ACN:THF with 0.1% HCOOH] at 11.00 min and hold this mobile phase composition for up to 12.10 min. Flow rate = 1.2 ml / min, RT = 5.18 min, calculated m / z [M + H] = 729.1, measured 729.7.

[0539] [Example 86] 2-((2-((3r,5r,7r)-Adamantan-1-yl)acetoxy)methyl)-4-(2-(4-methylpiperazin-1-yl)acetoxy)butyl (9Z,12Z)-octadeca-9,12-dienoate (86)

[0540] [Chemical formula]

[0541] In Step 3, 4-(pyrrolidin-1-yl)butanoic acid was replaced with 2-(4-methylpiperazin-1-yl)acetic acid and prepared by the same procedure as exemplified in Example 83. Yield of the final step: 14 mg, 5%. LCMS: Column - XTERRA RP 18 (4.6×50 mm), 5 μ, (Mobile phase: Initially 80% [water with 0.1% HCOOH] and 20% [(70:30) ACN:THF with 0.1% HCOOH]; Hold this initial condition for 0.75 minutes, then to 65% [water with 0.1% HCOOH] and 35% [(70:30) ACN:THF with 0.1% HCOOH] in 3.0 minutes, then to 2% [water with 0.1% HCOOH] and 98% [(70:30) ACN:THF with 0.1% HCOOH] in 6.0 minutes, hold this mobile phase composition for 9.0 minutes, and finally return to the initial condition, i.e., 80% [water with 0.1% HCOOH] and 20% [(70:30) ACN:THF with 0.1% HCOOH] at 11.00 minutes and hold this mobile phase composition for 12.10 minutes. Flow rate = 1.2 ml / min, RT = 2.51 minutes, calculated m / z [M + H] = 699.5, measured value: 699.3.

[0542] Lipid nanoparticles Screening F.LNP formulation The lipid nanoparticle components were dissolved in 100% ethanol at the specified molar ratios of the lipid components. The nucleic acid (NA) cargo was dissolved in 10 mM citrate, 100 mM NaCl, pH 4.0, resulting in a concentration of the NA cargo of approximately 0.22 mg / mL. In some embodiments, the NA cargo consists of both functional NA (e.g., siRNA, antisense, expression DNA, mRNA) mixed at a mass ratio of 1:10 to 10:1 with respect to the barcode of the functional NA, and a reporter DNA barcode (Sago, 2018 PNAS described above).

[0543] The LNPs were formulated at a total lipid to NA mass ratio of 11.7. The LNPs were formed by microfluidic mixing of lipids and NA solution using a Precision Nanosystems NanoAssemblr Spark or Benchtop Instrument according to the manufacturer's protocol. A 2:1 ratio of aqueous solvent to organic solvent was maintained during mixing using differential pressure flow. After mixing, the LNPs were collected, diluted with PBS (approximately 1:1 v / v), and further buffer exchange was performed using dialysis against a 20 kDa filter at 4 °C for 8 - 24 hours in PBS. After this first dialysis, each individual LNP formulation was characterized via DLS to measure size and polydispersity, and the pKa of subpopulations of LNPs was measured via the TNS assay. LNPs falling within a specific diameter and polydispersity range were pooled and further dialyzed against PBS at 4 °C for 1 - 4 hours against a 100 kDa dialysis cassette. After the second dialysis, the LNPs were filter sterilized using a 0.22 μM filter and stored at 4 °C for further use.

[0544] G. LNP Characterization DLS - The hydrodynamic diameter and polydispersity percent (PDI%) of the LNPs were measured using high - throughput dynamic light scattering (DLS) (DynaPro Plate Reader II, Wyatt). The LNPs were diluted to an appropriate concentration with 1×PBS and analyzed.

[0545] Concentration and Encapsulation Efficiency - The concentration of NA was determined using a Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. The encapsulation efficiency was determined by measuring non - lysed and lysed LNPs.

[0546] Stock solutions of pKa - 10 mM HEPES (Sigma Aldrich), 10 mM MES (Sigma Aldrich), 10 mM sodium acetate (Sigma), and 140 mM sodium chloride (Sigma Aldrich) were prepared and the pH was adjusted to the range of pH 4 - 10 using hydrogen chloride and sodium hydroxide. For each pH, 140 μL of the pH - adjusted buffer was added to a 96 - well plate using four replicate samples, followed by the addition of 5 μL of 2-(p - toluidino)-6 - naphthalenesulfonic acid (60 μg / mL). 5 μL of LNP was added to each well. After incubation for 5 minutes under gentle shaking, fluorescence was measured using an excitation wavelength of 325 nm and an emission wavelength of 435 nm (BioTek Synergy H4 Hybrid).

[0547] LNP administration - For all studies, male and female mice approximately 8 - 12 weeks of age were used. Each mouse was temporarily restrained and pooled LNP was administered IV via tail vein injection to a maximum of 5 animals per experiment. Vehicle (1×PBS) was also administered via tail vein injection to a maximum of 3 animals per experiment using age - matched mice. At 72 hours post - administration, tissues including the liver, spleen, bone marrow, and blood were collected for analysis.

[0548] The LNP formulation and information related to the LNP property evaluation can be seen in Figure 1. The lipid numbers correspond to the numbering in the Examples section.

[0549] Flow - Liver tissue was mechanically digested and then enzymatically digested using a mixture of proteinases, and then passed through a 70 μM filter to generate a single - cell suspension. Spleen tissue was mechanically digested to generate a single - cell suspension. All tissues were treated with ACK buffer to lyse red blood cells and then stained with fluorescent - labeled antibodies for flow cytometry and fluorescence - activated cell sorting (FACS). All antibodies were commercially available antibodies. Using a BD FACSMelody (Becton Dickinson), all samples were acquired by flow cytometry to create gates prior to sorting. Overall, the gating structure was size → single cell → live cell → cell of interest. T cells were defined as CD45+CD3+, monocytes were defined as CD45+CD11b+, and B cells were defined as CD45+CD19+. In the liver, endothelial cells were defined as CD31+, Kupffer cells were defined as CD45+CD11b+, and hepatocytes were defined as CD31 - / CD45 -. For siRNA studies, down - regulation of the target gene was gated, while for mRNA studies, up - regulation of the target gene was gated. Tissues from vehicle - dosed mice were used to set the gates for sorting. Up to 20,000 cells were sorted for each cell subset with the correct phenotype and added to 1×PBS. After sorting, the cells were pelleted via centrifugation and DNA was extracted using Quick Extract DNA Extraction Solution (Lucigen) according to the manufacturer's protocol. The DNA was stored at - 20 °C.

[0550] Barcode array determination - DNA (genome and DNA barcode) was isolated using QuickExtract (Lucigen) and sequenced using an Illumina MiniSeq as previously described (Sago et al. PNAS 2018, Sago et al. JACs 2018, Sago, Lokugamage et al. Nano Letters 2018), and the frequency DNA barcode counts in the FACS-isolated samples were normalized to the frequency of the injected input. These data are plotted as "Normalized Fold Above Input" and are in vivo data selected from experiments 71, 72, 73, and 74 that can be seen in Figures 2-5.

[0551] H. Confirmation LNP formulation The lipid nanoparticle components were dissolved in 100% ethanol at the specified molar ratios of the lipid components. The nucleic acid (NA) cargo was dissolved in 10 mM citrate, 100 mM NaCl, pH 4.0, resulting in a concentration of the NA cargo of approximately 0.22 mg / mL. In some embodiments, the NA cargo consists of both functional NA (e.g., siRNA, antisense, expression DNA, mRNA) mixed at a mass ratio of 1:10 to 10:1 with respect to the barcode of the functional NA, and a reporter DNA barcode (Sago, 2018 PNAS as described above). The LNP was formulated at a mass ratio of total lipid to NA of 11.7. The LNP was formed by microfluidic mixing of the lipid and the NA solution using a Precision Nanosystems NanoAssemblr Spark or Benchtop Instrument according to the manufacturer's protocol. A ratio of 3:1 of the aqueous solvent to the organic solvent was maintained during mixing using differential pressure flow. After mixing, the LNP was collected, diluted with PBS (approximately 1:1 v / v), and further buffer exchange was performed using dialysis against a 20 kDa filter at 4 °C for 8 - 24 hours. After this first dialysis, each individual LNP formulation was characterized via DLS to measure size and polydispersity, and the pKa of subpopulations of the LNP was measured via the TNS assay. After dialysis, the LNP was filter sterilized using a 0.22 micron sterile filter and stored at 4 °C for further use.

[0552] LNP Characterization DLS - The hydrodynamic diameter and polydispersity percentage (PDI%) of the LNP were measured using high - throughput dynamic light scattering (DLS) (DynaPro Plate Reader II, Wyatt). The LNP was diluted to an appropriate concentration with 1×PBS and analyzed.

[0553] Concentration and Encapsulation Efficiency - The concentration of NA was determined by the Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. The encapsulation efficiency was determined by measuring non - dissolved and dissolved LNP.

[0554] Stock solutions of pKa - 10 mM HEPES (Sigma Aldrich), 10 mM MES (Sigma Aldrich), 10 mM sodium acetate (Sigma), and 140 nM sodium chloride (Sigma Aldrich) were prepared and the pH was adjusted to the range of pH 4 - 10 using hydrogen chloride and sodium hydroxide. For each pH, 140 μL of the pH - adjusted buffer was added to a 96 - well plate using four replicate samples, followed by the addition of 5 μL of 2-(p - toluidino)-6 - naphthalenesulfonic acid (60 μg / mL). 5 μL of LNP was added to each well. After incubation for 5 minutes under gentle shaking, fluorescence was measured using an excitation wavelength of 325 nm and an emission wavelength of 435 nm (BioTek Synergy H4 Hybrid).

[0555] LNP administration - In all studies, male and female mice approximately 8 - 12 weeks of age were used. Each mouse was temporarily restrained and pooled LNP was administered IV via tail vein injection to a maximum of 5 animals per experiment. Vehicle (1×PBS) was also administered via tail vein injection to a maximum of 3 animals per experiment using age - matched mice. At 72 hours after administration, tissues including the liver, spleen, bone marrow, and blood were collected for analysis.

[0556] Flow - The liver tissue was mechanically digested and then enzymatically digested using a mixture of proteases, and then passed through a 70 μM filter to generate a single - cell suspension. The spleen tissue was mechanically digested to generate a single - cell suspension. All tissues were treated with ACK buffer to lyse red blood cells and then stained with fluorescent - labeled antibodies for flow cytometry and FACS sorting. All antibodies were commercially available antibodies. Using a BD FACSMelody (Becton Dickinson), all samples were acquired by flow cytometry to create gates before sorting. Overall, the gating structure was size → single - cell → live - cell → target cell. T cells were defined as CD45+CD3+, monocytes were defined as CD45+CD11b+, and B cells were defined as CD45+CD19+. In the liver, LSECs were defined as CD31+, Kupffer cells were defined as CD45+CD11b+, and hepatocytes were defined as CD31 - / CD45 -. For siRNA studies, down - regulation of the target gene was gated, while for mRNA studies, up - regulation of the target gene was gated. Tissues from vehicle - dosed mice were used to set the gates for sorting. Data were recorded as MFI by flow cytometry. Selected in vivo data on CD45 protein expression in CD3 - positive cells isolated from mouse spleen corresponding to the formulations shown in Table 2 are shown in Figure 3. The lipid numbers correspond to the numbering of the examples section. In Figure 6, group 1 is PBS - treated animals, while groups 2 - 8 are LNP1 - 7, respectively. In each of LNP1 - 7, the cholesterol used was cholesterol, the PEG used was DMG - PEG2000, the phospholipid used was DSPC, and the ratio of lipid:cholesterol:PEG:phospholipid was 35:46.5:3.5:16. The ratio of lipid to nucleic acid was 11.7 to 1.

[0557]

Table 1

[0558] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to be limiting, and the true scope and spirit are indicated by the following claims.

Claims

1. A compound of formula (I): 【Chemical 1】 (wherein: R １ is C ９ to C ２０ alkyl or C having unsaturation of 1 to 3 units ９ to C ２０ alkenyl; X １ and X ２ is each independently absent or —O—, NR ２ , and [Chemical 2] selected from, where R ２ is C １ to C ６ alkyl, and X １ and X ２ are both not -O- or NR ２ ; a is an integer between 1 and 6; X ３ and X ４ are each independently absent or are a 4- to 7-membered heterocyclyl optionally substituted with one or two C １ -C ６ alkyl groups, a 5- to 6-membered heteroaryl optionally substituted with one or two C １ -C ６ alkyl groups, and -NR ３ - selected from the group consisting of, where each R ３ is a hydrogen atom or C １ -C ６ alkyl; X ５ is -(CH ２ ) ｂ -, where b is an integer between 0 and 6; X ６ is hydrogen, C １ to C ６ alkyl, a 5- to 6-membered heteroaryl optionally substituted with one or two C １ to C ６ alkyl groups, or -NR ４ R ５ wherein R ４ and R ５ are each independently hydrogen or C １ to C ６ alkyl; alternatively, R ４ and R ５ together with the nitrogen to which they are attached form a 4- to 7-membered heterocyclyl optionally substituted with one or two C １ to C ６ alkyl groups, wherein said heterocyclyl optionally contains a further heteroatom selected from oxygen, sulfur and nitrogen; X ７ is hydrogen or -NR ６ R ７ wherein R ６ and R ７ are each independently hydrogen or C １ to C ６ alkyl; alternatively, R ６ and R ７ together with the nitrogen to which they are attached form a 4- to 7-membered heterocyclyl optionally substituted with one or two C １ to C ６ alkyl groups, wherein said heterocyclyl optionally contains an additional heteroatom selected from oxygen, sulfur and nitrogen; X １ , X ２ , X ３ , X ４ , and X ５ at least one of which exists; provided that X １ or X ２ If either is -O-, then X ３ is also X ４ or both [Chemical 3] shall not be X １ or X ２ In the case where any of them is -O-, R ４ and R ５ shall not both be ethyl, and 【Chemical 4】 is, 【Chemical Formula 5】 on the condition that it is not selected from the group consisting of).

2. R １ is 【Chemical Formula 6】 The compound according to claim 1, wherein is.

3. The compound is selected from the group consisting of: 【Chemical Formula 7】 [Chemical] [Chemical] [Chemical] 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 The compound according to claim 1.

4. The compound is [Chemical Formula 8] The compound according to claim 1.

5. 【Fig. 22】 A compound selected from the group consisting of.

6. Ionizable lipid: Phospholipid; Polyethylene glycol-lipid; Cholesterol; and optionally Nucleic acid A lipid nanoparticle composition comprising, wherein the ionizable lipid comprises the structure according to any one of claims 1 to 5, said lipid nanoparticle composition.

7. The lipid nanoparticle composition according to claim 6, wherein the amount of the ionizable lipid is present in the range of 35 to 65 mole percent based on the total moles.

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

9. The lipid nanoparticle composition according to any one of claims 6 to 8, for delivering a nucleic acid to a subject.

10. The lipid nanoparticle composition according to any one of claims 6 to 9, wherein the nucleic acid is siRNA, miRNA, antisense oligonucleotide, or immunostimulatory oligonucleotide.

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