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

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480047392.9 (22) Application Date 2024.07.19 (66) Domestic Priority Data PCT / CN2023 / 108217 2023.07.19 CN PCT / CN2023 / 108216 2023.07.19 CN PCT / CN2023 / 120617 2023.09.22 CN PCT / CN2023 / 120619 2023.09.22 CN (85) PCT International Application Entering National Phase Date 2026.01.15 (86) PCT International Application Application Data PCT / CN2024 / 106309 2024.07.19 (87) Publication data of PCT international application WO2025 / 016446 EN 2025.01.23 (71) Applicant Zhengji Gene Technology Co., Ltd. Address Room 101-102, 1 / F, 6W, Science Avenue West, Pak Shek Kok Science Park, New Territories, Hong Kong, China (72) Inventor Hu Sijun (74) Patent Agency Beijing Baishansong Intellectual Property Agency (General Partnership) 11413 Patent Attorneys Yin Haoming Hui Zhenhai (51) Int.Cl. C07C 323 / 52 (2006.01) C07C 323 / 12 (2006.01) A61K 47 / 18 (2006.01) A61K 47 / 20 (2006.01) A61P 35 / 00 (2006.01) (54) Title of Invention: Lipids and Lipid Nanoparticles (57) Abstract This article discloses an ionizable lipid and its salts (e.g., pharmaceutically acceptable salts thereof), as well as compositions containing such lipids that can be used to deliver bioactive agents. Claims 21 pages, Description 80 pages, Sequence List (electronic publication), Drawings 7 pages, CN 121532376 A 2026.02.13 CN 1 21 53 23 76 A 1. A compound (I) having the following general formula (I), or its N-oxide, pharmaceutically acceptable salt, isomer or prodrug: wherein: R1 is hydrogen; phenyl; 3 to 7-membered aliphatic group; 3 to 7-membered heterocyclic group containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur; 5 to 6-membered monocyclic heteroaryl group containing 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur; 8 to 10-membered bicyclic heteroaryl group containing 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur; -OR'; or; wherein phenyl, cyclic aliphatic group, 3 to 7 The 5- to 6-membered monocyclic heteroaryl group, or the 8- to 10-membered bicyclic heteroaryl group, is optionally substituted by one or more substituents independently selected from halogen, hydroxyl or mercapto.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, -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 are optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or mercapto; R9, R10, and R11 are each 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 optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or mercapto; R8 is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 ynyl; X1 is selected from bond, -S-, -O-, -S(O)2-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -OS(O)2N(R12)-, and -(R12)NS. (O)2O‑,‑N(R12)S(O)2N(R13)‑,‑C(=O)O‑,‑OC(=O)‑,‑OC(=O)O‑,‑OC(=S)O‑,‑SC(=O)O‑,‑OC (=O)S‑,‑OC(=O)N(R12)‑,‑(R12)NC(=O)O‑,‑OC(=S)N(R12)‑,‑(R12)NC(=S)O‑,‑SC(=O)N (R12)‑or‑(R12)NC(=O)S‑; R12 and R13 are each independently selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, and -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-, and -SC(=O)-; L0 and L1 are each independently selected from a bond, C1-C8 alkylene, C2-C8 alkenylene, or C2-C8 ynynylene; wherein the C1-C8 alkylene, C2-C8 alkenylene, and C2-C8 ynynylene 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 alkenyl, and C2-C18 ynynyl, wherein the C1-C18 alkylene, C2-C18 alkenyl, and C2-C18 ynynyl 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'; wherein R' is selected from hydrogen or C1-C8 alkyl; 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; R2, R3, R4, and R5 are each independently selected from hydrogen, C5-C18 alkyl, C5-C18 alkenyl, -S-C3-13 alkyl, and -CH2-S-C3-13 alkyl; wherein each of the C5-C18 alkyl, C5-C18 alkenyl, and C3-C13 alkyl groups is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or mercapto; R9, R10, and R11 are each independently selected from hydrogen or linear C1 alkyl; R8 is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl; X1 is selected from -C1-C5 alkyl, -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 -C1-C8 alkylene; wherein the C1-C8 alkylene 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 -C1-C18 alkylene or C2-C18 alkenyl, wherein the C1-C18 alkylene, C2-C18 alkenyl ... Each of the C18 alkenyl groups is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or mercapto. 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', wherein 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', wherein R' is hydrogen. 6. The compound according to any of the preceding claims, wherein R1 is; and R9, R10, and R11 are each independently selected from hydrogen or C1-C5 alkyl. Claims 2 / 21 pages 3 CN 121532376 A7. The compound according to any of the preceding claims, wherein R1 is alkyl; and R9, R10, and R11 are each independently selected from hydrogen or linear C1-C3 alkyl. 8. The compound according to any of the preceding claims, wherein R1 is alkyl; and R9, R10, and R11 are each independently selected from hydrogen or linear C1 alkyl. 9. The compound according to any of the preceding claims, wherein L0 is selected from C1-C8 alkylene. 10. The compound according to any of the preceding claims, wherein L0 is linear C1-C5 alkylene. 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 C1-C5 alkylene. 13. The compound according to any of the preceding claims, wherein L1 is selected from C1-C8 alkylene. 14. The compound according to any of the preceding claims, wherein L1 is linear C1-C5 alkylene. 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 C4 alkylene. 17. The compound according to any of the preceding claims, wherein L2 and L3 are independently selected from C3-C18 alkylene. 18. The compound according to any of the preceding claims, wherein L2 and L3 are independently C3-C10 alkylene. 19. The compound according to any of the preceding claims, wherein L2 and L3 are independently C3-C10 alkylene. 20. The compound according to any of the preceding claims, wherein L2 and L3 are independently C5-C8 alkylene. 21. The compound according to any of the preceding claims, wherein L2 and L3 are independently C7 alkylene. 22. The compound according to any of the preceding claims, wherein L2 and L3 are independently C6 alkylene. 23. The compound according to any of the preceding claims, wherein L4 and L5 are independently selected from C1-C5 alkylene. 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-. Claims 3 / 21, page 4, CN 121532376 A 30. The compound according to any of the preceding claims, wherein R2 and R3 are independently selected from hydrogen, C5-C12 alkyl, -S-C3-13 alkyl, or -CH2-S-C3-13 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. 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 -S-C3-13 alkyl; 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 -S-C3-13 alkyl; 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-S-C3-13 alkyl; 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 -S-C3-13 alkyl; 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, wherein: R2, R3, R4 and R5 are each independently selected from hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl or -S-(CH2)2-10CH3; X1 is a bond;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 C1-C8 alkylene groups; L2 and L3 are independently C3-C10 alkylene groups; L4 and L5 are independently selected from C1-C3 alkylene groups. 40. A compound having formula (IV): Claims 4 / 21, page 5, CN 121532376 A (IV), or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug, wherein: 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)2-10CH3; R9, R10, and R11 are each independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8; R8 is C1-C5 alkyl; X2 is selected from -C(=O)O-, -OC(=O)-, or -OC(=O)O-; L1 is C1-C8 alkylene; L2 and L3 are independently C3-C10 alkylene; L4 and L5 are independently selected from C1-C3 alkylene. 41. A compound having formula (V): (V) or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug, wherein: 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)2-10CH3; R9, R10, and R11 are each independently selected from hydrogen, C1-C5 alkyl, or -C(=O)R8; R8 is C1-C5 alkyl; X2 is selected from -C(=O)O-, -OC(=O)-, -OC(=O)O-, -SC(=O)O-, or -OC(=O)S-; L1 is C1-C8 alkylene; L2 and L3 are independently C3-C10 alkylene; Claims 5 / 21 pages 6 CN 121532376 A L4 and L5 are independently selected from a bond or C1-3 alkylene. 42. A compound having formula (VI): (VI), or its N-oxide, pharmaceutically acceptable salt, isomer or prodrug, wherein: 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)2-10CH3; R9, R10 and R11 are each independently selected from hydrogen, C1-C5 alkyl or -C(=O)R8; R8 is C1-C5 alkyl; X1 is a bond;L1 is a C1-C8 alkylene; L2 and L3 are independently C3-C10 alkylene; L4 and L5 are independently selected from C1-C3 alkylene. 43. The compound according to any one of claims 39-42, wherein R1 is; R9, R10, and R11 are each independently selected from hydrogen or straight-chain C1-C3 alkyl. 44. The compound according to any one of claims 39-43, wherein L0 is a C1-C8 alkylene. 45. The compound according to any one of claims 39-43, wherein L0 is a straight-chain C2 alkylene. 46. The compound according to any one of claims 39-43, wherein L0 is a straight-chain C4 alkylene. 47. The compound according to any one of claims 39-43, wherein L0 is a straight-chain C3 alkylene. 48. The compound according to any one of claims 39-47, wherein L1 is a C1-C10 alkylene. 49. The compound according to any one of claims 39-47, wherein L1 is a straight-chain C2 alkylene. 50. The compound according to any one of claims 39-47, wherein L1 is a straight-chain C4 alkylene. 51. The compound according to any one of claims 39-47, wherein L1 is a straight-chain C3 alkylene. 52. The compound according to any one of claims 39-51, wherein L2 and L3 are independently C5-C8 alkylene. 53. The compound according to any one of claims 39-51, wherein L2 and L3 are independently C6 alkylene. 54. The compound according to any one of claims 39-53, wherein L4 and L5 are independently C1 alkylene. 55. The compound according to any one of claims 39-53, wherein L4 and L5 are independently C1 alkylene. 56. The compound according to any one of claims 39-53, wherein L4 and L5 are independently C2 alkylene. 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 is selected from the group consisting of free bonds, -S-, or -O-; X2 and X3 are each independently selected from the group 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 group is 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 L5Each of the following groups is independently selected from the group consisting of 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, halogen, hydroxyl, or thiol groups; R4 is selected from the group consisting of C1-C18 alkyl and 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 groups; L6, L7, L10, and L11 are each independently a single bond; R2, R3, R5, and R6 Each is independently selected from the group consisting of C1-C18 alkyl or 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. 58. A compound having formula (VIII): (VIII) or its N-oxide, pharmaceutically acceptable salt, isomer or prodrug, wherein: L0 and L1 are each independently a single bond or C1-C5 alkylene; X1 is a single bond; 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; L4 and L5 are each independently C1-C5 alkylene; R4 is selected from C1-C18 alkyl or C2-C18 alkenyl; L6, L7, L10 and L11 are each independently a single bond; R2, R3, R5 and R6 Each is independently selected from C1-C18 alkyl or C2-C18 alkenyl. 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 to 62, wherein R4 is selected from, L10 and L11 are each independently a bond; and R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl groups. 64. A compound having the formula (IX): (IX) or its N-oxide, pharmaceutically acceptable salt, isomer, or prodrug, wherein: L0 and L1Each of the following is independently 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 C1-C5 alkylene groups, -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 C1-C5 alkylene groups, wherein the C3-C8 alkylene group is optionally substituted by one or more substituents independently selected from C1-C3 alkylene groups, halogens, hydroxyl groups, or thiols; L4 and L5 are each independently selected from C1-C5 alkylene groups, wherein the C1-C5 alkylene group is optionally substituted by one or more substituents independently selected from C1-C3 alkylene groups, halogens, hydroxyl groups, or thiols; R4 is selected from [missing information - likely a specific group or group]. 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; wherein L6, L7, L10 and L11 are each independently bonded; 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, wherein: L0 and L1 are each independently bonded or C1-C5 alkylene; X1 is a bond; 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; L4 and L5 are each independently C1-C5 alkylene; L6, L7, L10, and L11 are each independently bonded; R2, R3, R5, and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl. 66. The compound according to claim 64 or 65, wherein L0 is a bond and L1 is a C1-C5 alkylene. 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 a bond; and R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl. 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); 121532376 A YZL431 (17); YZL432 (18); YZL433 (19); YZL439 (20); Claims 13 / 21 pages 14 CN 121532376 A YZL440 (21); YZL441 (22); YZL442 (23); YZL443 (24); Claims 14 / 21 pages 15 CN 121532376 A YZL444 (25); YZL445 (26); YZL446 (27); YZL447 (28); YZL448 (29); Claims 15 / 21 pages 16 CN 121532376 A YZL449 (30); YZL450 (31); YZL452 (32); YZL453 (33); YZL455 (34); YZL456 (35); Claims 16 / 21, page 17, CN 121532376 A YZL457 (36); YZL458 (37); YZL459 (38); YZL460 (39); YZL501 (40); (YZL429) (41); (YZL428) (42); (YZL418) (43); Claims 17 / 21, page 18, CN 121532376 A (YZL419)(44); (YZL422) (45); (YZL423) (46); (YZL424) (47); (YZL425) (48); (YZL473) (49); (YZL434) (50); Claims 18 / 21 pages 19 CN 121532376 A (YZL451) (51); (YZL449) (52); YZL 479 (53); YZL480 (54); YZL491(55); YZL495 (56); Claims 19 / 21 pages 20 CN 121532376 A YZL499 (57); YZL701 (58); YZL702 (59); YZL703 (60); YZL704 (61); or YZL705 (62). Claims 20 / 21 pages 21 CN 121532376 A 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 a non-cationic lipid and a PEGylated lipid. 74. The composition of claim 72 or 73, wherein the composition further comprises a bioactive agent. 75. The composition of claim 74, wherein the bioactive agent comprises a nucleic acid. 76. The composition of claim 75, wherein the nucleic acid comprises a sequence encoding a Cas nuclease. 77. The composition of claim 75 or 76, wherein the nucleic acid comprises gRNA. 78. The composition of 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 cells are hepatocytes, such as hepatocytes. 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 cells with the composition of any one of claims 72-78. Claims 21 / 21 pages 22 CN 121532376 A Lipids and Lipid Nanoparticles 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 Art

[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) to cells and other intracellular compartments. There remains a continued need to develop new lipids for encapsulating therapeutic agents and to improve the safety, effectiveness, and specificity of such nanoparticle-based delivery vehicles.

[0004] The present 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 comprising 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; 5- to 6-membered monocyclic heteroaryl group comprising 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur; 8- to 10-membered bicyclic heteroaryl group comprising 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur; -OR'; or; wherein the phenyl, cycloalkyl, 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 thiol group;

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

[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 the C1-C18 alkyl, C2-C18 alkenyl, C2-C18 ynyl, or C3-13 alkyl portion is each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or thiol;

[0010] R9, R10, and R11 are each independently selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 ynyl, or -C(=O)R8; in the specification, page 1 / 80, 23 CN 121532376 A, 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;

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

[0012] X1 is selected from key, ‑S‑, ‑O‑, ‑S(O)2‑, ‑OS(O)2‑, ‑S(O)2O‑, ‑OS(O)2O‑, ‑OS(O)2N(R12)‑, ‑ (R12)NS(O)2O‑,‑N(R12)S(O)2N(R13)‑,‑C(=O)O‑,‑OC(=O)‑,‑OC(=O)O‑,‑OC(=S)O‑,‑SC(=O) O‑,‑OC(=O)S‑,‑OC(=O)N(R12)‑,‑(R12)NC(=O)O‑,‑OC(=S)N(R12)‑,‑(R12)NC(=S)O‑,‑SC(=O) N(R12)‑ or‑(R12)NC(=O)S‑;

[0013] R12 and R13 are each 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;

[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 bond, C1-C8 alkylene, C2-C8 alkenyl, or C2-C8 ynylene; wherein the C1-C8 The alkylene, C2-C8 alkenylene, and C2-C8 alkyne 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 alkyne, wherein the C1-C18 alkylene, C2-C18 alkenylene, and C2-C18 alkyne are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxyl, or thiol groups.

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

[0018] (II)

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

[0020] wherein

[0021] R1 is selected from -OR';

[0022] wherein 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, -S-C3-13 alkyl and -CH2-S-C3-13 alkyl; wherein the C5-C18 alkyl, C5-C18 alkenyl, -S-(CH2)2-10CH3 and C3-13 alkyl portions are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, cycloalkyl, aryl, halogen, hydroxy or thiol groups;

[0024] R9, R10 and R11 are each independently selected from hydrogen or linear C1 alkyl.

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

[0026] X1 is selected from -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 -C1-C8 alkylene; wherein the C1-C8 alkylene 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 C1-C18 alkylene or C2-C18 alkenyl groups, wherein the C1-C18 alkylene and C2-C18 alkenyl groups are 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 certain embodiments, R1 is -OR', where R' is selected from hydrogen or C1-C8 alkyl. In certain embodiments, R1 is -OR', where R' is hydrogen. In certain embodiments, R1 is, and R9, R10, and R11 are each independently selected from hydrogen or C1-C5 alkyl. In certain embodiments, R1 is, and R9, R10, and R11 are independently selected from hydrogen or linear C1-C3 alkyl. In some embodiments, R1 is selected from, and R9, R10, and R11 are each independently selected from hydrogen or linear C1 alkyl.

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

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

[0034] In some embodiments, L2 and L3 are independently selected from C1-C18 alkylene groups. In some embodiments, L2 and L3 are independently C1-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-C18 alkylene groups. In some embodiments, L4 and L5 are independently C1 alkylene, C2 alkylene, or C3 alkylene.

[0036] In some embodiments, X1 is a bond. 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, -S-C3-13 alkyl, or -CH2-S-C3-13 alkyl. 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-C12 alkyl, -S-C3-13 alkyl, or -CH2-S-C3-13 alkyl. 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-S-C3-13 alkyl. 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-C3-13 alkyl; 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] The present invention provides a compound as shown in formula (III),

[0041] (III)

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

[0043] wherein

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

[0045] X1 is a bond; 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 is independently selected from C1-C8 alkylene groups;

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

[0048] L4 and L5 are independently selected from C1-C3 alkylene groups.

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

[0050] (IV),

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

[0052] wherein the specification 4 / 80 pages 26 CN 121532376 A

[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)2-10CH3;

[0055] R9, R10 and R11 are each 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 C1-C3 alkylene groups.

[0061] The present invention provides a compound as shown in formula (V),

[0062] (V)

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

[0064] wherein

[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)2-10CH3;

[0067] R9, R10 and R11 are each independently selected from hydrogen, C1-C5 alkyl or -C(=O)R8;

[0068] R8 is C1-C5 alkyl;

[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 a C3-C10 alkylene group;

[0072] L4 and L5 are each independently selected from C1-C3 alkylene groups.

[0073] The present invention provides a compound as shown in formula (VI), specification 5 / 80 pages 27 CN 121532376 A

[0074] (VI),

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

[0076] wherein

[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)2-10CH3;

[0079] R9, R10 and R11 are each independently selected from hydrogen, C1-C5 alkyl or -C(=O)R8;

[0080] R8 is C1-C5 alkyl;

[0081] X1 is a bond;

[0082] L1 is C1-C8 alkylene;

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

[0084] L4 and L5 are each independently selected from C1-C3 alkylene groups.

[0085] In some embodiments, R1 is; R9, R10, and R11 are each independently selected from hydrogen or straight-chain C1-C3 alkyl groups.

[0086] In some embodiments, L0 is a C1-C3 alkylene group. In some embodiments, L0 is a straight-chain C2 alkylene group. In some embodiments, L0 is a straight-chain C4 alkylene group. In some embodiments, L0 is a straight-chain C3 alkylene group.

[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. In some embodiments, L2 and L3 are each independently C6 alkylene.

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

[0090] The present invention provides a compound as shown in formula (VII), specification 6 / 80 pages 28 CN 121532376 A

[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, said substituents being independently selected from halogens, hydroxyl groups or thiols;

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

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

[0095] L2 and L3 Each is independently selected from C1-C3 alkylene groups, wherein the C3-C9 alkylene group is optionally substituted by one or more substituents, wherein the substituents are independently selected from C1-C3 alkyl, halogen, hydroxyl or thiol;

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

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

[0098] wherein L6, L7, L10 L10 and L11 are each independently bonded or C6-C9 alkenyl; preferably, L6, L7, L10 and L11 are each independently bonded.

[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, said substituents being independently selected from C1-C3 alkyl, halogen, hydroxyl or thiol.

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

[0101] (VIII)

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

[0103] wherein

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

[0105] X1 is a bond;

[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 a C3-C9 alkylene group;

[0108] L4 and L5 are each independently a C1-C5 alkylene group; Specification 7 / 80 page 29 CN 121532376 A

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

[0110] L6, L7, L10 and L11 are each independently a bond;

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

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

[0113] In some embodiments, X2 and X3 are each 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. In some embodiments, L2 and L3 are each independently C3 alkylene. In some embodiments, L2 and L3 are each independently C4 alkylene. In some embodiments, L2 and L3 are each independently C5 alkylene. In some embodiments, L2 and L3 are each independently C6 alkylene. In some embodiments, L2 and L3 are each independently C7 alkylene. In some embodiments, L2 and L3 are each independently C8 alkylene. In some embodiments, L2 and L3 are each independently C9 alkylene.

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

[0116] In some embodiments, R4 is selected from C1-C18 alkyl or C2-C18 alkenyl; L10 and L11 are each independently bonded; R5 and R6 are each independently selected from C1-C18 alkyl or C2-C18 alkenyl.

[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 groups. In some embodiments, R2, R3, R5, and R6 are each...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] The present invention provides a compound as shown in formula (IX),

[0119] (IX)

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

[0121] wherein

[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, the substituents being independently selected from the group consisting of halogens, hydroxyl groups or mercapto groups;

[0123] X1 is selected from the group consisting of 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-; Specification 8 / 80 pages 30 CN 121532376 A

[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, halogen, hydroxyl, 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, halogen, hydroxyl, or mercapto groups;

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

[0127] wherein L6, L7, L10, and L11 are each independently free bonds;

[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, the substituents being independently selected from the group consisting of C1-C3 alkyl, halogen, hydroxyl or mercapto.

[0129] The present invention provides a compound as shown in formula (X),

[0130] (X)

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

[0132] wherein

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

[0134] X1 is a bond;

[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 a C3-C8 alkylene group;

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

[0138] L6, L7, L10 and L11 are each independently a bond;

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

[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.

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

[0144] In some embodiments, R4 is selected from C1-C18 alkyl or C2-C18 alkenyl.

[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 groups (page 9 / 80, CN 121532376 A). In some embodiments, R2, R3, R5, and R6 are each C5 alkyl groups. In some embodiments, R2, R3, R5, and R6 are each C6 alkyl groups. In some embodiments, R2, R3, R5, and R6 are each C7 alkyl groups. In some embodiments, R2, R3, R5, and R6 are each C8 alkyl groups. In some embodiments, R2, R3, R5, and R6 are each C9 alkyl groups. In some embodiments, R2, R3, R5, and R6 are each C10 alkyl groups. In some embodiments, R2, R3, R5, and R6 are each C11 alkyl groups. In some embodiments, R2, R3, R5, and R6 are each C12 alkyl groups.

[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); Specification 10 / 80 pages 32 CN 121532376 A

[0153]

[0154] YZL413 (4);

[0155]

[0156] YZL424 (5);

[0157]

[0158] YZL427 (6);

[0159]

[0160] YZL400 (7);

[0161]

[0162] YZL409 (8); Specification 11 / 80 pages 33 CN 121532376 A

[0163]

[0164] YZL411 (9);

[0165]

[0166] YZL414 (10);

[0167]

[0168] YZL415 (11);

[0169]

[0170] YZL416 (12);

[0171]

[0172] YZL417 (13); Specification 12 / 80 pages 34 CN 121532376 A

[0173]

[0174] YZL425 (14);

[0175]

[0176] YZL426 (15);

[0177]

[0178] YZL430 (16);

[0179]

[0180] YZL431 (17);

[0181] Specification 13 / 80 pages 35 CN 121532376 A

[0182] YZL432 (18);

[0183]

[0184] YZL433 (19);

[0185]

[0186] YZL439 (20);

[0187]

[0188] YZL440 (21);

[0189]

[0190] YZL441 (22);

[0191]

[0192] YZL442 (23); Specification 14 / 80 pages 36 CN 121532376 A

[0193]

[0194] YZL443 (24);

[0195]

[0196] YZL444 (25);

[0197]

[0198] YZL445 (26);

[0199]

[0200] YZL446 (27); Specification 15 / 80 Page 37 CN 121532376 A

[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); Specification 16 / 80 pages 38 CN 121532376 A

[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] Specification 17 / 80 pages 39 CN 121532376 A

[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); Specification 18 / 80 pages 40 CN 121532376 A

[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); Specification 19 / 80 pages 41 CN 121532376 A

[0257]

[0258] YZL495 (56);

[0259]

[0260] YZL499 (57);

[0261]

[0262] YZL701 (58);

[0263]

[0264] YZL702 (59);

[0265]

[0266] YZL703 (60);

[0267] Specification 20 / 80 pages 42 CN 121532376 A

[0268] YZL704 (61); or

[0269]

[0270] YZL705 (62).

[0271] The present invention discloses lipids containing amine groups, which can be used to prepare lipid nanoparticle (LNP) compositions.This LNP composition may have properties that facilitate the delivery of 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 a non-cationic lipid and a PEGylated lipid. In some embodiments, the non-cationic lipid is a neutral lipid.

[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 a 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 of delivering a bioactive agent to a cell, comprising contacting the cell with the composition disclosed herein. In some embodiments, the cell is a hepatocyte, such as a liver cell.

[0277] The present invention also provides a gene editing method comprising contacting cells with the compositions 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 reagent is preferably a drug.

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

[0281] In some embodiments, the method described herein comprises administering the composition to a human. In some embodiments, the method comprises administering the composition to cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, the method comprises 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 comprises administering mRNA and gRNA nucleic acids formulated in a single LNP composition. Specification 21 / 80 pages 43 CN 121532376 A Brief Description of the Drawings

[0282] Figures 1A-1C show the editing efficiency of targeting TTR after administration of various LNPs in mice.

[0283] Figures 2A-2C show the editing efficiency of various LNPs targeting TTR in PCH.

[0284] Figures 3A-3C show the editing efficiency of targeting TTR after administration of various LNPs in NHP.

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

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

[0287] Figures 6A-6B show the circulating ALT and AST levels in rats after administration of various LNPs.

[0288] Detailed Description of the Invention

[0289] General Definitions

[0290] It should be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as “comprising,” “including,” “having,” etc., may have the meanings given to them by U.S. patent law; for example, they may mean “comprising,” “being included,” “having,” etc.; while 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,” “described,” and similar terms used in the context of this invention (particularly in the context of the claims) should be interpreted to cover both singular and plural forms unless otherwise stated herein or the context clearly contradicts. Furthermore, it should be noted that a plural form does not necessarily mean that it is plural and needs to be understood according to the context in the text.

[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 specified 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 cover a range of specified values ​​and variations thereof, provided that such variations are appropriate to 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 it 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 the case where the event occurs and the case where the event does not occur.

[0298] Unless otherwise defined herein, scientific and technical terms relating to this disclosure shall have the meanings generally 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 take precedence over any dictionary or foreign definition. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms.

[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 technology considers any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variant thereof, such as methylation, hydroxylation, or glycosylation of these bases. Polymers or oligomers 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.Spiral, peptide nucleic acid (PNA) (see Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002) and U.S. Patent No. 5,034,506), locked nucleic acid (LNA; see Wahllestedt et al., Proc. Natl. Acad. Sci. USA, 97: 5633-5638 (2000)), cyclohexenyl nucleic acid (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 or ribonucleotides, or analogs thereof.

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

[0301] All publications, published patent documents, and patent applications are incorporated herein by reference in their entirety as if each individual publication, published patent document, or patent application were specifically and individually indicated as incorporated by reference.

[0302] Ionizable Lipids

[0303] This invention is based in part on the discovery of novel ionizable or amino lipids, which are used in the delivery of active or therapeutic agentsLipid nanoparticles (such as nucleic acids) offer advantages when delivered to mammalian cells in vivo or in vitro.

[0304] “Ionizable lipids” are a key component of lipid nanoparticles (LNPs) used in delivery systems. These lipids are characterized by their ability to ionize, meaning they can change 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 promoting 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, significantly increasing the therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions. Specification 23 / 80 pages 45 CN 121532376 A

[0306] In some embodiments, the present invention provides novel ionizable lipids that enable the formulation of improved compositions for 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 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 of the present invention are as follows:

[0308] Formula (I):

[0309] (I),

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

[0311] wherein:

[0312] R1 is hydrogen; phenyl; 3- to 7-membered aliphatic; 3- to 7-membered heterocyclic group comprising 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; 5- to 6-membered monocyclic heteroaryl group comprising 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur; 8- to 10-membered bicyclic heteroaryl group comprising 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur; -OR'; or; wherein the phenyl, aliphatic, 3- to 7-membered heterocyclic group, 5- to 6-membered monocyclic heteroaryl group, and 8- to 10-membered bicyclic heteroaryl group are optionally separated by one or moreThe substituent is selected from the group consisting of halogen, hydroxyl, or thiol;

[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 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 portions 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, R10 and R11 are each 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, 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;

[0317] (R12)NS(O)2O‑,‑N(R12)S(O)2N(R13)‑,‑C(=O)O‑,‑OC(=O)‑,‑OC(=O)O‑,‑OC(=S)O‑,‑SC(=O)

[0318] R12 and R13 are each 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 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 bond, C1-C8 alkylene, C2-C8 alkenyl or C2-C8 alkyne; wherein each of the C1-C8 alkylene, C2-C8 alkenyl and C2-C8 alkyne is optionally substituted by one or more substituents independently selected from the group consisting of: halogen, hydroxyl or thiol;

[0321] L2 and L3 are each independently selected from bond, C1-C18 alkylene, C2-C18 alkenyl, C2-C18 alkyne, wherein each of the C1-C18 alkylene, C2-C18 alkenyl and C2-C18 alkyne is 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 an N-oxide, pharmaceutically acceptable salt, isomer or prodrug of the compound,

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

[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, -S-(CH2)2-10CH3 or -(CH2)2-S-(CH2)2-10CH3; wherein C5-C18 alkyl, C5-C18 alkenyl, -S-(CH2)2-10CH3 and -(CH2)2-S-(CH2)2-10CH3 are each optionally substituted by one or more substituents independently selected from halogen, hydroxyl or mercapto;

[0328] R9, R10 and R11 are each independently selected from hydrogen or linear C1 alkyl.

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

[0330] X1 is selected from -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 -C1-C8 alkylene; wherein the C1-C8 alkylene 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 C1-C18 alkylene or C2-C18 alkenyl groups, wherein the C1-C18 alkylene and C2-C18 alkenyl groups are each optionally represented by one or more independently selected C1-C6 alkyl, cycloalkyl, aryl, halogen, or alkyl groups.Substituents of 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', wherein R' is hydrogen. In some embodiments, R1 is, and R9, R10 and R11 are each independently selected from hydrogen or C1-C5 alkyl. In some embodiments, R1 is, and R9, R10 and R11 are each independently selected from hydrogen or linear C1-C3 alkyl. In some embodiments, R1 is selected, and R9, R10 and R11 are each independently selected from hydrogen or linear C1 alkyl.

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

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

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

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

[0340] In some embodiments, X1 is C1 alkylene. 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)O-, or -OC(=O)O-.X1, 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)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, -S-(CH2)2-10CH3, 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, -S-(CH2)2-10CH3, or -(CH2)2-S-(CH2)2-10CH3. 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 an N-oxide, pharmaceutically acceptable salt, isomer or prodrug of the compound,

[0347] wherein

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

[0349] X1 is a bond;

[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 C1-C8 alkylene;

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

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

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

[0355] (IV),

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

[0357] wherein

[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)2-10CH3;

[0360] R9, R10 and R11 are each independently selected from hydrogen, C1-C5 alkyl or -C(=O)R8;

[0361] R8 is C1-C5 alkyl;

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

[0363] L1 It is a C1-C8 alkylene; Specification 27 / 80 pages 49 CN 121532376 A

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

[0365] L4 and L5 are each independently selected from C1-C3 alkylene.

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

[0367] (V)

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

[0369] wherein

[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)2-10CH3;

[0372] R9, R10 and R11 are each independently selected from hydrogen, C1-C5 alkyl or -C(=O)R8;

[0373] R8 is C1-C5 alkyl;

[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 an N-oxide, pharmaceutically acceptable salt, isomer or prodrug of the compound,

[0381] wherein

[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)2-10CH3; Specification 28 / 80 pages 50 CN 121532376 A

[0384] R9, R10 and R11 are each independently selected from hydrogen, C1-C5 alkyl or -C(=O)R8;

[0385] R8 is C1-C5 alkyl;

[0386] X1 is a bond;

[0387] L1 is C1-C8 alkylene;

[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, R10, and R11 are each independently selected from hydrogen or C1-3 linear alkylene groups.

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

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

[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 C2 linear alkylene group. In some embodiments, L4 and L5 are each independently C1 alkylene. In some embodiments, L4 and L5 are each independently C2 alkylene.

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

[0396] (VII)

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

[0398] X1 is selected from C1-, -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 C3-C9 alkylene, wherein the C3-C9 alkylene is optionally substituted by one or more substituents.Substituents are selected from C1-C3 alkyl, halogen, hydroxy or mercapto;

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

[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 selected from C1-C3 alkyl, halogen, hydroxy or mercapto; Specification 29 / 80 pages 51 CN 121532376 A

[0403] wherein L6, L7, L10 and L11 are each independently C1-C9 alkenyl; preferably, L6, L7, L10 and L11 are each independently C1-C9 alkenyl.

[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 an N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound,

[0408] wherein

[0409] L0 and L1 are each independently C1-C5 alkylene;

[0410] X1 is C1-C5 alkylene;

[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;

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

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

[0415] L6, L7, L10, and L11 are each independently a bond;

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

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

[0418] In some embodiments, X2 and X3 are each 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. In some embodiments, L2 and L3 are each independently C3 alkylene. In some embodiments, L2 and L3 are each independently C4 alkylene. In some embodiments, L2 andL3 is each independently C5 alkylene. In some embodiments, L2 and L3 are each independently C6 alkylene. In some embodiments, L2 and L3 are each independently C7 alkylene. In some embodiments, L2 and L3 are each independently C8 alkylene. In some embodiments, L2 and L3 are each independently C9 alkylene.

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

[0421] In some embodiments, R4 is selected from C1-C18 alkyl or C2-C18 alkenyl.

[0422] In some embodiments, R2, R3, R5 and R6 are each independently selected from C4-C18 alkyl or C4-C18 alkenyl. In some embodiments described on pages 30 / 80 of CN 121532376 A, R2, R3, R5, and R6 are each independently selected from C4-C12 alkyl groups. 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 an N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound,

[0426] wherein

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

[0428] X1 is selected from C1-, -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, wherein the C3-C8 alkylene is optionally substituted by one or more substituents.

[0431] L4 and L5 are each independently selected from C1-C3 alkyl, halogen, hydroxyl, or thiol;

[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] wherein L6, L7, L10, and L11 are each independently C1-C3 alkyl;

[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 an N-oxide, pharmaceutically acceptable salt, isomer, or prodrug of the compound,

[0438] wherein the specification is on page 31 / 80 of CN 121532376 A

[0439] L0 and L1 are each independently C1-C5 alkylene;

[0440] X1 is C1;

[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;

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

[0444] L6, L7, L10, and L11 are each independently bonded;

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

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

[0447] In some embodiments, X2 and X3 are each 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.

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

[0450] In some embodiments, 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.

[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 comprising 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, the alkenyl group can be substituted with one or more alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups. Exemplary examples...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 ynyl, C4 ynyl, C5 ynyl, C6 ynyl, C7 ynyl, C8 ynyl, C9 ynyl, C10 ynyl, C11 ynyl, C12 ynyl, C13 ynyl, C14 ynyl, C15 ynyl, C16 ynyl, C17 ynyl, or C18 ynyl. In some embodiments, the ynyl group is a branched ynyl or a straight-chain ynyl.

[0456] In this invention, "alkylene" refers to a divalent group obtained by removing two hydrogen atoms from a saturated hydrocarbon, which may be branched or straight-chain (e.g., linear). Any of the above-described monovalent alkyl groups can be converted into alkylene groups by removing a second hydrogen atom from the 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-, and -CH2CH2CH2CH2-. 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 groups described herein may include C3 alkylene, C4 alkylene, C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene, C9 alkylene, C10 alkylene, C11 alkylene, C12 alkylene, C13 alkylene, C14 alkylene, C15 alkylene, C16 alkylene, C17 alkylene, or C18 alkylene.

[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 to 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), mean 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, -S-C3-13 alkyl as described herein 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 or a straight-chain alkyl.

[0463] In some embodiments, the -CH2-S-C3-13 alkyl group described herein 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, -CH2-S-C11 alkyl, -CH2-S-C12 alkyl, or -CH2-S-C13 alkyl. In some embodiments, the alkyl group is a branched alkyl or a straight-chain alkyl. Specification 33 / 80 pages 55 CN 121532376 A

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

[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. The 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)-), sulfur (e.g., -S-, -S(=O)- or -S(=O)2-).

[0466] Compositions

[0467] The present invention provides a composition comprising the ionizable lipids disclosed herein. The lipid compositions may be provided in the form of particulate compositions. 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 comprise a water core, such as comprising a majority RNA molecule), dispersed phases in emulsions, micelles, or internal phases in suspensions.

[0468] According to an exemplary embodiment, the particles comprise ionizable lipids (compounds as 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 comprises non-cationic lipids and PEGylated lipids. In some embodiments, the non-cationic lipids are neutral lipids.

[0470] "Neutral lipids" suitable for the lipid compositions of the present invention include, 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), and 1,2-bisstearoyl-sn-glycerol-3-phosphatidylcholine.(DAPC), phosphatidylethanolamine (PE), egg yolk phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), l-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), l,2-diarachidonico-sn-glycerol 3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-docosahexaenoyl-sn-glycerol-3-phosphocholine (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 herein and refer to lipid molecules that have been covalently linked to one or more polyethylene glycol (PEG) chains, a process referred to as PEGylation. This modification significantly alters the physicochemical and biological properties of the lipid. The 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, page 34 / 80 of the specification, 56 CN 121532376 A.

[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, for example, siRNA,Nucleic acids such as miRNA, shRNA, and antisense RNA. In some embodiments, the nucleic acid component includes DNA, which may be referred to as the DNA component. In some embodiments, the nucleic acid component includes RNA. In some embodiments, the RNA component may include 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 contain mRNA encoding Cas9, Cpf1, c2c2, cas12i, cas12f, cas12b, cas12c, cas12d, cas12e, cas12g, cas12j, or cas12k. In some embodiments, the composition may contain gRNA. In some compositions containing mRNA encoding an RNA-guided DNA binder, the composition further contains gRNA nucleic acid such as gRNA. In some embodiments, the composition contains an RNA-guided DNA binder and gRNA. In some embodiments, the composition contains Cas nuclease mRNA and gRNA. In some embodiments, the composition contains two types of Cas nuclease mRNA and gRNA. In some embodiments, the composition contains (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 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] The modified nucleoside or nucleotide 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 the 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 a naturally occurring nucleobase, including with an atypical nucleobase (an exemplary base modification); (v) replacing or modifying the ribose-phosphate backbone (an exemplary backbone modification); (vi) modifying the 3' or 5' end of the 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 the sugar (an exemplary sugar modification). Some embodiments include modification of the 5' end of mRNA, gRNA, or nucleic acid. Some embodiments include modifications to mRNA, gRNA, or nucleic acids. Some embodiments include modifications to the 3' end of mRNA, gRNA, or nucleic acid. The modified RNA may contain 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. Specification 35 / 80 pages 57 CN 121532376 A 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 to RNA or nucleic acids include the consumption of bases (e.g., by deletion or replacement of one nucleotide with another) or modifications of bases, such as chemical modifications of bases. As used herein, "chemical modification" includes the introduction of different chemical properties 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 skeletal 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 complete replacement of the unmodified phosphate moiety with the modified phosphate group described herein. In some embodiments, skeletal 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, phosphoselenyl, boron phosphate, boron phosphate, hydrophosphonate, phosphoramide ester, alkyl or aryl phosphonate, and phosphate triester. The phosphorus atom in the 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 here) or an "S" configuration (Sp here). The backbone can also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate and the nucleoside) with nitrogen (bridged phosphoramide), sulfur (bridged thiophosphonophosphate), and carbon (bridged methylenephosphonate). The substitution can occur at any or both oxygen links. In some backbone modifications, the phosphate group can be replaced with a phosphorus-free linker. In some embodiments, the charged phosphate group can be replaced by a neutral portion. Examples of portions that can replace the phosphate group include, but are not limited to: methylphosphonates, hydroxylamino groups, siloxanes, carbonates, carboxymethyl groups, 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 one of the two types of Cas nucleases 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 applied. The mRNA may comprise one or more 5' caps, 5' untranslated regions (UTRs), 3' UTRs, and polyadenylated tails. The mRNA may comprise modified open reading frames, such as encoding nuclear localization sequences or encoding proteins using alternative codons.

[0481] The mRNA in the disclosed LNP compositions 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 nucleotides of a codon such that the codon encodesThe same amino acids but more stable codons than those found in the wild-type version of 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 is also considered in the mRNA nucleotides of the present invention. Incorporation of pseudouracil into the mRNA nucleotides of the present invention can improve stability and translational ability and reduce immunogenicity in vivo. Substitutions and modifications to the mRNAs of the present invention can be made by 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 a secondary structure as described on pages 36 / 80 of the specification, CN 121532376 A).

[0484] The poly A tail is considered to stabilize natural messengers. Therefore, in one embodiment, a long poly A tail can be added to the mRNA molecule to make the mRNA more stable. A variety of recognized techniques can be used to add the 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 transcriptional level of the protein. 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 temporal course of protein expression in the cell. 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 one embodiment, 3' and / or 5' flanking sequences that are naturally located on both sides of the mRNA and encode a second, unrelated protein can be incorporated into nucleotides of mRNA molecules encoding therapeutic or functional proteins.The sequence can be modified. For example, a 3' or 5' sequence can be obtained from a stable mRNA molecule and incorporated into the 3' and / or 5' region of a positive mRNA nucleic acid molecule to increase the stability of the positive mRNA molecule.

[0486] The compositions and methods disclosed herein may include a template nucleic acid. The 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, e.g., 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 a cell, or different templates may be provided for editing two different genes in a cell.

[0487] LNPs can be prepared using any method disclosed in the 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 include: 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) may be used for maintenance.

[0488] In some embodiments, the pH of the LNP-containing composition is maintained at or above 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 using a buffer. In some embodiments, the pH of the LNP-containing composition is maintained at or above 7.0 using a buffer. 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, for example, 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 the Tris buffer. In some exemplary embodiments, the composition contains about 5% w / v sucrose, about 45 mM NaCl, and about 50 mM Tris, with a pH of 7.5. The amounts 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.The particle size (diameter) ranges from approximately 50 nm to approximately 150 nm.

[0492] In some embodiments, the average particle size (diameter) ranges from approximately 10 nm. In some embodiments, the average particle size (diameter) exceeds approximately 20 nm. In some embodiments, the average particle size (diameter) exceeds approximately 30 nm. In some embodiments, the average particle size (diameter) exceeds approximately 40 nm. In some embodiments, the average particle size (diameter) exceeds approximately 50 nm. In some embodiments, the average particle size (diameter) exceeds approximately 60 nm. In some embodiments, the average particle size (diameter) exceeds approximately 70 nm. In some embodiments, the average particle size (diameter) exceeds approximately 80 nm. In some embodiments, the average particle size (diameter) exceeds approximately 90 nm. In some embodiments, the average particle size (diameter) exceeds approximately 100 nm. In some embodiments, the average particle size (diameter) exceeds approximately 200 nm. In some embodiments, the average particle size (diameter) does not exceed approximately 500 nm. In some embodiments, the average particle size (diameter) of the particles (including encapsulated nucleic acids) ranges from about 5 nm to about 200 nm. In some embodiments, the average particle size (diameter) of the particles (including encapsulated nucleic acids) 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 about 55 nm to about 58 nm. In some embodiments, this size is a 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 approximately 30-60% (molar) of ionizable lipids. For example, ionizable lipids may comprise approximately 40-50% of the lipid phase.

[0495] In some embodiments, the lipid phase may comprise approximately 20-70% (molar) of membrane-stabilizing lipids. For example, membrane-stabilized lipids can comprise approximately 40-60% of the lipid phase. In some embodiments, one or more membranes can be used in the lipid phase.Stabilizing lipids. For example, membrane-stabilizing lipids may include cholesterol (approximately 30-50% of the lipid phase) and phospholipids (e.g., DSPC), whose specification 38 / 80 pages 60 CN 121532376 A may comprise approximately 5-15% of the lipid phase.

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

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

[0498] In some embodiments, the nucleic acids may be mixed with the lipid mixture in a microfluidic mixer to form particles that encapsulate / carry the 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, the nanoparticle composition may be contacted with mammalian cells located within a mammal via various routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous), and may involve varying amounts of the nanoparticle composition. Furthermore, one or more mammalian cells may 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 medicament to a subject may involve administering to the subject a nanoparticle composition comprising the therapeutic and / or preventative medicament (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0501] The composition may also comprise a salt of one or more compounds. The salt may be a pharmaceutically acceptable salt. As used herein, “pharmaceutically acceptable salt” refers to a derivative of the disclosed compound, wherein the parent compound is altered by converting an existing acid or base portion into its salt form (e.g., by reacting a 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 acetates, adipates, alginates, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, hydrogen sulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionate, dodecyl sulfates, ethanesulfonates, fumarates, gluconates, glycerophosphates, hemisulfates, heptahydrates, hexanoates, hexafluoroacetate, hydrochlorides, hydroiodates, 2-hydroxyethanesulfonates, lactobionates, and lactates.Laurate, lauryl sulfate, malate, maleate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, 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, formed 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 can be prepared by reacting the free acid or base form of these compounds with an appropriate amount of base or acid in water or 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, “polydispersity index” or “PDI” is a ratio describing the uniformity of the particle size distribution of a system. Smaller values, such as less than 0.3, indicate a narrower particle size distribution. In some embodiments, the polydispersity index may be less than 0.1.

[0503] In some embodiments, the polydispersity index (PDI) of the LNPs disclosed herein may be between about 0.005 and about 0.75. In some embodiments, the PDI of the LNP may be between about 0.01 and about 0.5. In some embodiments, the PDI of the LNP may be between about zero and about 0.4. In some embodiments, the PDI of the LNP may 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] Although 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 teachings. The subheadings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. Unless otherwise stated, all scopes given in this application include endpoints. Examples

[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 chlorosulfonic acid-4-nitrobenzene ester (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 again at room temperature for 2–24 hours. The reaction mixture was extracted with hexane (20 mL) and washed with water. The resulting aqueous layer was 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 (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] 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 a solution of 6-bromohexane-1-thiol (2 g, 10 mmol, 1 equivalent) in acetonitrile (0.04–0.1 M) 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 (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 Specification 40 / 80 pages 62 CN 121532376 A

[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 chlorosulfonic acid-4-nitro nitrate were added sequentially.Benzyl ester (2.1 g, 10 mmol, 1.5 equivalence) was added at room temperature. After stirring for at least 2 hours, 2-heptyldecane-1-ol (2.56 g, 10 mmol, 1 equivalence) 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 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 (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 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: 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, 1.5 equivalents). After stirring for at least 2 hours, (E)-decyl-2-en-1-ol (1.5 g, 1.5 mmol, 1.5 equivalents) was added.The reaction mixture was stirred for 2–24 hours at room temperature. 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 with 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] This intermediate was synthesized by the same method as intermediate a, with a yield of 51%. MS: 353.11 m / z [M+H].

[0527]

[0528] Intermediate h

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

[0530]

[0531] Intermediate i

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

[0533]

[0534] Intermediate j

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

[0536]

[0537] Intermediate k

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

[0539]

[0540] Intermediate l

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

[0542]

[0543] Intermediate m

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

[0545] Specification 42 / 80 pages 64 CN 121532376 A

[0546] Intermediate n

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

[0548]

[0549] Intermediate o

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

[0551]

[0552] Intermediate p

[0553] This intermediate was synthesized by 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] The 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]. Instructions for Use, pages 43 / 80, 65 CN 121532376 A

[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. 1H 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] This compound was synthesized from intermediate b using the same method as compound 1, with a yield of 35%. ¹H NMR (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] This compound was synthesized from intermediates b and a using the same method as compound 1, in a yield of 38%. 1H NMR (400 MHz, Chloroform‑d) δ 4.78 (tt, J = 10.8, 1.5 Hz, 1H), 4.23 (t, J = 4.6 Hz, 2H), 3.58 (td, J = 7.2, 5.0 Hz, 2H), 2.85 (t, J = 7.8 Hz, 4H), 2.55 (t, J = 7.2 Hz, 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%. 1H NMR (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, in a yield of 37%. 1H NMR (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, 4.5 Hz, 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, in a yield of 51%. 1H NMR (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]. Manual 45 / 80Page 67 CN 121532376 A

[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%. 1H NMR (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, in a yield of 38%. 1H NMR (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] This compound was synthesized from intermediate b using the same method as compound 1, with a yield of 39%. ¹H NMR (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, Specification 46 / 80 pages 68 CN 121532376 A 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%. 1H 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 – 1.21 (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%. ¹H NMR (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, in a yield of 27%. 1H NMR (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 = 7.9 Hz, 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]. Specification 47 / 80 pages 69 CN 121532376 A

[0599]

[0600] Compound: YZL417

[0601] This compound was synthesized from intermediate m using the same method as compound 1, in a yield of 32%. 1H NMR (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] The compound was synthesized from intermediates i and d using the same method as compound 1, with a yield of 24%. 1H NMR (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, in a yield of 25%. 1H NMR (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]. Specification 48 / 80 pages 70 CN 121532376 A

[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%. 1H NMR (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, in a yield of 49%. 1H NMR (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 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%. 1H NMR (400 MHz, Chloroform-d) δ 4.47 (p, J = 7.0 Hz, 4H), 3.46 (td, J = 7.1, 5.0 Hz, 12H).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]. Specification 49 / 80 pages 71 CN 121532376 A

[0617]

[0618] Compound: YZL433

[0619] This compound was synthesized from intermediates b and j using the same method as compound 1, in a yield of 42%. 1H NMR (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%. 1H NMR (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, in a yield of 39%. 1HNMR (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, 2.0 Hz, 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: 800.62 Specification 50 / 80 pages 72 CN 121532376 A m / z [M+H].

[0626]

[0627] Compound: YZL441

[0628] This compound was synthesized from intermediate j using the same method as compound 1, in a yield of 29%. 1H NMR (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%. ¹H NMR (400 MHz, Chloroform-d) δ 4.56 (td, J = 10.9, 2.3 Hz, ¹H), 4.23 (t, J = 7.5 Hz).Hz, 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%. 1H NMR (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]. Specification 51 / 80 pages 73 CN 121532376 A

[0635]

[0636] Compound: YZL444

[0637] This compound was synthesized from intermediate n using the same method as compound 1, with a yield of 41%. 1H NMR (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%. 1H NMR (400 MHz, Chloroform‑d) δ 4.32 – 4.20 (m, 2H), 3.86 (tdd, J = 12.0, 5.5, 3.6 Hz, 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, in a yield of 25%. ¹H NMR (400 MHz, Chloroform-d) δ 4.39 (td, J = 10.4, 3.0 Hz, 1H), 4.23 (t, J = 4.2 Hz, 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 = 10.4, 3.0 Hz, 1H), 4.23 (t, J = 4.2 Hz, 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), 1.18 (tt, J = 10.4, 3.0 Hz, 4H), 0.89 (t, J = 10.4, 3.0 Hz, 4H), 1.03 (m, 3H), 0.89 (t, J = 10.4, 3.0 Hz, 1H), 1.03 (m, 3H), 0.89 (t, J = 10.4, 3.0 Hz, 1H), 1.03 (t, J = 10.4, 3. 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, in a yield of 45%. 1H NMR (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, in a yield of 23%. 1H NMR (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, in a yield of 29%. ¹H NMR (400 MHz, Chloroform-d) δ 4.50 (td, J = 12.1, 3.7 Hz, 1H), 4.25 (t, J = 7.0 Hz, 1H), 4.17 (dd, J = 12.3, 7.0 Hz, 1H).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]. Specification 53 / 80 pages 75 CN 121532376 A

[0653]

[0654] Compound: YZL450

[0655] This compound was synthesized from intermediates b and o using the same method as compound 1, in a yield of 27%. 1H NMR (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, in a yield of 28%. 1H NMR (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, in a yield of 19%. 1H NMR (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] Specification 54 / 80 pages 76 CN 121532376 A

[0663] Compound: YZL455

[0664] This compound was synthesized from intermediates r and i using the same method as compound 1, in a yield of 15%. 1H NMR (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 = 7.6 Hz, 4H), 1.94 – 1.65 (m, 7H), 1.62 – 1.10 (m, 45H), 0.98 – 0.77 (m, 9H). MS: 732.57 m / 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 (400 MHz, 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, 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, in a yield of 43%. 1H NMR (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, 3.1 Hz, 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 Specification 55 / 80 pages 77 CN 121532376 A

[0673] This compound was synthesized from intermediate a using the same method as compound 1, with a yield of 28%. 1H NMR (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%. 1H NMR (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 Hz1H), 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, in a yield of 41%. 1H NMR (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: 698.571 m / z [M+H].

[0680]

[0681] Compound: YZL460

[0682] This compound was synthesized from intermediates g and q using the same method as compound 1, in a yield of 43%. 1H NMR (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, 2.6 Hz, 1H), 0.89 (td, J = 8.0, 4.6 Hz, ... (Instruction manual, pages 56 / 80, CN 121532376 A)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%. 1H NMR (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] py-TsOH (1.46 g, 5.81 mmol, 0.05 eq.) was added 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.). The mixture was stirred at 110 °C for 24 hours. 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, and KOH (7.3 g, 129.57 mmol, 3 eq) was added. The mixture was stirred at room temperature for 16 hours. EA (30 mL) and H2O were added to the reaction mixture, and then extracted with EA (300 mL * 3). The crude product was obtained by drying and concentration, and 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: Specification 57 / 80 pages 79 CN 121532376 A 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] Prepared from dodecanoic acid using a method similar to that used for intermediate d', to give 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 intermediate d', to give 3.8 g of the desired product as a clear oil. MS: 453.2 m / z [M+H].

[0704]

[0705] Intermediate g':

[0706] in 2-hexyldecanoic acid (1.8 g, 10 mmol, 1 equiv) and 6-bromohexane-1-ol (2.56 g, 10 mmol, 1 equiv),DCM (20 mL) was added to a mixture of 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 mixture was reacted 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] Prepared from dodecanoic acid using a method similar to that used for intermediate g, to give 3.8 g of the desired product as a clear oil. MS: 405.2 m / z [M+H]. Instructions for Use, pages 58 / 80, CN 121532376 A

[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) 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), and 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 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 added to 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) and reacted 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]. Specification 59 / 80 pages 81 CN 121532376 A

[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, 3.0 Hz, 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 = 7.9 Hz, 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 method similar to that used to synthesize compound YZL473, yielding YZL418 (220 mg, 44%), a pale yellow oil. 1H 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 = 7.8 Hz, 12H). MS: 866.5 m / z [M+H].

[0737]

[0738] Compound YZL419:

[0739] It is used in conjunction with the synthetic compound YZL473A similar method was used to prepare YZL419 (282 mg, 45%) from intermediate e' (2 eq) and 2-aminoethanol (1 eq), which was a light yellow oil. 1H 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 method similar to that used to synthesize compound YZL473, to obtain compound YZL422 (420 mg, 45%), which is a light 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 = 3.8 Hz, 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]. Specification 61 / 80 pages 83 CN 121532376 A

[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, to obtain 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 Hz, 3H), 1.66 – 1.33 (m, 14H), 1.36 – 1.24 (m, 9H), 1.28 – 1.18 (m, 6H), 1.22 – 1.03 (m, 6H), 0.89 (t, J = 7.8 Hz, 12H). MS: 816.6 m / z [M+H].

[0746]

[0747] Compound YZL428:

[0748] It was prepared from intermediate d' and 2-aminoethanol using a method similar to that used in the synthesis of compound YZL473, yielding compound YZL428 (220 mg, 21%), a pale yellow oil. ¹H NMR (400 MHz, Chloroform-d) δ 4.15 (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 method similar to that used to synthesize compound YZL473, yielding compound YZL429 (120 mg, 21%), a pale yellow oil. ¹H NMR (400 MHz, Chloroform-d) δ 4.15 (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 (m, 2H), 3.10 – 2.98 (m, 2H), 2.54 (dd, J = 12.6, 5.8 Hz, 1H), 2.46 – 2.31 (m, 14H), 2.29 – 2.06 (m, 3H) , 2.00 – 1.78 (m, 4H), 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 method similar to that used to synthesize compound YZL422, to obtain the compoundYZL434 (410mg, 45%) is a light yellow oily substance. 1H 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, to obtain compound YZL451 (550 mg, 55%), which is a light 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]. Specification 63 / 80 pages 85 CN 121532376 A

[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, to obtain 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 method similar to that used to synthesize compound ZL22, yielding compound ZL25 (360 mg, 52%), a pale yellow oil. 1H NMR (400 MHz, Chloroform‑d) δ 4.57 (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 = 7.0Hz, 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, to obtain compound YZL449 (29% yield). 1H NMR (400 MHz, Chloroform‑d) δ 4.50 (td, J = 12.1, 3.7 Hz, 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 is used in the synthesis of compound YZL449Compound YZL489 (21% yield) was prepared using a similar method.

[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]+. H 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 method similar 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). Instruction manual, pages 66 / 80, CN 121532376 A

[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, 2.3 Hz, 2.45 Hz, 2.74 (td, J = 12.3, 4.3 Hz, 2.45 Hz, 2.74 Hz, 2.74 Hz, 2.85 ... 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, 7.6 Hz, 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 method similar 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 = 7.9 Hz, 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; Specification 67 / 80 pages 89 CN 121532376 A

[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 method similar to that used to synthesize compound YZL449, yielding compound YZL480 (33% yield).

[0800] YZL480;

[0801] 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, 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 method similar 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). Instruction manual, pages 68 / 80, CN 121532376 A

[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] 1H 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 0.5, 7.3 Hz5H), 1.37 – 1.22 (m, 38H), Specification 69 / 80 pages 91 CN 121532376 A 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] 1H 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, 1H), 1.63 – 1.46 (m, 4H), 1.38 (tdd, J = 11.2, 6.2, 2.9 Hz, 5H), 1.27 (tdd, J = 9.9, 7.0, 3.7 Hz,19H), 0.95 – 0.79 (m, 9H). 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). Instruction manual, pages 70 / 80, 92, CN 121532376 A

[0828]

[0829] 1H 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] 1H 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, 7.0 Hz, 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 effective lipid nanoparticles containing siRNA by controlled microfluidic methods. Journal of the American Chemical Society 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-dimyristicoyl-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] The 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 LNP samples. The average particle size and polydispersity were measured using dynamic light scattering (DLS) on a Malvern Zetasizer DLS instrument. Prior to 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 LNPs was also measured using a Malvern Zetasizer. Prior to measurement, 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 encapsulated using 1x PBS containing 1% Triton-X 100.TE buffer was diluted to an appropriate concentration to determine total RNA, or 1x TE buffer was diluted 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, encapsulation efficiency >80%, particle size <150 nm, and PDI <0.2 are required. The average particle size, polydispersity, and %EE values ​​for various LNP combinations are listed in Tables 1-3.

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

[0843]

[0844] Table 2: Summary of LNP formulation data for PCH and NHP (Instruction manual, pages 72 / 80, 94, CN 121532376 A)

[0845] (Instruction manual, pages 73 / 80, 95, CN 121532376 A)

[0846]

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

[0848]

[0849] Table 4: sgRNA in this invention

[0850]

[0851] Table 5: DNA sequences encoded by Cas9 and their corresponding RNA (Instruction manual, pages 74 / 80, 96, CN 121532376 A)

[0852] (Instruction manual, pages 75 / 80, 97, CN 121532376 A)

[0853] (Instruction manual, pages 76 / 80, 98, CN 121532376 A)

[0854] Instructions for Use, pages 77 / 80, 99 CN 121532376 A

[0855]

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

[0857] 3. In vitro delivery of LNPs

[0858] Primary rhesus monkey hepatocytes (TPCS, CCH-100CYS-PQ) were cultured according to the manufacturer's protocol. Cells were seeded into 96-well plates at a density of 30,000 cells per well. After 24 hours, the cells were treated with LNPs containing 6.25 nM. After 72 hours of transfection, the 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 are derived from in vitro transcribed Cas9 mRNA (SEQ ID NO. 5) and chemically modified sgRNA (targeting NHP).The TTR (SEQ ID NO. 1) was formulated as described in Example 2. Details of these formulations are shown in Table 2, including average particle size, polydispersity, and encapsulation efficiency. As shown in Figures 2A-2C, in vitro editing was observed for each formulation. Including Lipid 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), YZL431 (LNP031), YZL432 (LNP032), YZL433 (LNP033), YZL439 (LNP034), YZL449 (LNP035), YZL450 (LNP036), YZL452 (LNP037), YZL453 (LNP038), YZL455 (LNP039), YZL456 (LNP040), YZL457 (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 in vitro delivery of CRISPR / Cas9 cargo to rhesus monkey hepatocytes 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 dissected 168 hours after administration.Examination. Clinical symptoms, body weight, organ weight, and histopathology were assessed. Liver segments 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 (targeted mouse instruction manual page 78 / 80 100 CN 121532376 A 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. As shown in Figures 1A-1C, in vivo editing (approximately 50%-68% editing) was observed in the livers of animals receiving LNPs targeting TTR in each formulation. The lipids included are YZL410 (LNP001), YZL411 (LNP002), YZL412 (LNP003), YZL413 (LNP004), YZL414 (LNP005), YZL415 (LNP006), YZL416 (LNP007), YZL417 (LNP008), YZL424 (LNP009), YZL425 (LNP010), YZL426 (LNP011), YZL427 (LNP012), YZL430 (LNP013), YZL431 (LNP014), YZL432 (LNP015), and YZL433. YZL416 (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 have excellent in vivo delivery efficiency of CRISPR / Cas9 cargo, achieving saturation editing even at very low doses.

[0865] The 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. Detailed information on these formulations is shown in Table 2, including average particle size, polydispersity, and encapsulation efficiency. Similarly, LNPs targeting NHP TTR showed a potent and sustained reduction in rhesus monkey serum TTR protein in in vivo experiments in a non-human primary (NHP) study (data not shown). 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 by 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 and serum separated every 7 days post-dose as instructed. In vivo editing was observed in the livers of animals receiving LNPs targeting TTR in each formulation, as shown in Figures 3A-3C. It contains 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), YZL431 (LNP031), YZL432 (LNP032), and YZL433 (LNP033). YZL439 (LNP034), YZL449 (LNP035), YZL450 (LNP036), YZL452 (LNP037), YZL453 (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 a single sample was encouraging, exceeding 40%.

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

[0867] The 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...The dose was administered via tail vein injection at a dose of mL / kg body weight. 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 was performed 168 hours post-administration. Clinical signs, body weight, organ weight, and histopathology were evaluated. As shown in Figures 4A and 4B, 5A and 5B, and 6A and 6B, 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, compared to rats treated with the commercial lipid ALC-0315, animals treated with LNP046, LNP047, LNP048, LNP049, LNP050, and LNP051 had lower ALT and AST levels. Furthermore, at 18 hours post-administration, ALT and AST levels in LNP113 were also lower than in LNP114. ALT and AST levels in these LNP215 and LNP216 rats were relatively low. 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 repaired after 7 days.

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

[0869] 5. PCR Amplification

[0870] All samples were amplified and sequenced using a two-step nested PCR. 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 performed for 20 cycles. The second PCR was used to add Nextera XT chemistry, indexing, and i5 / i7 adapter regions, running 5-10 cycles with the product of "PCR 1" as a template. Double-indexed samples were run on a 2% agarose gel 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 human Prealbumin (Transthyretin) ELISA kit (Ai) was used.BAK (product number ab231920) determined the total serum level of NHP TTR according to the manufacturer's protocol. Briefly, serum was serially diluted to a final dilution of 300,000 times using 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 3 times with the provided wash buffer. Then 100 μL of detection antibody was added to each well, incubated at room temperature for 20 min, and washed 3 times. 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. The serum TTR level was calculated from 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 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 Pearson correlation coefficients. R² values ​​(ranging from 0 to 1) were calculated using the squared Pearson correlation coefficients. Instruction manual, 80 / 80 pages, 102 CN 121532376 A, Figure 1A, Figure 1B; Instruction manual drawing, 1 / 7 pages, 103 CN 121532376 A, Figure 1C, Figure 2A, Figure 2B; Instruction manual drawing, 2 / 7 pages, 104 CN 121532376 A, Figure 2C, Figure 3A; Instruction manual drawing, 3 / 7 pages, 105 CN 121532376 A, Figure 3B, Figure 3C; Instruction manual drawing, 4 / 7 pages, 106 CN 121532376 A, Figure 4A, Figure 4B; Instruction manual drawing, 5 / 7 pages, 107 CN 121532376 A, Figure 5A, Figure 5B, Figure 6A; Instruction manual drawing, 6 / 7 pages, 108 CN 121532376 A, Figure 6B; Instruction manual drawing, 7 / 7 pages, 109 CN 121532376 A

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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