Lipids and lipid nanoparticles

Abstract

Disclosed herein are ionizable lipids and salts thereof (e.g., pharmaceutically acceptable salts thereof), as well as compositions comprising such lipids, which are useful for the delivery of bioactive agents.

Description

Technical Field

[0001] This application claims priority to PCT applications PCT / CN2023 / 108217, PCT / CN2023 / 108216, PCT / CN2023 / 120617 and PCT / CN2023 / 120619. The entire contents of the foregoing applications are incorporated herein by reference.

[0002] This invention relates to ionizable lipid compounds and compositions comprising such compounds. The invention also relates to processes for preparing these compounds and compositions, as well as methods of using and applications of such compounds and compositions, for example, for delivering bioactive agents (such as RNA agents) to cells and tissues. Background Technology

[0003] Lipid-containing particles have been used to encapsulate therapeutic agents and serve as carriers for transporting therapeutic agents, such as nucleic acids, small molecule compounds, and proteins, into cells and other intracellular compartments. There remains an ongoing need to develop novel lipids for encapsulating therapeutic agents and to improve the safety, efficacy, and specificity of these nanoparticle-based delivery systems. Summary of the Invention

[0004] This invention provides a compound as shown in formula (I),

[0005] (I),

[0006] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0007] Wherein R1 is hydrogen; phenyl; 3- to 7-membered cycloalkyl; 3- to 7-membered heterocyclic group containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 5- to 6-membered monocyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 8- to 10-membered bicyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; -OR'; or ; wherein the phenyl, cycloalkyl, 3 to 7-membered heterocyclic, 5 to 6-membered monocyclic heteroaryl, and 8 to 10-membered bicyclic heteroaryl are optionally substituted by one or more substituents independently selected from halogen, hydroxyl or thiol groups;

[0008] R' is selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, and C2-C8 alkynyl, wherein the C1-C8 alkyl, C2-C8 alkenyl, and C2-C8 alkynyl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or thiol groups.

[0009] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 ynyl, -S-C3-13 alkyl, and -CH2-S-C3-13 alkyl; wherein C1-C18 alkyl, C2-C18 alkenyl, C2-C18 ynyl, or C 3-13 Each alkyl moiety is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxy or thiol groups;

[0010] R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 ynyl or -C(=O)R8; wherein the C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 ynyl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxy or thiol groups;

[0011] R8 is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl groups;

[0012] X1 is selected from the group consisting of -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R 12 )-、-(R 12 )NS(O)2O-、-N(R 12 )S(O)2N(R 13 )-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)N(R 12 )-、-(R 12 )NC(=O)O-、-OC(=S)N(R 12 )-、-(R 12 )NC(=S)O-、-SC(=O)N(R 12 )- or -(R 12 )NC(=O)S-;

[0013] R 12 and R 13 Each is independently selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 ynyl, -C(=O)R8; wherein the C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 ynyl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or thiol groups;

[0014] X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)O-, -C(=O)S-, -SC(=O)-;

[0015] L0 and L1 are each independently selected from a bond, C1-C8 alkylene, C2-C8 alkenyl or C2-C8 ynynyl; wherein the C1-C8 alkylene, C2-C8 alkenyl and C2-C8 ynynyl are each optionally substituted by one or more substituents independently selected from halogen, hydroxyl or thiol groups;

[0016] L2 and L3 are each independently selected from C1-C18 alkylene, C2-C18 alkenylene, and C2-C18 ynynylene, wherein each of the C1-C18 alkylene, C2-C18 alkenylene, and C2-C18 ynynylene is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or thiol groups.

[0017] This invention provides a compound as shown in formula (II),

[0018] (II)

[0019] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0020] in

[0021] R1 is selected from -OR', ;

[0022] R' is selected from hydrogen or C1-C8 alkyl; wherein each of the C1-C8 alkyl groups is optionally substituted by one or more substituents independently selected from halogen, hydroxyl or thiol groups;

[0023] R2, R3, R4, and R5 are each independently selected from hydrogen, C5-C18 alkyl, C5-C18 alkenyl, and -SC. 3-13 Alkyl and -CH2-SC 3-13 Alkyl groups; including C5-C18 alkyl groups, C5-C18 alkenyl groups, and -S-(CH2) groups. 2-10 CH3 and C 3-13 Each alkyl moiety is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxy or thiol groups;

[0024] R9, R 10 and R 11 Each is independently selected from hydrogen or linear C1 alkyl groups.

[0025] R8 is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl groups;

[0026] X1 is selected from the key, -S-, or -O-;

[0027] X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)O-, -C(=O)S-, -SC(=O)-;

[0028] L0 and L1 are each independently selected from a bond or a C1-C8 alkylene group; wherein the C1-C8 alkylene group is optionally substituted by one or more substituents independently selected from halogen, hydroxyl or thiol groups;

[0029] L2, L3, L4 and L5 are each independently selected from a bond, a C1-C18 alkylene group or a C2-C18 alkenyl group, wherein the C1-C18 alkylene group and the C2-C18 alkenyl group are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol groups.

[0030] In certain specific embodiments, at least one of X2 and X3 is -OC(=O)S- or -SC(=O)O-.

[0031] In some embodiments, R1 is -OR', where R' is selected from hydrogen or C1-C8 alkyl. In some embodiments, R1 is -OR', where R' is hydrogen. In some embodiments, R1 is... R9, R 10 and R 11 Each is independently selected from hydrogen or C1-C5 alkyl groups. In some embodiments, R1 is... R9, R 10 and R 11 R1 is independently selected from hydrogen or linear C1-C3 alkyl groups. In some embodiments, R1 is selected from... R9, R 10 and R 11 Each is independently selected from hydrogen or linear C1 alkyl groups.

[0032] In some embodiments, L0 is selected from C1-C8 alkylene groups. In some embodiments, L0 is a linear C1-C5 alkylene group. In some embodiments, L0 is selected from linear C4 alkylene groups, linear C3 alkylene groups, or linear C2 alkylene groups. In some embodiments, L0 is a C1-C8 alkylene group.

[0033] In some embodiments, L1 is selected from C1-C8 alkylene groups. In some embodiments, L1 is a linear C1-C5 alkylene group. In some embodiments, L1 is selected from linear C4 alkylene groups, linear C3 alkylene groups, or linear C2 alkylene groups. In some embodiments, L1 is a C1-C8 alkylene group.

[0034] In some embodiments, L2 and L3 are independently selected from C3-C18 alkylene groups. In some embodiments, L2 and L3 are independently C3-C10 alkylene groups. In some embodiments, L2 and L3 are independently C5-C8 alkylene groups. In some embodiments, L2 and L3 are independently C8 alkylene groups. In some embodiments, L2 and L3 are independently C7 alkylene groups. In some embodiments, L2 and L3 are independently C6 alkylene groups. In some embodiments, L2 and L3 are independently C5 alkylene groups. In some embodiments, L2 and L3 are independently C4 alkylene groups. In some embodiments, L2 and L3 are independently C3 alkylene groups.

[0035] In some embodiments, L4 and L5 are independently selected from C1-C5 alkylene groups. In some embodiments, L4 and L5 are independently C1, C2, or C3 alkylene groups.

[0036] In some embodiments, X1 is a key. In some embodiments, X2 and X3 are independently selected from -OC(=O)S-, -SC(=O)O-, -C(=O)O-, -OC(=O)-, or -OC(=O)O-. In some embodiments, X2 and X3 are independently selected from -OC(=O)S-, -SC(=O)O-, -C(=O)O-, -OC(=O)-, or -OC(=O)O-; and at least one X2 and X3 are selected from -OC(=O)S- or -SC(=O)O-. In some embodiments, X2 and X3 are independently selected from -OC(=O)S- or -SC(=O)O-. In some embodiments, X2 and X3 are independently selected from -OC(=O)S-, -SC(=O)O-, -C(=O)O-, -OC(=O)-, or -OC(=O)O-. X3 is selected from -OC(=O)S- or -SC(=O)O-; while X2 is selected from -OC(=O)- or -C(=O)O-. In some embodiments, X2 and X3 are independently selected from -OC(=O)- or -C(=O)O-.

[0037] In some embodiments, R2 and R3 are independently selected from hydrogen, C5-C12 alkyl, and -SC. 3-13 Alkyl or -CH2-SC 3-13Alkyl group. In some embodiments, R2 and R3 are independently C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, C5 alkyl, or C4 alkyl. In some embodiments, R2 and R3 are independently -S-(CH2)5CH3.

[0038] In some embodiments, R4 and R5 are each independently selected from hydrogen, C5-C 12 Alkyl, -SC 3-13 Alkyl or -CH2-SC 3-13 Alkyl group. In some embodiments, R4 and R5 are independently C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, C5 alkyl, or C4 alkyl. In some embodiments, R4 and R5 are independently -S-(CH2)5CH3.

[0039] In some embodiments, R3 and R5 are independently -CH2-SC 3-13 Alkyl group. In some embodiments, R3 and R5 are independently –CH2S-(CH2)8CH3, –CH2S-(CH2)7CH3, –CH2S-(CH2)6CH3, –CH2S-(CH2)5CH3, –CH2S-(CH2)4CH3 or –CH2S-(CH2)3CH3; R2 and R4 are independently -SC 3-13 Alkyl group; in some embodiments, R2 and R4 are independently -S-(CH2)8CH3, -S-(CH2)7CH3, -S-(CH2)6CH3, -S-(CH2)5CH3, -S-(CH2)4CH3 or -S-(CH2)3CH3.

[0040] This invention provides a compound as shown in formula (III),

[0041] (III)

[0042] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0043] in

[0044] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2-10 CH3;

[0045] X1 is the key; X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)O-, -C(=O)S-, -SC(=O-); and at least one X2 and X3 are selected from -OC(=O)S- or -SC(=O)O-;

[0046] L1 or L0 are each independently selected from a bond or a C1-C8 alkylene group;

[0047] L2 and L3 are each independently C3-C10 alkylene groups;

[0048] L4 and L5 are each independently selected from the key or C. 1-3 Alkylene.

[0049] This invention provides a compound as shown in formula (IV),

[0050] (IV),

[0051] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0052] in

[0053] R1 is -OH or ;

[0054] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2-10 CH3;

[0055] R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8;

[0056] R8 is a C1-C5 alkyl group;

[0057] X2 is selected from -C(=O)O-, -OC(=O-, or -OC(=O)O-;

[0058] L1 is a C1-C8 alkylene group;

[0059] L2 and L3 are each independently C3-C10 alkylene groups;

[0060] L4 and L5 are each independently selected from the key or C. 1-3 Alkylene.

[0061] This invention provides a compound as shown in formula (V).

[0062] (V)

[0063] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0064] in

[0065] R1 is -OH or ;

[0066] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2-10 CH3;

[0067] R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8;

[0068] R8 is a C1-C5 alkyl group;

[0069] X2 is selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -SC(=O)O- or -OC(=O)S-;

[0070] L1 is a C1-C8 alkylene group;

[0071] L2 and L3 are each independently C3-C10 alkylene groups;

[0072] L4 and L5 are each independently selected from the key or C. 1-3 Alkylene.

[0073] This invention provides a compound as shown in formula (VI),

[0074] (VI),

[0075] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0076] in

[0077] R1 is -OH or ;

[0078] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2-10 CH3;

[0079] R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8;

[0080] R8 is a C1-C5 alkyl group;

[0081] X1 is the key;

[0082] L1 is a C1-C8 alkylene group;

[0083] L2 and L3 are each independently C3-C10 alkylene groups;

[0084] L4 and L5 are each independently selected from the key or C. 1-3 Alkylene.

[0085] In some embodiments, R1 is R9, R 10 and R 11 Each is independently selected from hydrogen or straight-chain C1-3 alkyl groups.

[0086] In some embodiments, L0 is a bond. In some embodiments, L0 is a straight-chain C2 alkylene. In some embodiments, L0 is a straight-chain C4 alkylene. In some embodiments, L0 is a straight-chain C3 alkylene.

[0087] In some embodiments, L1 is a bond. In some embodiments, L1 is a straight-chain C2 alkylene. In some embodiments, L1 is a straight-chain C4 alkylene. In some embodiments, L1 is a straight-chain C3 alkylene.

[0088] In some embodiments, L2 and L3 are each independently C5-C8 alkylene groups. In some embodiments, L2 and L3 are each independently C6 alkylene groups.

[0089] In some embodiments, L4 and L5 are each independently a bond. In some embodiments, L4 and L5 are each independently a C1 alkylene group. In some embodiments, L4 and L5 are each independently a C2 alkylene group.

[0090] This invention provides a compound as shown in formula (VII),

[0091] (VII)

[0092] L0 and L1 are each independently a bond or a C1-C5 alkylene group, wherein the C1-C5 alkylene group is optionally substituted by one or more substituents, which are independently selected from halogens, hydroxyl groups or thiols;

[0093] X1 is selected from key, -S-, or -O-;

[0094] X2 and X3 are independently selected from -C(=O)O-, -OC(=O-, -OC(=O)O-, -OC(=O)S-, or -SC(=O)O-;

[0095] L2 and L3 are each independently selected from C3-C9 alkylene groups, wherein the C3-C9 alkylene groups are optionally substituted by one or more substituents, wherein the substituents are independently selected from C1-C3 alkyl groups, halogens, hydroxyl groups, or thiols;

[0096] L4 and L5 are each independently selected from C1-C5 alkylene groups, wherein the C1-C5 alkylene groups are optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl groups, halogens, hydroxyl groups or thiols;

[0097] R4 is selected from C1-C18 alkyl, C2-C18 alkenyl; wherein the C1-C18 alkyl and C2-C18 alkenyl are each optionally substituted by one or more substituents, said substituents being independently selected from C1-C3 alkyl, halogen, hydroxyl or thiol;

[0098] Among them, L6, L7, L 10 and L 11 Each is independently a bond or a C6-C9 sub-alkenyl group; preferably, L6, L7, L... 10 and L 11 Each is a key independently.

[0099] R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl; wherein the C1-C18 alkyl and C2-C18 alkenyl are each optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl, halogen, hydroxyl or thiol.

[0100] This invention provides a compound as shown in formula (VIII),

[0101] (VIII)

[0102] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0103] in

[0104] L0 and L1 are each independently a bond or a C1-C5 alkylene group;

[0105] X1 is the key;

[0106] X2 and X3 are independently selected from -C(=O)O-, -OC(=O-, -OC(=O)O-, -OC(=O)S- or -SC(=O)O-;

[0107] L2 and L3 are each independently C3-C9 alkylene groups;

[0108] L4 and L5 are each independently C1-C5 alkylene groups;

[0109] R4 is selected from C1-C18 alkyl, C2-C18 alkenyl;

[0110] L6, L7, L 10 and L 11 Each is an independent key;

[0111] R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

[0112] In some embodiments, L0 is a bond, and L1 is a C1-C5 alkylene group, preferably, L1 is a C2, C3 or C4 alkylene group.

[0113] In some embodiments, X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-.

[0114] In some embodiments, L2 and L3 are each independently C3-C9 alkylene oxides. In some embodiments, L2 and L3 are each independently C3 alkylene oxides. In some embodiments, L2 and L3 are each independently C4 alkylene oxides. In some embodiments, L2 and L3 are each independently C5 alkylene oxides. In some embodiments, L2 and L3 are each independently C6 alkylene oxides. In some embodiments, L2 and L3 are each independently C7 alkylene oxides. In some embodiments, L2 and L3 are each independently C8 alkylene oxides. In some embodiments, L2 and L3 are each independently C9 alkylene oxides.

[0115] In some embodiments, L4 and L5 are each independently C1-C3 alkylene oxides. In some embodiments, L4 and L5 are each independently C1 alkylene oxides. In some embodiments, L4 and L5 are each independently C2 alkylene oxides. In some embodiments, L4 and L5 are each independently C3 alkylene oxides.

[0116] In some embodiments, R4 is selected from C1-C18 alkyl or C2-C18 alkenyl; L 10 and L 11 Each is an independent bond; R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

[0117] In some embodiments, R2, R3, R5, and R6 are each independently selected from C4-C18 alkyl or C4-C18 alkenyl. In some embodiments, R2, R3, R5, and R6 are each independently selected from C4-C12 alkyl. In some embodiments, R2, R3, R5, and R6 are each C4 alkyl. In some embodiments, R2, R3, R5, and R6 are each C5 alkyl. In some embodiments, R2, R3, R5, and R6 are each C6 alkyl. In some embodiments, R2, R3, R5, and R6 are each C7 alkyl. In some embodiments, R2, R3, R5, and R6 are each C8 alkyl. In some embodiments, R2, R3, R5, and R6 are each C9 alkyl. In some embodiments, R2, R3, R5, and R6 are each C10 alkyl. In some embodiments, R2, R3, R5, and R6 are each C11 alkyl. In some embodiments, R2, R3, R5, and R6 are each C12 alkyl.

[0118] This invention provides a compound as shown in formula (IX),

[0119] (IX)

[0120] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0121] in

[0122] L0 and L1 are each independently a bond or a C1-C5 alkylene group, wherein the C1-C5 alkylene group is optionally substituted by one or more substituents, which are independently selected from the group consisting of halogens, hydroxyl groups or mercapto groups;

[0123] X1 is selected from the group consisting of free bonds, -S-, or -O-; X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=O)S-, or -SC(=O)O-;

[0124] L2 and L3 are each independently selected from the group consisting of free bonds and C3-C8 alkylene groups, wherein the C3-C8 alkylene groups are optionally substituted by one or more substituents, and the substituents are independently selected from the group consisting of C1-C3 alkyl groups, halogens, hydroxyl groups or mercapto groups;

[0125] L4 and L5 are each independently selected from the group consisting of free bonds and C1-C5 alkylene groups, wherein the C1-C5 alkylene groups are optionally substituted by one or more substituents, and the substituents are independently selected from the group consisting of C1-C3 alkyl groups, halogens, hydroxyl groups or mercapto groups;

[0126] R4 selects freedom , , The group consisting of C1-C18 alkyl and C2-C18 alkenyl groups, wherein each of the C1-C18 alkyl and C2-C18 alkenyl groups is optionally substituted by one or more substituents, the substituents being independently selected from the group consisting of C1-C3 alkyl, halogen, hydroxyl or mercapto groups;

[0127] Among them, L6, L7, L 10 and L 11 Each is an independent key;

[0128] R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl, wherein the C1-C18 alkyl and C2-C18 alkenyl are each optionally substituted by one or more substituents, which are independently selected from the group consisting of C1-C3 alkyl, halogen, hydroxyl or mercapto.

[0129] This invention provides a compound as shown in formula (X).

[0130] (X)

[0131] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0132] in

[0133] L0 and L1 are each independently a bond or a C1-C5 alkylene group;

[0134] X1 is the key;

[0135] X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=O)S- or -SC(=O)O-;

[0136] L2 and L3 are each independently C3-C8 alkylene groups;

[0137] L4 and L5 are each independently C1-C5 alkylene groups;

[0138] L6, L7, L 10 and L 11 Each is an independent key;

[0139] R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

[0140] In some embodiments, L0 is a bond and L1 is a C1-C5 alkylene group.

[0141] In some embodiments, X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-.

[0142] In some embodiments, L2 and L3 are each independently C5-C8 alkylene groups.

[0143] In some embodiments, L4 and L5 are each independently C1-C3 alkylene groups.

[0144] In some embodiments, R4 is freely selectable. , L 10 and L 11 Each is an independent bond; R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

[0145] In some embodiments, R2, R3, R5, and R6 are each independently selected from C4-C18 alkyl or C4-C18 alkenyl. In some embodiments, R2, R3, R5, and R6 are each independently selected from C4-C12 alkyl. In some embodiments, R2, R3, R5, and R6 are each C4 alkyl. In some embodiments, R2, R3, R5, and R6 are each C5 alkyl. In some embodiments, R2, R3, R5, and R6 are each C6 alkyl. In some embodiments, R2, R3, R5, and R6 are each C7 alkyl. In some embodiments, R2, R3, R5, and R6 are each C8 alkyl. In some embodiments, R2, R3, R5, and R6 are each C9 alkyl. In some embodiments, R2, R3, R5, and R6 are each C10 alkyl. In some embodiments, R2, R3, R5, and R6 are each C11 alkyl. In some embodiments, R2, R3, R5, and R6 are each C12 alkyl.

[0146] In some embodiments, the compounds disclosed herein are selected from the following compounds:

[0147]

[0148] YZL408 (1);

[0149]

[0150] YZL410 (2);

[0151]

[0152] YZL412 (3);

[0153]

[0154] YZL413 (4);

[0155]

[0156] YZL424 (5);

[0157]

[0158] YZL427 (6);

[0159]

[0160] YZL400 (7);

[0161]

[0162] YZL409 (8);

[0163]

[0164] YZL411 (9);

[0165]

[0166] YZL414 (10);

[0167]

[0168] YZL415 (11);

[0169]

[0170] YZL416 (12);

[0171]

[0172] YZL417 (13);

[0173]

[0174] YZL425 (14);

[0175]

[0176] YZL426 (15);

[0177]

[0178] YZL430 (16);

[0179]

[0180] YZL431 (17);

[0181]

[0182] YZL432 (18);

[0183]

[0184] YZL433 (19);

[0185]

[0186] YZL439 (20);

[0187]

[0188] YZL440 (21);

[0189]

[0190] YZL441 (22);

[0191]

[0192] YZL442 (23);

[0193]

[0194] YZL443 (24);

[0195]

[0196] YZL444 (25);

[0197]

[0198] YZL445 (26);

[0199]

[0200] YZL446 (27);

[0201]

[0202] YZL447 (28);

[0203]

[0204] YZL448 (29);

[0205]

[0206] YZL449 (30);

[0207]

[0208] YZL450 (31);

[0209]

[0210] YZL452 (32);

[0211]

[0212] YZL453 (33);

[0213]

[0214] YZL455 (34);

[0215]

[0216] YZL456 (35);

[0217]

[0218] YZL457 (36);

[0219]

[0220] YZL458 (37);

[0221]

[0222] YZL459 (38);

[0223]

[0224] YZL460 (39);

[0225]

[0226] YZL501 (40);

[0227]

[0228] (YZL429) (41);

[0229]

[0230] (YZL428) (42);

[0231]

[0232] (YZL418) (43);

[0233]

[0234] (YZL419) (44);

[0235]

[0236] (YZL422) (45);

[0237]

[0238] (YZL423) (46);

[0239]

[0240] (YZL424) (47);

[0241]

[0242] (YZL425) (48);

[0243]

[0244] (YZL473) (49);

[0245]

[0246] (YZL434) (50);

[0247]

[0248] (YZL451) (51);

[0249]

[0250] (YZL449) (52);

[0251]

[0252] YZL 479 (53);

[0253]

[0254] YZL480 (54);

[0255]

[0256] YZL491(55);

[0257]

[0258] YZL495 (56);

[0259]

[0260] YZL499 (57);

[0261]

[0262] YZL701 (58);

[0263]

[0264] YZL702 (59);

[0265]

[0266] YZL703 (60);

[0267]

[0268] YZL704 (61); or

[0269]

[0270] YZL705 (62).

[0271] This invention discloses amine-containing lipids that can be used to prepare lipid nanoparticle (LNP) compositions. Such LNP compositions may possess properties favorable for delivering nucleic acid cargoes to cells, such as CRISPR / Cas gene editing components.

[0272] The present invention also provides a composition comprising one of the compounds disclosed herein as a lipid component, preferably an LNP composition.

[0273] In some embodiments, the lipid component comprises non-cationic lipids and PEGylated lipids. In some embodiments, the non-cationic lipids are neutral lipids.

[0274] In some embodiments, the composition further comprises a buffer.

[0275] In some embodiments, the composition further comprises a bioactive agent. In some embodiments, the bioactive agent comprises a polypeptide. In some embodiments, the bioactive agent comprises a nucleic acid. In some embodiments, the nucleic acid component comprises RNA. In some embodiments, the RNA component comprises modified RNA. In some embodiments, the RNA component comprises a sequence encoding an RNA-guided DNA binder, such as Cas nuclease mRNA. In some embodiments, the nucleic acid component comprises RNA, and the RNA component comprises mRNA. In some embodiments, the RNA component comprises Class 2 Cas nuclease mRNA. In some embodiments, the RNA component comprises mRNA encoding Cas9, Cpf1, c2c2, cas12i, cas12f, cas12b, cas12c, cas12d, cas12e, cas12g, cas12j, or cas12k nucleases. In some embodiments, the RNA component comprises gRNA. In some embodiments, the nucleic acid component comprises a sequence encoding gRNA. In some embodiments, the RNA component comprises Cas nuclease mRNA and gRNA. In some embodiments, the gRNA is a two-way guide RNA (dgRNA). In some embodiments, the gRNA is a one-way guide RNA (sgRNA). In some embodiments, the gRNA is modified gRNA. In some embodiments, the composition comprises donor DNA. In some embodiments, the composition further comprises at least one template nucleic acid. In some embodiments, the bioactive agent is a ribonucleoprotein (RNP) complex. In some embodiments, the bioactive agent comprises siRNA, miRNA, or ASO. In some embodiments, the bioactive agent comprises a small molecule.

[0276] The present invention also provides a method for delivering a bioactive agent to cells, comprising contacting the cells with the compositions disclosed herein. In some embodiments, the cells are hepatocytes, such as liver cells.

[0277] The present invention also provides a gene editing method comprising contacting cells with the composition disclosed herein.

[0278] The present invention also provides the use of the compositions disclosed herein for gene editing or DNA cutting.

[0279] The present invention also provides the use of the compositions disclosed herein in the manufacture of reagents for gene editing or DNA cutting; the reagents are preferably pharmaceuticals.

[0280] The present invention also provides a method for cutting DNA, comprising contacting cells with the composition disclosed herein.

[0281] In some embodiments, the methods described herein include administering the composition to a human. In some embodiments, the method includes administering the composition to cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, the method includes administering an mRNA formulated in a first LNP composition and an LNP composition comprising one or more mRNAs, gRNAs, or donors in a second LNP composition. In some embodiments, the method includes administering mRNA and gRNA nucleic acids formulated in a single LNP composition. Attached Figure Description

[0282] Figure 1A-1C (Fig. 1A-1C) shows the editing efficiency of targeting TTR after administration of various LNPs in mice.

[0283] Figure 2A-2C (Fig. 2A-2C) shows the editing efficiency of various LNPs targeting TTR in PCH.

[0284] Figures 3A-3C (Fig. 3A-3C) shows the editing efficiency of targeting TTR after applying various LNPs in NHP.

[0285] Figures 4A-4B (Fig. 4A-4B) shows the circulating ALT and AST levels in rats after administration of various LNPs.

[0286] Figures 5A-5B (Fig. 5A-5B) shows the circulating ALT and AST levels in rats after administration of various LNPs.

[0287] Figures 6A-6B (Fig. 6A-6B) shows the circulating ALT and AST levels in rats after administration of various LNPs. Invention Details

[0289] General definition

[0290] It should be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as “comprising,” “including,” and “having” may have the meanings given to them by U.S. patent law; for example, they may mean “comprising,” “being included,” “having,” etc.; and terms such as “consistently composed of” and “composed of” have the meanings given to them by U.S. patent law.

[0291] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of this invention (especially in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise stated herein or the context clearly contradicts this. Furthermore, it should be noted that a plural form does not necessarily imply that it is plural and should be understood in accordance with the context of the article.

[0292] The use of “or” or “ / ” is inclusive and means “and / or” unless otherwise stated; or it may be interpreted differently depending on the context.

[0293] When “t” or “T” appears in the sequence of this invention as a nucleotide of an RNA sequence, it should be understood as “u” or “U”.

[0294] In the context of two or more nucleic acid or polypeptide sequences, the term "identity" refers to two or more sequences or subsequences being identical, or having the same percentage of amino acid residues or nucleotides when measured using a sequence comparison algorithm such as BLAST or BLAST 2.0 or FASTA with default parameters.

[0295] As used herein, when referring to measurable values ​​(such as parameters, quantities, durations of time, etc.), the term “about” means to encompass the range of the specified value and its variations, provided that such variations are appropriate for the disclosed information. It should be understood that the value referred to by the modifier “about” is itself specifically and preferably disclosed.

[0296] As used herein, the term "exemplary" is used to indicate that something is used as an example, instance, or illustration. Any aspect or design described herein as "exemplary" should not be construed as being more preferred or advantageous than other aspects, embodiments, or designs.

[0297] As used herein, the terms “optional” or “optionally” mean that the events, situations, or substituents described below may or may not occur, and the description includes both cases where the events occur and cases where the events do not occur.

[0298] Unless otherwise defined herein, scientific and technical terms relating to this disclosure shall have meanings as commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms.

[0299] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, as well as adenine and guanine (see Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). This technique considers any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variant thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The polymer or oligomer may be heterogeneous or homogeneous in composition and may be isolated from natural sources or produced artificially or synthetically. Furthermore, nucleic acids may be DNA or RNA, or mixtures thereof, and may exist permanently or temporarily in single-stranded or double-stranded form, including homodouble helices, heterodouble helices, and hybrid states. In some embodiments, nucleic acids or nucleic acid sequences include other types of nucleic acid structures, such as, for example, DNA / RNA helices, peptide nucleic acids (PNAs) (see Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002) and U.S. Patent No. 5,034,506), locked nucleic acids (LNAs; see Wahllestedt et al., Proc. Natl. Acad. Sci. USA, 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122:8595-8602 (2000)), and / or ribozymes. Therefore, "nucleic acid" or "nucleic acid sequence" can also encompass chains containing non-natural nucleotides, modified nucleotides, and / or non-nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., "nucleotide analogs"); further, as used herein, "nucleic acid sequence" refers to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, as well as DNA or RNA of genomic or synthetic origin, which can be single-stranded or double-stranded and represent a sense or antisense strand. The terms "nucleic acid," "polynucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or their analogs.

[0300] It should be noted that the specific embodiments are not intended as an exhaustive description or limitation of the broader aspects discussed herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and may be practiced in conjunction with other embodiments(s). Throughout this specification, references to “in some embodiments,” “in some preferred embodiments,” “in some typical embodiments,” “in typical embodiments,” or similar expressions mean that a particular feature, structure, or characteristic associated with that embodiment is included in at least one embodiment of this disclosure. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner, as will be apparent to those skilled in the art from this disclosure, in one or more embodiments. Moreover, while some embodiments described herein include, but are not limited to, features contained in other embodiments, combinations of features from different embodiments are intended to fall within the scope of this disclosure. For example, any embodiment claimed in the appended claims may be used in any combination.

[0301] All publications, published patent documents and patent applications are incorporated herein by reference to the extent that each individual publication, published patent document or patent application is specifically and separately indicated as incorporated by reference.

[0302] Ionizable lipids

[0303] This invention is partly based on the discovery of novel ionizable or amino lipids that are advantageous for delivering active or therapeutic agents (such as nucleic acids) into in vivo or in vitro lipid nanoparticles for mammalian cells.

[0304] Ionizable lipids are key components of lipid nanoparticles (LNPs) used in delivery systems. These lipids are characterized by their ability to ionize, meaning they can alter their charge state in response to pH changes. This typically involves the protonation or deprotonation of functional groups (such as amines or carboxylic acids) present in the lipid structure. Ionizable lipids play a crucial role in the formation and stability of LNPs and are important in facilitating the encapsulation, protection, and cellular uptake of nucleic acids such as RNA and DNA. Due to their pH-sensitive properties, ionizable lipids facilitate endosome escape of cargo molecules, improving the efficiency of gene silencing or expression in target cells. Therefore, they are essential for developing LNP-based delivery systems for applications in gene therapy, vaccines, and other nucleic acid-based interventions.

[0305] In some embodiments, the present invention provides nucleic acid-lipid nanoparticle compositions comprising one or more novel ionizable lipids described herein, which enhance the activity of nucleic acids and improve the tolerability of the compositions in vivo or in vitro, and significantly increase the therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions.

[0306] In some embodiments, the present invention provides novel ionizable lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions can be used for the expression of proteins encoded by mRNA. In other embodiments, the lipid nanoparticles can also be used to deliver mRNA and plasmids to express transgenes. In yet another embodiment, the lipid nanoparticle compositions can be used to induce pharmacological effects resulting from protein expression, such as increasing red blood cell production by delivering appropriate erythropoietin mRNA, or combating infection by delivering mRNA encoding appropriate antigens or antibodies.

[0307] The cationic lipids or ionizable lipids described in this invention are as follows:

[0308] Formula (I):

[0309] (I),

[0310] Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug.

[0311] in:

[0312] R1 is hydrogen; phenyl; 3- to 7-membered aliphatic ring; 3- to 7-membered heterocyclic group containing 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 5- to 6-membered monocyclic heteroaryl group containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; 8- to 10-membered bicyclic heteroaryl group containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; -OR'; or ; wherein the phenyl, cycloaliphatic, 3- to 7-membered heterocyclic, 5- to 6-membered monocyclic heteroaryl, and 8- to 10-membered bicyclic heteroaryl groups are optionally substituted by one or more substituents independently selected from the group consisting of halogens, hydroxyl groups, or thiols;

[0313] R' is selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, wherein the C1-C8 alkyl, C2-C8 alkenyl and C2-C8 alkynyl are each optionally substituted by one or more substituents independently selected from the group consisting of: C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or thiol.

[0314] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, -S-C3-13 alkyl, and -CH2-S-C3-13 alkyl; wherein the C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, and C3-13 alkyl moieties are each optionally substituted by one or more substituents independently selected from the group consisting of: C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or thiol;

[0315] R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, or -C(=O)R8; wherein the C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl are each optionally substituted by one or more substituents independently selected from the group consisting of: C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or thiol;

[0316] R8 is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl groups;

[0317] X1 is selected from the group consisting of -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R 12 )-、-(R 12 )NS(O)2O-、-N(R 12 )S(O)2N(R 13 )-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)N(R 12 )-、-(R 12 )NC(=O)O-、-OC(=S)N(R 12 )-、-(R 12 )NC(=S)O-、-SC(=O)N(R 12 )- or -(R 12 )NC(=O)S-;

[0318] R 12 and R 13 Each is independently selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, -C(=O)R8; wherein the C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl are each optionally substituted by one or more substituents independently selected from the group consisting of: C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or thiol;

[0319] X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)O-, -C(=O)S-, -SC(=O)-;

[0320] L0 and L1 are each independently selected from a bond, a C1-C8 alkylene group, a C2-C8 alkenyl group, or a C2-C8 ynynyl group; wherein the C1-C8 alkylene group, the C2-C8 alkenyl group, and the C2-C8 ynynyl group are each optionally substituted by one or more substituents independently selected from the group consisting of halogens, hydroxyl groups, or thiols;

[0321] L2 and L3 are each independently selected from the group consisting of a bond, a C1-C18 alkylene group, a C2-C18 alkenyl group, or a C2-C18 ynynyl group, wherein the C1-C18 alkylene group, the C2-C18 alkenyl group, and the C2-C18 ynynyl group are each optionally substituted by one or more substituents independently selected from the group consisting of C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or thiol.

[0322] The present invention also provides a compound as shown in formula (II):

[0323]

[0324] Or the N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound.

[0325] R1 is selected from -OR', R' is selected from hydrogen or C1-C8 alkyl groups;

[0326] R' is selected from hydrogen or C1-C8 alkyl; wherein each of the C1-C8 alkyl groups is optionally substituted by one or more substituents independently selected from halogen, hydroxyl or mercapto.

[0327] R2, R3, R4, and R5 are each independently selected from hydrogen, C5-C18 alkyl, C5-C18 alkenyl, and -S-(CH2). 2-10 CH3 or -(CH2)2-S-(CH2) 2-10 CH3; including C5-C18 alkyl, C5-C18 alkenyl, -S-(CH2) 2-10 CH3 and -(CH2)2-S-(CH2) 2- 10 Each of the CH3 groups is optionally substituted by one or more independent substituents selected from halogen, hydroxyl or thiol groups;

[0328] R9, R 10 and R 11 Each is independently selected from hydrogen or linear C1 alkyl groups.

[0329] R8 is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl groups;

[0330] X1 is selected from the key, -S-, or -O-;

[0331] X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)O-, -C(=O)S-, -SC(=O)-;

[0332] L0 and L1 are each independently selected from a bond or a C1-C8 alkylene group; wherein the C1-C8 alkylene group is optionally substituted by one or more substituents independently selected from halogen, hydroxyl or mercapto.

[0333] L2, L3, L4 and L5 are each independently selected from a bond, a C1-C18 alkylene group or a C2-C18 alkenyl group, wherein the C1-C18 alkylene group and the C2-C18 alkenyl group are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto groups.

[0334] In some specific embodiments, at least one of X2 and X3 is -OC(=O)S- or -SC(=O)O-.

[0335] In some embodiments, R1 is -OR', and R' is selected from hydrogen or C1-C8 alkyl. In some embodiments, R1 is -OR', where R' is hydrogen. In some embodiments, R1 is... R9, R 10 and R 11 Each is independently selected from hydrogen or C1-C5 alkyl groups. In some embodiments, R1 is... R9, R 10 and R 11 R1 is independently selected from hydrogen or linear C1-C3 alkyl groups. In some embodiments, R1 is selected from... R9, R 10 and R 11 Each is independently selected from hydrogen or linear C1 alkyl groups.

[0336] In some embodiments, L0 is selected from a bond or a C1-C8 alkylene group. In some embodiments, L0 is a linear C1-C5 alkylene group. In some embodiments, L0 is selected from a linear C4 alkylene group, a linear C3 alkylene group, or a linear C2 alkylene group. In some embodiments, L0 is a bond.

[0337] In some embodiments, L1 is selected from a bond or a C1-C8 alkylene group. In some embodiments, L1 is a linear C1-C5 alkylene group. In some embodiments, L1 is selected from a linear C4 alkylene group, a linear C3 alkylene group, or a linear C2 alkylene group. In some embodiments, L1 is a bond.

[0338] In some embodiments, L2 and L3 are independently selected from C3-C18 alkylene groups. In some embodiments, L2 and L3 are independently C3-C10 alkylene groups. In some embodiments, L2 and L3 are independently C5-C8 alkylene groups. In some embodiments, L2 and L3 are independently C8 alkylene groups. In some embodiments, L2 and L3 are independently C7 alkylene groups. In some embodiments, L2 and L3 are independently C6 alkylene groups. In some embodiments, L2 and L3 are independently C5 alkylene groups. In some embodiments, L2 and L3 are independently C4 alkylene groups. In some embodiments, L2 and L3 are independently C3 alkylene groups.

[0339] In some embodiments, L4 and L5 are independently selected from C1-C5 alkylene groups. In some embodiments, L4 and L5 are independently C1 alkylene groups. In some embodiments, L4 and L5 are independently C1 alkylene groups, C2 alkylene groups, or C3 alkylene groups.

[0340] In some embodiments, X1 is a key. In some embodiments, X2 and X3 are independently selected from -OC(=O)S-, -SC(=O)O-, -C(=O)O-, -OC(=O)-, or -OC(=O)O-. In some embodiments, X2 and X3 are independently selected from -OC(=O)S-, -SC(=O)O-, -C(=O)O-, -OC(=O)-, or -OC(=O)O-; and at least one of X2 and X3 is selected from -OC(=O)S- or -SC(=O)O-. In some embodiments, X2 and X3 are independently selected from -OC(=O)S- or -SC(=O)O-. In some embodiments, X2 and X3 are independently selected from -OC(=O)S-, -SC(=O)O-, -C(=O)O-, -OC(=O)-, or -OC(=O)O-; and X3 is selected from -OC(=O)S- or -SC(=O)O-; and X2 is selected from -OC(=O)- or -C(=O)O-. In some embodiments, X2 and X3 are independently selected from -OC(=O)- or -C(=O)O-.

[0341] In some embodiments, R2 and R3 are independently selected from hydrogen, C5-C12 alkyl groups, and -S-(CH2). 2-10 CH3 or -(CH2)2-S-(CH2) 2-10CH3. In some embodiments, R2 and R3 are independently C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, C5 alkyl, or C4 alkyl. In some embodiments, R2 and R3 are independently -S-(CH2)5CH3.

[0342] In some embodiments, R4 and R5 are each independently selected from hydrogen, C5-C12 alkyl, and -S-(CH2). 2-10 CH3 or -(CH2)2-S-(CH2) 2-10 CH3. In some embodiments, R4 and R5 are each independently C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, C5 alkyl, or C4 alkyl. In some embodiments, R4 and R5 are each independently -S-(CH2)5CH3.

[0343] In some embodiments, R3 and R5 are independently –CH2S-(CH2)8CH3, –CH2S-(CH2)7CH3, –CH2S-(CH2)6CH3, –CH2S-(CH2)5CH3, –CH2S-(CH2)4CH3 or –CH2S-(CH2)3CH3; R2 and R4 are independently -S-(CH2)8CH3, -S-(CH2)7CH3, -S-(CH2)6CH3, -S-(CH2)5CH3, -S-(CH2)4CH3 or -S-(CH2)3CH3.

[0344] The present invention also provides a compound as shown in formula (III):

[0345] (III)

[0346] Or the N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound.

[0347] in

[0348] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2-10 CH3;

[0349] X1 is the key;

[0350] X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)O-, -C(=O)S-, -SC(=O-); and at least one of X2 and X3 is selected from -OC(=O)S- or -SC(=O)O-;

[0351] L1 or L0 is independently selected from a bond or a C1-C8 alkylene group;

[0352] L2 and L3 are independently C3-C10 alkylene groups;

[0353] L4 and L5 are each independently selected from C1-3 alkylene groups.

[0354] The present invention also provides a compound as shown in formula (IV):

[0355] (IV),

[0356] Or the N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound.

[0357] in

[0358] R1 is -OH or ;

[0359] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2-10 CH3;

[0360] R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8;

[0361] R8 is a C1-C5 alkyl group;

[0362] X2 is selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-;

[0363] L1 is a C1-C8 alkylene group;

[0364] L2 and L3 are independently C3-C10 alkylene groups;

[0365] L4 and L5 are each independently selected from C1-3 alkylene groups.

[0366] The present invention also provides a compound as shown in formula (V):

[0367] (V)

[0368] Or the N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound.

[0369] in

[0370] R1 is -OH or ;

[0371] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2-10 CH3;

[0372] R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8;

[0373] R8 is a C1-C5 alkyl group;

[0374] X2 is selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -SC(=O)O- or -OC(=O)S-;

[0375] L1 is a C1-C8 alkylene group;

[0376] L2 and L3 are independently C3-C10 alkylene groups;

[0377] L4 and L5 are each independently selected from C1-3 alkylene groups.

[0378] The present invention also provides a compound as shown in formula (VI):

[0379] (VI),

[0380] Or the N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound.

[0381] in

[0382] R1 is -OH or ;

[0383] R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2-10 CH3;

[0384] R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8;

[0385] R8 is a C1-C5 alkyl group;

[0386] X1 is the key;

[0387] L1 is a C1-C8 alkylene group;

[0388] L2 and L3 are independently C3-C10 alkylene groups;

[0389] L4 and L5 are each independently selected from C1-3 alkylene groups.

[0390] In some embodiments, R1 is R9, R 10 and R 11 Each is independently selected from hydrogen or C1-3 linear alkyl groups.

[0391] In some embodiments, L0 is a bond. In some embodiments, L0 is a C2 linear alkylene. In some embodiments, L0 is a C4 linear alkylene. In some embodiments, L0 is a C3 linear alkylene.

[0392] In some embodiments, L1 is a bond. In some embodiments, L1 is a C2 linear alkylene. In some embodiments, L1 is a C4 linear alkylene. In some embodiments, L1 is a C3 linear alkylene.

[0393] In some embodiments, L2 and L3 are independently C5-C8 alkylene groups. In some embodiments, L2 and L3 are independently C6 alkylene groups.

[0394] In some embodiments, L4 and L5 are each independently a bond. In some embodiments, L4 and L5 are each independently a C1 alkylene group. In some embodiments, L4 and L5 are each independently a C2 alkylene group.

[0395] The present invention also provides a compound as shown in formula (VII):

[0396] (VII)

[0397] L0 and L1 are each independently a bond or a C1-C5 alkylene group, wherein the C1-C5 alkylene group is optionally substituted by one or more substituents, which are independently selected from halogens, hydroxyl groups or mercapto groups;

[0398] X1 is selected from the key, -S-, or -O-;

[0399] X2 and X3 are independently selected from -C(=O)O-, -OC(=O-, -OC(=O)O-, -OC(=O)S- or -SC(=O)O-;

[0400] L2 and L3 are each independently selected from C1-C3 alkyl groups, C3-C9 alkylene groups, wherein the C3-C9 alkylene groups are optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl groups, halogens, hydroxyl groups or mercapto groups;

[0401] L4 and L5 are each independently selected from C1-C5 alkylene groups, wherein the C1-C5 alkylene groups are optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl, halogen, hydroxy, or mercapto groups;

[0402] R4 is selected from C1-C18 alkyl, C2-C18 alkenyl; wherein the C1-C18 alkyl and C2-C18 alkenyl are each optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl, halogen, hydroxy or mercapto;

[0403] Among them, L6, L7, L 10 and L 11 Each is independently a bond or a C6-C9 sub-alkenyl group; preferably, L6, L7, L... 10 and L 11 Each is a key independently.

[0404] R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl; wherein the C1-C18 alkyl and C2-C18 alkenyl are each optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl, halogen, hydroxy or mercapto.

[0405] The present invention also provides a compound as shown in formula (VIII):

[0406] (VIII)

[0407] Or the N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound.

[0408] in

[0409] L0 and L1 are each independently a bond or a C1-C5 alkylene group;

[0410] X1 is the key;

[0411] X2 and X3 are independently selected from -C(=O)O-, -OC(=O-, -OC(=O)O-, -OC(=O)S- or -SC(=O)O-;

[0412] L2 and L3 are each independently C3-C9 alkylene groups;

[0413] L4 and L5 are each independently C1-C5 alkylene groups;

[0414] R4 is selected from C1-C18 alkyl, C2-C18 alkenyl;

[0415] L6, L7, L 10 and L 11 Each is an independent key;

[0416] R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

[0417] In some embodiments, L0 is a bond, and L1 is a C1-C5 alkylene group, preferably, L1 is a C2, C3 or C4 alkylene group.

[0418] In some embodiments, X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-.

[0419] In some embodiments, L2 and L3 are each independently C3-C9 alkylene oxides. In some embodiments, L2 and L3 are each independently C3 alkylene oxides. In some embodiments, L2 and L3 are each independently C4 alkylene oxides. In some embodiments, L2 and L3 are each independently C5 alkylene oxides. In some embodiments, L2 and L3 are each independently C6 alkylene oxides. In some embodiments, L2 and L3 are each independently C7 alkylene oxides. In some embodiments, L2 and L3 are each independently C8 alkylene oxides. In some embodiments, L2 and L3 are each independently C9 alkylene oxides.

[0420] In some embodiments, L4 and L5 are each independently C1-C3 alkylene oxides. In some embodiments, L4 and L5 are each independently C1 alkylene oxides. In some embodiments, L4 and L5 are each independently C2 alkylene oxides. In some embodiments, L4 and L5 are each independently C3 alkylene oxides.

[0421] In some embodiments, R4 is selected from L 10 and L 11 Each is an independent bond; R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

[0422] In some embodiments, R2, R3, R5, and R6 are each independently selected from C4-C18 alkyl or C4-C18 alkenyl. In some embodiments, R2, R3, R5, and R6 are each independently selected from C4-C12 alkyl. In some embodiments, R2, R3, R5, and R6 are each C4 alkyl. In some embodiments, R2, R3, R5, and R6 are each C5 alkyl. In some embodiments, R2, R3, R5, and R6 are each C6 alkyl. In some embodiments, R2, R3, R5, and R6 are each C7 alkyl. In some embodiments, R2, R3, R5, and R6 are each C8 alkyl. In some embodiments, R2, R3, R5, and R6 are each C9 alkyl. In some embodiments, R2, R3, R5, and R6 are each C10 alkyl. In some embodiments, R2, R3, R5, and R6 are each C11 alkyl. In some embodiments, R2, R3, R5, and R6 are each C12 alkyl.

[0423] The present invention also provides a compound as shown in formula (IX):

[0424] (IX)

[0425] Or the N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound.

[0426] in

[0427] L0 and L1 are each independently a bond or a C1-C5 alkylene group, wherein the C1-C5 alkylene group is optionally substituted by one or more substituents, which are independently selected from halogens, hydroxyl groups or thiols;

[0428] X1 is selected from the key, -S-, or -O-;

[0429] X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=O)S- or -SC(=O)O-;

[0430] L2 and L3 are each independently selected from C3-C8 alkylene groups, wherein the C3-C8 alkylene groups are optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl groups, halogens, hydroxyl groups or thiols;

[0431] L4 and L5 are each independently selected from C1-C5 alkylene groups, wherein the C1-C5 alkylene groups are optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl groups, halogens, hydroxyl groups or thiols;

[0432] R4 is selected from , , C1-C18 alkyl, C2-C18 alkenyl; wherein the C1-C18 alkyl and C2-C18 alkenyl are each optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl, halogen, hydroxyl or thiol;

[0433] Among them, L6, L7, L 10 and L 11 Each is an independent key;

[0434] R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl; wherein the C1-C18 alkyl and C2-C18 alkenyl are each optionally substituted by one or more substituents, which are independently selected from C1-C3 alkyl, halogen, hydroxyl or thiol.

[0435] The present invention also provides a compound as shown in formula (X):

[0436] (X)

[0437] Or the N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound.

[0438] in

[0439] L0 and L1 are each independently a bond or a C1-C5 alkylene group;

[0440] X1 is the key;

[0441] X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=O)S- or -SC(=O)O-;

[0442] L2 and L3 are each independently C3-C8 alkylene groups;

[0443] L4 and L5 are each independently C1-C5 alkylene groups;

[0444] L6, L7, L 10 and L 11 Each is an independent key;

[0445] R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

[0446] In some embodiments, L0 is a bond and L1 is a C1-C5 alkylene group.

[0447] In some embodiments, X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-.

[0448] In some embodiments, L2 and L3 are each independently C5-C8 alkylene groups.

[0449] In some embodiments, L4 and L5 are each independently C1-C3 alkylene groups.

[0450] In some embodiments, R4 is selected from L 10 and L 11 Each is an independent bond; R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

[0451] In some embodiments, R2, R3, R5, and R6 are each independently selected from C4-C18 alkyl or C4-C18 alkenyl. In some embodiments, R2, R3, R5, and R6 are each independently selected from C4-C12 alkyl. In some embodiments, R2, R3, R5, and R6 are each C4 alkyl. In some embodiments, R2, R3, R5, and R6 are each C5 alkyl. In some embodiments, R2, R3, R5, and R6 are each C6 alkyl. In some embodiments, R2, R3, R5, and R6 are each C7 alkyl. In some embodiments, R2, R3, R5, and R6 are each C8 alkyl. In some embodiments, R2, R3, R5, and R6 are each C9 alkyl. In some embodiments, R2, R3, R5, and R6 are each C10 alkyl. In some embodiments, R2, R3, R5, and R6 are each C11 alkyl. In some embodiments, R2, R3, R5, and R6 are each C12 alkyl.

[0452] In this invention, "alkyl" refers to a monovalent saturated hydrocarbon group obtained from an aliphatic hydrocarbon by removing a hydrogen atom, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, etc. Alkyl groups can be cyclic or acyclic. Alkyl groups can be branched or linear (e.g., linear). Alkyl groups can also be substituted or unsubstituted. For example, an alkyl group can be substituted by one or more groups including, but not limited to, the following: alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halogen, hydroxyl, nitro, silyl, sulfoxide, sulfonate, carboxylate, or thiol, as described herein.

[0453] In some embodiments, the C1-C18 alkyl groups described herein may include C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, or C18 alkyl. In some embodiments, the alkyl group is branched or straight-chain alkyl. The term "alkenyl," as used herein, refers to an aliphatic group containing at least one carbon-carbon double bond and is intended to include both unsubstituted alkenyl and substituted alkenyl groups, the latter referring to alkenyl groups having substituents that replace one or more hydrogen atoms on one or more carbons. Such substituents may occur with or without substituents on one or more carbons of one or more double bonds. Furthermore, such substituents include all those substituents considered for the alkyl group, as discussed below, except where stability does not permit. For example, an alkenyl group may be substituted with one or more alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups. Exemplary alkenyl groups include, but are not limited to, vinyl (-CH=CH2-), propenyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

[0454] In some embodiments, the C2-C18 alkenyl groups described herein may include C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl, C14 alkenyl, C15 alkenyl, C16 alkenyl, C17 alkenyl, or C18 alkenyl. In some embodiments, the alkenyl group is a branched alkenyl or a straight-chain alkenyl.

[0455] In some embodiments, the C2-C18 ynyl group described herein may include C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 ynyl groups. In some embodiments, the ynyl group is a branched ynyl group or a straight-chain ynyl group.

[0456] In this invention, "alkylene" refers to a divalent group obtained by removing two hydrogen atoms from a saturated hydrocarbon, which can be branched or linear (e.g., linear). Any monovalent alkyl group described above can be converted into an alkylene group by removing a second hydrogen atom from an alkyl group. Representative alkylene groups include C2-4 alkylene groups and / or C2-3 alkylene groups. Typical alkylene groups include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc. Alkylene groups can also be substituted or unsubstituted. For example, an alkylene group can be substituted by one or more of the following groups, including but not limited to: alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halogen, hydroxyl, nitro, silyl, sulfoxide, sulfonate, carboxylate, or thiol, as described herein.

[0457] In some embodiments, the C1-C18 alkylene compounds described herein may include C3 alkylene compounds, C4 alkylene compounds, C5 alkylene compounds, C6 alkylene compounds, C7 alkylene compounds, C8 alkylene compounds, C9 alkylene compounds, C10 alkylene compounds, C11 alkylene compounds, C12 alkylene compounds, C13 alkylene compounds, C14 alkylene compounds, C15 alkylene compounds, C16 alkylene compounds, C17 alkylene compounds, or C18 alkylene compounds.

[0458] In this invention, the term "alkenyl" includes a divalent, straight-chain or branched, unsaturated acyclic hydrocarbon group having at least one carbon-carbon double bond, and in one embodiment, no carbon-carbon triple bond. Any of the above-described monovalent alkenyl groups can be converted into an alkenyl group by removing a second hydrogen atom from the alkenyl group. Representative alkenyl groups include C2-C6 alkenyl groups.

[0459] In some embodiments, the C2-C18 alkylene groups described herein may include C3 alkylene groups, C4 alkylene groups, C5 alkylene groups, C6 alkylene groups, C7 alkylene groups, C8 alkylene groups, C9 alkylene groups, C10 alkylene groups, C11 alkylene groups, C12 alkylene groups, C13 alkylene groups, C14 alkylene groups, C15 alkylene groups, C16 alkylene groups, C17 alkylene groups, or C18 alkylene groups.

[0460] In some embodiments, the C2-C18 ynylene group described herein may include C3 ynylene, C4 ynylene, C5 ynylene, C6 ynylene, C7 ynylene, C8 ynylene, C9 ynylene, C10 ynylene, C11 ynylene, C12 ynylene, C13 ynylene, C14 ynylene, C15 ynylene, C16 ynylene, C17 ynylene, or C18 ynylene.

[0461] In this invention, the terms "Cx-y", "Cx-Cy", or "Cx" (where x or y independently represents an integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18…) when used in conjunction with a chemical moiety (such as alkyl or alkylene), refer to a group containing x to y carbon atoms, or a group containing x carbon atoms in a chain. In some embodiments, it may include any integer value of carbon atoms from x to y (e.g., in some embodiments, C1-8 alkyl may include C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, or C8 alkyl). In some embodiments, for example, the terms "Cx-y alkyl" (such as C1-8 alkyl or C1-18 alkyl) or "Cx-Cy alkyl" (such as C1-C8 alkyl or C1-C18 alkyl) herein include substituted or unsubstituted saturated hydrocarbon groups, including straight-chain and branched alkyl groups containing x to y carbons. For example, C8 alkyl refers to a substituted or unsubstituted saturated hydrocarbon group containing 8 carbons, including straight-chain and branched alkyl groups.

[0462] In some embodiments, the -SC described herein 3-13 The alkyl group may include -S-C3 alkyl, -S-C4 alkyl, -S-C5 alkyl, -S-C6 alkyl, -S-C7 alkyl, -S-C8 alkyl, -S-C9 alkyl, -S-C10 alkyl, -S-C11 alkyl, -S-C12 alkyl, or -S-C13 alkyl. In some embodiments, the alkyl group is a branched alkyl group or a straight-chain alkyl group.

[0463] In some embodiments, the -CH2-SC described herein 3-13 Alkyl groups may include -CH2-S-C3 alkyl, -CH2-S-C4 alkyl, -CH2-S-C5 alkyl, -CH2-S-C6 alkyl, -CH2-S-C7 alkyl, -CH2-S-C8 alkyl, -CH2-S-C9 alkyl, -CH2-S-C10 alkyl, -CH 2- S-C11 alkyl, -CH2-S-C12 alkyl, or -CH2-S-C13 alkyl. In some embodiments, the alkyl group is a branched alkyl group or a straight-chain alkyl group.

[0464] In this invention, the term "alkoxy" refers to any alkyl moiety linked by an oxygen bridge (i.e., -OC). 1-3 Alkyl groups, wherein C1-3 alkyl groups are as defined herein. Examples of such groups include, but are not limited to, methoxy, ethoxy, and propoxy groups.

[0465] In this invention, the term "cycloalkyl" refers to a saturated monocyclic, bicyclic, or tricyclic hydrocarbon ring having a specified number of carbon atoms. For example, C3.7 cycloalkyl refers to a cycloalkyl ring having 3 to 7 carbon atoms. A cycloalkyl group may optionally be substituted with one or more substituents. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, adamantyl, etc. In some embodiments, the atoms forming the cycloalkyl group may be substituted with atoms other than carbon, such as oxygen, nitrogen (e.g., -NH-, -N(alkyl)-, or -N(aryl)-), and sulfur (e.g., -S-, -S(=O)-, or -S(=O)2-).

[0466] Composition

[0467] This invention provides a composition comprising the ionizable lipids disclosed herein. The lipid composition may be provided in the form of a particulate composition. These particles may be, for example, microspheres (including monolayer and multilayer vesicles, such as "liposomes"—in some embodiments, substantially spherical lamellar lipid bilayers, and in more specific embodiments, may contain a water core, such as containing predominantly RNA molecules), a dispersed phase in an emulsion, a micelle, or an internal phase in a suspension.

[0468] According to an exemplary embodiment, the particles comprise ionizable lipids (such as compounds described herein), cholesterol, neutral lipids (e.g., DSPC), and PEGylated lipids (e.g., DMG-PEG); mixed in various molar:molar ratios.

[0469] In some embodiments, the composition is a lipid nanoparticle (LNP) composition. In some embodiments, the lipid component includes non-cationic lipids and PEGylated lipids. In some embodiments, the non-cationic lipids are neutral lipids.

[0470] The term "neutral lipid" suitable for the lipid compositions of the present invention includes, for example, various neutral, uncharged, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present invention include, but are not limited to: 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), bisstearoylphosphatidylcholine (DSPC), phosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-bisstearoyl-sn-glycerol-3-phosphatidylcholine (DAPC), phosphatidylethanolamine (PE), egg yolk phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), l-myristoyl-2-palmitoylphosphatidylcholine (MPPC), l-palmitoyl-2-myristoylphosphatidylcholine (… PMPC, 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-disarachidonico-sn-glycerol-3-phosphatecholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-docosahexaenooyl-sn-glycerol-3-phosphatecholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), isophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), isophosphatidylethanolamine, and combinations thereof. In one embodiment, the neutral phospholipid is selected from bisstearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE).

[0471] PEGylated lipids or PEG derivatives are used interchangeably in this document and refer to lipid molecules that have been covalently linked to one or more polyethylene glycol (PEG) chains; this process is called PEGylation. This modification significantly alters the physicochemical and biological properties of lipids. PEG linkage offers several advantages, including increased solubility, improved stability, reduced immunogenicity, and / or enhanced circulating half-life.

[0472] In some embodiments, the PEG derivative or PEGylated lipid may be coupled to one or more other molecules, such as lipids. In some embodiments, the PEGylated lipid is selected from, but not limited to: PEG-DMG, 3-N-(methoxy polyethylene glycol 2000)carbamoyl-1,2-dimyristylglycerol, PEG-CDMA, 3-N-(methoxy polyethylene glycol 2000)carbamoyl-1,2-dimyristyloxypropylamine, PEG-CDSA, 3-N-(methoxy polyethylene glycol 2000)carbamoyl-1,2-distearate, DSPE-PEG, PEG-maleimide, DSPE-PEG-maleimide, or combinations thereof. Each possibility represents a single embodiment of the invention.

[0473] In some embodiments, the composition further comprises a bioactive agent. In some embodiments, the bioactive agent comprises a polypeptide, optionally used in combination with a nucleic acid. In some embodiments, the bioactive agent comprises a nucleic acid, such as RNA. In some embodiments, the composition comprises a nucleic acid component. In some representative embodiments, the composition comprises a nucleic acid such as siRNA, miRNA, shRNA, antisense RNA, etc. In some embodiments, the nucleic acid component comprises DNA, which may be referred to as a DNA component. In some embodiments, the nucleic acid component comprises RNA. In some embodiments, the RNA component may comprise mRNA, such as mRNA encoding an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is a Cas nuclease. In some embodiments, the composition may comprise mRNA encoding Cas9, Cpf1, c2c2, cas12i, cas12f, cas12b, cas12c, cas12d, cas12e, cas12g, cas12j, or cas12k. In some embodiments, the composition may comprise gRNA. In some compositions comprising mRNA encoding an RNA-guided DNA binder, the composition further comprises a gRNA nucleic acid such as gRNA. In some embodiments, the composition comprises an RNA-guided DNA binder and gRNA. In some embodiments, the composition comprises Cas nuclease mRNA and gRNA. In some embodiments, the composition comprises two types of Cas nuclease mRNA and gRNA. In some embodiments, the composition comprises (e.g., an expression cassette) a nucleic acid encoding the gRNA described herein.

[0474] The term “Cas nuclease” is used in this application and includes Cas lysin, Cas cleavage enzyme, and dCas DNA binder. “Guide RNA” and “gRNA” are used interchangeably herein to refer to the conjugated guide nucleic acid of an RNA-guided DNA binder. Guide RNA may include modified RNA as described herein. gRNA may be crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. trRNA may be a naturally occurring sequence or a trRNA sequence modified or mutated compared to a naturally occurring sequence. In some embodiments, sgRNA is “Cas9 sgRNA” capable of RNA-guided DNA cleavage mediated by the Cas9 protein. In some embodiments, sgRNA is “Cpf1 sgRNA” capable of RNA-guided DNA cleavage mediated by the Cpf1 protein. In some embodiments, the gRNA includes crRNA and tracrRNA sufficient to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In some embodiments, the gRNA includes crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage.

[0475] In some embodiments, lipid compositions such as LNP compositions comprise modified nucleic acids, including modified RNA. Modified nucleosides or nucleotides, such as gRNA or mRNA, may be present in the RNA. gRNA or mRNA containing one or more modified nucleosides or nucleotides, for example, is referred to as “modified” RNA, to describe the presence of one or more non-natural and / or naturally occurring components or configurations used to replace or add to typical A, G, C, and U residues. In some embodiments, the modified RNA is synthesized using atypical nucleosides or nucleotides, referred to herein as “modified.”

[0476] Modified nucleosides or nucleotides may include one or more of the following: (i) altering, for example, replacing one or two unlinked phosphate groups and / or one or more linked phosphate groups in a phosphodiester backbone (an exemplary backbone modification); (ii) altering, for example, replacing a portion of the ribose, such as the 2' hydroxyl group on the ribose (an exemplary sugar modification); (iii) completely replacing the phosphate portion with a "dephosphated" linker (an exemplary backbone modification); (iv) modifying or replacing naturally occurring nucleobases, including with atypical nucleobases (an exemplary base modification); (v) replacing or modifying the ribose-phosphate backbone (an exemplary backbone modification); (vi) modifying the 3' or 5' end of a polynucleotide, for example, by removing, modifying, or replacing the terminal phosphate group or linker, cap, or adapter (such 3' or 5' cap modification may include sugar and / or backbone modifications); and (vii) modifying or replacing sugars (an exemplary sugar modification). Some embodiments include modifications to the 5' end of mRNA, gRNA, or nucleic acid. Some embodiments include modifications to mRNA, gRNA, or nucleic acid. Some embodiments include modification of the 3' end of mRNA, gRNA, or nucleic acid. The modified RNA may contain both 5' and 3' end modifications. The modified RNA may contain one or more modified residues at non-terminal positions. In some embodiments, the gRNA includes at least one modified residue. In some embodiments, the mRNA includes at least one modified residue.

[0477] In some embodiments, RNA or nucleic acids are chemically or biologically modified to make them more stable. Exemplary modifications of RNA or nucleic acids include the depletion of bases (e.g., by deletion or replacement of one nucleotide with another) or modification of bases, such as chemical modification of bases. As used herein, “chemical modification” includes the introduction of chemical properties different from those seen in naturally occurring RNA or nucleic acids, such as covalent modifications, such as the introduction of modified nucleotides (e.g., nucleotide analogs, or side chain groups contained in such RNA or nucleic acid molecules that are not naturally present).

[0478] In some embodiments of backbone modification, the phosphate group of the modified residue can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, the modified residue, such as those present in the modified nucleic acid, can include the complete replacement of the unmodified phosphate moiety with the modified phosphate group described herein. In some embodiments, backbone modification of the phosphate backbone can include changes resulting in uncharged links or charged links with an asymmetric charge distribution.

[0479] Examples of modified phosphate groups include: thiophosphoryl, selenophosphoryl, borophosphate, borophosphate, hydrophosphonate, phosphoramide, alkyl or aryl phosphonates, and phosphate triesters. The phosphorus atom in an unmodified phosphate group is achiral. However, replacing a non-bridging oxygen atom with one of the aforementioned atoms or groups can make the phosphorus atom chiral. Stereoisomeric phosphorus atoms can have an "R" configuration (Rp in this case) or an "S" configuration (Sp in this case). The skeleton can also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate to the nucleoside) with nitrogen (bridging phosphoramide), sulfur (bridging thiophosphoryl), and carbon (bridging methylene phosphonate). Substitution can occur at any or both bridging oxygen sites. In some skeleton modifications, the phosphate group can be replaced with a phosphorus-free connector. In some embodiments, the charged phosphate group can be replaced by a neutral portion. Examples of parts that can replace the phosphate group include, but are not limited to: methylphosphonates, hydroxyamino, siloxanes, carbonates, carboxymethyl, carbamates, amides, thioethers, ethylene oxide linkers, sulfonates, sulfonamides, thioacetals, oximes, methyleneimine, methyleneimine, methylene hydrazine, methylene dimethylhydrazine, and methylene oxomethyleneimine.

[0480] In some embodiments, the compositions or formulations disclosed herein comprise mRNA containing an open reading frame (ORF) encoding an RNA-guided DNA binder (e.g., a Cas nuclease or a class 2 Cas nuclease described herein). In some embodiments, mRNA comprising an ORF encoding an RNA-guided DNA binder (e.g., a Cas nuclease or a class 2 Cas nuclease) is provided, used, or administered. The mRNA may contain one or more 5' caps, 5' untranslated regions (UTRs), 3' UTRs, and polyadenylated tails. The mRNA may contain modified open reading frames, such as those encoding nuclear localization sequences or encoding proteins using alternative codons.

[0481] The mRNA in the disclosed LNP composition may encode, for example, secretory hormones, enzymes, receptors, polypeptides, peptides, or other commonly secreted proteins. In one embodiment of the invention, the mRNA may optionally have chemical or biological modifications that, for example, improve the stability and / or half-life of such mRNA, or improve or otherwise promote protein production.

[0482] Furthermore, suitable modifications include altering one or more codons of nucleotides such that the codon encodes the same amino acid but is more stable than the codons found in the wild-type version of the mRNA. In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by replacing another codon encoding the same or related amino acid with a codon encoding a specific amino acid. The incorporation of pseudouracil into the mRNA nucleotides of the present invention is also considered. Incorporating pseudouracil into the mRNA nucleotides of the present invention can improve stability and translational efficiency, and reduce immunogenicity in vivo. Substitutions and modifications to the mRNAs of the present invention can be made using methods readily known to those skilled in the art.

[0483] The term "modification" also includes incorporating non-nucleotide linkers or modified nucleotides into the mRNA sequence of the present invention, for example, modifying the 3' and 5' ends of mRNA molecules encoding functional secretory proteins or enzymes. These modifications include adding bases to the mRNA sequence (e.g., adding a poly A tail or a longer poly A tail), altering the 3' UTR or 5' UTR, complexing the mRNA with reagents (e.g., proteins or complementary nucleic acid molecules), and adding elements that alter the structure of the mRNA molecule (e.g., forming secondary structures).

[0484] Poly-A tails are considered to stabilize natural messengers. Therefore, in one embodiment, a long poly-A tail can be added to an mRNA molecule to make the mRNA more stable. A variety of well-established techniques can be used to add a poly-A tail. For example, a long poly-A tail can be added to in vitro transcribed mRNA or synthetic mRNA using a poly-A polymerase. Transcription vectors can also encode a long poly-A tail. In one embodiment, the length of the poly-A tail is adjusted as needed to control the stability of the modified mRNA molecule of the present invention, thereby controlling the level of protein transcription. For example, since the length of the poly-A tail can affect the half-life of the mRNA molecule, the length of the poly-A tail can be adjusted to modify the degree of resistance of the mRNA to nucleases, and thus control the time course of protein expression in cells. In one embodiment, stable mRNA molecules are sufficiently resistant to in vivo degradation (e.g., caused by nucleases) such that they can be delivered to target cells without a transfer vector.

[0485] In one embodiment, mRNA can be modified by incorporating 3' and / or 5' untranslated region (UTR) sequences that are not naturally present in wild-type mRNA. In another embodiment, 3' and / or 5' flanking sequences naturally located on either side of the mRNA and encoding a second, unrelated protein can be incorporated into the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein for modification. For example, 3' or 5' sequences can be obtained from stable mRNA molecules and incorporated into the 3' and / or 5' regions of a positive-sense mRNA molecule to increase the stability of the positive-sense mRNA molecule.

[0486] The compositions and methods disclosed herein may include a template nucleic acid. This template can be used to modify or insert a nucleic acid sequence near a target site of an RNA-guided DNA-binding protein, such as a Cas nuclease, for example, a type 2 Cas nuclease. In some embodiments, the method includes introducing the template into a cell. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided for editing at two or more target sites. For example, different templates may be provided for editing a single gene in the cell, or different templates may be provided for editing two different genes in the cell.

[0487] LNPs can be prepared using any method disclosed in existing literature. In some embodiments, LNPs are formed by mixing a water-soluble RNA solution with an organic solvent-based lipid solution. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. For example, the organic solvent may be 100% ethanol. Pharmaceutically acceptable buffers (e.g., for in vivo administration of LNPs) can be used for maintenance.

[0488] In some embodiments, a buffer solution is used to maintain the pH of the LNP-containing composition at or above 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In some embodiments, a buffer solution is used to maintain the pH of the LNP-containing composition at or above 7.0. In some embodiments, the pH range of the composition is from about 7.2 to about 7.7. In additional embodiments, the pH range of the composition is from about 7.3 to about 7.7 or from about 7.4 to about 7.6. In further embodiments, the pH of the composition is from about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH value of the composition can be measured using a micro pH probe.

[0489] In some embodiments, the composition includes a lyophilization protectant. Examples of non-limiting lyophilization protectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may contain up to 10% of a lyophilization protectant, such as sucrose. In some embodiments, the composition may include TSS (tristanosodium citrate sucrose). In some embodiments, the LNP composition may contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of a lyophilization protectant. In some embodiments, the LNP composition may contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of sucrose.

[0490] In some embodiments, the LNP composition may contain a buffer. In some embodiments, the buffer may include phosphate-buffered saline (PBS), Tris buffer, citrate buffer, and mixtures thereof. In some exemplary embodiments, the buffer includes NaCl. In some embodiments, the buffer does not contain NaCl. Exemplary concentrations of NaCl range from about 20 mM to about 45 mM. Exemplary concentrations of NaCl range from about 40 mM to about 50 mM. In some embodiments, the concentration of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Exemplary concentrations of Tris range from about 20 mM to about 60 mM. Exemplary concentrations of Tris range from about 40 mM to about 60 mM. In some embodiments, the concentration of Tris is about 50 mM. In some embodiments, the buffer contains both NaCl and Tris. The LNP composition in some exemplary embodiments contains 5% sucrose and 45 mM NaCl in a Tris buffer. In some exemplary embodiments, the composition contains about 5% w / v sucrose, about 45 mM NaCl, and about 50 mM Tris at a pH of 7.5. The contents of salt, buffer, and antifreeze can be varied to maintain the overall osmotic pressure of the composition.

[0491] In some embodiments, the particle size (diameter) of the particles (surface particles with or without targeting groups) ranges from about 10 nm to about 500 nm. In some embodiments, the particle size (diameter) ranges from about 10 nm to about 350 nm. In some embodiments, the particle size (diameter) ranges from about 50 nm to about 250 nm. In some embodiments, the particle size (diameter) ranges from about 10 nm to about 200 nm. In some embodiments, the particle size (diameter) ranges from about 20 nm to about 200 nm. In some embodiments, the particle size (diameter) ranges from about 50 nm to about 200 nm. In some embodiments, the particle size (diameter) ranges from about 50 nm to about 100 nm; in some embodiments, the particle size (diameter) ranges from about 75 nm to about 200 nm. In some embodiments, the particle size (diameter) ranges from about 75 nm to about 100 nm; in some embodiments, the particle size (diameter) ranges from about 75 nm to about 150 nm; in some embodiments, the particle size (diameter) ranges from about 90 nm to about 200 nm. In some embodiments, the particle size (diameter) ranges from about 100 nm to about 200 nm. In some embodiments, the particle size (diameter) ranges from about 120 nm to about 200 nm. In some embodiments, the particle size (diameter) ranges from about 150 nm to about 200 nm. In some embodiments, the particle size (diameter) ranges from about 50 nm to about 150 nm.

[0492] In some embodiments, the average particle size (diameter) ranges from about 10 nm. In some embodiments, the average particle size (diameter) ranges from about 20 nm. In some embodiments, the average particle size (diameter) ranges from about 30 nm. In some embodiments, the average particle size (diameter) ranges from about 40 nm. In some embodiments, the average particle size (diameter) ranges from about 50 nm. In some embodiments, the average particle size (diameter) ranges from about 60 nm. In some embodiments, the average particle size (diameter) ranges from about 70 nm. In some embodiments, the average particle size (diameter) ranges from about 80 nm. In some embodiments, the average particle size (diameter) ranges from about 90 nm. In some embodiments, the average particle size (diameter) ranges from about 100 nm. In some embodiments, the average particle size (diameter) ranges from about 200 nm. In some embodiments, the average particle size (diameter) ranges from about 50 nm to about 60 nm. In some embodiments, the average particle size (diameter) of the particles (including encapsulated nucleic acids) ranges from approximately 55 nm to approximately 58 nm. In some embodiments, this size is the hydrodynamic diameter.

[0493] In some embodiments, the average particle size (diameter) is approximately 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, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, or 200 nm.

[0494] In some embodiments, the lipid phase may comprise about 30-60% (molar) of ionizable lipids. For example, ionizable lipids may comprise about 40-50% of the lipid phase.

[0495] In some embodiments, the lipid phase may include about 20-70% (molar) of membrane-stabilizing lipids. For example, membrane-stabilizing lipids may comprise about 40-60% of the lipid phase. In some embodiments, one or more membrane-stabilizing lipids may be used in the lipid phase. For example, membrane-stabilizing lipids may include cholesterol (comprising about 30-50% of the lipid phase) and phospholipids (e.g., DSPC), which may comprise about 5-15% of the lipid phase.

[0496] In some embodiments, the additional PEG derivative (conjugated with lipids) may comprise about 0.5-10% of the lipid phase composition.

[0497] In some embodiments, lipids are suspended in an organic solution, such as ethanol. In some embodiments, nucleic acids are in an acetate buffer.

[0498] In some embodiments, nucleic acids can be mixed with a lipid mixture in a microfluidic mixer to form particles that encapsulate / carry nucleic acids.

[0499] As used herein, “contact” refers to establishing a physical connection between two or more entities. For example, contacting mammalian cells with a nanoparticle composition means that the mammalian cells and nanoparticles are made to share a physical connection. In the biological field, well-known methods involve contacting cells with external entities both in vivo and in vitro. For example, nanoparticle compositions are contacted with mammalian cells located within a mammalian body via various routes of administration, such as intravenous, intramuscular, intradermal, and subcutaneous, and can involve varying amounts of nanoparticle composition. Furthermore, one or more mammalian cells can be contacted by a single nanoparticle composition.

[0500] As used herein, “delivery” means providing an entity to a destination. For example, delivering a therapeutic and / or preventative medicine to a subject may involve administering to the subject a nanoparticle composition containing the therapeutic and / or preventative medicine (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cells may involve contacting one or more cells with the nanoparticle composition.

[0501] The composition may also include a salt of one or more compounds. The salt may be a pharmaceutically acceptable salt. As used herein, a “pharmaceutically acceptable salt” refers to a derivative of the disclosed compound, wherein the parent compound is altered by converting an existing acid or base moiety into its salt form (e.g., by reacting the free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, basic residues of mineral or organic acid salts, such as amines; and acidic residues of basic or organic salts, such as carboxylic acids. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, dodecyl sulfate, ethanesulfonate, transbutenedioate, gluconate, glyceryl phosphate, hemisulfate, heptanate, hexanoate, hexafluoroacetate, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecenoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Pharmaceutically acceptable salts of this disclosure include conventional non-toxic salts formed from parent compounds, for example, from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of this disclosure can be synthesized from parent compounds containing basic or acidic moieties using conventional chemical methods. Typically, such salts are prepared by reacting the free acidic or basic forms of these compounds with an appropriate amount of base or acid in water, in an organic solvent, or in a mixture of both; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0502] As used herein, the polydispersity index (PDI) is a ratio that describes the uniformity of the particle size distribution in a system. Smaller values, such as less than 0.3, indicate a narrower particle size distribution. In some embodiments, the PDI may be less than 0.1.

[0503] In some embodiments, the polydispersity index (PDI) of the LNPs disclosed herein can be between about 0.005 and about 0.75. In some embodiments, the PDI of the LNP can be between about 0.01 and about 0.5. In some embodiments, the PDI of the LNP can be between about zero and about 0.4. In some embodiments, the PDI of the LNP can be between about zero and about 0.35. In some embodiments, the PDI of the LNP can be between about zero and about 0.35. In some embodiments, the PDI of the LNP can be between about zero and about 0.3. In some embodiments, the PDI of the LNP can be between about zero and about 0.25. In some embodiments, the PDI of the LNP can be between about zero and about 0.2. In some embodiments, the PDI of the LNP can be less than about 0.02, 0.05, 0.08, 0.1, 0.15, 0.2, or 0.4.

[0504] While the invention has been described in conjunction with the illustrated embodiments, it should be understood that they are not intended to limit the invention to these embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents, including equivalents of specific features that may be included within the invention as defined by the appended claims.

[0505] The foregoing general and detailed descriptions, as well as the following examples, are exemplary and illustrative, and do not constitute a limitation on the teaching content. The subheadings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. Unless otherwise stated, all scopes given in this application include endpoints. Example

[0506] Example 1: Synthesis of the compound

[0507] intermediate a

[0508] To a solution of 6-bromohexane-1-thiol (2 g, 10 mmol, 1 equivalent) in acetonitrile (0.04–0.1 M), pyridine (160 mg, 20 mmol, 2 equivalents), DMAP (122 mg, 1 mmol, 0.1 equivalents), and 4-nitrobenzene chlorosulfonate (2 g, 10 mmol, 1.5 equivalents) were added sequentially at room temperature. After stirring for at least 2 hours, decane-1-ol (1.80 g, 10 mmol, 1 equivalent) was added, and the resulting reaction mixture was stirred at room temperature for another 2–24 hours. The reaction mixture was extracted with hexane (20 mL) and washed with water. The resulting aqueous layer was then back-extracted with hexane. The combined hexane layers were dried over anhydrous magnesium sulfate or sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (a gradient of ethyl acetate in hexane or methanol in dichloromethane) to give 1.2 g of product as a clear oil. Mass spectrometry: 380 m / z [M+H].

[0509]

[0510] intermediate b

[0511] To a solution of 6-bromohexane-1-thiol (2 g, 10 mmol, 1 equivalent) in acetonitrile (0.04–0.1 M), pyridine (160 mg, 20 mmol, 2 equivalents), DMAP (122 mg, 1 mmol, 0.1 equivalents), and 4-nitrobenzene chlorosulfonate (2.1 g, 10 mmol, 1.5 equivalents) were added sequentially at room temperature. After stirring for at least 2 hours, heptadecanol (2.42 g, 10 mmol, 1 equivalent) was added, and the resulting reaction mixture was stirred at room temperature for another 2–24 hours. The reaction mixture was extracted with hexane (20 mL) and washed with water. The resulting aqueous layer was then back-extracted with hexane. The combined hexane layers were dried over anhydrous magnesium sulfate or sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (a gradient of ethyl acetate in hexane or methanol in dichloromethane) to give 1.5 g of product as a clear oil. Mass spectrometry: 480.6 m / z [M+H].

[0512]

[0513] intermediate c

[0514] To a solution of 6-bromohexane-1-thiol (2 g, 10 mmol, 1 equivalent) in acetonitrile (0.04–0.1 M), pyridine (160 mg, 20 mmol, 2 equivalents), DMAP (122 mg, 1 mmol, 0.1 equivalents), and 4-nitrobenzene chlorosulfonate (2.1 g, 10 mmol, 1.5 equivalents) were added sequentially at room temperature. After stirring for at least 2 hours, 2-heptyldecane-1-ol (2.56 g, 10 mmol, 1 equivalent) was added, and the resulting reaction mixture was stirred at room temperature for another 2–24 hours. The reaction mixture was extracted with hexane (20 mL) and washed with water. The resulting aqueous layer was then back-extracted with hexane. The combined hexane layers were dried over anhydrous magnesium sulfate or sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (a gradient of ethyl acetate in hexane or methanol in dichloromethane) to give 2.2 g of product as a clear oil. Mass spectrometry: 466.6 m / z [M+H].

[0515]

[0516] intermediate d

[0517] To a solution of 6-bromohexane-1-ol (2.32 g, 10 mmol, 1.0 equivalence) and 3,3-bis(hexylthio)propionic acid (3.06 g, 10 mmol, 1.0 equivalence) in dichloromethane (20 mL), DMAP (122 mg, 1 mmol, 0.1 equivalence), DIPEA (3.9 g, 30 mmol, 3 equivalence), and EDC HCl (1.91 g, 10 mmol, 1.0 equivalence) were added sequentially at room temperature. The reaction mixture was stirred at room temperature for at least 5 hours, concentrated under reduced pressure, and purified directly by silica gel chromatography (ethyl acetate gradient in hexane) to give 4.1 g of product as a clear oil. Mass spectrometry: 470.6 m / z [M+H].

[0518]

[0519] intermediate e

[0520] To a solution of 6-bromohexane-1-ol (2.32 g, 10 mmol, 1.0 equivalence) and 2-hexyldecanoic acid (3.06 g, 10 mmol, 1.0 equivalence) in dichloromethane (20 mL), DMAP (122 mg, 1 mmol, 0.1 equivalence), DIPEA (3.9 g, 30 mmol, 3 equivalence), and EDC HCl (1.91 g, 10 mmol, 1.0 equivalence) were added sequentially at room temperature. The reaction mixture was stirred at room temperature for at least 5 h, concentrated under reduced pressure, and purified directly by silica gel chromatography (ethyl acetate gradient in hexane) to give 4.1 g of product as a clear oil. Mass spectrometry: 420.6 m / z [M+H].

[0521]

[0522] intermediate f

[0523] At room temperature, pyridine (160 mg, 20 mmol, 2 equivalents), DMAP (122 mg, 1 mmol, 0.1 equivalents), and chlorosulfonic acid-4-nitrobenzene ester (2.1 g, 10 mmol, 1.5 equivalents) were added sequentially to an acetonitrile solution (0.04–0.1 M) of 6-bromohexane-1-thiol (2 g, 10 mmol, 0.1 equivalents). After stirring for at least 2 hours, (E)-decyl-2-en-1-ol (1.56 g, 10 mmol, 1 equivalent) was added, and the resulting reaction mixture was stirred at room temperature for 2–24 hours. The reaction mixture was extracted with n-hexane (20 mL) and washed with water. The resulting aqueous layer was extracted again with n-hexane. The n-hexane layers were combined, dried over anhydrous magnesium sulfate or sodium sulfate, filtered, and concentrated under vacuum. The crude residue was purified by silica gel chromatography (gradient of ethyl acetate in n-hexane or gradient of methanol in dichloromethane) to give 2.6 g of product as a clear oil. MS: 380.6 m / z [M+H].

[0524]

[0525] intermediate g

[0526] The intermediate was synthesized using the same method as intermediate a, with a yield of 51%. MS: 353.11 m / z [M+H].

[0527]

[0528] intermediate h

[0529] The intermediate was synthesized using the same method as intermediate a, with a yield of 45%. MS: 367.11 m / z [M+H].

[0530]

[0531] intermediate i

[0532] The intermediate was synthesized using the same method as intermediate a, with a yield of 47%. MS: 381.11 m / z [M+H].

[0533]

[0534] intermediate j

[0535] The intermediate was synthesized using the same method as intermediate a, with a yield of 38%. MS: 367.11 m / z [M+H].

[0536]

[0537] intermediate k

[0538] The intermediate was synthesized using the same method as intermediate a, with a yield of 44%. MS: 397.12 m / z [M+H].

[0539]

[0540] intermediate l

[0541] The intermediate was synthesized using the same method as intermediate a, with a yield of 33%. MS: 395.12 m / z [M+H].

[0542]

[0543] intermediate m

[0544] The intermediate was synthesized using the same method as intermediate a, with a yield of 46%. MS: 379.11 m / z [M+H].

[0545]

[0546] intermediate n

[0547] The intermediate was synthesized using the same method as intermediate a, with a yield of 51%. MS: 507.28 m / z [M+H].

[0548]

[0549] intermediate o

[0550] The intermediate was synthesized using the same method as intermediate a, with a yield of 40%. MS: 479.25 m / z [M+H].

[0551]

[0552] intermediate p

[0553] The intermediate was synthesized using the same method as intermediate a, with a yield of 42%. MS: 465.23 m / z [M+H].

[0554]

[0555] intermediate q

[0556] The intermediate was synthesized using the same method as intermediate e, with a yield of 39%. MS: 405.23 m / z [M+H].

[0557]

[0558] intermediate r

[0559] This intermediate was synthesized using the same method as intermediate d, with a yield of 55%. MS: 437.22 m / z [M+H].

[0560]

[0561] intermediate s

[0562] The intermediate was synthesized using the same method as intermediate a, with a yield of 32%. MS: 437.20 m / z [M+H].

[0563]

[0564] Compound: YZL408

[0565] Intermediate a (960 mg, 2 mmol, 2 equiv) was dissolved in acetonitrile (10 mL), and 2-aminoethanol (61 mg, 1 mmol, 1 equiv) and potassium carbonate (411 mg, 3 mmol, 3 equiv) were added at 80°C. After stirring for at least 12 hours, the reaction mixture was extracted with hexane (20 mL) and washed with water. The resulting aqueous layer was then re-extracted with hexane. The combined hexane layers were dried over anhydrous magnesium sulfate or anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in hexane or gradient of methanol in dichloromethane) to give 210 mg of the desired product as a clear oil. 1 H NMR (400 MHz, Chloroform-d) δ 3.58 (td, J = 7.3, 5.1 Hz, 2H), 2.85 (t, J = 7.9 Hz, 8H), 2.55 (t, J = 7.2 Hz, 2H), 2.42 (t, J = 5.0 Hz, 4H), 1.94 (p, J = 7.9 Hz, 8H), 1.50 (tt, J = 7.6, 5.1 Hz, 4H), 1.38 – 1.14 (m, 37H), 0.96 – 0.80 (m, 6H). MS: 695 m / z [M+H].

[0566]

[0567] Compound: YZL410

[0568] The compound was synthesized from intermediate b using the same method as compound 1, with a yield of 35%. 1 HNMR (400 MHz, Chloroform-d) δ 4.73 – 4.44 (m, 1H), 3.58 (td, J = 7.2, 5.0 Hz, 1H), 2.85 (t, J = 7.9 Hz, 2H), 2.55 (t, J = 7.2 Hz, 1H), 2.49 – 2.38 (m, 2H), 2.26 (ddt,J = 13.2, 11.4, 2.6 Hz, 0H), 2.10 – 1.60 (m, 5H), 1.60 – 1.03 (m, 23H), 0.89 (t, J= 7.4 Hz, 6H). MS: 858.70 m / z [M+H].

[0569]

[0570] Compound: YZL412

[0571] The compound was synthesized from intermediates b and a using the same method as compound 1, with a yield of 38%. 1 HNMR (400 MHz, Chloroform-d) δ 4.78 (tt, J = 10.8, 1.5 Hz, 1H), 4.23 (t, J = 4.6Hz, 2H), 3.58 (td, J = 7.2, 5.0 Hz, 2H), 2.85 (t, J = 7.8 Hz, 4H), 2.55 (t, J = 7.2Hz, 2H), 2.49 – 2.37 (m, 4H), 1.94 (p, J = 7.9 Hz, 4H), 1.89 – 1.68 (m, 7H), 1.61 (td, J = 11.7, 5.8 Hz, 1H), 1.57 – 1.39 (m, 8H), 1.39 – 0.94 (m, 31H), 0.89 (t, J =7.7 Hz, 9H). MS:746.55 m / z [M+H].

[0572]

[0573] Compound: YZL413

[0574] This compound was synthesized from intermediate c using the same method as compound 1, with a yield of 28%. 1 HNMR (400 MHz, Chloroform-d) δ 4.16 – 4.07 (m, 2H), 4.00 – 3.89 (m, 2H), 3.85 – 3.70 (m, 2H), 3.41 (tt, J = 12.5, 3.4 Hz, 2H), 3.30 – 3.15 (m, 2H), 2.99 (tdd, J = 12.1,9.9, 2.8 Hz, 2H), 2.49 – 2.38 (m, 1H), 2.36 – 2.20 (m, 5H), 2.23 – 1.98 (m, 2H), 1.96 – 1.78 (m, 3H), 1.78 (td, J = 6.5, 6.0, 2.7 Hz, 3H), 1.78 – 1.49 (m, 11H), 1.52 – 1.31 (m, 9H), 1.31 – 1.13 (m, 27H), 1.12 – 0.93 (m, 5H), 0.89 (t, J = 7.9 Hz, 13H). MS: 859 m / z [M+H].

[0575]

[0576] Compound: YZL424

[0577] This compound was synthesized from intermediates s and d using the same method as compound 1, with a yield of 37%. 1 HNMR (400 MHz, Chloroform-d) δ 4.49 (t, J = 7.0 Hz, 1H), 4.23 (t, J = 7.5 Hz, 2H), 4.15 (t, J = 7.5 Hz, 2H), 3.56 – 3.42 (m, 3H), 3.19 (td, J = 12.2, 1.3 Hz, 1H), 2.95 (d, J = 6.9 Hz, 2H), 2.85 (t, J = 8.0 Hz, 2H), 2.71 (dtd, J = 48.9, 12.1, 4.5Hz, 2H), 2.42 (t, J = 7.5 Hz, 6H), 2.17 (d, J = 12.5 Hz, 1H), 1.94 (p, J = 8.0 Hz, 2H), 1.79 – 1.61 (m, 8H), 1.62 – 1.48 (m, 6H), 1.46 (dd, J = 15.6, 7.8 Hz, 6H), 1.45– 1.08 (m, 23H), 0.89 (t, J = 7.9 Hz, 9H). MS: 751 m / z [M+H].

[0578]

[0579] Compound: YZL427

[0580] This compound was synthesized from intermediates b and e using the same method as compound 1, with a yield of 51%. 1HNMR (400 MHz, Chloroform-d) δ 4.34 (ddd, J = 11.4, 9.1, 2.0 Hz, 1H), 4.14 – 3.99 (m, 2H), 3.98 – 3.87 (m, 1H), 3.53 (ddt, J = 10.8, 5.4, 2.4 Hz, 2H), 2.94 (td, J =12.3, 3.2 Hz, 4H), 2.42 (qd, J = 12.6, 2.3 Hz, 1H), 2.34 – 2.22 (m, 2H), 2.16 –1.95 (m, 2H), 1.96 – 1.66 (m, 9H), 1.63 – 1.50 (m, 8H), 1.49 – 1.09 (m, 54H), 1.00 –0.78 (m, 16H). MS: 841 m / z [M+H].

[0581]

[0582] Compound: YZL400

[0583] This compound was synthesized from intermediates e and f using the same method as compound 1, with a yield of 44%. 1 HNMR (400 MHz, Chloroform-d) δ 6.98 – 6.80 (m, 1H), 5.67 (t, J = 4.9 Hz, 1H), 5.55 (dt, J = 15.0, 6.1 Hz, 1H), 4.15 (t, J = 4.6 Hz, 2H), 3.46 (q, J = 4.7 Hz, 2H), 2.85 (t, J = 7.9 Hz, 2H), 2.47 – 2.36 (m, 8H), 2.28 (dtt, J = 7.1, 4.8, 2.5 Hz, 1H), 2.07 (qd, J = 7.6, 6.9, 1.0 Hz, 2H), 1.94 (tt, J = 7.9, 5.4 Hz, 2H), 1.80 – 1.71 (m, 3H), 1.72 – 1.59 (m, 6H), 1.63 – 1.54 (m, 3H), 1.56 – 1.42 (m, 8H), 1.46 – 1.27 (m, 11H), 1.30 – 1.15 (m, 11H), 1.09 – 0.85 (m, 11H). MS: 741 m / z [M+H].

[0584]

[0585] Compound: YZL409

[0586] This compound was synthesized from intermediate f using the same method as compound 1, with a yield of 38%. 1 HNMR (400 MHz, Chloroform-d) δ 5.71 – 5.57 (m, 4H), 4.86 – 4.80 (m, 4H), 3.58 (td, J = 7.3, 5.1 Hz, 2H), 2.85 (t, J = 7.9 Hz, 4H), 2.55 (t, J = 7.2 Hz, 2H), 2.42 (s, 2H), 2.07 (td, J = 7.8, 5.2 Hz, 4H), 1.94 (p, J = 7.9 Hz, 4H), 1.54 – 1.45 (m, 4H), 1.40 – 1.21 (m, 31H), 0.93 – 0.84 (m, 6H). MS: 658.45 m / z [M+H].

[0587]

[0588] Compound: YZL411

[0589] The compound was synthesized from intermediate b using the same method as compound 1, with a yield of 39%. 1 HNMR (400 MHz, Chloroform-d) δ 4.72 – 4.62 (m, 2H), 3.58 (td, J = 7.1, 5.0 Hz, 2H), 2.85 (t, J = 7.9 Hz, 4H), 2.55 (t, J = 7.1 Hz, 2H), 2.42 (t, J = 6.9 Hz, 4H), 2.39– 2.21 (m, 2H), 2.11 – 2.01 (m, 1H), 1.94 (p, J = 7.8 Hz, 4H), 1.81 (tdd, J = 12.2,8.8, 3.1 Hz, 1H), 1.70 – 1.38 (m, 16H), 1.38 – 1.19 (m, 25H), 1.14 (dddt, J = 14.5,10.0, 6.6, 2.2 Hz, 3H), 0.99 (dddd, J = 17.9, 13.2, 11.5, 8.8 Hz, 2H), 0.89 (t, J= 7.9 Hz, 12H). MS: 858.70 m / z [M+H].

[0590]

[0591] Compound: YZL414

[0592] This compound was synthesized from intermediate k using the same method as compound 1, with a yield of 21%. 1 HNMR (400 MHz, Chloroform-d) δ 3.58 (td, J = 7.3, 5.1 Hz, 2H), 2.85 (t, J = 7.9 Hz, 8H), 2.55 (t, J = 7.2 Hz, 2H), 2.42 (t, J = 5.0 Hz, 4H), 1.94 (p, J = 7.9 Hz, 8H), 1.50 (tt, J = 7.6, 5.1 Hz, 4H), 1.38 – 1.21 (m, 23H), 0.93 – 0.85 (m, 6H). MS: 694.43 m / z [M+H].

[0593]

[0594] Compound: YZL415

[0595] This compound was synthesized from intermediate i using the same method as compound 1, with a yield of 22%. 1 HNMR (400 MHz, Chloroform-d) δ 4.23 (t, J = 7.4 Hz, 4H), 3.58 (td, J = 7.3, 5.1 Hz, 2H), 2.85 (t, J = 8.0 Hz, 4H), 2.55 (t, J = 7.2 Hz, 2H), 2.42 (s, 2H), 2.42 (dd, J =12.3, 2.5 Hz, 2H), 1.94 (p, J = 8.0 Hz, 4H), 1.74 (p, J = 7.6 Hz, 4H), 1.48 (dq, J= 15.7, 7.7 Hz, 8H), 1.38 – 1.21 (m, 19H), 0.96 – 0.82 (m, 6H). MS: 662.48 m / z [M+H].

[0596]

[0597] Compound: YZL416

[0598] This compound was synthesized from intermediate 1 using the same method as compound 1, with a yield of 27%. 1HNMR (400 MHz, Chloroform-d) δ 5.74 (dt, J = 15.2, 6.1 Hz, 2H), 5.59 (dt, J = 15.0, 6.0 Hz, 1H), 5.50 (dt, J = 15.2, 6.1 Hz, 1H), 3.62 – 3.51 (m, 6H), 2.85 (t, J = 7.9Hz, 4H), 2.55 (t, J = 7.2 Hz, 2H), 2.42 (dd, J = 12.2, 2.4 Hz, 2H), 2.11 – 2.03 (m, 4H), 1.94 (p, J = 7.9 Hz, 4H), 1.54 – 1.45 (m, 4H), 1.40 – 1.22 (m, 31H), 0.93 –0.84 (m, 6H). MS: 690.40 m / z [M+H].

[0599]

[0600] Compound: YZL417

[0601] This compound was synthesized from intermediate m using the same method as compound 1, with a yield of 32%. 1 HNMR (400 MHz, Chloroform-d) δ 5.79 – 5.69 (m, 2H), 5.59 – 5.44 (m, 2H), 4.23 (t, J =7.4 Hz, 4H), 3.62 – 3.51 (m, 6H), 2.55 (t, J = 7.2 Hz, 2H), 2.44 – 2.35 (m, 4H), 2.06 (dd, J = 6.8, 5.5 Hz, 3H), 1.74 (t, J = 7.5 Hz, 3H), 1.55 – 1.42 (m, 8H), 1.40 – 1.22 (m, 15H), 0.94 – 0.84 (m, 6H). MS: 658.45 m / z [M+H].

[0602]

[0603] Compound: YZL425

[0604] This compound was synthesized from intermediates i and d using the same method as compound 1, with a yield of 24%. 1HNMR (400 MHz, Chloroform-d) δ 4.46 (t, J = 7.1 Hz, 1H), 4.23 (t, J = 7.5 Hz, 2H), 4.15 (t, J = 7.5 Hz, 2H), 3.46 (td, J = 7.4, 5.0 Hz, 2H), 3.29 (dtd, J = 18.1,12.5, 1.9 Hz, 2H), 3.04 (ddd, J = 12.3, 3.7, 2.3 Hz, 1H), 2.95 (d, J = 7.0 Hz, 2H), 2.85 (t, J = 8.0 Hz, 2H), 2.42 (t, J = 7.5 Hz, 6H), 2.01 – 1.89 (m, 3H), 1.72 (dp, J = 22.1, 7.6 Hz, 7H), 1.62 – 1.42 (m, 13H), 1.43 – 1.10 (m, 21H), 0.89 (t, J = 7.8 Hz, 9H). MS: 750.51 m / z [M+H].

[0605]

[0606] Compound: YZL426

[0607] This compound was synthesized from intermediates i and e using the same method as compound 1, with a yield of 25%. 1 HNMR (400 MHz, Chloroform-d) δ 4.29 (td, J = 11.9, 3.5 Hz, 1H), 4.13 – 4.00 (m, 2H), 3.89 (td, J = 12.2, 2.6 Hz, 1H), 3.54 (tdd, J = 7.7, 5.9, 2.2 Hz, 2H), 3.46 – 3.36 (m, 2H), 2.74 – 2.65 (m, 2H), 2.38 (ddd, J = 7.7, 4.3, 2.0 Hz, 2H), 2.09 – 1.99 (m, 3H), 1.94 – 1.45 (m, 12H), 1.44 – 1.24 (m, 39H), 1.26 – 1.18 (m, 5H), 1.16 – 1.01 (m, 1H), 0.94 – 0.80 (m, 9H). MS:728.61 m / z [M+H].

[0608]

[0609] Compound: YZL430

[0610] This compound was synthesized from intermediates f and b using the same method as compound 1, with a yield of 52%. 1 HNMR (400 MHz, Chloroform-d) δ 5.69 – 5.55 (m, 2H), 4.83 (d, J = 5.0 Hz, 2H), 4.46 – 4.23 (m, 1H), 3.46 (td, J = 7.4, 5.0 Hz, 2H), 2.85 (t, J = 7.9 Hz, 4H), 2.46 – 2.39 (m, 6H), 2.27 (ddtd, J = 17.8, 13.8, 6.5, 5.8, 1.6 Hz, 2H), 2.07 (td, J = 7.7,5.0 Hz, 2H), 1.94 (p, J = 7.9 Hz, 4H), 1.78 – 1.47 (m, 15H), 1.46 – 1.07 (m, 32H), 0.89 (t, J = 7.9 Hz, 9H), 0.84 – 0.74 (m, 1H). MS:786.60 m / z [M+H].

[0611]

[0612] Compound: YZL431

[0613] This compound was synthesized from intermediate c using the same method as compound 1, with a yield of 49%. 1 HNMR (400 MHz, Chloroform-d) δ 4.21 (dd, J = 12.4, 7.0 Hz, 2H), 3.96 (dd, J = 12.4, 7.0 Hz, 2H), 3.46 (td, J = 7.0, 5.4 Hz, 2H), 2.85 (t, J = 7.1 Hz, 4H), 2.42 (t, J =7.1 Hz, 6H), 1.92 (dp, J = 24.7, 7.0 Hz, 6H), 1.73 – 1.63 (m, 3H), 1.61 – 1.52 (m, 2H), 1.51 – 1.40 (m, 6H), 1.37 – 1.14 (m, 49H), 0.94 (q, J = 7.1 Hz, 4H), 0.91 –0.85 (m, 12H). MS: 886.73 m / z [M+H].

[0614]

[0615] Compound: YZL432

[0616] This compound was synthesized from intermediate b using the same method as compound 1, with a yield of 51%. 1 HNMR (400 MHz, Chloroform-d) δ 4.47 (p, J = 7.0 Hz, 4H), 3.46 (td, J = 7.1, 5.0 Hz, 4H), 2.85 (t, J = 7.1 Hz, 8H), 2.42 (t, J = 7.1 Hz, 12H), 1.94 (p, J = 7.1 Hz, 8H), 1.78 – 1.65 (m, 14H), 1.53 (dp, J = 34.5, 7.2 Hz, 12H), 1.42 – 1.17 (m, 105H), 0.94 – 0.85 (m, 12H). MS: 886.73 m / z [M+H].

[0617]

[0618] Compound: YZL433

[0619] The compound was synthesized from intermediates b and j using the same method as compound 1, with a yield of 42%. 1 HNMR (400 MHz, Chloroform-d) δ 4.59 – 4.43 (m, 1H), 4.23 (t, J = 7.5 Hz, 2H), 3.46 (td, J = 7.4, 5.0 Hz, 2H), 2.85 (t, J = 7.9 Hz, 4H), 2.46 – 2.39 (m, 6H), 2.01 – 1.79 (m, 6H), 1.79 – 1.37 (m, 21H), 1.36 – 1.08 (m, 27H), 1.00 – 0.85 (m, 10H). MS:774.60 m / z [M+H].

[0620]

[0621] Compound: YZL439

[0622] This compound was synthesized from intermediate e using the same method as compound 1, with a yield of 40%. 1HNMR (400 MHz, Chloroform-d) δ 5.71 – 5.58 (m, 4H), 4.83 (d, J = 5.2 Hz, 4H), 3.57 (td, J = 7.5, 5.1 Hz, 2H), 2.85 (t, J = 7.8 Hz, 6H), 2.42 (dd, J = 12.3, 1.8 Hz, 2H), 2.11 – 2.03 (m, 4H), 1.94 (p, J = 7.9 Hz, 4H), 1.77 (p, J = 7.6 Hz, 2H), 1.53 – 1.45 (m, 4H), 1.40 – 1.21 (m, 21H), 0.93 – 0.84 (m, 6H). MS: 672.46 m / z [M+H].

[0623]

[0624] Compound: YZL440

[0625] This compound was synthesized from intermediates e and n using the same method as compound 1, with a yield of 39%. 1 HNMR (400 MHz, Chloroform-d) δ 5.87 (ddd, J = 15.2, 6.8, 5.6 Hz, 1H), 5.67 (dt, J =14.9, 5.9 Hz, 1H), 4.83 (dd, J = 6.1, 1.2 Hz, 2H), 4.43 (ddt, J = 10.4, 8.2, 2.0Hz, 1H), 3.57 (q, J = 4.8 Hz, 2H), 2.85 (t, J = 7.8 Hz, 6H), 2.48 – 2.40 (m, 4H), 2.31 (qt, J = 12.5, 2.2 Hz, 1H), 2.11 – 2.02 (m, 2H), 2.01 – 1.89 (m, 7H), 1.88 – 1.79 (m, 1H), 1.77 – 1.67 (m, 3H), 1.67 – 1.45 (m, 11H), 1.45 – 1.20 (m, 24H), 1.12 (dddt, J = 18.0, 15.3, 12.6, 2.4 Hz, 3H), 0.89 (t, J = 7.9 Hz, 10H). MS:800.62m / z [M+H].

[0626]

[0627] Compound: YZL441

[0628] This compound was synthesized from intermediate j using the same method as compound 1, with a yield of 29%. 1 HNMR (400 MHz, Chloroform-d) δ 4.23 (t, J = 7.5 Hz, 8H), 3.57 (td, J = 7.5, 5.1 Hz, 4H), 2.85 (t, J = 7.8 Hz, 12H), 2.49 – 2.37 (m, 8H), 1.94 (p, J = 7.8 Hz, 8H), 1.76 (dp, J = 15.3, 7.6 Hz, 12H), 1.48 (dq, J = 15.7, 7.7 Hz, 16H), 1.37 – 1.22 (m, 58H), 0.89 (s, 6H). MS: 648.46 m / z [M+H].

[0629]

[0630] Compound: YZL442

[0631] This compound was synthesized from intermediates j and n using the same method as compound 1, with a yield of 45%. 1 HNMR (400 MHz, Chloroform-d) δ 4.56 (td, J = 10.9, 2.3 Hz, 1H), 4.23 (t, J = 7.5Hz, 2H), 3.57 (td, J = 7.5, 5.0 Hz, 2H), 2.85 (t, J = 7.9 Hz, 6H), 2.42 (dd, J =12.3, 2.5 Hz, 2H), 2.29 – 2.13 (m, 2H), 1.94 (p, J = 7.8 Hz, 4H), 1.82 – 1.66 (m, 6H), 1.65 – 1.43 (m, 11H), 1.43 – 1.24 (m, 27H), 1.24 – 1.07 (m, 3H), 0.89 (t, J =7.8 Hz, 9H), 0.87 – 0.77 (m, 1H). MS: 787.62 m / z [M+H].

[0632]

[0633] Compound: YZL443

[0634] This compound was synthesized from intermediate k using the same method as compound 1, with a yield of 48%. 1HNMR (400 MHz, Chloroform-d) δ 4.23 (t, J = 4.6 Hz, 8H), 3.57 (td, J = 7.5, 5.1 Hz, 4H), 2.89 – 2.81 (m, 12H), 2.42 (s, 4H), 2.42 (dd, J = 12.7, 2.3 Hz, 4H), 1.98 – 1.89 (m, 7H), 1.82 – 1.70 (m, 12H), 1.55 – 1.42 (m, 16H), 1.38 – 1.20 (m, 40H), 0.93 – 0.85 (m, 6H). MS: 676.49 m / z [M+H].

[0635]

[0636] Compound: YZL444

[0637] This compound was synthesized from intermediate n using the same method as compound 1, with a yield of 41%. 1 HNMR (400 MHz, Chloroform-d) δ 4.47 (p, J = 7.0 Hz, 2H), 3.57 (td, J = 7.1, 5.1 Hz, 2H), 2.85 (t, J = 7.1 Hz, 6H), 2.42 (t, J = 7.1 Hz, 4H), 1.94 (p, J = 7.1 Hz, 4H), 1.80 – 1.67 (m, 7H), 1.50 (p, J = 7.1 Hz, 4H), 1.42 – 1.20 (m, 60H), 0.94 – 0.81 (m, 12H). MS: 928.77 m / z [M+H].

[0638]

[0639] Compound: YZL445

[0640] This compound was synthesized from intermediate c using the same method as compound 1, with a yield of 29%. 1HNMR (400 MHz, Chloroform-d) δ 4.32 – 4.20 (m, 2H), 3.86 (tdd, J = 12.0, 5.5, 3.6Hz, 1H), 3.80 – 3.62 (m, 3H), 3.47 (td, J = 12.4, 3.3 Hz, 1H), 3.40 – 3.28 (m, 1H), 3.22 (td, J = 12.5, 3.2 Hz, 1H), 3.09 – 2.94 (m, 2H), 2.89 (dt, J = 12.4, 3.1 Hz, 1H), 2.78 (td, J = 12.3, 2.6 Hz, 1H), 2.15 – 1.97 (m, 2H), 1.96 – 1.79 (m, 4H), 1.79 – 1.43 (m, 15H), 1.43 – 1.10 (m, 20H), 1.10 – 0.91 (m, 3H), 0.89 (t, J = 7.9 Hz, 15H). MS: 872.71 m / z [M+H].

[0641]

[0642] Compound: YZL446

[0643] This compound was synthesized from intermediates k and n using the same method as compound 1, with a yield of 25%. 1 HNMR (400 MHz, Chloroform-d) δ 4.39 (td, J = 10.4, 3.0 Hz, 1H), 4.23 (t, J = 4.2Hz, 2H), 3.57 (td, J = 7.5, 5.1 Hz, 2H), 2.94 – 2.78 (m, 6H), 2.42 (t, J = 5.4 Hz, 4H), 1.94 (tt, J = 7.7, 5.3 Hz, 5H), 1.89 – 1.70 (m, 8H), 1.68 – 1.19 (m, 36H), 1.18 – 1.03 (m, 3H), 0.89 (t, J = 7.9 Hz, 9H). MS: 802.63 m / z [M+H].

[0644]

[0645] Compound: YZL447

[0646] This compound was synthesized from intermediates i and e using the same method as compound 1, with a yield of 45%. 1HNMR (400 MHz, Chloroform-d) δ 4.30 (td, J = 12.0, 3.6 Hz, 1H), 4.20 – 3.96 (m, 2H), 3.90 (td, J = 12.2, 2.5 Hz, 1H), 3.61 – 3.32 (m, 4H), 2.70 (dtd, J = 15.1,12.3, 2.7 Hz, 2H), 2.43 (td, J = 12.3, 2.8 Hz, 1H), 2.31 (td, J = 12.3, 3.0 Hz, 1H), 2.15 – 1.50 (m, 15H), 1.47 – 1.16 (m, 38H), 1.09 (dtd, J = 12.8, 10.6, 2.3 Hz, 1H), 0.99 – 0.78 (m, 10H). MS: 714.60 m / z [M+H].

[0647]

[0648] Compound: YZL448

[0649] This compound was synthesized from intermediates p and e using the same method as compound 1, with a yield of 23%. 1 HNMR (400 MHz, Chloroform-d) δ 4.44 (ddd, J = 11.3, 9.1, 2.1 Hz, 1H), 4.27 (ddd, J = 12.5, 10.2, 4.4 Hz, 1H), 3.94 – 3.73 (m, 1H), 3.61 – 3.45 (m, 3H), 3.40 (td, J =12.5, 3.3 Hz, 1H), 2.77 – 2.62 (m, 2H), 2.44 (td, J = 12.2, 3.2 Hz, 1H), 2.29 (td,J = 12.2, 3.0 Hz, 1H), 2.18 – 2.08 (m, 3H), 2.07 – 1.95 (m, 2H), 1.94 – 1.55 (m, 13H), 1.54 – 0.99 (m, 30H), 0.89 (t, J = 7.7 Hz, 12H). MS: 798.63 m / z [M+H].

[0650]

[0651] Compound: YZL449

[0652] This compound was synthesized from intermediates d and o using the same method as compound 1, with a yield of 29%. 1 HNMR (400 MHz, Chloroform-d) δ 4.50 (td, J = 12.1, 3.7 Hz, 1H), 4.25 (t, J = 7.0Hz, 1H), 4.17 (dd, J = 12.3, 7.0 Hz, 1H), 4.04 (tdd, J = 12.4, 5.5, 2.8 Hz, 1H), 3.96 – 3.87 (m, 1H), 3.80 (td, J = 12.5, 4.0 Hz, 1H), 3.68 – 3.41 (m, 3H), 3.40 –3.14 (m, 3H), 2.98 (td, J = 12.6, 2.0 Hz, 1H), 2.82 – 2.65 (m, 2H), 2.63 (dt, J =12.4, 3.1 Hz, 1H), 2.53 (td, J = 12.1, 3.7 Hz, 1H), 2.28 (tdd, J = 12.5, 8.1, 2.3Hz, 2H), 2.22 – 1.95 (m, 6H), 1.95 – 1.64 (m, 9H), 1.64 – 0.97 (m, 23H), 0.89 (t, J =7.8 Hz, 12H). MS: 862.64 m / z [M+H].

[0653]

[0654] Compound: YZL450

[0655] This compound was synthesized from intermediates b and o using the same method as compound 1, with a yield of 27%. 1HNMR (400 MHz, Chloroform-d) δ 4.49 (tt, J = 11.5, 2.5 Hz, 1H), 4.22 (dd, J = 12.2, 6.9 Hz, 1H), 3.80 (dd, J = 12.5, 6.9 Hz, 1H), 3.68 (td, J = 12.3, 3.5 Hz, 1H), 3.61 – 3.44 (m, 3H), 3.24 (td, J = 12.5, 3.5 Hz, 1H), 3.11 (tt, J = 12.6, 2.7 Hz, 2H), 2.75 (dtd, J = 31.6, 12.5, 2.6 Hz, 2H), 2.30 – 2.00 (m, 4H), 1.99 – 1.04 (m, 52H), 0.99 (dddd, J = 20.1, 12.9, 6.4, 3.4 Hz, 2H), 0.89 (t, J = 7.9 Hz, 12H). MS: 872.71 m / z [M+H].

[0656]

[0657] Compound: YZL452

[0658] This compound was synthesized from intermediates o and q using the same method as compound 1, with a yield of 28%. 1 HNMR (400 MHz, Chloroform-d) δ 4.59 (td, J = 12.0, 2.8 Hz, 1H), 4.24 (ddd, J =12.7, 5.7, 2.1 Hz, 1H), 3.92 – 3.78 (m, 2H), 3.53 (td, J = 12.5, 3.3 Hz, 1H), 3.41 (dtd, J = 17.3, 12.4, 3.2 Hz, 3H), 3.05 – 2.80 (m, 3H), 2.37 (q, J = 6.9 Hz, 1H), 2.19 (ddt, J = 13.0, 6.9, 3.4 Hz, 1H), 2.08 (dt, J = 12.4, 3.2 Hz, 1H), 2.01 (td,J = 12.4, 2.5 Hz, 2H), 1.96 – 1.71 (m, 8H), 1.71 – 0.95 (m, 41H), 0.89 (td,J =7.9, 4.9 Hz, 13H). MS: 798.69 m / z [M+H].

[0659]

[0660] Compound: YZL453

[0661] This compound was synthesized from intermediates b and q using the same method as compound 1, with a yield of 19%. 1 HNMR (400 MHz, Chloroform-d) δ 4.37 (ddd, J = 11.6, 9.9, 2.0 Hz, 1H), 4.15 – 3.97 (m, 2H), 3.60 – 3.31 (m, 4H), 2.69 (tt, J = 12.2, 2.6 Hz, 2H), 2.46 – 2.15 (m, 4H), 2.13 – 2.00 (m, 3H), 1.95 – 1.03 (m, 62H), 0.97 – 0.71 (m, 15H). MS: 798.69 m / z [M+H].

[0662]

[0663] Compound: YZL455

[0664] This compound was synthesized from intermediates r and i using the same method as compound 1, with a yield of 15%. 1 HNMR (400 MHz, Chloroform-d) δ 5.45 (t, J = 6.2 Hz, 1H), 4.23 (t, J = 7.4 Hz, 2H), 4.15 (t, J = 7.5 Hz, 2H), 3.55 (dd, J = 7.1, 5.0 Hz, 2H), 3.44 – 3.23 (m, 3H), 3.02– 2.92 (m, 1H), 2.85 (t, J = 7.3 Hz, 4H), 2.55 (d, J = 6.0 Hz, 2H), 2.42 (t, J = 7.6Hz, 4H), 1.94 – 1.65 (m, 7H), 1.62 – 1.10 (m, 45H), 0.98 – 0.77 (m, 9H). MS: 732.57m / z [M+H].

[0665]

[0666] Compound: YZL500

[0667] This compound was synthesized using a method similar to that used for compound YZL449, with a yield of 23%. 1H NMR (400MHz, Chloroform-d) δ 4.73 (t, J = 5.8 Hz, 2H), 4.23 (t, J = 7.5 Hz, 4H), 4.15 (t, J = 7.4 Hz, 4H), 3.57 (td, J = 7.5, 5.0 Hz, 4H), 3.19 – 3.00 (m, 8H), 2.85 (t, J =7.9 Hz, 8H), 2.42 (t, J = 7.5 Hz, 8H), 2.35 (t, J = 8.3 Hz, 4H), 2.05 – 1.89 (m, 10H), 1.76 (dp, J = 15.2, 7.6 Hz, 12H), 1.65 – 1.56 (m, 2H), 1.48 (dq, J = 15.6, 7.7 Hz, 24H), 1.37 – 1.21 (m, 47H), 1.11 (dddt, J = 26.4, 19.4, 13.3, 3.1 Hz, 4H), 0.89 (dt, J = 11.3, 2.1 Hz, 9H). LCMS: m / z = 732.6 [M+H]+.

[0668]

[0669] Compound: YZL456

[0670] This compound was synthesized from intermediates i and q using the same method as compound 1, with a yield of 43%. 1HNMR (400 MHz, Chloroform-d) δ 4.51 (dt, J = 12.4, 2.8 Hz, 1H), 4.25 (dt, J = 12.4, 2.9 Hz, 1H), 4.03 – 3.91 (m, 1H), 3.77 (tdd, J = 12.1, 5.5, 3.6 Hz, 1H), 3.69 (dt,J = 12.2, 3.1 Hz, 1H), 3.51 (t, J = 12.1 Hz, 1H), 3.41 (tdd, J = 12.2, 5.6, 3.1Hz, 1H), 3.23 (td, J = 12.3, 3.2 Hz, 1H), 3.02 (td, J = 12.4, 1.6 Hz, 1H), 2.84 (dd, J = 12.4, 2.9 Hz, 1H), 2.67 – 2.58 (m, 1H), 2.56 – 2.33 (m, 4H), 2.22 (p, J =7.0 Hz, 1H), 2.07 – 1.03 (m, 42H), 1.04 – 0.94 (m, 1H), 0.89 (td, J = 7.9, 4.9 Hz, 9H). MS: 700.58 m / z [M+H].

[0671]

[0672] Compound: YZL457

[0673] This compound was synthesized from intermediate a using the same method as compound 1, with a yield of 28%. 1 HNMR (400 MHz, Chloroform-d) δ 4.31 – 4.18 (m, 1H), 3.57 (td, J = 7.5, 5.0 Hz, 0H), 2.98 – 2.78 (m, 1H), 2.59 – 2.37 (m, 1H), 1.94 (p, J = 7.9 Hz, 1H), 1.88 – 1.67 (m, 2H), 1.48 (dq, J = 15.7, 7.6 Hz, 2H), 1.38 – 1.15 (m, 8H), 0.98 – 0.78 (m, 1H). MS: 676.49 m / z [M+H].

[0674]

[0675] Compound: YZL458

[0676] This compound was synthesized from intermediates h and q using the same method as compound 1, with a yield of 20%. 1 HNMR (400 MHz, Chloroform-d) δ 4.24 (td, J = 12.3, 5.9 Hz, 2H), 3.96 (dt, J = 12.5, 2.5 Hz, 1H), 3.71 (ddd, J = 12.4, 8.8, 5.2 Hz, 1H), 3.61 – 3.44 (m, 2H), 3.42 –3.33 (m, 2H), 2.84 (dtd, J = 22.2, 12.1, 2.7 Hz, 2H), 2.65 (td, J = 12.3, 3.8 Hz, 1H), 2.17 (td, J = 12.3, 2.6 Hz, 1H), 2.08 (p, J = 7.0 Hz, 1H), 2.02 – 1.72 (m, 8H), 1.72 – 1.52 (m, 5H), 1.52 – 1.09 (m, 29H), 1.08 – 0.94 (m, 1H), 0.89 (td, J =7.8, 4.7 Hz, 9H). MS: 686.57 m / z [M+H].

[0677]

[0678] Compound: YZL459

[0679] This compound was synthesized from intermediates f and q using the same method as compound 1, with a yield of 41%. 1 HNMR (400 MHz, Chloroform-d) δ 5.77 – 5.53 (m, 2H), 4.92 (dd, J = 12.4, 5.5 Hz, 1H), 4.60 – 4.52 (m, 1H), 4.50 – 4.37 (m, 1H), 3.81 – 3.68 (m, 1H), 3.60 – 3.45 (m, 3H), 3.32 (td, J = 12.5, 3.7 Hz, 1H), 2.84 – 2.57 (m, 2H), 2.49 – 2.10 (m, 5H), 2.10 – 1.92 (m, 3H), 1.92 – 1.01 (m, 42H), 0.89 (td, J = 7.9, 4.5 Hz, 9H). MS: 698.571m / z [M+H].

[0680]

[0681] Compound: YZL460

[0682] This compound was synthesized from intermediates g and q using the same method as compound 1, with a yield of 43%. 1 HNMR (400 MHz, Chloroform-d) δ 4.31 – 4.16 (m, 1H), 4.13 – 4.00 (m, 2H), 3.89 (td, J = 12.2, 2.3 Hz, 1H), 3.52 (dt, J = 9.6, 5.5 Hz, 2H), 3.48 – 3.32 (m, 2H), 2.70 (ddd, J = 13.6, 11.0, 2.7 Hz, 2H), 2.43 – 2.32 (m, 2H), 2.17 – 1.99 (m, 3H), 1.90 – 1.54 (m, 11H), 1.54 – 1.44 (m, 1H), 1.43 – 1.17 (m, 37H), 1.11 (tt, J = 12.8, 2.6Hz, 1H), 0.89 (td, J = 8.0, 4.6 Hz, 9H). MS: 672.55 m / z [M+H].

[0683]

[0684] Compound: YZL501

[0685] This compound was synthesized from intermediates n and q using the same method as compound 1, with a yield of 19%. 1 HNMR (400 MHz, Chloroform-d) δ 4.60 (td, J = 12.1, 3.2 Hz, 1H), 4.37 (ddd, J =11.5, 9.7, 2.0 Hz, 1H), 3.60 – 3.40 (m, 4H), 3.35 (ddd, J = 12.4, 9.2, 6.5 Hz, 1H), 3.01 – 2.88 (m, 3H), 2.36 – 2.16 (m, 2H), 2.13 – 2.03 (m, 1H), 1.96 – 1.76 (m, 6H), 1.77 – 1.07 (m, 59H), 0.89 (td, J = 8.0, 4.5 Hz, 12H), 0.84 – 0.72 (m, 2H). MS: 826.72 m / z [M+H].

[0686]

[0687] Intermediate a':

[0688] To a solution of 4,4-dimethoxybutyronitrile (15.0 g, 116 mmol, 1.0 eq.) in hexane-1-thiol (41.2 g, 349 mmol, 3.0 eq.), py-TsOH (1.46 g, 5.81 mmol, 0.05 eq.) was added. The mixture was stirred at 110 °C for 24 h. The crude product was purified by silica gel column chromatography, eluting with (PE:EA = 50:1 → 40:1 → 20:1) to give a yellow oily 4,4-bis(hexylthio)butyronitrile (13 g, yield 37.1%). MS: 302 m / z [M+H].

[0689]

[0690] Intermediate b':

[0691] A solution of intermediate a' (13 g, 43.19 mmol, 1.0 eq.) was added to EtOH:H2O = 1:1 (120 mL) at room temperature, followed by the addition of KOH (7.3 g, 129.57 mmol, 3 eq). The mixture was stirred at room temperature for 16 hours. EA (30 mL) and H2O were added to the reaction mixture, followed by extraction with EA (300 mL x 3). The mixture was dried and concentrated to give a crude product, which was further purified by com-flash (EA at PE = 0-10%) to give a yellow oily product b' (8 g, yield: 57.9%). TLC (PE:EA = 30:1, Rf = 0.7, KMnO4). MS: 321.3 m / z [M+H].

[0692]

[0693] Intermediate c':

[0694] A solution of 4,4-bis(hexylthio)butyric acid (3 g, 9.4 mmol, 1.0 eq.) was added to DCM (50 mL) with hexane-1,6-diol (1.4 g, 14 mmol, 1.5 eq.), DCC (2.3 g, 11.3 mmol, 1.2 eq.), and DMAP (1.5 g, 12.2 mmol, 1.3 eq.). The mixture was stirred at room temperature for 16 hours. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (PE:EA = 10:1) to give the title compound 5-hydroxypentyl 4,4-bis(hexylthio)butyrate (1.8 g, yield: 47%) as a yellow oil. TLC (PE:EA = 5:1, Rf = 0.5, KMnO4). MS: 421.2 m / z [M+H].

[0695]

[0696] Intermediate d':

[0697] A solution of 5-hydroxypentyl 4,4-bis(hexylthio)butyrate (1.8 g, 4.4 mmol, 1.0 eq.) was added to DCM (50 mL) with Ms₂O (1.1 g, 6.6 mmol, 1.5 eq.) and Et₃N (1.1 g, 13.2 mmol, 3.0 eq). The mixture was stirred at room temperature for 16 hours. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (PE:EA = 10:1) to give the title compound 5-((methanesulfonyl)oxy)pentyl 4,4-bis(hexylthio)butyrate (1.4 g, yield: 79.7%) as a yellow oil. TLC (PE:EA = 5:1, Rf = 0.5, KMnO₄). MS: 499.3 m / z [M+H].

[0698]

[0699] Intermediate e':

[0700] The product was prepared from dodecanoic acid using a method similar to that used for the synthesis of intermediate d', yielding 3.3 g of the desired product as a clear oil. MS: 485.2 m / z [M+H].

[0701]

[0702] intermediate f':

[0703] Prepared from dodecanoic acid using a method similar to that used for synthesizing intermediate d', yielding 3.8 g of the desired product as a clear oil. MS: 453.2 m / z [M+H].

[0704]

[0705] intermediate g':

[0706] DCM (20 mL) was added to a mixture of 2-hexyldecanoic acid (1.8 g, 10 mmol, 1 equiv) and 6-bromohexane-1-ol (2.56 g, 10 mmol, 1 equiv), followed by EDCI (1.91 g, 10 mmol, 1 equiv), DMAP (122 mg, 1 mmol, 0.1 equiv), and DIEA (2.02 g, 20 mmol, 2 equiv), and the reaction was carried out at room temperature. The reaction mixture was stirred for at least 18 hours. The reaction mixture was purified directly by silica gel column chromatography (EtOAc gradient in hexane) to give 3.4 g of the desired product as a clear oil. MS: 419.2 m / z [M+H].

[0707]

[0708] intermediate h':

[0709] The product was prepared from dodecanoic acid using a method similar to that used for intermediate g, yielding 3.8 g of the desired product as a clear oil. MS: 405.2 m / z [M+H].

[0710]

[0711] Intermediate i':

[0712] A solution of 6-bromohexane-1-ol (2.6 g, 14.51 mmol, 1.0 eq.) was added to ACN (50 mL) along with octane-1-thiol (4.2 g, 29.02 mmol, 2.0 eq.), pyridine (2.3 g, 29.02 mmol, 2.0 eq.), and DMAP (531 mg, 4.35 mmol, 0.3 eq.). The mixture was stirred at 85°C for 16 hours. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (PE:EA = 10:1) to give the title compound ZL24-2 (0.2 g, yield: 3.85%) as a yellow oil. MS: 352.11 [M+H].

[0713]

[0714] Intermediate j':

[0715] Pyridine (2.9 g, 37.4 mmol, 2 eq) and 4-nitrophenylcarbonyl chloride (7.5 g, 37.4 mmol, 2.0 eq), along with 5-bromopentane-1-thiol (6.8 g, 37.4 mmol, 2.0 eq), were added to a DCM solution of tetradecane-7-ol (2.7 g, 18.7 mmol, 1.0 eq). The mixture was stirred at room temperature for 16 hours. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (PE:EA = 10:1) to give intermediate j (2 g, yield: 38.5%) as a yellow oil. MS: 422.19 m / z [M+H].

[0716]

[0717] Intermediate k':

[0718] It was synthesized in 32% yield using the same method as the synthetic intermediate j. MS: 479.25 m / z [M+H].

[0719]

[0720] Compound YZL473:

[0721] A solution of intermediate d' (1.4 g, 2.8 mmol, 2.2 eq) was reacted in ACN (30 mL) with 3-aminopropane-1-ol (96 mg, 1.27 mmol, 1.0 eq), K₂CO₃ (1.1 g, 8.4 mmol, 3.0 eq), and KI (465 mg, 2.8 mmol, 1.0 eq) at room temperature. The mixture was stirred at 85 °C for 16 hours. The mixture was filtered and concentrated under vacuum, and purified by silica gel column chromatography (DCM: MeOH = 10:1) to give the residue ((3-hydroxypropyl)azadiene)bis(hexane-6,1-diene)bis(4,4-di(hexylthio)butyrate) (312 mg, yield: 15.0%) as a yellow oil. 1H NMR (400 MHz, chloroform-d) δ 4.07 (td, J = 7.1, 3.9 Hz, 4H), 3.89 – 3.39 (m, 4H), 2.66 (dd, J = 12.4, 7.0 Hz, 5H), 2.56 (ddd, J = 30.6, 12.4, 6.9 Hz, 9H), 2.11 (m,4H), 1.78–1.61 (m, 2H), 1.57 (ddd, J = 20.2, 13.4, 6.5 Hz, 19H), 1.46-1.23(m, J = 14.5, 7.3 Hz, 34H), 0.94 – 0.84 (m, 12H). MS: 880.6 m / z [M+H].

[0722]

[0723] Compound YZL474:

[0724] It was synthesized in 19% yield using a method similar to that used to synthesize compound YZL473.

[0725]

[0726] 1H NMR (500 MHz, Chloroform-d) δ 4.15 (t, J = 7.5 Hz, 4H), 4.05 –3.92 (m, 2H), 3.57 (td, J = 7.5, 5.0 Hz, 2H), 3.40 (dtd, J = 17.0, 12.4, 3.0Hz, 2H), 3.32 – 3.13 (m, 3H), 2.85 (t, J = 7.6 Hz, 2H), 2.67 (dtd, J = 19.0,12.5, 1.6 Hz, 2H), 2.55 – 2.45 (m, 1H), 2.38 – 2.32 (m, 6H), 2.16 – 2.03 (m,2H), 1.86 – 1.77 (m, 2H), 1.77 – 1.70 (m, 6H), 1.67 – 1.46 (m, 10H), 1.46 – 1.37 (m, 8H), 1.37 – 1.31 (m, 8H), 1.31 – 1.25 (m, 6H), 1.25 – 1.07 (m, 6H),0.89 (t, J = 7.9 Hz, 12H). LCMS: m / z =852.6 [M+H]+.

[0727] Compound YZL475:

[0728] It was synthesized in 39% yield using a method similar to that used to synthesize compound YZL473.

[0729]

[0730] 1H NMR (500 MHz, Chloroform-d) δ 4.15 (t, J = 7.4 Hz, 4H), 3.90 (t, J= 3.4 Hz, 1H), 3.64 – 3.51 (m, 5H), 3.38 – 3.24 (m, 2H), 3.07 (td, J = 12.3,1.6 Hz, 1H), 2.93 (td, J = 12.4, 1.9 Hz, 1H), 2.89 – 2.79 (m, 3H), 2.49 –2.32 (m, 13H), 2.26 – 2.07 (m, 2H), 2.05 – 1.94 (m, 1H), 1.85 – 1.79 (m, 2H),1.79 – 1.68 (m, 9H), 1.68 – 1.60 (m, 4H), 1.60 – 1.40 (m, 15H), 1.40 – 1.30 (m, 4H), 1.30 – 1.27 (m, 9H), 1.27 – 1.19 (m, 5H), 1.10 (dddd, J = 20.8,15.7, 11.8, 5.2 Hz, 4H), 1.00 (qt, J = 13.0, 2.4 Hz, 1H), 0.89 (t, J = 7.9Hz, 12H). LCMS: m / z =908.6 [M+H]+.

[0731] Compound YZL476:

[0732] It was synthesized in 28% yield using a method similar to that used to synthesize compound YZL473.

[0733]

[0734] 1H NMR (500 MHz, Chloroform-d) δ 4.15 (t, J = 7.1 Hz, 4H), 3.57 (td,J = 7.1, 5.0 Hz, 2H), 3.52 – 3.44 (m, 2H), 2.85 (t, J = 7.1 Hz, 2H), 2.77 (t,J = 7.0 Hz, 4H), 2.65 (t, J = 7.1 Hz, 4H), 2.45 – 2.39 (m, 5H), 2.39 – 2.31(m, 7H), 1.82 – 1.58 (m, 16H), 1.49 (dp, J = 14.4, 7.1 Hz, 8H), 1.37 – 1.22(m, 37H), 0.93 – 0.85 (m, 12H). LCMS: m / z =936.7 [M+H]+.

[0735] Compound YZL418:

[0736] It was prepared from intermediate e' and 3-aminopropanol using a similar method to that used to synthesize compound YZL473, yielding YZL418 (220 mg, 44%), a pale yellow oil. 1 H NMR (400 MHz, Chloroform-d) δ 4.45(t, J = 7.6 Hz, 1H), 4.26 (t, J = 7.0 Hz, 1H), 4.15 (t, J = 7.5 Hz, 4H), 3.57– 3.40 (m, 5H), 3.33 (td, J = 12.3, 1.6 Hz, 1H), 3.21 – 3.03 (m, 3H), 2.98 –2.87 (m, 5H), 2.74 (td, J = 12.3, 1.5 Hz, 1H), 2.42 (t, J = 7.5 Hz, 6H), 2.36– 2.23 (m, 1H), 1.82 – 1.63 (m, 9H), 1.64 – 1.11 (m, 37H), 0.89 (t, J = 7.8Hz, 12H). MS: 866.5 m / z [M+H].

[0737]

[0738] Compound YZL419:

[0739] It was prepared from intermediate e' (2 eq) and 2-aminoethanol (1 eq) using a similar method to that used to synthesize compound YZL473, yielding YZL419 (282 mg, 45%), a pale yellow oil. 1 H NMR (400 MHz, Chloroform-d) δ 4.45 (t, J = 7.6 Hz, 1H), 4.27 (t, J = 6.9 Hz, 1H), 4.15 (t,J = 7.5 Hz, 4H), 3.62 – 3.40 (m, 4H), 3.23 – 3.03 (m, 3H), 2.98 – 2.86 (m,5H), 2.75 (td, J = 12.3, 1.6 Hz, 1H), 2.55 (t, J = 7.2 Hz, 2H), 2.42 (t, J =7.6 Hz, 4H), 2.35 – 2.23 (m, 1H), 1.74 (p, J = 7.6 Hz, 7H), 1.62 – 1.53 (m,2H), 1.49 (dp, J = 15.5, 7.6 Hz, 12H), 1.43 – 1.35 (m, 2H), 1.38 – 1.21 (m,15H), 1.24 – 1.11 (m, 2H), 0.89 (t, J = 7.8 Hz, 12H). MS: 838.4 m / z [M+H].

[0740]

[0741] Compound YZL422:

[0742] It was prepared from intermediates e', f' and 3-aminopropanol using a similar method to that used to synthesize compound YZL473, yielding compound YZL422 (420 mg, 45%), which is a pale yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 5.50 (t, J = 4.0 Hz, 1H), 4.40 (t, J = 6.9 Hz, 1H), 4.15 (t, J = 7.5 Hz, 5H), 3.46 (td, J = 7.4, 5.0 Hz, 2H), 3.38 – 3.30 (m, 1H), 3.20 – 2.99 (m,6H), 2.98 – 2.87 (m, 4H), 2.72 (td, J = 12.3, 3.4 Hz, 1H), 2.55 (d, J = 3.8Hz, 2H), 2.42 (t, J = 7.5 Hz, 7H), 1.84 – 1.61 (m, 11H), 1.64 – 1.46 (m,11H), 1.49 – 1.33 (m, 7H), 1.37 – 1.09 (m, 19H), 0.89 (t, J = 7.8 Hz, 12H).MS: 834.6 m / z [M+H].

[0743]

[0744] Compound YZL423:

[0745] It was prepared from intermediates e', g' and 2-aminoethanol using a method similar to that used to synthesize compound YZL473, yielding compound YZL423 (320 mg, 41%), which is a pale yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 4.53 – 4.41 (m, 2H), 4.30 (dtd,J = 25.5, 12.3, 2.4 Hz, 2H), 3.99 (dtd,J = 31.0, 11.9, 3.9 Hz, 2H), 3.33 – 3.21 (m, 2H), 3.11 – 2.86 (m, 6H), 2.54 –2.34 (m, 2H), 2.17 – 2.02 (m, 2H), 2.01 – 1.82 (m, 6H), 1.85 – 1.70 (m, 3H),1.69 (dddd, J = 14.5, 9.1, 4.4, 2.3 MS: 816.6 m / z [M+H].

[0746]

[0747] Compound YZL428:

[0748] It was prepared from intermediate d' and 2-aminoethanol using a similar method to that used to synthesize compound YZL473, yielding compound YZL428 (220 mg, 21%), which is a pale yellow oil. 1H NMR (400 MHz, Chloroform-d) δ4.15 (s, 2H), 4.15 (dd, J = 12.3, 2.6 Hz, 2H), 3.78 (q, J = 6.5, 5.6 Hz, 2H), 3.58 (td, J = 7.1, 5.0 Hz, 2H), 3.34 – 2.98 (m, 7H), 2.64 – 2.52 (m, 3H), 2.46 – 2.35 (m, 7H), 2.35 (q, J = 4.9, 4.3 Hz, 5H), 1.93 – 1.61 (m, 9H), 1.64– 1.47 (m, 7H), 1.49 – 1.42 (m, 4H), 1.45 – 1.31 (m, 2H), 1.29 (tdd, J =16.1, 8.3, 3.1 Hz, 8H), 1.26 – 1.05 (m, 5H), 0.89 (t, J = 7.9 Hz, 12H). MS: 866.6 m / z [M+H].

[0749]

[0750] Compound YZL429:

[0751] It was prepared from intermediate d' and 4-aminobutanol using a similar method to that used to synthesize compound YZL473, yielding compound YZL429 (120 mg, 21%), which is a pale yellow oil. 1H NMR (400 MHz, Chloroform-d) δ4.15 (t, J = 4.6 Hz, 4H), 4.11 – 3.94 (m, 3H), 3.77 (td, J = 12.6, 2.0 Hz,1H), 3.49 – 3.38 (m, 3H), 3.37 – 3.29 1.82 – 1.55 (m, 11H), 1.58 – 1.38 (m, 15H), 1.30 (dtdd, J = 16.5,8.8, 6.3, 2.7 Hz, 11H), 0.89 (t, J = 7.9 Hz, 12H). MS: 894.6 m / z [M+H].

[0752]

[0753] Compound YZL434:

[0754] It was prepared from intermediates e', f' and 3-aminopropanol using a similar method to that used to synthesize compound YZL422, yielding compound YZL434 (410 mg, 45%), which is a pale yellow oil. 11H NMR (400 MHz, Chloroform-d) δ 5.42 (t, J = 4.9 Hz, 1H), 4.43 (t, J = 7.0 Hz, 1H), 4.15 (t, J = 7.5 Hz, 4H), 3.57 (td, J = 7.5, 5.0 Hz, 2H), 3.32 – 3.13 (m, 4H), 3.09 – 2.99 (m, 1H), 2.95 (d, J = 7.0 Hz, 2H), 2.90 – 2.77 (m, 4H), 2.67 (s, 0H), 2.63 (d, J = 3.5 Hz, 1H), 2.55 (d, J = 4.9 Hz, 2H), 2.42 (t, J = 7.6 Hz, 4H), 1.88 – 1.74 (m, 4H), 1.75 (d, J = 7.5 Hz, 4H), 1.75 – 1.54 (m, 4H), 1.49 (dp, J = 15.6, 7.8 Hz, 12H), 1.43 – 1.00 (m, 19H), 0.89 (t, J = 7.8 Hz, 12H). MS: 820.6 m / z [M+H].

[0755]

[0756] Compound YZL451:

[0757] It was prepared from intermediates d', h' and 3-aminopropanol using a method similar to that used to synthesize compound YZL422, yielding compound YZL451 (550 mg, 55%), which is a pale yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 4.44 – 4.35 (m, 2H), 4.17 – 4.00 (m, 2H), 3.90 – 3.75 (m, 2H), 3.46 –3.36 (m, 2H), 3.12 – 3.03 (m, 2H), 3.06 – 2.79 (m, 4H), 2.78 – 2.68 (m, 2H), 2.29 – 2.16 (m, 2H), 2.14 – 2.03 (m, 2H), 2.00 – 1.70 (m, 6H), 1.73 – 1.52(m, 7H), 1.56 – 1.33 (m, 12H), 1.37 – 1.27 (m, 2H), 1.30 – 1.16 (m, 4H), 1.19 – 0.99 (m, 2H), 0.89 (td, J = 7.9, 5.0 Hz, 12H). MS: 788.6 m / z [M+H].

[0758]

[0759] Compound YZL424:

[0760] It was prepared from intermediates d', j' and 4-aminobutanol using a method similar to that used to synthesize compound YZL422, yielding compound YZL424 (285 mg, 35%), which is a pale yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 4.57 (t, J = 7.0 Hz, 1H), 4.33 (td, J = 9.1, 3.7 Hz, 1H), 4.14 – 3.96(m, 2H), 3.58 – 3.34 (m, 4H), 3.28 – 3.13 (m, 2H), 3.12 – 3.02 (m, 2H), 2.76 – 2.61 (m, 3H), 2.49 – 2.32 (m, 3H), 2.08 – 1.99 (m, 2H), 1.90 – 1.48 (m,16H), 1.44 – 1.16 (m, 46H), 1.16 – 0.98 (m, 2H), 0.95 – 0.82 (m, 12H). MS: 834.6 m / z [M+H].

[0761]

[0762] Compound YZL425:

[0763] It was prepared from intermediates d', i' and 4-aminobutanol using a similar method to that used to synthesize compound ZL22, yielding compound ZL25 (360 mg, 52%), which is a pale yellow oil. 1 H NMR (400 MHz, Chloroform-d) δ4.57 (t, J = 7.0 Hz, 1H), 4.23 (t, J = 7.5 Hz, 2H), 4.15 (t, J = 7.5 Hz, 2H), 3.46 (td, J = 7.4, 5.0 Hz, 2H), 3.24 (td, J = 12.4, 2.9 Hz, 1H), 3.07 (ddd, J= 10.8, 4.4, 1.4 Hz, 2H), 2.95 (d, J = 7.0 Hz, 2H), 2.85 (t, J = 8.0 Hz, 2H), 2.49 – 2.39 (m, 7H), 1.94 (p, J = 8.0 Hz, 2H), 1.89 – 1.79 (m, 1H), 1.78 –1.62 (m, 6H), 1.52 (ddp, J = 38.5, 15.6, 7.5 Hz, 12H), 1.42 – 1.15 (m, 26H), 0.98 – 0.78 (m, 9H). MS: 750.5 m / z [M+H].

[0764]

[0765] Compound YZL449:

[0766] It was prepared from intermediates d' and k' using a method similar to that used to synthesize compound YZL422, yielding compound YZL449 (29% yield). 1 H NMR (400 MHz, Chloroform-d) δ 4.50 (td, J = 12.1, 3.7Hz, 1H), 4.25 (t, J = 7.0 Hz, 1H), 4.17 (dd, J = 12.3, 7.0 Hz, 1H), 4.04(tdd, J = 12.4, 5.5, 2.8 Hz, 1H), 3.96 – 3.87 (m, 1H), 3.80 (td, J = 12.5, 4.0 Hz, 1H), 3.68 – 3.41 (m, 3H), 3.40 – 3.14 (m, 3H), 2.98 (td, J = 12.6,2.0 Hz, 1H), 2.82 – 2.65 (m, 2H), 2.63 (dt, J = 12.4, 3.1 Hz, 1H), 2.53 (td,J = 12.1, 3.7 Hz, 1H), 2.28 (tdd, J = 12.5, 8.1, 2.3 Hz, 2H), 2.22 – 1.95 (m,6H), 1.95 – 1.64 (m, 9H), 1.64 – 0.97 (m, 23H), 0.89 (t, J = 7.8 Hz, 12H). MS: 862.64 m / z [M+H].

[0767]

[0768] Compound YZL489:

[0769] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL489 (21% yield).

[0770]

[0771] 1H NMR (400 MHz, Chloroform-d) δ 4.45 (t, J = 7.0 Hz, 1H), 4.23 (t, J= 7.4 Hz, 2H), 4.15 (t, J = 7.4 Hz, 2H), 3.57 (td, J = 7.5, 5.1 Hz, 2H), 3.36– 3.27 (m, 1H), 3.21 (td, J = 12.5, 1.4 Hz, 1H), 3.04 – 2.92 (m, 3H), 2.85(t, J = 7.8 Hz, 4H), 2.64 (td, J = 12.5, 3.6 Hz, 1H), 2.42 (t, J = 7.4 Hz, 4H), 1.99 – 1.85 (m, 3H), 1.76 (dp, J = 15.2, 7.6 Hz, 7H), 1.66 – 1.40 (m,16H), 1.38 (p, J = 2.8 Hz, 2H), 1.37 – 1.26 (m, 18H), 1.26 – 1.16 (m, 4H), 1.16 – 1.02 (m, 4H), 0.89 (t, J = 7.9 Hz, 9H). LCMS: m / z =778.5 [M+H]+.

[0772] Compound YZL490:

[0773] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL490 (33% yield).

[0774]

[0775] LCMS: m / z = 764.5 [M+H]+.

[0776] 1H NMR (400 MHz, Chloroform-d) δ 4.23 (t, J = 7.4 Hz, 2H), 4.15 (t, J= 7.4 Hz, 2H), 3.91 (t, J = 5.9 Hz, 1H), 3.57 (td, J = 7.6, 4.9 Hz, 2H), 3.44(tt, J = 12.0, 2.8 Hz, 2H), 2.85 (t, J = 7.7 Hz, 4H), 2.54 (d, J = 1.8 Hz,1H), 2.40 – 2.31 (m, 4H), 2.12 – 2.00 (m, 1H), 1.99 – 1.82 (m, 4H), 1.82 –1.69 (m, 7H), 1.63 – 1.39 (m, 14H), 1.39 – 1.21 (m, 22H), 1.21 – 1.03 (m,4H), 0.89 (t, J = 6.0 Hz, 9H).

[0777] Compound YZL493:

[0778] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL493 (22% yield).

[0779]

[0780] 1H NMR (400 MHz, Chloroform-d) δ 4.35 (t, J = 6.9 Hz, 1H), 4.23 (t, J= 7.5 Hz, 2H), 4.15 (t, J = 4.6 Hz, 2H), 3.68 – 3.54 (m, 3H), 3.07 (td, J =12.4, 3.7 Hz, 1H), 2.98 – 2.91 (m, 2H), 2.91 – 2.81 (m, 5H), 2.56 (td, J =12.5, 2.7 Hz, 1H), 2.42 (t, J = 5.3 Hz, 4H), 1.94 (tt, J = 7.6, 5.6 Hz, 2H),1.86 – 1.78 (m, 2H), 1.78 – 1.70 (m, 6H), 1.70 – 1.60 (m, 2H), 1.60 – 1.52(m, 5H), 1.52 – 1.46 (m, 7H), 1.46 – 1.39 (m, 3H), 1.39 – 1.34 (m, 3H), 1.34– 1.30 (m, 6H), 1.30 – 1.18 (m, 15H), 1.00 (qt, J = 12.8, 2.7 Hz, 1H), 0.89(t, J = 7.9 Hz, 9H). LCMS: m / z =778.5 [M+H]+.

[0781] Compound YZL494:

[0782] It was prepared using a similar method to that used to synthesize compound YZL449, yielding compound YZL494 (38% yield).

[0783]

[0784] 1H NMR (400 MHz, Chloroform-d) δ 4.23 (t, J = 7.5 Hz, 2H), 4.15 (t, J= 7.5 Hz, 2H), 3.87 – 3.81 (m, 1H), 3.61 – 3.51 (m, 3H), 3.17 (td, J = 12.4,1.4 Hz, 1H), 2.96 – 2.81 (m, 5H), 2.65 (td, J = 11.9, 4.7 Hz, 1H), 2.45 –2.31 (m, 8H), 2.18 (qdd, J = 12.4, 4.5, 2.1 Hz, 1H), 1.94 (p, J = 8.0 Hz,2H), 1.76 (dp, J = 15.3, 7.6 Hz, 7H), 1.65 – 1.48 (m, 8H), 1.48 – 1.39 (m,4H), 1.39 – 1.33 (m, 5H), 1.29 (dddd, J = 19.6, 8.8, 5.1, 3.4 Hz, 20H), 1.23– 1.10 (m, 3H), 0.89 (t, J = 6.0 Hz, 9H). LCMS: m / z =779.6 [M+H]+.

[0785] Compound YZL497:

[0786] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL497 (19% yield).

[0787]

[0788] 1H NMR (400 MHz, Chloroform-d) δ 4.34 (t, J = 7.1 Hz, 1H), 4.23 (t, J= 7.6 Hz, 2H), 4.15 (t, J = 7.5 Hz, 2H), 3.61 – 3.45 (m, 3H), 3.27 (td, J =12.2, 1.3 Hz, 1H), 2.95 (d, J = 7.0 Hz, 2H), 2.85 (t, J = 7.7 Hz, 4H), 2.74(td, J = 12.3, 4.3 Hz, 1H), 2.60 (td, J = 12.2, 4.6 Hz, 1H), 2.45 – 2.39 (m,4H), 2.17 – 2.05 (m, 1H), 1.94 (p, J = 7.9 Hz, 2H), 1.76 (dp, J = 15.3, 7.6Hz, 7H), 1.68 – 1.54 (m, 3H), 1.54 – 1.48 (m, 7H), 1.47 (d, J = 1.9 Hz, 3H),1.44 (dt, J = 10.8, 2.1 Hz, 4H), 1.42 – 1.32 (m, 6H), 1.32 – 1.22 (m, 19H),1.22 – 1.07 (m, 3H), 0.89 (t, J = 7.9 Hz, 9H). LCMS: m / z =778.5 [M+H]+.

[0789] Compound YZL498:

[0790] It was prepared using a similar method to that used to synthesize compound YZL449, yielding compound YZL498 (39% yield).

[0791]

[0792] 1H NMR (400 MHz, Chloroform-d) δ 4.23 (t, J = 7.5 Hz, 2H), 4.15 (t, J= 7.5 Hz, 2H), 3.92 (t, J = 6.7 Hz, 1H), 3.57 (td, J = 7.6, 4.9 Hz, 2H), 3.48– 3.39 (m, 1H), 3.01 (td, J = 12.4, 1.4 Hz, 1H), 2.85 (t, J = 7.8 Hz, 4H), 2.74 – 2.64 (m, 1H), 2.55 (td, J = 12.1, 4.6 Hz, 1H), 2.46 – 2.32 (m, 8H),2.22 – 2.07 (m, 1H), 1.94 (p, J = 7.9 Hz, 2H), 1.76 (dp, J = 15.3, 7.7 Hz,6H), 1.64 – 1.53 (m, 1H), 1.57 – 1.38 (m, 15H), 1.38 – 1.26 (m, 20H), 1.26 –1.15 (m, 8H), 1.11 (dddd, J = 21.7, 12.8, 6.4, 3.9 Hz, 2H), 0.89 (t, J = 7.9Hz, 9H). LCMS: m / z =792.6 [M+H]+.

[0793] Compound YZL479:

[0794] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL479 (42% yield).

[0795]

[0796] YZL 479;

[0797] 1H NMR (400 MHz, Chloroform-d) δ 4.18 – 4.11 (m, 4H), 3.57 (td, J =7.1, 5.5 Hz, 2H), 2.92 – 2.82 (m, 4H), 2.76 – 2.59 (m, 4H), 2.52 – 2.32 (m,14H), 2.26 (dt, J = 16.8, 6.9 Hz, 4H), 1.82 – 1.58 (m, 17H), 1.49 (dp, J =14.3, 7.1 Hz, 8H), 1.37 – 1.23 (m, 32H), 0.94 – 0.84 (m, 12H). LCMS: m / z =926.7 [M+H]+.

[0798] Compound YZL480:

[0799] It was prepared using a similar method to that used to synthesize compound YZL449, yielding compound YZL480 (33% yield).

[0800] YZL480;

[0801] 1 H NMR (400 MHz, Chloroform-d) δ 4.18 – 4.11 (m, 4H), 3.57 (td, J =7.1, 5.5 Hz, 2H), 2.92 – 2.82 (m, 4H), 2.76 – 2.59 (m, 4H), 2.52 – 2.32 (m,14H), 2.26 (dt, J = 16.8, 6.9 Hz, 4H), 1.82 – 1.58 (m, 18H), 1.49 (dp, J =14.4, 7.1 Hz, 8H), 1.37 – 1.22 (m, 37H), 0.94 – 0.84 (m, 12H). LCMS: m / z =964.7 [M+H]+.

[0802] Compound YZL491:

[0803] It was prepared using a similar method to that used to synthesize compound YZL449, yielding compound YZL491 (19% yield).

[0804] YZL491;

[0805] 1H NMR (400 MHz, Chloroform-d) δ 4.44 (td, J = 12.1, 2.8 Hz, 1H), 4.25 – 4.10 (m, 1H), 3.96 – 3.81 (m, 1H), 3.63 – 3.33 (m, 6H), 3.33 – 3.19(m, 3H), 3.03 – 2.78 (m, 4H), 2.72 – 2.59 (m, 2H), 2.53 – 2.42 (m, 2H), 2.06– 1.91 (m, 2H), 1.88 – 1.80 (m, 2H), 1.80 – 1.68 (m, 5H), 1.68 – 1.64 (m, 3H), 1.64 – 1.61 (m, 2H), 1.61 – 1.46 (m, 4H), 1.46 – 1.43 (m, 1H), 1.43 –1.00 (m, 35H), 0.89 (t, J = 7.9 Hz, 9H). LCMS: m / z =964.5 [M+H]+.

[0806] Compound YZL495:

[0807] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL495 (25% yield).

[0808] YZL495;

[0809] 1H NMR (400 MHz, Chloroform-d) δ 4.53 (td, J = 12.0, 3.8 Hz, 1H), 4.24 (ddt, J = 12.1, 8.8, 3.5 Hz, 1H), 3.89 (td, J = 12.5, 2.5 Hz, 1H), 3.62– 3.45 (m, 4H), 3.22 (dtd, J = 37.4, 12.6, 2.4 Hz, 2H), 3.03 – 2.94 (m, 2H), 2.83 (dd, J = 12.4, 7.0 Hz, 1H), 2.75 – 2.62 (m, 3H), 2.62 – 2.41 (m, 4H),2.26 (td, J = 12.4, 3.6 Hz, 1H), 2.09 – 1.98 (m, 3H), 1.98 – 1.78 (m, 2H), 1.78 – 1.69 (m, 2H), 1.69 – 1.54 (m, 5H), 1.54 – 1.08 (m, 39H), 0.89 (t, J =7.9 Hz, 9H). LCMS: m / z =764.5 [M+H]+.

[0810] Compound YZL499:

[0811] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL499 (26% yield).

[0812] YZL499;

[0813] 1H NMR (400 MHz, Chloroform-d) δ 5.04 (ddt, J = 12.3, 10.3, 2.5 Hz,1H), 4.50 – 4.32 (m, 1H), 4.09 – 3.96 (m, 1H), 3.92 – 3.78 (m, 1H), 3.57 –3.44 (m, 3H), 3.43 – 3.14 (m, 4H), 3.02 (dd, J = 12.3, 7.0 Hz, 1H), 2.85 –2.73 (m, 3H), 2.70 – 2.56 (m, 2H), 2.46 – 2.22 (m, 3H), 2.10 – 1.99 (m, 3H),1.99 – 1.86 (m, 2H), 1.86 – 0.95 (m, 44H), 0.89 (t, J = 7.9 Hz, 9H). LCMS: m / z =764.5 [M+H]+.

[0814] Compound YZL701:

[0815] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL701 (39% yield).

[0816]

[0817] 1 H NMR (500 MHz, Chloroform-d) δ 4.15 (td, J = 7.1, 3.9 Hz, 4H), 3.50– 3.39 (m, 4H), 2.88 (dd, J = 12.4, 7.0 Hz, 2H), 2.66 (ddd, J = 30.6, 12.4,6.9 Hz, 4H), 2.52 – 2.32 (m, 18H), 1.78 – 1.61 (m, 15H), 1.57 (ddd, J = 20.2,13.4, 6.5 Hz, 1H), 1.46 (dd, J = 14.5, 7.3 Hz, 5H), 1.37 – 1.22 (m, 38H), 0.94 – 0.84 (m, 12H). MS: 951.6 m / z [M+H]+.

[0818] Compound YZL702:

[0819] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL702 (44% yield).

[0820]

[0821] 1 H NMR (500 MHz, Chloroform-d) δ 4.18 – 4.11 (m, 4H), 3.46 (td, J =7.0, 5.4 Hz, 2H), 2.88 (dd, J = 12.1, 6.8 Hz, 2H), 2.76 – 2.59 (m, 4H), 2.52– 2.32 (m, 16H), 2.26 (dt, J = 16.8, 6.9 Hz, 4H), 1.79 – 1.42 (m, 30H), 1.37– 1.24 (m, 28H), 0.94 – 0.84 (m, 12H). MS: 923.5 m / z [M+H]+.

[0822] Compound YZL703:

[0823] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL703 (45% yield).

[0824]

[0825] 1H NMR (500 MHz, Chloroform-d) δ 4.16 – 3.96 (m, 4H), 3.53 (dq, J =10.4, 5.3 Hz, 2H), 3.11 (dd, J = 12.3, 7.0 Hz, 1H), 3.05 – 2.91 (m, 5H), 2.83– 2.68 (m, 3H), 2.62 (td, J = 12.5, 3.1 Hz, 1H), 2.57 – 2.43 (m, 3H), 2.29 –2.18 (m, 2H), 2.14 – 2.05 (m, 1H), 1.93 – 1.79 (m, 4H), 1.78 – 1.67 (m, MS: 789.3 m / z [M+H]+.

[0826] Compound YZL704:

[0827] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL704 (25% yield).

[0828]

[0829] 1 H NMR (500 MHz, Chloroform-d) δ 4.15 (td, J = 7.1, 3.9 Hz, 4H), 3.57(td, J = 7.1, 5.5 Hz, 2H), 3.44 (p, J = 7.0 Hz, 2H), 2.92 – 2.82 (m, 4H), 2.66 (ddd, J = 30.6, 12.4, 6.9 Hz, 4H), 2.52 – 2.32 (m, 15H), 1.82 – 1.65 (m,9H), 1.68 – 1.58 (m, 8H), 1.49 (dp, J = 14.3, 7.1 Hz, 9H), 1.37 – 1.22 (m,38H), 0.94 – 0.84 (m, 12H). MS: 937.6 m / z [M+H]+.

[0830] Compound YZL705:

[0831] It was prepared using a method similar to that used to synthesize compound YZL449, yielding compound YZL705 (37% yield).

[0832]

[0833] 1 H NMR (500 MHz, Chloroform-d) δ 4.15 (td, J = 7.1, 3.9 Hz, 4H), 3.58 (td, J = 7.1, 5.6 Hz, 2H), 3.44 (p, J = 7.0 Hz, 2H), 2.88 (dd, J = 12.4, 7.0Hz, 2H), 2.66 (ddd, J = 30.6, 12.4, 7.0 Hz, 4H), 2.55 (t, J = 7.1 Hz, 2H), 2.47 – 2.32 (m, 14H), 1.79 – 1.69 (m, 4H), 1.70 – 1.58 (m, 9H), 1.49 (dp, J =14.3, 7.1 Hz, 8H), 1.37 – 1.22 (m, 38H), 0.94 – 0.84 (m, 12H). MS: 922.6 m / z[M+H]+.

[0834] Example 2:

[0835] Materials and methods:

[0836] 1. Nanoparticle formulation

[0837] LNPs were prepared by mixing an aqueous phase containing nucleic acids with an ethanol phase containing lipids and excipients using a microfluidic chip device as described previously (Chen D et al., (2012) Rapid discovery of siRNA-containing effective lipid nanoparticles by controlled microfluidic methods. JAMA 134(16): 6948-6951). Specifically, the ethanol phase contained a mixture of ionizable lipids, 1,2-dioctanoyl-sn-glycerol-3-phosphorylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000). The molar ratio of ionizable lipids / DSPC / cholesterol / DMG-PEG2000 was 50 / 10 / 38.5 / 1.5. The ethanol phase was prepared by dissolving the mixture. The aqueous phase was prepared by adding RNA to 25 mM citrate buffer. The resulting LNPs were dialyzed for 2 hours at room temperature against a buffer containing 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS), and then extruded through a 0.22 μm sterile filter. The final LNPs were stored at -80°C for further use. ALC-0315 and LP01 are commercial lipids with good performance characteristics.

[0838] 2. LNP characterization

[0839] LNP particle diameter and polydispersity were measured by dynamic light scattering (DLS). Dynamic light scattering ("DLS") was used to determine the average particle size and polydispersity index ("PDI") of the LNP samples. The average particle size and polydispersity were measured using dynamic light scattering (DLS) on a Malvern Zetasizer DLS instrument. Before testing, diluted LNP samples were diluted 1:99 in 1x PBS by volume. The average hydrodynamic diameter of each sample was measured by reporting the average particle size and PDI. The zeta potential of the LNP was also measured by the Malvern Zetasizer. Before measurement, the samples were diluted 1:19 in 10 mM NaCl by volume.

[0840] Encapsulation efficiency (EE (%)) was determined using a fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) calculated as (total RNA - free RNA) / total RNA. LNP samples were diluted to an appropriate concentration with 1xTE buffer containing 1% Triton-X 100 to determine total RNA, or diluted with 1xTE buffer to determine free RNA. A standard curve was prepared using the starting RNA solution according to the manufacturer's instructions. Ribogreen® dye was then added to each standard and sample, and incubated at room temperature in the dark for approximately 5 minutes. Samples were read using a Tecan INFINITE 200 PRO with excitation and emission wavelengths of 480 nm and 520 nm, respectively. Total RNA and free RNA were calculated from the appropriate standard curve, and the fluorescence values ​​for each sample were subtracted from the reagent blank.

[0841] Typically, when preparing LNPs, the encapsulation efficiency is >80%, the particle size is <150 nm, and the PDI is <0.2. The average particle size, polydispersity, and %EE values ​​of various LNP combinations are listed in Tables 1-3.

[0842] Table 1: Summary of LNP formulation data in mice

[0843]

[0844] Table 2: Summary of LNP formulation data for PCH and NHP

[0845]

[0846]

[0847] Table 3: Summary of LNP formulation data for rats

[0848]

[0849] Table 4: sgRNA in this invention

[0850]

[0851] Table 5: DNA sequence encoded by Cas9 and its corresponding RNA

[0852]

[0853]

[0854]

[0855]

[0856] The mRNA used in the example is uracil modified with N1-methylpseuuridine. ALC-0315 and LP01 in different LNPs are from different batches.

[0857] 3. In vitro delivery of LNP

[0858] Primary rhesus monkey hepatocytes (TPCS, CCH-100CYS-PQ) were cultured according to the manufacturer's protocol. Cells were seeded at a density of 30,000 cells per well in 96-well plates. After 24 hours, cells were treated with LNP containing 6.25 nM. 72 hours after transfection, cells were washed with PBS, and genomic DNA was extracted using QuickExtract DNA extraction solution (Epicentre, QE09050) according to the manufacturer's recommended protocol.

[0859] LNPs were formulated using in vitro transcribed Cas9 mRNA (SEQ ID NO. 5) and chemically modified sgRNA (targeting NHPTTR) (SEQ ID NO. 1) as described in Example 2. Detailed information on these formulations, including average particle size, polydispersity, and encapsulation efficiency, is shown in Table 2. Figure 2A-2CAs shown, in vitro editing was observed in each formulation. These formulations include LipidYZL410 (LNP018), YZL411 (LNP019), YZL412 (LNP020), YZL413 (LNP021), YZL414 (LNP022), YZL415 (LNP023), YZL416 (LNP024), YZL417 (LNP025), YZL424 (LNP026), YZL425 (LNP027), YZL426 (LNP028), and YZL4... 27 (LNP029), YZL430 (LNP030), YZL431 (LNP031), YZL432 (LNP032), YZL433 (LNP033), YZL439 (LNP034), YZ L449 (LNP035), YZL450 (LNP036), YZL452 (LNP037), YZL453 (LNP038), YZL455 (LNP039), YZL456 (LNP040), Y ZL457 (LNP041), YZL457 (LNP041), YZL458 (LNP042), YZL459 (LNP043), YZL460 (LNP044), YZL418 (LNP107) , YZL419 (LNP108), YZL422 (LNP109), YZL423 (LNP110), YZL428 (LNP111), YZL429 (LNP112), YZL474 (LNP201 YZL475 (LNP202), YZL476 (LNP203), YZL479 (LNP204), YZL480 (LNP205), YZL497 (LNP206), YZL498 (LNP207), YZL499 (LNP208), YZL500 (LNP209), YZL489 (LNP210), YZL490 (LNP211), and YZL501 (LNP212) successfully delivered Cas9 mRNA and edited the TTR gene.

[0860] These results demonstrate the promise of delivering CRISPR / Cas9 cargoes to rhesus monkey hepatocytes in vitro using LNPs containing novel lipids.

[0861] 4. Animal experiments

[0862] All mice were purchased from Charles River Labs. For in vivo nanoparticle screening, 6-week-old female BALB / c mice were injected with LNPs via tail vein at a dose of 0.3 mg / kg. All animals were euthanized and necropsy performed 168 hours post-administration. Clinical signs, body weight, organ weight, and histopathology were assessed. Liver sections were cut into small pieces, and genomic DNA was extracted using the TIANamp Genomic DNA Kit according to the manufacturer's recommended protocol.

[0863] LNPs were formulated using in vitro transcribed Cas9 mRNA (SEQ ID NO. 5) and chemically modified sgRNA (targeting mouse TTR) (SEQ ID NO. 2), as described in Example 2. Details of these formulations are shown in Table 1, including average particle size, polydispersity, and encapsulation efficiency. Animals in each group were euthanized 7 days after administration. Figure 1A-1C As shown, in vivo editing (approximately 50%-68% editing) was observed in the livers of animals receiving TTR-targeted LNPs in each formulation. The formulations contain lipids YZL410 (LNP001), YZL411 (LNP002), YZL412 (LNP003), YZL413 (LNP004), YZL414 (LNP005), YZL415 (LNP006), YZL416 (LNP007), YZL417 (LNP008), YZL424 (LNP009), YZL425 (LNP010), YZL426 (LNP011), YZL427 (LNP012), and YZL414 (LNP009). L430 (LNP013), YZL431 (LNP014), YZL432 (LNP015), YZL433 (LNP016), YZL418 (LNP101), YZL419 (LNP102), YZL422 (LNP103), YZL423 (LNP104), YZL428 (LNP105), YZL429 (LNP106), YZL479 (LNP214), and YZL480 (LNP215) successfully delivered Cas9 mRNA to the liver and edited the TTR gene.

[0864] These results demonstrate that these LNPs using novel lipids exhibit excellent in vivo delivery efficiency for CRISPR / Cas9 cargo, achieving saturation editing effects even at very low doses.

[0865] LNPs were formulated using in vitro transcribed Cas9 mRNA (SEQ ID NO. 5) and chemically modified sgRNA (targeting NHP TTR) (SEQ ID NO. 1), as described in Example 2. Details of these formulations are shown in Table 2, including average particle size, polydispersity, and encapsulation efficiency. Similarly, LNPs targeting NHP TTR showed a robust and sustained reduction in rhesus monkey serum TTR protein (data not shown) in in vivo experiments in non-human primary (NHP) studies. For in vivo screening, LNPs were administered intravenously at a dose of 2 mg / kg. For in vivo editing, liver tissue was collected on days 14 and 28 post-dose via ultrasound-guided biopsy. The liver was sliced, and genomic DNA was extracted using the TIANamp Genomic DNA Kit according to the manufacturer's recommended protocol. Blood was collected every 7 days post-dose, and serum was separated as instructed. Figures 3A-3C As shown, in vivo editing was observed in the livers of animals receiving LNPs targeting TTR in each formulation. The formulations included lipids YZL410 (LNP018), YZL411 (LNP019), YZL412 (LNP020), YZL413 (LNP021), YZL414 (LNP022), YZL415 (LNP023), YZL416 (LNP024), YZL417 (LNP025), YZL424 (LNP026), YZL425 (LNP027), YZL426 (LNP028), YZL427 (LNP029), YZL430 (LNP030), and YZL434 (LNP030). 1 (LNP031), YZL432 (LNP032), YZL433 (LNP033), YZL439 (LNP034), YZL449 (LNP035), YZL450 (LNP036), YZL452 (LNP037), YZ L453 (LNP038), YZL455 (LNP039), YZL456 (LNP040), YZL457 (LNP041), YZL457 (LNP041), YZL458 (LNP042), YZL459 (LNP043), YZL460 (LNP044), YZL418 (LNP107), YZL419 (LNP108), YZL423 (LNP109), YZL428 (LNP111), YZL429 (LNP112), YZL479 (LNP217), and YZL480 (LNP218) successfully delivered Cas9 mRNA to the liver and edited the TTR gene. Even the delivery efficiency for single samples was encouraging, exceeding 40%.

[0866] These results demonstrate that these LNPs using the novel lipids exhibit excellent CRISPR / Cas9 cargo delivery efficiency in NHP.

[0867] LNPs were formulated using in vitro transcribed Cas9 mRNA (SEQ ID NO. 5) and chemically modified sgRNA (targeting rat TTR) (SEQ ID NO. 3), as described in Example 2. Details of these formulations are shown in Table 3, including average particle size, polydispersity, and encapsulation efficiency. All rats were purchased from Charles River Labs. Female Sprague-Dawley rats aged 6–8 weeks were used. The LNP dose was 2.5 mg / kg, with a dose volume of 7 mL / kg body weight, administered via tail vein injection. Plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), and other blood biochemical parameters were assessed using a Cobas c701 analyzer (Roche GmbH, Mannheim, Germany) at 18 and 168 hours post-administration. All animals were euthanized and necropsy performed 168 hours post-administration. Clinical signs, body weight, organ weight, and histopathology were assessed. Figure 4A and 4B , Figure 5A and 5B , Figure 6A and 6B As shown, ALT and AST were detected in the serum of animals receiving each formulation of LNPs targeting TTR. Mice treated with LNPs showed significantly increased AST and ALT activities compared to negative control animals (TSS). However, at 18 hours post-administration, animals treated with LNP046, LNP047, LNP048, LNP049, LNP050, and LNP051 had lower ALT and AST levels compared to rats treated with the commercial lipid ALC-0315. Furthermore, at 18 hours post-administration, ALT and AST levels in LNP113 were also lower than in LNP114. ALT and AST levels were relatively low in these LNP215 and LNP216 formulations. After 7 days, ALT and AST levels returned to normal. These data show that lipids YZL412, YZL425, YZL427, YZL449, YZL452, YZL453, YZL428, YZL479, and YZL480 caused less acute hepatotoxicity than ALC-0315, and the acute liver injury was manageable and could be repaired after 7 days.

[0868] These results indicate that these LNPs using the new lipids have better safety profiles compared to previous lipids.

[0869] 5. PCR amplification

[0870] All samples were amplified and sequenced using a two-step nested PCR process. Specifically, 1 μL of primers (final concentration 5 μM, Final Reverse / Forward) were added to 5 μL of Kapa HiFi 2X master mix (Roche), followed by 4 μL of template DNA / water. The first PCR reaction was run for 20 cycles. The second PCR, used to add Nextera XT chemistry, indexing, and i5 / i7 adapter regions, was run for 5–10 cycles, using the product of “PCR 1” as a template. Double-indexed samples were run on 2% agarose gels to ensure merging and gel purification after the PCR reaction.

[0871] 6. Transthyretin (TTR) ELISA analysis used in animal studies

[0872] Blood was collected and serum was separated. The total serum NHP TTR level was determined using the Human Prealbumin (Transthyretin) ELISA Kit (Aibike, product number ab231920) according to the manufacturer's protocol. Briefly, serum was serially diluted to a final dilution of 300,000 times with the diluent provided in the kit. 100 μL of the prepared standard curve or diluted serum sample was added to the ELISA plate and incubated at room temperature for 30 min, followed by washing three times with the provided wash buffer. Then, 100 μL of detection antibody was added to each well, and the plate was incubated at room temperature for 20 min, followed by three washes. After adding 100 μL of substrate, the plate was incubated at room temperature for 10 min, followed by adding 100 μL of stop solution. The absorbance of the contents in the wells was measured using an INFINITE 200 PRO plate reader and analyzed using SoftmaxPro version 7.0 software. Serum TTR levels were calculated based on the standard curve and expressed as μg / mL serum.

[0873] 7. Deep sequencing

[0874] PCR samples were purified using AMPure XP magnetic beads. Final library quality control was performed using an Agilent Bioanalyzer 2100. Illumina deep sequencing was performed on an Illumina MiniSeq™. Primers were designed based on Nextera XT adapter sequences.

[0875] 8. Data Analysis

[0876] Sequencing results were processed using a custom Python-based tool to extract raw barcode counts for each tissue. These raw counts were then normalized using an R script. Correlation analysis was performed assuming a Gaussian distribution to obtain the Pearson correlation coefficient. The R² value (range 0-1) was calculated using the squared Pearson correlation coefficient.

Claims

1. A compound having the following general formula (I) (I), Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug: in: R1 is hydrogen; phenyl; a 3- to 7-membered aliphatic ring; a 3- to 7-membered heterocyclic group containing 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur; a 5- to 6-membered monocyclic heteroaryl group containing 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur; an 8- to 10-membered bicyclic heteroaryl group containing 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur; -OR'; or ; wherein the phenyl, cyclic aliphatic group, 3 to 7-membered heterocyclic group, 5 to 6-membered monocyclic heteroaryl group, and 8 to 10-membered bicyclic heteroaryl group are optionally substituted by one or more substituents independently selected from halogen, hydroxyl or mercapto group; R' is selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, and C2-C8 alkynyl, wherein the C1-C8 alkyl, C2-C8 alkenyl, and C2-C8 alkynyl are optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or mercapto. R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, and -SC. 3-13 Alkyl and -CH2-SC 3-13 Alkyl groups; wherein C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl and C 3-13 The alkyl group is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxy or mercapto groups; R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl or -C(=O)R8; wherein the C1-C5 alkyl, C2-C5 alkenyl and C2-C5 alkynyl are optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxy or mercapto; R8 is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl groups; X1 is selected from a bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R 12 )-, -(R 12 )NS(O)2O-, -N(R 12 )S(O)2N(R 13 )-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)N(R 12 )-, -(R 12 )NC(=O)O-, -OC(=S)N(R 12 )-, -(R 12 )NC(=S)O-, -SC(=O)N(R 12 )- or -(R 12 )NC(=O)S-; R 12 and R 13 Each is independently selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, -C(=O)R8; wherein the C1-C5 alkyl, C2-C5 alkenyl and C2-C5 alkynyl are optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto; X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)O-, -C(=O)S-, -SC(=O-); L0 and L1 are each independently selected from a bond, C1-C8 alkylene, C2-C8 alkenyl, or C2-C8 ynynyl; wherein the C1-C8 alkylene, C2-C8 alkenyl, and C2-C8 ynynyl are optionally substituted by one or more substituents independently selected from halogen, hydroxyl, or mercapto. L2 and L3 are each independently selected from C1-C18 alkylene, C2-C18 alkenylene, and C2-C18 ynynylene, wherein the C1-C18 alkylene, C2-C18 alkenylene, and C2-C18 ynynylene are optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or mercapto.

2. A compound having the following general formula (II): (II) Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug: R1 is selected from -OR', R' is selected from hydrogen or C1-C8 alkyl groups; R' is selected from hydrogen or C1-C8 alkyl, wherein each of the C1-C8 alkyl groups is optionally substituted by one or more substituents selected independently from halogen, hydroxyl or mercapto; R2, R3, R4, and R5 are each independently selected from hydrogen, C5-C18 alkyl, C5-C18 alkenyl, and -SC. 3-13 Alkyl and -CH2-SC 3-13 Alkyl; wherein the C5-C18 alkyl, C5-C18 alkenyl and C3-C13 alkyl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxy or mercapto; R9, R 10 and R 11 Each is independently selected from hydrogen or linear C1 alkyl groups; R8 is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl groups; X1 is selected from the key, -S-, or -O-; X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)O-, -C(=O)S-, -SC(=O-); L0 and L1 are each independently selected from a bond or a C1-C8 alkylene group; wherein the C1-C8 alkylene group is optionally substituted by one or more substituents independently selected from halogen, hydroxyl or mercapto. L2, L3, L4 and L5 are each independently selected from a bond, a C1-C18 alkylene group or a C2-C18 alkenyl group, wherein the C1-C18 alkylene group and the C2-C18 alkenyl group are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl or mercapto groups.

3. The compound according to claim 1 or 2, wherein at least one of X2 and X3 is -OC(=O)S- or -SC(=O)O-.

4. The compound according to any one of claims 1 to 3, wherein R1 is -OR', and R' is selected from hydrogen or C1-C8 alkyl.

5. The compound according to any one of claims 1 to 4, wherein R1 is -OR', and R' is hydrogen.

6. The compound according to any of the preceding claims, wherein R1 is And R9, R 10 and R 11 Each is independently selected from hydrogen or C1-C5 alkyl groups.

7. The compound according to any of the preceding claims, wherein R1 is And R9, R 10 and R 11 Each is independently selected from hydrogen or linear C1-C3 alkyl groups.

8. The compound according to any of the preceding claims, wherein R1 is And R9, R 10 and R 11 Each is independently selected from hydrogen or linear C1 alkyl groups.

9. The compound according to any of the preceding claims, wherein L0 is selected from C1-C8 alkylene groups.

10. The compound according to any of the preceding claims, wherein L0 is a linear C1-C5 alkylene group.

11. The compound according to any of the preceding claims, wherein L0 is selected from linear C4 alkylene, linear C3 alkylene, or linear C2 alkylene.

12. The compound according to any of the preceding claims, wherein L0 is a bond.

13. The compound according to any of the preceding claims, wherein L1 is selected from a bond or a C1-C8 alkylene group.

14. The compound according to any of the preceding claims, wherein L1 is a linear C1-C5 alkylene group.

15. The compound according to any of the preceding claims, wherein L1 is selected from linear C4 alkylene, linear C3 alkylene, or linear C2 alkylene.

16. The compound according to any of the preceding claims, wherein L1 is a bond.

17. The compound according to any of the preceding claims, wherein L2 and L3 are independently selected from C3-C18 alkylene groups.

18. The compound according to any of the preceding claims, wherein L2 and L3 are independently bonds.

19. The compound according to any of the preceding claims, wherein L2 and L3 are independently C3-C10 alkylene groups.

20. The compound according to any of the preceding claims, wherein L2 and L3 are independently C5-C8 alkylene groups.

21. The compound according to any of the preceding claims, wherein L2 and L3 are independently C7 alkylene groups.

22. The compound according to any of the preceding claims, wherein L2 and L3 are independently C6 alkylene groups.

23. The compound according to any of the preceding claims, wherein L4 and L5 are independently selected from C1-C5 alkylene groups.

24. The compound according to any of the preceding claims, wherein L4 and L5 are independently bonds.

25. The compound according to any of the preceding claims, wherein L4 and L5 are independently C1 alkylene, C2 alkylene, or C3 alkylene.

26. The compound according to any of the preceding claims, wherein X1 is a bond.

27. The compound according to any of the preceding claims, wherein X2 and X3 are independently selected from -OC(=O)S-, -SC(=O)O-, -C(=O)O-, -OC(=O)-, or -OC(=O)O-; and at least one of X2 and X3 is selected from -OC(=O)S- or -SC(=O)O-.

28. The compound according to any of the preceding claims, wherein X2 and X3 are independently selected from -OC(=O)S- or -SC(=O)O-.

29. The compound according to any of the preceding claims, wherein X3 is selected from -OC(=O)S- or -SC(=O)O-; and X2 is selected from -OC(=O)- or -C(=O)O-.

30. The compound according to any of the preceding claims, wherein R2 and R3 are independently selected from hydrogen, C5-C12 alkyl, and -SC. 3-13 Alkyl or -CH2-SC 3-13 alkyl.

31. The compound according to any of the preceding claims, wherein R2 and R3 are independently C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, C5 alkyl or C4 alkyl.

32. The compound according to any of the preceding claims, wherein R4 and R5 are each independently selected from hydrogen and C5-C12 alkyl groups.

33. The compound according to any of the preceding claims, wherein R4 and R5 are independently C10 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, C5 alkyl or C4 alkyl.

34. The compound according to any of the preceding claims, wherein R2, R3, R4 and R5 are all unbranched.

35. The compound according to any of the preceding claims, wherein R4 and R5 are independently -SC. 3-13 Alkyl group; optionally, R4 and R5 are independently -S-(CH2)5CH3.

36. The compound according to any of the preceding claims, wherein R2 and R3 are independently -SC. 3-13 Alkyl group; optionally, R4 and R5 are independently -S-(CH2)5CH3.

37. The compound according to any of the preceding claims, wherein R3 and R5 are independently -CH2-SC 3-13 Alkyl group; optionally, R3 and R5 are independently –CH2S-(CH2)8CH3, –CH2-S-(CH2)7CH3, –CH2-S-(CH2)6CH3, –CH2S-(CH2)5CH3, –CH2S-(CH2)4CH3, or –CH2S-(CH2)3CH3.

38. The compound according to any of the preceding claims, wherein R2 and R4 are independently -SC. 3-13 Alkyl group; optionally, R2 and R4 are independently -S-(CH2)8CH3, -S-(CH2)7CH3, -S-(CH2)6CH3, -S-(CH2)5CH3, -S-(CH2)4CH3, or -S-(CH2)3CH3.

39. A compound having formula (III): (III) Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug. in: R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2- 10 CH3; X1 is the key; X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=S)O-, -SC(=O)O-, -OC(=O)S-, -OC(=O)O-, -C(=O)S-, -SC(=O-); and at least one of X2 and X3 is selected from -OC(=O)S- or -SC(=O)O-; L1 or L0 is independently selected from a bond or a C1-C8 alkylene group; L2 and L3 are independently C3-C10 alkylene groups; L4 and L5 are independently selected from the bond or C. 1-3 Alkylene.

40. A compound having formula (IV): (IV), Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug. in: R1 is -OH or ; R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2- 10 CH3; R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8; R8 is a C1-C5 alkyl group; X2 is selected from -C(=O)O-, -OC(=O-, or -OC(=O)O-; L1 is a C1-C8 alkylene group; L2 and L3 are independently C3-C10 alkylene groups; L4 and L5 are independently selected from the bond or C. 1-3 Alkylene.

41. A compound having formula (V): (V) Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug. in: R1 is -OH or ; R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2- 10 CH3; R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8; R8 is a C1-C5 alkyl group; X2 is selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -SC(=O)O- or -OC(=O)S-; L1 is a C1-C8 alkylene group; L2 and L3 are independently C3-C10 alkylene groups; L4 and L5 are independently selected from the bond or C. 1-3 Alkylene.

42. A compound having formula (VI): (WE), Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug. in: R1 is -OH or ; R2, R3, R4, and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or -S-(CH2). 2- 10 CH3; R9, R 10 and R 11 Each is independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8; R8 is a C1-C5 alkyl group; X1 is the key; L1 is a C1-C8 alkylene group; L2 and L3 are independently C3-C10 alkylene groups; L4 and L5 are independently selected from the bond or C. 1-3 Alkylene.

43. The compound according to any one of claims 39-42, wherein R1 is R9, R 10 and R 11 Each is independently selected from hydrogen or straight-chain C1-3 alkyl groups.

44. The compound according to any one of claims 39-43, wherein L0 is a bond.

45. The compound according to any one of claims 39-43, wherein L0 is a straight-chain C2 alkylene group.

46. ​​The compound according to any one of claims 39-43, wherein L0 is a straight-chain C4 alkylene group.

47. The compound according to any one of claims 39-43, wherein L0 is a straight-chain C3 alkylene group.

48. The compound according to any one of claims 39-47, wherein L1 is a bond.

49. The compound according to any one of claims 39-47, wherein L1 is a straight-chain C2 alkylene group.

50. The compound according to any one of claims 39-47, wherein L1 is a straight-chain C4 alkylene group.

51. The compound according to any one of claims 39-47, wherein L1 is a straight-chain C3 alkylene group.

52. The compound according to any one of claims 39-51, wherein L2 and L3 are independently C5-C8 alkylene groups.

53. The compound according to any one of claims 39-51, wherein L2 and L3 are independently C6 alkylene groups.

54. The compound according to any one of claims 39-53, wherein L4 and L5 are independently bonds.

55. The compound according to any one of claims 39-53, wherein L4 and L5 are independently C1 alkylene groups.

56. The compound according to any one of claims 39-53, wherein L4 and L5 are independently C2 alkylene groups.

57. A compound having formula (VII): (VII) L0 and L1 are each independently a single bond or a C1-C5 alkylene group, wherein the C1-C5 alkylene group is optionally substituted by one or more substituents, which are independently selected from the group consisting of halogens, hydroxyl groups or thiols; X1 selects a group consisting of a free key, -S-, or -O-; X2 and X3 can each be independently selected from groups consisting of -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=O)S-, or -SC(=O)O-; L2 and L3 are each independently selected from the group consisting of free bonds and C3-C9 alkylene groups, wherein the C3-C9 alkylene groups are optionally substituted by one or more substituents, which are independently selected from the group consisting of C1-C3 alkyl groups, halogens, hydroxyl groups or thiols; L4 and L5 are each independently selected from the group consisting of free bonds and C1-C5 alkylene groups, wherein the C1-C5 alkylene groups are optionally substituted by one or more substituents, which are independently selected from the group consisting of C1-C3 alkyl groups, halogens, hydroxyl groups or thiols; R4 selects freedom The group consisting of C1-C18 alkyl and C2-C18 alkenyl groups, wherein each of the C1-C18 alkyl and C2-C18 alkenyl groups is optionally substituted by one or more substituents, which are independently selected from the group consisting of C1-C3 alkyl, halogen, hydroxyl or thiol; L6, L7, L 10 and L 11 Each is an independent key; R2, R3, R5 and R6 are each independently selected from the group consisting of C1-C18 alkyl or C2-C18 alkenyl groups, wherein the C1-C18 alkyl and C2-C18 alkenyl groups are each optionally substituted by one or more substituents, which are independently selected from the group consisting of C1-C3 alkyl, halogen, hydroxyl or thiol.

58. A compound having formula (VIII): (VIII) Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug. in: L0 and L1 are each independently a single bond or a C1-C5 alkylene group; X1 is the key X2 and X3 are each independently selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=O)S-, or -SC(=O)O-; L2 and L3 are each independently C3-C9 alkylene groups; L4 and L5 are each independently C1-C5 alkylene groups; R4 is selected from C1-C18 alkyl or C2-C18 alkenyl; L6, L7, L 10 and L 11 Each is an independent key; R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

59. The compound of claim 57 or 58, wherein L0 is a bond and L1 is a C1-C5 alkylene group.

60. The compound of any one of claims 57 to 59, wherein X2 and X3 are each independently selected from the group consisting of -C(=O)O-, -OC(=O)- or -OC(=O)O-.

61. The compound of any one of claims 57 to 60, wherein L2 and L3 are each independently C4-C8 alkylene groups.

62. The compound of any one of claims 57 to 61, wherein L4 and L5 are each independently C1-C3 alkylene groups.

63. The compound of any one of claims 57-62, wherein R4 is selected from... L 10 and L 11 Each is an independent bond; R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

64. A compound having formula (IX): (IX) Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug. in: L0 and L1 are each independently a bond or a C1-C5 alkylene group, wherein the C1-C5 alkylene group is optionally substituted by one or more substituents independently selected from halogens, hydroxyl groups or thiols; X1 is selected from the key, -S-, or -O-; X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=O)S- or -SC(=O)O-; L2 and L3 are each independently selected from C3-C8 alkylene groups, wherein the C3-C8 alkylene groups are optionally substituted by one or more substituents independently selected from C1-C3 alkyl groups, halogens, hydroxyl groups or thiols; L4 and L5 are each independently selected from C1-C5 alkylene groups, wherein the C1-C5 alkylene groups are optionally substituted by one or more substituents independently selected from C1-C3 alkyl groups, halogens, hydroxyl groups, or thiols; R4 is selected from , , C1-C18 alkyl, C2-C18 alkenyl; wherein the C1-C18 alkyl and the C2-C18 alkenyl are each optionally substituted by one or more substituents independently selected from C1-C3 alkyl, halogen, hydroxyl or thiol; Among them, L6, L7, L 10 and L 11 Each is an independent key; R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl; wherein the C1-C18 alkyl and the C2-C18 alkenyl are each optionally substituted by one or more substituents independently selected from C1-C3 alkyl, halogen, hydroxyl or thiol.

65. A compound having formula (X): (X) Or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug. in: L0 and L1 are each independently a bond or a C1-C5 alkylene group; X1 is the key; X2 and X3 are independently selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -OC(=O)S- or -SC(=O)O-; L2 and L3 are each independently C3-C8 alkylene groups; L4 and L5 are each independently C1-C5 alkylene groups; L6, L7, L 10 and L 11 Each is an independent key; R2, R3, R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

66. The compound according to claim 64 or 65, wherein L0 is a bond and L1 is a C1-C5 alkylene group.

67. The compound according to any one of claims 64-66, wherein X2 and X3 are independently selected from -C(=O)O-, -OC(=O)- or -OC(=O)O-.

68. The compound according to any one of claims 64-67, wherein L2 and L3 are each independently C5-C8 alkylene groups.

69. The compound according to any one of claims 64-68, wherein L4 and L5 are each independently C1-C3 alkylene groups.

70. The compound according to any one of claims 64-69, wherein R4 is selected from... L10 and L11 are each independently bonded; R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups.

71. The compound of any one of the preceding claims, wherein the compound is selected from: YZL408 (1); YZL410 (2); YZL412 (3); YZL413 (4); YZL424 (5); YZL427 (6); YZL400 (7); YZL409 (8); YZL411 (9); YZL414 (10); YZL415 (11); YZL416 (12); YZL417 (13); YZL425 (14); YZL426 (15); YZL430 (16); YZL431 (17); YZL432 (18); YZL433 (19); YZL439 (20); YZL440 (21); YZL441 (22); YZL442 (23); YZL443 (24); YZL444 (25); YZL445 (26); YZL446 (27); YZL447 (28); YZL448 (29); YZL449 (30); YZL450 (31); YZL452 (32); YZL453 (33); YZL455 (34); YZL456 (35); YZL457 (36); YZL458 (37); YZL459 (38); YZL460 (39); YZL501 (40); (YZL429) (41); (YZL428) (42); (YZL418) (43); (YZL419) (44); (YZL422) (45); (YZL423) (46); (YZL424) (47); (YZL425) (48); (YZL473) (49); (YZL434) (50); (YZL451) (51); (YZL449) (52); YZL 479 (53); YZL480 (54); YZL491(55); YZL495 (56); YZL499 (57); YZL701 (58); YZL702 (59); YZL703 (60); YZL704 (61); or YZL705 (62).

72. A composition comprising the compound of any of the preceding claims in a lipid component, preferably, the composition being an LNP composition.

73. The composition of claim 72, wherein the lipid component comprises non-cationic lipids and PEGylated lipids.

74. The composition according to claim 72 or 73, wherein the composition further comprises a bioactive agent.

75. The composition of claim 74, wherein the bioactive agent comprises nucleic acid.

76. The composition of claim 75, wherein the nucleic acid comprises a sequence encoding a Cas nuclease.

77. The composition according to claim 75 or 76, wherein the nucleic acid comprises gRNA.

78. The composition according to any one of claims 75-77, wherein the nucleic acid comprises Cas nuclease mRNA and gRNA.

79. A method of delivering a bioactive agent to cells, comprising contacting the cells with the composition of any one of claims 72-78.

80. The method of claim 79, wherein the cell is a hepatocyte, such as a hepatocyte.

81. A gene editing method comprising contacting cells with the composition of any one of claims 72-78.

82. A method for cutting DNA, comprising contacting a cell with the composition of any one of claims 72-78.

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