Lipid compound for delivering therapeutic agent as well as preparation method and application of lipid compound

Abstract

The invention discloses a lipid compound for delivering a therapeutic agent as well as a preparation method and application of the lipid compound, and particularly discloses a compound shown in a formula I or pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor of the compound, the ionizable lipid compound can effectively deliver nucleic acid molecules, small molecule compounds and other drugs, and through comparison, the lipid nanoparticles are good in particle size distribution and high in encapsulation efficiency, the delivery effect can be obviously better than that of contrast lipid nanoparticles, and the requirement of in-vivo delivery can be met. (I)

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to lipid compounds for delivering therapeutic agents, their preparation methods, and their uses. Background Technology

[0002] Gene therapy is a hot research topic in modern biomedicine. Nucleic acid drugs can prevent and treat cancer, bacterial and viral infections, and diseases with genetic causes. Because nucleic acid drugs are easily degraded and difficult to enter cells, they usually need to be encapsulated and delivered to target cells using vectors. Therefore, developing safe and efficient delivery vectors is a prerequisite for the clinical application of gene therapy.

[0003] Lipid nanoparticles (LNPs) are currently a research hotspot in the field of non-viral gene vectors. In 2018, the FDA approved LNP delivery of patisiran (onpattro) for the treatment of hereditary transthyretin amyloidosis, leading to an explosive growth in research using LNP technology to deliver nucleic acid drugs. Particularly noteworthy is the FDA's approval in late 2020 of Moderna's and BioNTech & Pfizer's COVID-19 vaccines, both of which utilize LNP technology to deliver mRNA drugs for the prevention of SARS-CoV-2. LNPs typically contain ionizable lipids, and their selection significantly impacts delivery efficacy.

[0004] There is still a need to develop new lipid compounds to facilitate the in vitro or in vivo delivery of nucleic acid molecules for therapeutic and / or preventative purposes. Summary of the Invention

[0005] The purpose of this invention is to provide novel lipid compounds that facilitate the in vitro or in vivo delivery of nucleic acid molecules for therapeutic and / or preventative purposes.

[0006] Another object of the present invention is to provide a lipid compound or a pharmaceutically acceptable form thereof (e.g., salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug), which can be used in combination with other lipid compounds (e.g., neutral lipids, charged lipids, steroids) to prepare lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acid molecules, specifically including mRNA) to improve the delivery efficiency of nucleic acid drugs in vivo. The lipid compound with a specific structure can be selected as a lipid carrier according to the organ where the nucleic acid drug needs to be enriched.

[0007] Another object of the present invention is to provide a lipid carrier comprising the above-described compounds or in a pharmaceutically acceptable form thereof.

[0008] Another object of the present invention is to provide a nucleic acid lipid nanoparticle composition comprising the above-described compound or a pharmaceutically acceptable form thereof or the above-described lipid carrier.

[0009] Another object of the present invention is to provide pharmaceutical formulations comprising the above-described compounds or their pharmaceutically acceptable forms, or the above-described lipid carriers, or the above-described nucleic acid lipid nanoparticle compositions.

[0010] Another object of the present invention is to provide a method for delivering a therapeutic or preventative agent to subject cells.

[0011] Another object of the present invention is to provide a method for generating a target protein or target polypeptide in a subject's cells.

[0012] Another object of the present invention is to provide the use of the above-described compounds or their pharmaceutically acceptable forms, or the above-described lipid carriers, or the above-described nucleic acid lipid nanoparticle compositions, or the above-described pharmaceutical preparations in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs.

[0013] Another object of the present invention is to provide a method for in vivo delivery of a nucleic acid drug, the method comprising administering the above-described nucleic acid lipid nanoparticle composition or the above-described pharmaceutical preparation to a subject in need.

[0014] In a first aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof is provided having the following structure of formula I:

[0015] (I)

[0016] in,

[0017] R1 is selected from -OH and R a (R b )N-, where R a and R b Each independently is hydrogen, C1-C 10 Alkyl or C1-C 10 Halogenated alkyl groups;

[0018] R2 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 acetylin

[0019] R3 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, or R c-(CH2)n-, where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, and more preferably a positive integer from 1 to 10;

[0020] R c Selected from the structure shown below:

[0021] , , , , and ,in, Represents a connection key;

[0022] L1, L2, L3, L4, and L5 do not exist, or each can be independently selected from the following groups:

[0023] , , , , , ;

[0024] X does not exist, or is -CH- or N;

[0025] R4 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0026] R5 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0027] R6 is C1-C 30 Alkyl, C2-C 30 alkenyl, C2-C 30 alkynyl group;

[0028] R7 is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group;

[0029] R8 and R9 do not exist, or they are each independently C1-C. 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C14 alkynyl group, wherein R h For O or S;

[0030] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h It can be O or S.

[0031] In another implementation, R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl.

[0032] In another embodiment, R2 is selected from -(CH2)m-, where m is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, and even more preferably a positive integer from 1 to 6.

[0033] In another embodiment, R3 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0034] In another embodiment, R3 is a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl.

[0035] In another embodiment, R6 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group.

[0036] In another embodiment, R2 is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group.

[0037] In another implementation, R2 and R7 are each independently C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0038] In another embodiment, R4 and R5 are each independently -(CH2)q-CH3, where q is selected from positive integers from 1 to 20, preferably positive integers from 1 to 12, and more preferably positive integers from 1 to 8.

[0039] In another embodiment, R4 and R5 each have an independent structure -R d -R e -;

[0040] Where R d The following functional groups are absent or selected: -(CH2)-(CH=CH)-;

[0041] R e It is -(CH2)p-CH3, where p is selected from positive integers from 0 to 20, preferably from positive integers from 0 to 12, and even more preferably from positive integers from 0 to 8.

[0042] In another embodiment, R8 and R9 are each independently -(CH2)q-CH3, where q is selected from positive integers from 1 to 14, preferably positive integers from 1 to 12, and more preferably positive integers from 1 to 8.

[0043] In another embodiment, R7 is -(CH2)q-CH3, where q is a positive integer selected from 1 to 14, preferably a positive integer from 1 to 12, and more preferably a positive integer from 1 to 8.

[0044] In another embodiment, R6 has a structure -R f -R g -;

[0045] Where R f Selected from the following functional group: -(CH2) s1 -(CH=CH)-CH2-(CH=CH)-(CH2) s2 -CH3, s1 is a positive integer from 1 to 14, preferably a positive integer from 1 to 10, s2 is a positive integer from 1 to 8, preferably a positive integer from 1 to 6;

[0046] R g It is -(CH2)m-, where m is a positive integer from 1 to 14, preferably a positive integer from 1 to 10, and more preferably a positive integer from 1 to 6.

[0047] In another implementation, R 10 R 11 Each independently is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, or -R h -C1-C 10 Alkyl, -Rh -C2-C 10 alkenyl, -R h -C2-C 10 alkynyl group, wherein R h It can be O or S.

[0048] In another implementation, R 11 It is C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl.

[0049] In another implementation, R8 and R9 are each independently C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, or -R h -C1-C 10 Alkyl, -R h -C2-C 10 alkenyl, -R h -C2-C 10 alkynyl group, wherein R h It can be O or S.

[0050] In another embodiment, R9 is a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl.

[0051] In another implementation, X is absent or is -CH-.

[0052] In another embodiment, the compound of formula I has the structure of formula I-1 as follows:

[0053] (I-1),

[0054] in,

[0055] R1-R2, R6-R 11 L2-L5 are as defined above;

[0056] R3 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group.

[0057] In another embodiment, R3 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0058] In another embodiment, R3 is a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl.

[0059] In another embodiment, the compound of formula I has the structure of formula I-2 as follows:

[0060] (I-2)

[0061] in,

[0062] R1-R2, R6-R7, R 10 -R 11 L1-L5 are as defined above;

[0063] R3 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group, or Rc-(CH2)n-, where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, and Rc is as defined above;

[0064] R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0065] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group.

[0066] In another embodiment, R5 is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group.

[0067] In another embodiment, R5 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0068] In another embodiment, R3 is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group.

[0069] In another embodiment, R3 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0070] In another embodiment, the compound of formula I has the structure of formula I-3 as follows:

[0071] (I-3)

[0072] in,

[0073] R1-R3, R6, R7, R 10 R 11 L1-L5 are as defined above;

[0074] R4 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0075] R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0076] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group.

[0077] In another embodiment, R4 is a C1-C14 alkyl group, C2-C... 14 alkenyl, C2-C 14 Alkyne group.

[0078] In another embodiment, R4 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0079] In another embodiment, R5 is a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl.

[0080] In another implementation, R8 and R9 are each independently C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0081] In another embodiment, R9 is a C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl.

[0082] In another embodiment, the compound of formula I has the structure of formula I-4 as follows:

[0083] (I-4)

[0084] Among them, R1-R7, R 10 -R 11 L1-L3 and L5 are as defined above.

[0085] In a second aspect of the invention, a compound of formula II or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof is provided having the following structure:

[0086] (II)

[0087] Wherein, R1 is selected from -OH and Ra(R b )N-, where Ra and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl;

[0088] R3 is C1-C 15 Alkyl, C2-C 15 alkenyl, C2-C 15 Alkyne group, or Rc-(CH2)n-, wherein n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, and even more preferably a positive integer from 1 to 6;

[0089] Rc is selected from the following structure:

[0090] , , , , and ,in Indicates a connection key;

[0091] L1, L2, L3, L4, and L5 do not exist, or each can be independently selected from the following groups:

[0092] , , , , , ;

[0093] R4 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0094] R5 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0095] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0096] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0097] r is 2 or 3;

[0098] u is a positive integer from 1 to 10;

[0099] t is a positive integer from 1 to 10;

[0100] Furthermore, when R1 is selected from R a (R b When N-, r is 2.

[0101] In another embodiment, R3 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0102] In another implementation, R4 is absent or is C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0103] In another implementation, R5 is absent or is C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0104] In another implementation, R8 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0105] In another embodiment, R9 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0106] In another implementation, R 10 For C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0107] In another implementation, R 11 For C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0108] In another embodiment, u is a positive integer from 3 to 6, preferably from 3 to 5, and more preferably 4.

[0109] In another embodiment, t is a positive integer from 3 to 6, preferably from 3 to 5, and more preferably 4.

[0110] In another implementation, L1, R4, and R5 are not present.

[0111] In another implementation, L1, L2, L3, L4, and L5 are each independently selected from the following groups:

[0112] , , , , , ;

[0113] In another embodiment, the compound of formula II has the following structure:

[0114] (II-1), or

[0115] (II-2),

[0116] in,

[0117] In equation II-1, R3, r, R8-R 11 L2-L5 are as defined above;

[0118] In equation II-2, r, R3-R5, R8-R 11 L1-L5 are as defined above.

[0119] In another embodiment, the compound of formula II has the following structure:

[0120] (II-3),

[0121] Among them, Ra, R b R3-R5, R8-R 11 r, L1-L5 are as defined above.

[0122] In another embodiment, the compound is a specific compound prepared in this embodiment, preferably selected from the group consisting of:

[0123]

[0124] In a third aspect of the invention, a compound of formula III or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof is provided having the following structure:

[0125] (III),

[0126] in,

[0127] R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl;

[0128] Rc is selected from the following structure:

[0129] , , , , and ,in Indicates a connection key;

[0130] s is a positive integer from 1 to 10;

[0131] L1, L2, L3, L4, and L5 either do not exist or are independently selected from the following groups:

[0132] , , , , , ;

[0133] R4 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0134] R5 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0135] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0136] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0137] r is a positive integer of at least 3;

[0138] u is a positive integer from 1 to 10;

[0139] t is a positive integer from 1 to 10.

[0140] In another implementation, R4 and R5 do not exist simultaneously.

[0141] In another implementation, R4 and R5 are both present.

[0142] In another implementation, when R4 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group or C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 When R5 is alkynyl, R5 is absent; or when R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group or C1-C 10Alkyl, C2-C 10 alkenyl, C2-C 10 When the group is alkynyl, R4 is not present.

[0143] In another implementation, R4 is absent or is C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0144] In another implementation, R5 is absent or is C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0145] In another implementation, R8 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0146] In another embodiment, R9 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0147] In another implementation, R 10 For C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0148] In another implementation, R 11 For C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0149] In another implementation, s is a positive integer from 1 to 8.

[0150] In another embodiment, r is a positive integer from 3 to 10, preferably from 3 to 8.

[0151] In another embodiment, u is a positive integer from 3 to 6, preferably from 3 to 5, and more preferably 4.

[0152] In another embodiment, t is a positive integer from 3 to 6, preferably from 3 to 5, and more preferably 4.

[0153] In another implementation, L1, L2, L3, L4, and L5 are each independently selected from the following group:

[0154] , , , , , ;

[0155] In another embodiment, the compound is a specific compound prepared in this embodiment, preferably selected from the group consisting of:

[0156]

[0157]

[0158]

[0159] In a fourth aspect of the invention, a compound of formula IV or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof is provided having the following structure:

[0160] (IV)

[0161] in

[0162] R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl;

[0163] R3 is C1-C 15 Alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl group, or R c -(CH2)n-, where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, and more preferably a positive integer from 1 to 10;

[0164] Rc is selected from the following structure:

[0165] , , , , and ,in Indicates a connection key;

[0166] L1, L2, L3, L4, and L5 either do not exist or are independently selected from the following groups.

[0167] , , , , , ;

[0168] R4 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0169] R5 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0170] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0171] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0172] r is a positive integer of at least 3;

[0173] u is a positive integer from 1 to 10;

[0174] t is a positive integer from 1 to 10.

[0175] In another embodiment, R3 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0176] In another implementation, R4 is absent or is C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0177] In another implementation, R5 is absent or is C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0178] In another implementation, R4 and R5 do not exist simultaneously.

[0179] In another implementation, R4 and R5 are both present.

[0180] In another implementation, when R4 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group or C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 When R5 is alkynyl, R5 is absent; or when R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group or C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 When the group is alkynyl, R4 is not present.

[0181] In another implementation, R8 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0182] In another embodiment, R9 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0183] In another implementation, R 10 For C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0184] In another implementation, R 11 For C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0185] In another embodiment, r is a positive integer from 3 to 10, preferably from 3 to 8.

[0186] In another embodiment, u is a positive integer from 3 to 6, preferably from 3 to 5, and more preferably 4.

[0187] In another embodiment, u is a positive integer from 3 to 6, preferably from 3 to 5, and more preferably 4.

[0188] In another implementation, L1, L2, L3, L4, and L5 are each independently selected from the following group:

[0189] , , , , , .

[0190] In another embodiment, the compound is a specific compound prepared in this embodiment, preferably selected from the group consisting of:

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] In a fifth aspect of the invention, a compound of formula V or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof is provided having the following structure:

[0197] (V)

[0198] Where R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; Rc is selected from the following structures:

[0199] , , , , and ,in Indicates a connection key;

[0200] L1, L2, L3, and L5 either do not exist or are independently selected from the following groups:

[0201] , , , , , ;

[0202] R4 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0203] R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0204] R6 is C1-C 30 Alkyl, C2-C 30 alkenyl, C2-C 30 alkynyl group;

[0205] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0206] r is a positive integer of at least 3;

[0207] t is a positive integer from 1 to 10.

[0208] In another embodiment, R4 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0209] In another embodiment, R5 is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0210] In another embodiment, R6 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group.

[0211] In another implementation, R 10 It does not exist or is C1-C 10Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0212] In another implementation, R 11 It does not exist or is C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group.

[0213] In another embodiment, r is a positive integer from 3 to 10, preferably from 3 to 8.

[0214] In another embodiment, t is a positive integer from 3 to 6, preferably from 3 to 5, and more preferably 4.

[0215] In another implementation, L1, L2, L3, and L5 are each independently selected from the following group:

[0216] , , , , , .

[0217] In another embodiment, the compound is a specific compound prepared in this embodiment, preferably selected from the group consisting of:

[0218]

[0219] In another implementation, R1-R 11 L1-L5 and X are specific functional groups corresponding to each specific compound in this embodiment.

[0220] In another embodiment, the compound is one of the specific compounds prepared in this embodiment, preferably selected from the group consisting of (Table 1):

[0221] Table 1

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228] In another embodiment, the compound preferably has the following structure:

[0229]

[0230]

[0231] In another embodiment, the compound of formula I preferably has the following structure:

[0232]

[0233]

[0234] In another embodiment, the compound of formula I, II, III, IV, or V can be used to prepare a drug delivery system, including lipid nanoparticles (LNPs), liposomes, polymer nanoparticles, etc., preferably for preparing lipid nanoparticles.

[0235] In a second aspect of the invention, a lipid carrier is provided comprising a compound of formula I, II, III, IV, or V as described in the first aspect of the invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof.

[0236] In another embodiment, the lipid carrier further comprises auxiliary lipids.

[0237] In another embodiment, the content of compound I in the lipid carrier accounts for 30-65% molar proportion of the total lipid content.

[0238] In another embodiment, the content of the compound of formula I in the lipid carrier accounts for 40-60% molar of the total lipid content; preferably 50% molar.

[0239] In another embodiment, the auxiliary lipid comprises one or more of anionic lipids, neutral lipids, steroids, and polymer-bound lipids.

[0240] In another embodiment, the lipid carrier also comprises other cationic or ionizable lipid compounds.

[0241] In another embodiment, the lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises a compound of formula I according to any one of claims 1-5 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, and optionally other ionizable lipids, and the second lipid compound comprises one or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids.

[0242] In another embodiment, the first lipid compound is a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug, and a combination of other cationic or ionizable lipids.

[0243] In another embodiment, the other cationic or ionizable lipid compounds include 1,2-diolenoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-diolenoyloxy-N,N-dimethylaminopropane (DODMA), DLin-MC2-MPZ, 2,2-diolenoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolanecyclopentane (DLin-KC2-DMA), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTA). P), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)di-dodecane-2-ol (C12-200), 3β[N-N'N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or a combination of two or more of these.

[0244] In another embodiment, the anionic lipid comprises one or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), and dimyristoylphosphatidylglycerol.

[0245] In another embodiment, the neutral lipid comprises at least one of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), dipalmitoylphosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), and 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or a lipid modified with an anionic or cationic modifying group. The anionic or cationic modifying group is not limited.

[0246] In another embodiment, the steroids include one or more of cholesterol, nonsteroidal, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatidine, ursolic acid, α-tocopherol, coccosterol, and corticosteroids.

[0247] In another embodiment, the polymer-bound lipids include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), α-(3'-{[1,2-di(myristoyloxy)propoxy]carbonylamino}propyl)-ω-methoxypolyoxyethylene (PEG-c-DMG), PEG-1,2-dimyristoylpropyl-3-amine (PEG-c-DMA), and 1,2-distearatel-sn-glycero-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] One or more of the following: (PEG-DSPE), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, 1,2-dipalmityl-sn-glycerol-methoxy polyethylene glycol (PEG-DPG), 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-s-DMG), PEG-dialkoxypropyl (PEG-DAA), mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol ester (PEG-c-DOMG), and N-acetylgalactosamine-((R)-2,3-bis(octadecanoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate) (GalNAc-PEG-DSG).

[0248] In another embodiment, in the lipid carrier, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid, and polymer-bound lipid is (20-65):(0-20):(5-25):(25-55):(0.3-15); exemplaryly, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid, and polymer-bound lipid can be 20:20:5:50:5, 30:5:25:30:10, or 20:5:5:5. 5:15, 65:0:9.7:25:0.3, etc.; wherein, in the first lipid compound, the molar ratio of the compound of formula I or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and other cationic or ionizable lipids is (1-10):(0-10); exemplaryly, the molar ratio can be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

[0249] In another embodiment, in the lipid carrier, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid and polymer-bound lipid is (20-55):(0-13):(5-25):(25-51.5):(0.5-15); wherein, in the first lipid compound, the molar ratio of the compound of formula I or its pharmaceutically acceptable form, such as salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and other cationic or ionizable lipid is (3-4):(0-5).

[0250] In another embodiment, the lipid carrier further comprises a bioactive substance encapsulated within the lipid carrier.

[0251] In another embodiment, the bioactive substance is selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0252] In another embodiment, the nucleic acid includes RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid, or PNA.

[0253] In another embodiment, the antisense nucleic acid is an antisense oligonucleic acid.

[0254] In another embodiment, the RNA is mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA.

[0255] In another embodiment, the DNA includes a plasmid.

[0256] In another embodiment, the mRNA includes a sequence encoding an RNA-directed DNA binder, and more specifically, includes mRNA encoding a nuclease or a base editor.

[0257] In another embodiment, the nucleic acid further includes guide RNA, specifically, the guide RNA includes gRNA nucleic acid.

[0258] In another embodiment, the nucleic acid includes mRNA and gRNA encoding a nuclease or a base editor.

[0259] In another embodiment, the gRNA is a modified or unmodified gRNA.

[0260] In another embodiment, the lipid carrier has high encapsulation efficiency for bioactive substances, which greatly improves the delivery efficiency of bioactive substances in vivo.

[0261] In another embodiment, the lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises a compound of formula I, II, III, IV or V of the present invention and optionally other ionizable lipids, and the second lipid compound comprises one or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids.

[0262] In another embodiment, the first lipid compound is any of the compounds described above or in a pharmaceutically acceptable form such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug.

[0263] In some embodiments, the first lipid compound is any of the compounds described above or in a pharmaceutically acceptable form such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug, and a combination of other cationic or ionizable lipids.

[0264] In some embodiments, the other cationic or ionizable lipid compounds include 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DODMA), DLin-MC2-MPZ, 2,2-dilinoleyloxy-4-(2-dimethylaminoethyl)-[1,3]-dioxolanecyclopentane (DLin-KC2-DMA), and 1,2-dioleoyl-3-trimethylammonium-propane (DOT). AP), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)di-dodecane-2-ol (C12-200), 3β[N-N'N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or a combination of two or more of these.

[0265] In some embodiments, the anionic lipid comprises one or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), and dimyristoylphosphatidylglycerol.

[0266] In some embodiments, the neutral lipid comprises at least one of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), dipalmitoylphosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), and 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or a lipid modified with an anionic or cationic modifying group. The anionic or cationic modifying group is not limited.

[0267] In some embodiments, the steroids include one or more of cholesterol, nonsteroidal, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, coccosterol, and corticosteroids.

[0268] In some embodiments, the polymer-bound lipids include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), α-(3'-{[1,2-di(myristoyloxy)propoxy]carbonylamino}propyl)-ω-methoxypolyoxyethylene (PEG-c-DMG), PEG-1,2-dimyristoylpropyl-3-amine (PEG-c-DMA), and 1,2-distearatel-sn-glycero-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] One or more of the following: (PEG-DSPE), polyethylene glycolated phosphatidylethanolamine (PEG-PE), PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, 1,2-dipalmityl-sn-glycerol-methoxy polyethylene glycol (PEG-DPG), 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-s-DMG), PEG-dialkoxypropyl (PEG-DAA), mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol ester (PEG-c-DOMG), and N-acetylgalactosamine ((R)-2,3-bis(octadecanoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate) (GalNAc-PEG-DSG).

[0269] In some embodiments, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid, and polymer-bound lipid in the lipid carrier is (20-65):(0-20):(5-25):(25-55):(0.3-15); exemplaryly, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid, and polymer-bound lipid can be 20:20:5:50:5, 30:5:25:30:10, or 20:5:5:55. :15, 65:0:9.7:25:0.3, etc.; wherein, in the first lipid compound, the molar ratio of any of the above compounds or their pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (1-10):(0-10); exemplaryly, the molar ratio can be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

[0270] In some embodiments, in the lipid carrier, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid and polymer-bound lipid is (20-55):(0-13):(5-25):(25-51.5):(0.5-15); wherein, in the first lipid compound, the molar ratio of any of the above compounds or their pharmaceutically acceptable forms, such as salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs, and other cationic or ionizable lipids is (3-4):(0-5).

[0271] In a third aspect of the invention, a lipid nanoparticle (LNP) is provided, comprising a compound of formula I, II, III, IV or V as described in the first aspect of the invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof.

[0272] In another embodiment, the lipid nanoparticles further comprise auxiliary lipids.

[0273] In another embodiment, the lipid nanoparticles further comprise bioactive substances encapsulated within the lipid nanoparticles.

[0274] In a fourth aspect of the invention, a lipid nanoparticle composition is provided, comprising a lipid carrier as described in the second aspect of the invention or a lipid nanoparticle as described in the third aspect of the invention, and a bioactive substance encapsulated within the lipid carrier or lipid nanoparticle.

[0275] In another embodiment, the mass ratio of the bioactive substance to the lipid carrier in the composition is 1:(3 to 40); exemplary, the mass ratios are 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, 1:40, etc.

[0276] In another embodiment, the mass ratio (w / w) of nuclease or base editor mRNA to gRNA in the bioactive substance is about 10:1 to about 1:10.

[0277] In another embodiment, the mass ratio (w / w) of nuclease or base editor mRNA to gRNA in the bioactive substance is about 3:1 to about 1:3.

[0278] In another embodiment, the mass ratio (w / w) of nuclease or base editor mRNA to gRNA in the bioactive substance is approximately 3:2.

[0279] In another embodiment, the composition comprises a pharmaceutical composition.

[0280] In a fifth aspect of the invention, a pharmaceutical composition is provided comprising a lipid carrier as described in the second aspect of the invention or lipid nanoparticles as described in the third aspect of the invention, a bioactive substance encapsulated in the lipid carrier or lipid nanoparticles, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0281] In a sixth aspect of the invention, a pharmaceutical formulation is provided comprising a lipid carrier of the second aspect of the invention or lipid nanoparticles of the third aspect of the invention, a bioactive substance encapsulated in the lipid carrier or lipid nanoparticles, and pharmaceutically acceptable excipients, carriers or diluents; or the pharmaceutical formulation comprises a nucleic acid lipid nanoparticle composition of the fourth aspect of the invention, and pharmaceutically acceptable excipients, carriers or diluents.

[0282] In another embodiment, the particle size of the pharmaceutical formulation is 30-500 nm. For example, the particle size can be 30 nm, 50 nm, 100 nm, 150 nm, 250 nm, 350 nm, 500 nm, etc.

[0283] In another embodiment, the encapsulation rate of the bioactive substance in the pharmaceutical formulation is greater than 50%. Exemplarily, the encapsulation rate can be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, etc.

[0284] In another embodiment, the hydrated particle size of the drug is 50-200 nm, preferably 70-150 nm, and most preferably 75-110 nm.

[0285] In another embodiment, the pharmaceutical preparation can be used for the treatment and / or prevention of a disease.

[0286] In another embodiment, the disease is selected from the group consisting of metabolic diseases, hereditary diseases, cancer, cardiovascular diseases, infectious diseases, and combinations thereof; preferably, metabolic diseases include familial hypercholesterolemia (FH), hereditary diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluria type 1 (PH1), and hereditary angioedema (HAE), and infectious diseases include hepatitis B.

[0287] In another embodiment, the dosage form of the pharmaceutical preparation is selected from the group consisting of injections, lyophilized preparations, nebulized inhalers, and topical medications.

[0288] In another embodiment, the drug is administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, duodenal, or intraperitoneal administration.

[0289] In another embodiment, the pharmaceutical preparation is administered by inhalation, such as intranasal administration.

[0290] In another embodiment, the pharmaceutical preparation is administered transdermally, such as by topical application or electrode delivery.

[0291] In a seventh aspect of the invention, the use of compounds of formula I, II, III, IV or V, or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or precursors thereof, as described in the first aspect of the invention, in the preparation of lipid nanoparticles.

[0292] In an eighth aspect of the invention, the use of compounds of formula I, II, III, IV or V as described in the first aspect of the invention, or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or precursors thereof, or lipid carriers as described in the second aspect of the invention, or lipid nanoparticles as described in the third aspect of the invention, or lipid nanoparticle compositions as described in the fourth aspect of the invention, or pharmaceutical compositions as described in the fifth aspect of the invention, or pharmaceutical formulations as described in the sixth aspect of the invention, in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs is provided.

[0293] In a ninth aspect of the present invention, a method for preparing the lipid nanoparticle composition described in the fourth aspect of the present invention is provided, comprising:

[0294] (a) The compound of formula I, II, III, IV or V or thereof, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or its precursor and optionally an auxiliary lipid, as described in the first aspect of the invention are mixed with an organic solvent to obtain a lipid organic phase.

[0295] (b) The bioactive substance is mixed with an aqueous solvent to obtain an aqueous phase containing the bioactive substance;

[0296] (c) The lipid organic phase from step (a) is mixed with the aqueous phase from step (b) to obtain the lipid nanoparticle composition.

[0297] In another embodiment, the organic solvent includes ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, or combinations thereof.

[0298] In another embodiment, the aqueous solvent is a buffer solution.

[0299] In another embodiment, the aqueous solvent is a buffer solution with a pH range of 3-7.

[0300] In another embodiment, the acidic buffer is a citrate buffer with a pH of 4.0.

[0301] In another embodiment, the volume ratio of the lipid organic phase to the aqueous phase containing bioactive substances is 1:(2-5), preferably 1:(3-4).

[0302] In another embodiment, in step (c), the lipid organic phase and the aqueous phase are mixed via a microfluidic chip.

[0303] In another embodiment, the method further includes step (d): purifying, concentrating, and filtering the nucleic acid lipid nanoparticle composition obtained in step (c) to remove bacteria.

[0304] In a tenth aspect of the invention, the use of compounds of formula I, II, III, IV or V of the invention, or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or precursors thereof, for the preparation of drug delivery systems is provided.

[0305] In another embodiment, the delivery system includes lipid nanoparticles (LNPs), liposomes, polymer nanoparticles, etc., and is preferably used to prepare lipid nanoparticles.

[0306] In another embodiment, the drug delivery system is used to deliver drugs for the treatment and / or prevention of tumors, infectious diseases, and rare diseases.

[0307] In an eleventh aspect of the invention, the use of a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, or a lipid carrier as described in the second aspect of the invention, or a lipid nanoparticle as described in the third aspect of the invention, or a lipid nanoparticle composition as described in the fourth aspect of the invention, or a pharmaceutical composition as described in the fifth aspect of the invention, or a pharmaceutical formulation as described in the sixth aspect of the invention, in the preparation of a medicament for treating and / or preventing diseases is provided.

[0308] In another embodiment, the disease is selected from the group consisting of metabolic diseases, hereditary diseases, cancer, cardiovascular diseases, infectious diseases, and combinations thereof; preferably, metabolic diseases include familial hypercholesterolemia (FH), hereditary diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluria type 1 (PH1), and hereditary angioedema (HAE), and infectious diseases include hepatitis B.

[0309] In a twelfth aspect of the invention, a method for delivering a therapeutic or preventative agent to the cells, tissues, or organs of a subject is provided, comprising contacting the subject's cells, tissues, or organs with the lipid nanoparticle composition of the fourth aspect of the invention, the pharmaceutical composition of the fifth aspect of the invention, or the pharmaceutical formulation of the sixth aspect of the invention.

[0310] In another embodiment, the therapeutic or preventative agent is a bioactive substance, preferably mRNA.

[0311] In another embodiment, the cells are selected from the group consisting of hepatocytes, lung cells, epithelial cells, hematopoietic cells, endothelial cells, osteocytes, stem cells, mesenchymal cells, nerve cells, photoreceptor cells, retinal pigment epithelial cells, secretory cells, cardiac cells, adipocytes, smooth muscle cells, cardiomyocytes, skeletal muscle cells, β cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, lymphocytes, B cells, T cells, antigen-presenting cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0312] In another embodiment, the tumor cells include lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendix cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, kidney cancer cells, central nervous system cancer cells, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteosarcoma tumor cells, and leukemia cells.

[0313] In another embodiment, the tissue or organ is selected from the group consisting of the heart, liver, spleen, lungs, kidneys, brain, lymph nodes, muscles, blood, spine, bones, and combinations thereof.

[0314] In another embodiment, the subject is a human or a non-human mammal (such as a mouse, rat, rabbit, or monkey).

[0315] In a thirteenth aspect of the invention, a method for generating a target protein or target polypeptide in subject cells is provided, comprising contacting the subject cells with the lipid nanoparticle composition of the fourth aspect of the invention, the pharmaceutical composition of the fifth aspect of the invention, or the pharmaceutical preparation of the sixth aspect of the invention.

[0316] In another embodiment, the bioactive substance in the lipid nanoparticle composition, pharmaceutical composition, or pharmaceutical formulation is mRNA, wherein the mRNA encodes a target protein or polypeptide, and the mRNA is capable of being translated in a cell to produce the target protein or target polypeptide.

[0317] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0318] Figure 1 This is a schematic diagram of the delivery strategy for PCSK9 gene base editing in mouse liver cells in this invention.

[0319] Figure 2 To assess the base editing efficiency of the PCSK9 gene in mouse hepatocytes using lipid nanoparticles (LNPs) containing different compounds. Detailed Implementation

[0320] Through long-term and in-depth research, the inventors unexpectedly discovered for the first time an ionizable lipid compound for delivering therapeutic agents. This ionizable lipid compound can effectively deliver drugs such as nucleic acid molecules and small molecule compounds. Furthermore, by comparison, the lipid nanoparticles of this invention exhibit better particle size distribution, higher encapsulation efficiency, and significantly better delivery performance than comparative lipid nanoparticles, thus meeting the requirements for in vivo delivery. Based on this, the present invention was completed.

[0321] For ease of understanding, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0322] In this specification, the range of values ​​referred to as “value A - value B” is the range that includes the endpoint values ​​A and B.

[0323] In this specification, the terms "substantially" or "truly" are used to indicate that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0324] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0325] In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0326] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0327] Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described herein; it should also be understood that the terminology used herein is for description only and not for limiting specific embodiments.

[0328] Unless otherwise stated, the following terms have the following meanings:

[0329] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention that is substantially non-toxic to organisms. Pharmaceutically acceptable salts generally include, but are not limited to, salts formed by reacting the compounds of this invention with pharmaceutically acceptable inorganic / organic acids or inorganic / organic bases; such salts are also known as acid addition salts or base addition salts. Common inorganic acids include (but are not limited to) hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.; common organic acids include (but are not limited to) trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.; common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, etc.; and common organic bases include (but are not limited to) diethylamine, triethylamine, ethylaminobutanol, etc.

[0330] The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular asymmetric plane due to having at least one chiral element (including a chiral center, chiral axis, chiral plane, etc.), thereby enabling the rotation of plane-polarized light. Since the compounds of this invention contain asymmetric centers and other chemical structures that may lead to stereoisomerism, this invention also includes these stereoisomers and mixtures thereof. Because the compounds of this invention and their salts include asymmetric carbon atoms, they can exist as single stereoisomers, racemates, or mixtures of enantiomers and diastereomers. Typically, these compounds can be prepared as racemic mixtures. However, if desired, such compounds can be prepared or isolated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched with single stereoisomers (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). The single stereoisomer of a compound is synthesized from an optically active starting material containing the desired chiral center, or obtained by preparing a mixture of enantiomers followed by separation or resolution, for example, by converting it into a mixture of diastereomers followed by separation or recrystallization, chromatographic processing, using chiral resolving reagents, or by direct separation of the enantiomers on a chiral column. Starting compounds with specific stereochemistry are either commercially available or prepared according to the methods described herein and then resolved by methods well known in the art.

[0331] The term "tautomer" (or "tautomer form") refers to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (or proton transfer tautomers) include (but are not limited to) interconversions via proton transfer, such as keto-enol isomerization, imine-enamine isomerization, amide-imine alcohol isomerization, etc. Unless otherwise stated, all tautomer forms of the compounds of this invention are within the scope of this invention.

[0332] The term "solvent" refers to a substance formed by the combination of a compound of the present invention or a pharmaceutically acceptable salt thereof with at least one solvent molecule through non-covalent intermolecular forces. Common solvates include (but are not limited to) hydrates, ethanol compounds, acetone compounds, etc.

[0333] The term "chelate" refers to a complex with a cyclic structure, obtained through the chelation of two or more ligands with the same metal ion to form a chelate ring.

[0334] The term "non-covalent complex" refers to a complex formed through the interaction of a compound with another molecule, where no covalent bond is formed between the two molecules. Complexation can occur, for example, through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

[0335] The term "prodrug" refers to a derived compound that, upon administration to a patient, can directly or indirectly provide the compounds of the present invention. Particularly preferred derived compounds or prodrugs are those that, when administered to a patient, can improve the bioavailability of the compounds of the present invention (e.g., facilitate absorption into the bloodstream) or promote the delivery of the parent compound to its site of action (e.g., the lymphatic system). Unless otherwise stated, all prodrug forms of the compounds of the present invention are within the scope of the present invention, and various prodrug forms are well known in the art.

[0336] The term "independently" means that at least two groups (or ring systems) in a structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl.

[0337] The terms “contain” and “include” are used in their open, non-restrictive sense.

[0338] The term "alkyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, without unsaturation, and linked to other segments by a single bond, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, and tert-butyl. For example, "C 1-30 "Alkyl" refers to a saturated monovalent straight-chain or branched hydrocarbon group containing 1 to 30 carbon atoms.

[0339] The term "alkylene" refers to a divalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, without saturation, and connected to other segments via two single bonds, including (but not limited to) methylene, 1,1-ethylene, and 1,2-ethylene. For example, "C 1-30 "Alkylene" refers to saturated divalent straight-chain or branched alkyl groups containing 1 to 30 carbon atoms.

[0340] The term "cycloalkyl" refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) non-aromatic hydrocarbon group consisting only of carbon and hydrogen atoms. Cycloalkyl groups can include fused, bridged, or spirocyclic systems. For example, the term "C" as used herein... 3-6"Cycloalkyl" refers to a cycloalkyl group having 3 to 6 carbon atoms. For example, cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or bicyclo[2.2.1]heptyl, etc.

[0341] The term "cycloalkylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group as defined above, including (but not limited to) cycloalkylene, cycloalkylene, cycloalkylene, cycloalkylene, and cycloalkylene, etc. For example, "C 3-30 "Cycloalkylene" refers to a divalent group obtained by removing hydrogen atoms from a cycloalkyl group containing 3 to 30 carbon atoms.

[0342] The term "branched alkyl" refers to an alkane radical that is attached to a parent molecule and forms at least two branches. For example... .

[0343] The term "alkenyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via a single bond. This includes (but is not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl, and isobutenyl groups. For example, "C..." 2-30 "Alkenyl" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

[0344] The term "alkenyl" refers to a divalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via two single bonds, including (but not limited to) vinylenes. For example, "C 2-30 "Alkenyl" refers to a divalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

[0345] The term "alkynyl" refers to a monovalent, straight-chain or branched alkane group composed only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other segments by a single bond. This includes (but is not limited to) ethynyl, propynyl, butynyl, and pentyynyl groups. For example, "C..." 2-30 "Alkyne" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.

[0346] The term "ethynyl" refers to a divalent, straight-chain or branched alkane group composed only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other segments via two single bonds, including (but not limited to) ethynyl groups. For example, "C 2-30 "Alynyl group" refers to a divalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.

[0347] The term "cycloalkenyl" refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) non-aromatic hydrocarbon group composed only of carbon and hydrogen atoms. Cycloalkenyl groups can include fused, bridged, or spirocyclic systems. Examples include cyclopropenyl and cyclobutenyl.

[0348] The term "cycloene-alkenyl" refers to a divalent group obtained by removing a hydrogen atom from a cycloene-alkenyl group as defined above, including (but not limited to) cycloene-propenyl and cycloene-butenyl groups. For example, "C 3-30 "Biopylene alkenyl" refers to a divalent group obtained by removing hydrogen atoms from a cycloalkenyl group containing 3 to 30 carbon atoms.

[0349] The term "branched alkenyl" refers to an alkene radical that is linked to a parent molecule and forms at least two branches. For example... .

[0350] The term "heterocyclic group" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused, bridged, or spirocyclic) non-aromatic group whose ring atoms comprise a carbon atom and at least one heteroatom selected from N, O, and S, wherein the S atom is optionally substituted to form S(=O), S(=O)2, or S(=O)(=NR). x ), R x Independently selected from H or C 1-4 Alkyl group. If the valence requirement is met, the heterocyclic group can be connected to the rest of the molecule via any one ring atom. For example, the term "3-8 membered heterocyclic group" as used in this invention refers to a heterocyclic group having 3 to 8 ring atoms. For example, the heterocyclic group can be ethylene oxide, aziridine propane, aziridine butane, oxadiazine, tetrahydrofuranyl, dioxolyl, pyrrolyl, pyrrolidone, imidazoalkyl, pyrazolyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithiaalkyl, or trithiaalkyl.

[0351] The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π-electron system. For example, the term "C" as used in this invention... 6-10 "Aryl" refers to an aryl group having 6 to 10 carbon atoms. For example, aryl can be phenyl, naphthyl, anthracene, phenanthryl, acenaphthene, azulene, fluorenyl, indene, pyrene, etc.

[0352] The term "heteroaryl" refers to an aromatic group consisting of a monocyclic or fused polycyclic ring with a conjugated π-electron system, wherein the ring atom is composed of a carbon atom and at least one heteroatom selected from N, O, and S. If the valence requirement is met, the heteroaryl group can be connected to the rest of the molecule through any one ring atom. For example, the term "5-10-membered heteroaryl" as used in this invention refers to a heteroaryl group having 5 to 10 ring atoms. Examples of heteroaryl groups include thiophene, furanyl, pyrroloyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiazolyl, pyridinyl, pyridinyl, triazinyl and their benzo[a] derivatives, pyrrolopyridyl, pyrrolopyridinyl, pyrazolopyridyl, imidazopyridyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purine, etc.

[0353] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0354] The term "hydroxyl group" refers to -OH.

[0355] The term "cyano" refers to -CN.

[0356] The term "amino" refers to -NH2.

[0357] The term "nitro" refers to -NO2.

[0358] The term "oxo group" refers to (=O).

[0359] The term "subject" includes both humans and non-human animals. Non-human animals include vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, cats, horses, cattle, chickens, dogs, mice, rats, goats, rabbits, and pigs. Preferably, the subject is a human. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably herein.

[0360] The term "nuclease" refers to an enzyme that catalyzes the breaking of phosphodiester bonds between nucleotides in a nucleic acid molecule. In some embodiments, the nuclease is selected from a wide range of nucleases, zinc-finger nucleases (ZFNs), TAL-effector DNA-binding domain-nuclease fusion proteins (TALENs), and RNA-guided nucleases, or variants thereof in which the nuclease activity has been reduced or inhibited.

[0361] In some implementations, the RNA-guided nuclease is a naturally occurring CRISPR-Cas protein or its active variant or fragment. CRISPR-Cas systems are classified into class I or class II systems. Class II systems contain a single-effect nuclease and include types II, V, and VI. Each class is further subdivided into types (I, II, III, IV, V, VI), some of which are further subdivided into subtypes (e.g., II-A, II-B, II-C, VA, VB).

[0362] The terms "type II CRISPR-Cas protein," "type II CRISPR-Cas effector protein," or "Cas9" refer to a CRISPR-Cas effector protein that requires trans-activation RNA (tracrRNA) and contains two nuclease domains (RuvC and HNH) (each responsible for cleaving a single strand of a double-stranded DNA molecule). In other embodiments, the CRISPR-Cas protein is a naturally occurring type V CRISPR-Cas protein or its active variant or fragment.

[0363] As used herein, the terms "Type V CRISPR-Cas protein," "Type V CRISPR-Cas effector protein," or "Cas12" refer to a CRISPR-Cas effector protein that cleaves dsDNA and contains a single RuvC nuclease domain or a split RuvC nuclease domain and lacks an HNH domain. In other embodiments, the CRISPR-Cas protein is a naturally occurring Type VI CRISPR-Cas protein or an active variant or fragment thereof. As used herein, the terms "Type VI CRISPR-Cas protein," "Type VI CRISPR-Cas effector protein," or "Cas13" refer to a CRISPR-Cas effector protein that does not require tracrRNA and contains two HEPN domains that cleave RNA.

[0364] The term "gRNA" refers to a nucleotide sequence that is sufficiently complementary to the target nucleotide sequence to hybridize with it and guide the sequence-specific binding of the associated nuclease to the target nucleotide sequence. For CRISPR-Cas enzymes, the corresponding guide RNA is one or more RNA molecules (usually one or two) that bind to the Cas enzyme and guide the Cas enzyme to bind to the specific target nucleotide sequence, and in those cases, the Cas enzyme also cleaves the target nucleotide sequence if it has nicking or nuclease activity.

[0365] Group definition

[0366] As used in this article, the term "C1-C" 30 Alkyl" or "C1-C" 20 Alkyl" or "C1-C" 14Alkyl" or "C1-C" 10 "Alkyl" refers to a straight-chain or branched alkyl group having 1-30, 1-20, 1-14, or 1-10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups.

[0367] As used in this article, the term "C2-C" 30 "Alkenyl" or "C2-C" 20 "Alkenyl" or "C2-C" 14 "Alkenyl" refers to alkenyl groups with straight or branched chains having 2-30, 2-20, or 2-14 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups.

[0368] As used in this article, the term "C2-C" 30 "Alkyne group" or "C2-C" 20 "Alkyne" or "C2-C" 14 "Alynyl" refers to a straight-chain or branched alkynyl group having 2-30, 2-20, or 2-14 carbon atoms, such as ethynyl, propynyl, or similar groups.

[0369] As used herein, the term "halogenated" refers to a group substituted with one or more of the same or different halogen atoms described above, which may be partially or fully halogenated, such as trifluoromethyl, pentafluoroethyl, heptafluoroisopropyl, or similar groups.

[0370] As used in this article, the term "C1-C" 10 "Halogenated alkyl" refers to a straight-chain or branched alkyl group having 1 to 10 carbon atoms in which one or more hydrogen atoms are replaced by a halogen, such as halogenated methyl, halogenated ethyl, halogenated propyl, halogenated isopropyl, or similar groups, preferably trifluoromethyl.

[0371] The compounds of this invention may contain one or more asymmetric centers and thus appear as racemates, racemic mixtures, single enantiomers, diastereomers, and single diastereomers. The asymmetric centers that can exist depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers, diastereomer mixtures, and pure or partially pure compounds are included within the scope of this invention. This invention encompasses all isomeric forms of the compounds.

[0372] Ionizable lipids

[0373] As used herein, the terms "ionizable lipids of the present invention" and "ionizable cationic lipids of the present invention" are used interchangeably and both refer to compounds of Formula I or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or precursors thereof.

[0374] Ionizable lipids protonate and transform into cationic lipids at low pH values, while at normal physiological pH values ​​they transform into helper phospholipids. Helper phospholipids interact less with the anionic cell membranes of blood cells, improving the biocompatibility of lipid nanoparticles. When lipid nanoparticles are endocytosed by cells, the pH value within the endosomes is low, causing the lipids to protonate and become positively charged. This reduces or even disrupts the membrane structure's stability, facilitating the escape of lipid nanoparticles from the endosomes. In summary, the pH-sensitive nature of lipids is beneficial for the in vivo delivery of lipid nanoparticles encapsulating bioactive components (such as mRNA molecules).

[0375] In one aspect of the invention, an ionizable lipid is provided having a structure as shown in Formula I:

[0376] (I);

[0377] In the formula,

[0378] R1 is selected from -OH and R a (R b )N-, where R a and R b Each independently is hydrogen, C1-C 10 Alkyl or C1-C 10 Halogenated alkyl groups;

[0379] R2 is C1-C 20 Alkyl, C2-C 20 alkenyl, or C2-C 20 alkynyl group;

[0380] R3 is C1-C 20 Alkyl, C2-C 20 alkenyl, or C2-C 20 alkynyl group, or R c -(CH2)n-, where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, and more preferably a positive integer from 1 to 10;

[0381] R c Selected from the structure shown below:

[0382] , , , , and ,in, Represents a connection key;

[0383] L1, L2, L3, L4, and L5 do not exist, or each can be independently selected from the following groups:

[0384] , , , and ;

[0385] X does not exist, or is -CH- or N;

[0386] R4 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, or C2-C 20 alkynyl group;

[0387] R5 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, or C2-C 20 alkynyl group;

[0388] R6 is C1-C 30 Alkyl, C2-C 30 alkenyl, or C2-C 30 alkynyl group;

[0389] R7 is C1-C 14 Alkyl, C2-C 14 alkenyl, or C2-C 14 alkynyl group;

[0390] R8 and R9 do not exist, or they are each independently C1-C. 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0391] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h It can be O or S.

[0392] In a preferred embodiment of the present invention, the ionizable lipids respectively have the substructures shown in formulas I-1, I-2, I-3, and I-4:

[0393] (I-1)

[0394] (I-2)

[0395] (I-3)

[0396] (I-4)

[0397] In the structure of Equation I-1, R1-R2, R6-R 11 The definitions of L2-L5 are as described above;

[0398] R3 is C1-C 20 Alkyl, C2-C 20 alkenyl, or C2-C 20 Alkyne group.

[0399] In the structure of Equation I-2, R1-R2, R6-R7, R 10 -R 11 The definitions of L1-L5 are as described above;

[0400] R3 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, or R c -(CH2)n-, where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, and Rc is defined as described above;

[0401] R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, or C2-C 20 alkynyl group;

[0402] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group.

[0403] In the structure of Equation I-3, R1-R3, R6, R7, R 10 R 11 The definitions of L1-L5 are as described above;

[0404] R4 is C1-C 20 Alkyl, C2-C 20 alkenyl, or C2-C 20alkynyl group;

[0405] R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, or C2-C 20 alkynyl group;

[0406] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h It can be O or S.

[0407] In the structure of formula I-4, R1-R7, R 10 -R 11 The definitions of L1-L3 and L5 are as described above.

[0408] In a second aspect of the invention, a compound of formula II or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof is provided having the following structure:

[0409] (II);

[0410] Wherein: R1 is selected from -OH and R a (R b )N-, where R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl;

[0411] R3 is C1-C 15 Alkyl, C2-C 15 alkenyl, C2-C 15 Alkyne group, or Rc-(CH2)n-, where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, and even more preferably a positive integer from 1 to 6;

[0412] Rc is selected from the structure shown below:

[0413] , , , , and , in Represents a connection key;

[0414] L1, L2, L3, L4, and L5 either do not exist or are independently selected from the following groups:

[0415] , , , , , ;

[0416] R4 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0417] R5 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0418] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0419] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0420] r is 2 or 3;

[0421] u is a positive integer from 1 to 10;

[0422] t is a positive integer from 1 to 10;

[0423] Furthermore, when R1 is selected from R a (R b When N-, r is 2.

[0424] In a third aspect of the invention, a compound of formula III or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof is provided having the following structure:

[0425] (III)

[0426] in,

[0427] R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl;

[0428] R c Selected from the structure shown below:

[0429] , , , , and ,in Represents a connection key;

[0430] s is a positive integer from 1 to 10;

[0431] L1, L2, L3, L4, and L5 either do not exist or are independently selected from the following groups:

[0432] , , , , , ;

[0433] R4 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0434] R5 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0435] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h - C2-C 14 alkenyl, -Rh -C2-C 14 alkynyl group, wherein R h For O or S;

[0436] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0437] r is a positive integer of at least 3;

[0438] u is a positive integer from 1 to 10;

[0439] t is a positive integer from 1 to 10.

[0440] In a fourth aspect of the invention, a compound of formula IV or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof is provided having the following structure:

[0441] (IV)

[0442] in,

[0443] R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl;

[0444] R3 is C1-C 15 Alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl group, or R c -(CH2)n-, where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, and more preferably a positive integer from 1 to 10;

[0445] Rc is selected from the structure shown below:

[0446] , , , , and ,in Represents a connection key;

[0447] L1, L2, L3, L4, and L5 either do not exist or are independently selected from the following groups:

[0448] , , , , , ;

[0449] R4 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0450] R5 does not exist or is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0451] R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -Rh-C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0452] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S;

[0453] r is a positive integer of at least 3;

[0454] u is a positive integer from 1 to 10;

[0455] t is a positive integer from 1 to 10.

[0456] A fifth aspect of the invention provides a compound of formula V or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof having the following structure:

[0457] (V)

[0458] Among them, R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; Rc is selected from the structures shown below:

[0459] , , , , and ,in Represents a connection key;

[0460] L1, L2, L3, and L5 either do not exist or are independently selected from the following groups:

[0461] , , , , , ;

[0462] R4 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0463] R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group;

[0464] R6 is C1-C 30 Alkyl, C2-C 30 alkenyl, C2-C 30 alkynyl group;

[0465] R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14alkynyl group, wherein R h For O or S;

[0466] r is a positive integer of at least 3;

[0467] t is a positive integer from 1 to 10.

[0468] In a more preferred embodiment of the invention, the ionizable lipid has a structure selected from those shown in Table 1, wherein the following compounds in Table 1 are preferred:

[0469]

[0470]

[0471] More preferably, the compounds are selected from the following compounds in Table 1:

[0472]

[0473]

[0475] assist lipids

[0476] As used herein, the term “auxiliary lipid” refers to one or more of the following types of lipids in lipid nanoparticles, other than ionizable lipids: anionic lipids, neutral lipids, steroid-bound lipids, and polymer-bound lipids.

[0477] In some implementations, auxiliary lipids can be used to improve the properties of lipid nanoparticles, such as enhancing nanoparticle stability, fusogenicity, and / or fluidity.

[0478] In another embodiment, the anionic lipid comprises one or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), and dimyristoylphosphatidylglycerol.

[0479] In another embodiment, the neutral lipid comprises at least one of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), dipalmitoylphosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), and 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or a lipid modified with an anionic or cationic modifying group. The anionic or cationic modifying group is not limited.

[0480] In another embodiment, the steroids include one or more of cholesterol, nonsteroidal, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, coccosterol, and corticosteroids.

[0481] In another embodiment, the polymer-bound lipids include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), α-(3'-{[1,2-di(myristoyloxy)propoxy]carbonylamino}propyl)-ω-methoxypolyoxyethylene (PEG-c-DMG), PEG-1,2-dimyristoylpropyl-3-amine (PEG-c-DMA), and 1,2-distearatel-sn-glycero-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] One or more of the following: (PEG-DSPE), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, 1,2-dipalmityl-sn-glycerol-methoxy polyethylene glycol (PEG-DPG), 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-s-DMG), PEG-dialkoxypropyl (PEG-DAA), mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol ester (PEG-c-DOMG), and N-acetylgalactosamine ((R)-2,3-bis(octadecanoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate) (GalNAc-PEG-DSG).

[0482] In another embodiment, in the lipid carrier, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid, and polymer-bound lipid is (20-65):(0-20):(5-25):(25-55):(0.3-15); exemplaryly, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid, and polymer-bound lipid can be 20:20:5:50:5, 30:5:25:30:10, or 20:5:5:5. 5:15, 65:0:9.7:25:0.3, etc.; wherein, in the first lipid compound, the molar ratio of the compound of formula I or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and other cationic or ionizable lipids is (1-10):(0-10); exemplaryly, the molar ratio can be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

[0483] In another embodiment, in the lipid carrier, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid and polymer-bound lipid is (20-55):(0-13):(5-25):(25-51.5):(0.5-15); wherein, in the first lipid compound, the molar ratio of the compound of formula I or its pharmaceutically acceptable form, such as salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and other cationic or ionizable lipid is (3-4):(0-5).

[0484] In a preferred embodiment of the present invention, the auxiliary lipid is selected from DSPC, cholesterol and PEG-DMG.

[0485] In another embodiment, the co-adjuvant lipids include DSPC, cholesterol, and PEG-DMG.

[0486] Lipid nanoparticles (LNP)

[0487] As used herein, the term "lipid nanoparticle" or "LNP" refers to particles with a diameter of approximately 5 to 500 nm. In some embodiments, the lipid nanoparticles contain one or more active agents (bioactive substances). In some embodiments, the lipid nanoparticles include nucleic acids. In some embodiments, the nucleic acids are condensed within the nanoparticles with cationic lipids, polymers, or multivalent small molecules, and an external lipid coating that interacts with the biological environment. Nucleic acids are naturally rigid polymers and tend to have elongated configurations due to the repulsive forces between phosphate groups. In cells, to cope with volume constraints, DNA can package itself under appropriate solution conditions with the help of ions and other molecules. Typically, DNA condensation is defined as the collapse of an elongated DNA strand into a compact, ordered particle containing only one or a few molecules. By binding to phosphate groups, cationic lipids can concentrate DNA and cause it to pack tightly together by neutralizing the phosphate charge.

[0488] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance can be anionic compounds, including but not limited to DNA (including plasmids), RNA (mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitors, microRNA activators, or shRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, aptamers, immunostimulatory nucleic acids or PNA, DNA-containing nucleoproteins such as intact or partially deproteinized viral particles (viral particles), and oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance can be mixed with an adjuvant. In a preferred embodiment, the mRNA includes a sequence encoding an RNA-directed DNA binder, more specifically, including mRNA encoding a nuclease or base editor.

[0489] In a preferred embodiment, the nucleic acid further includes guide RNA, specifically, the guide RNA includes gRNA nucleic acid.

[0490] In another embodiment, the nucleic acid includes mRNA and gRNA encoding a nuclease or a base editor.

[0491] In LNP vaccine products, the bioactive substance is typically contained within the LNP itself. In some embodiments, the bioactive substance includes nucleic acids. Typically, water-soluble nucleic acids are condensed within the particle with cationic lipids or polycationic polymers, and the particle surface is enriched with accessory phospholipids or PEG lipid derivatives. Additional ionizable cationic lipids may also be located on the surface; upon entering the lysosome, these ionizable cationic lipids become positively charged due to the acidic environment of the lysosome, interacting with the lysosomal membrane and facilitating endosome escape.

[0492] Regarding LNPs, ionizable lipids can have different properties or functions. Due to the pKa of amino groups, when the external pH is lower than the pKa of the lipid molecule, it can be protonated and become positively charged. Under these conditions, lipid molecules can electrostatically bind to the phosphate groups of nucleic acids, which leads to LNP formation and nucleic acid encapsulation, and the surface charge of LNPs in biological fluids (such as blood) at physiological pH is essentially neutral. High LNP surface charge is associated with toxicity, rapid clearance of circulating LNPs by fixed and free macrophages, hemolytic toxicity, including immune activation (Filion et al., Biochim Biophys Acta. 1997 Oct 23; 1329(2):345-56).

[0493] In some embodiments, the pKa can be high enough that ionizable cationic lipids can take a positively charged form at acidic endosomal pH. This allows the cationic lipids to bind to endogenous endosomal anionic lipids to promote membrane lysis of non-bilayer structures, such as the hexagonal HII phase, resulting in more efficient intracellular delivery. In some embodiments, the pKa ranges from 6.2 to 6.5. For example, the pKa can be about 6.2, about 6.3, about 6.4, or about 6.5. Unsaturated tails also contribute to the lipids' ability to take a non-bilayer form. (Jayaraman et al., Angew Chem Int Ed Engl. 20 Aug 2012; 51(34):8529-33).

[0494] The release of nucleic acids from LNP formulations, as well as other characteristics such as liposome clearance and circulating half-life, can be altered by the presence of polyethylene glycol and / or sterols (e.g., cholesterol) or other potential additives in the LNP, and by the overall chemical structure, including the pKa of any ionizable cationic lipids that are part of the formulation.

[0495] In one aspect of the invention, a lipid nanoparticle (LNP) is provided, the lipid nanoparticle comprising a compound of formula I as described in the first aspect of the invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof. Further, the lipid nanoparticle also comprises one or more auxiliary lipids, the auxiliary lipids comprising one or more combinations of anionic lipids, neutral lipids, steroid-bound lipids, and polymer-bound lipids.

[0496] pharmaceutical preparations

[0497] In another aspect of the invention, a pharmaceutical formulation (or lipid nanoparticle composition, or LNP composition) is provided, the pharmaceutical formulation comprising a lipid carrier as described in the third aspect of the invention, a bioactive substance encapsulated in the lipid carrier, and a pharmaceutically acceptable carrier. The pharmaceutical formulation is intended for delivering the bioactive substance to cells in a subject in need.

[0498] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance may be anionic compounds, including but not limited to DNA (including plasmids), RNA (mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitors, microRNA activators, or shRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, aptamers, immunostimulatory nucleic acids or PNAs, DNA-containing nucleoproteins, such as intact or partially deproteinized viral particles (viral particles), and oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance may be mixed with an adjuvant. In some embodiments, the LNP composition comprises: nucleic acids, ionizable lipids having the structure shown in Formula I, II, III, IV, or V, and optionally accessory phospholipids (e.g., distearate phosphatidylcholine). In some embodiments, the LNP composition comprises: nucleic acid; an ionizable lipid having the structure shown in Formula I, in an amount of 30-65% (molar percentage) of the total lipids of the composition; and optionally an accessory phospholipid (e.g., distearylphosphatidylcholine), in an amount of 1-10% of the total lipids of the composition.

[0499] Furthermore, in a preferred embodiment, in the LNP composition, the molar ratio of the first lipid compound (an ionizable lipid of the structure shown in Formula (I) and optionally other cationic or ionizable lipids), anionic lipid, neutral lipid, steroid, and polymer-bound lipid is (20-65):(0-20):(5-25):(25-55):(0.3-15); exemplaryly, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid, and polymer-bound lipid can be 20:20:5:50:5, 30:5:25:30:10, 20:5:5:55:15, 65:0:9.7:25:0.3, etc.

[0500] In a preferred embodiment, in the first lipid compound, the molar ratio of a compound of formula I, II, III, IV or V or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and other cationic or ionizable lipids is (1-10):(0-10); exemplaryly, this molar ratio can be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

[0501] In some embodiments, in the lipid carrier, the molar ratio of the first lipid compound, anionic lipid, neutral lipid, steroid and polymer-bound lipid is (20-55):(0-13):(5-25):(25-51.5):(0.5-15); wherein, in the first lipid compound, the molar ratio of any of the above compounds or their pharmaceutically acceptable forms, such as salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs, and other cationic or ionizable lipids is (3-4):(0-5).

[0502] As used herein, the terms “encapsulation” and “encapsulated” refer to mRNA, DNA, siRNA, or other nucleic acid drugs being contained within or bound to lipid nanoparticles. As used herein, the term “encapsulation” refers to complete or partial encapsulation. For example, mRNA may be selected to treat and / or prevent a target disease when administered to a subject in need of a lipid nanoparticle composition comprising mRNA.

[0503] As used herein, the term "pharmaceuticalally acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, scintillation agent, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the Food and Drug Administration for use in humans or livestock.

[0504] The present invention also provides a method for in vivo delivery of nucleic acid drugs, the method comprising administering the lipid nanoparticle composition or the drug formulation to a subject in need.

[0505] In some embodiments, the above-described nucleic acid lipid nanoparticle composition or pharmaceutical preparation is administered via one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the above-described nucleic acid lipid nanoparticle composition or pharmaceutical preparation is administered, for example, via enteral or parenteral administration. In some embodiments, the nucleic acid lipid nanoparticle composition or pharmaceutical preparation is administered to the subject at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0506] Preparation method of pharmaceutical formulation

[0507] In another aspect of the invention, a method for preparing a pharmaceutical formulation is provided, the method comprising: (a) mixing a compound of formula I, II, III, IV, or V as described in the first aspect of the invention, and optionally an auxiliary lipid, with an organic solvent to obtain a lipid organic phase; (b) mixing a bioactive substance with an aqueous solvent to obtain an aqueous phase containing the bioactive substance; and (c) mixing the lipid organic phase from step (a) with the aqueous phase from step (b) to obtain the lipid nanoparticle composition. Further, the method further comprises step (d): purifying, concentrating, and sterilizing the lipid nanoparticle drug obtained in step (c).

[0508] In some embodiments, the organic solvent includes (but is not limited to) ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, or tetrahydrofuran, or combinations thereof. In some embodiments, the lipid organic phase includes a small percentage of water or a pH buffer. The lipid organic phase may contain up to 60% by volume of water, for example, up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% by volume of water. In one embodiment, the lipid organic phase contains between about 0.05% and 60% by volume of water, for example, between about 0.05% and 50%, between about 0.05% and 40%, or between about 5% and 20% by volume of water.

[0509] In some embodiments, the aqueous solvent is water. In some embodiments, the aqueous solvent is an aqueous buffer solution with a pH between 3 and 8 (e.g., pH of about 3, about 4, about 5, or about 6, etc.). A bioactive substance, such as a nucleic acid (e.g., mRNA), is dissolved in the aqueous solvent to obtain an aqueous phase containing the bioactive substance. The aqueous phase may contain a small percentage of a water-miscible organic solvent. The aqueous phase may contain up to 60% by volume of at least one water-miscible organic solvent, such as up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or any volume percentage of an organic solvent (e.g., a water-miscible organic solvent) between two values. In one embodiment, the aqueous phase comprises between about 0.05% and 60% by volume an organic solvent, such as an organic solution (e.g., a water-miscible organic solvent) between about 0.05% and 50%, about 0.05% and 40%, or about 5% and 20% by volume. The aqueous buffer may be a citrate buffer, Tris-HCl buffer, sodium acetate buffer, PBS buffer, or a combination thereof. In some embodiments, the aqueous buffer is a citrate buffer with a pH between 4 and 6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer solution is a citrate buffer with a pH of about 4.

[0510] In some embodiments, a solution comprising a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance, including an LNP suspension, may be diluted. In some embodiments, the pH of the solution comprising the lipid organic phase and the aqueous phase containing the bioactive substance of the LNP suspension may be adjusted. The pH of the LNP suspension may be diluted or adjusted by adding water, acid, base, or an aqueous buffer. In some embodiments, the pH of the LNP suspension is not diluted or adjusted. In some embodiments, the pH of the LNP suspension is diluted and adjusted.

[0511] In some embodiments, excess reagents, solvents, and unencapsulated nucleic acids can be removed from the LNP suspension by tangential flow filtration (TFF) (e.g., percolation). Organic solvents (e.g., ethanol) and buffers can also be removed from the LNP suspension by TFF. In some embodiments, the LNP suspension is dialyzed. In some embodiments, the LNP suspension is subjected to TFF. In some embodiments, the LNP suspension is subjected to both dialyzed and TFF.

[0512] The main advantages of this invention include:

[0513] (1) This invention provides a series of novel Formula I compounds that can be used as ionizable lipids and co-prepared with other lipid compounds to prepare lipid carriers. The particle size is controllable, the distribution is uniform, and the encapsulation efficiency is very high.

[0514] (2) The lipid compound synthesis method of the present invention is simple, has a high yield, can be synthesized rapidly, and is low in cost. The compounds of the present invention can be used to deliver nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs, enriching the types of ionizable lipid compounds, and are of great significance for the development and application of nucleic acid prophylaxis and therapeutic agents.

[0515] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0516] Unless otherwise specified, all reagents and materials used in the embodiments of this invention are commercially available products.

[0517] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0518] In this invention, "appropriate amount" means that the amount of solvent or reagent added can be adjusted within a large range and has little impact on the synthesis result, and no specific limitation is required.

[0519] In the following examples, all solvents and reagents used were of analytical or chemical purity; all solvents were redistilled before use; and all anhydrous solvents were processed according to standard or literature methods.

[0520] Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the invention. Technical features involved in the various embodiments of the invention can be combined with each other as long as they do not conflict with each other. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0521] Example 1. Synthesis of Compound 1

[0522] 2-Butyloctanoic acid-8-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-13-hydroxy-5-oxane-10-propyl-10-aza-6-oxatridecane-1-yl ester

[0523] Intermediate 1-2: Benzyl 5-hydroxyvalerate

[0524] A solution of tetrahydro-2H-pyran-2-one (25.00 g, 249.70 mmol, 1.0 equivalent) and NaOH (10.99 g, 274.67 mmol, 1.1 equivalent) in H2O (20 mL) and ethanol (200 mL) was stirred at 70 °C for 3 hours and concentrated under reduced pressure. Then, acetone (200 mL), TBAI (4.61 g, 12.48 mmol, 0.05 equivalent), and BnBr (51.25 g, 299.64 mmol, 1.2 equivalent) were added. The resulting solution was stirred at 70 °C for 16 hours, diluted with H2O (500 mL), and extracted with EtOAc (250 mL × 2). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EtOAc = 10 / 1 to 2 / 1) to obtain the target product in the form of a colorless oil (37.00 g, yield 71.2%).

[0525] Intermediate 1-4: 5-(benzyloxy)-5-oxopentyl-2-butyloctanoate

[0526] A solution of benzyl 5-hydroxyvalerate (37.00 g, 177.67 mmol, 1.0 equivalent), 2-butyloctanoic acid (35.59 g, 177.67 mmol, 1.0 equivalent), DMAP (21.70 g, 177.67 mmol, 1.0 equivalent), and EDCI (51.09 g, 266.50 mmol, 1.5 equivalent) in DCM (250 mL) was stirred at 25 °C for 4 hours. The reaction solution was diluted with H2O (500 mL) and extracted with DCM (200 mL × 2). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EtOAc = 20 / 1 to 4 / 1) to give the target product (64.00 g, yield 92.2%) as a colorless oil.

[0527] Intermediate 1-5: 5-((2-Butyloctanoyl)oxy)valerate

[0528] A solution of 5-(benzyloxy)-5-oxopentyl-2-butyloctanoate (64.00 g, 163.87 mmol, 1.0 equivalent) and Pd / C (3.49 g, 32.78 mmol, 0.2 equivalent, 10% purity) in MeOH (75 mL) and THF (75 mL) was stirred at 25 °C for 16 hours under H2 balloon. The reaction solution was filtered and concentrated under reduced pressure to give the target product (45.00 g, 91.4% yield) as a colorless oil, which could be used for the next reaction without further purification.

[0529] Intermediates 1-7: ((2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate)

[0530] A solution of 5-((2-butyloctanoyl)oxy)valerate (10.00 g, 33.29 mmol, 1.0 equivalent), 2-(hydroxymethyl)propane-1,3-diol (3.53 g, 33.29 mmol, 1.0 equivalent), DMAP (0.81 g, 6.66 mmol, 0.2 equivalent), EDCI (9.57 g, 49.94 mmol, 1.5 equivalent), and DIEA (8.60 g, 66.58 mmol, 2.0 equivalent) in DCM (100 mL) was stirred at 25 °C for 4 hours. The reaction solution was diluted with H2O (100 mL) and extracted with DCM (100 mL × 2). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EtOAc = 20 / 1 to 3 / 1) to obtain the target product in the form of a colorless oil (7.80 g, yield 69.9%).

[0531] Intermediates 1-8: ((2-((((methanesulfonyl)oxy)methyl)propane-1,3-diyl)bis(oxy))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate)

[0532] A solution of ((2-(hydroxymethyl)propane-1,3-diyl)bis(oxo))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate) (3.90 g, 5.81 mmol, 1.0 equivalent) and TEA (1.76 g, 17.43 mmol, 3.0 equivalent) in DCM (30 mL) was cooled in an ice bath, and Ms2O (2.02 g, 11.62 mmol, 2.0 equivalent) was added dropwise. The reaction mixture was then heated and stirred at 25 °C for 4 hours. The solution was diluted with H2O (30 mL) and extracted with DCM (50 mL × 2). The organic layers were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EtOAc = 50 / 1 to 5 / 1) to give the target product (3.85 g, yield 88.4%) as a colorless oil.

[0533] Compound 1: ((2-(((3-hydroxypropyl)(propyl)amino)methyl)propane-1,3-diyl)bis(oxy))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate)

[0534] A solution of ((2-((((methanesulfonyl)oxy)methyl)propane-1,3-diyl)bis(oxy))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate) (410.0 mg, 0.55 mmol, 1.0 equivalent), 3-(propylamino)prop-1-ol (65.0 mg, 0.55 mmol, 1.0 equivalent), K₂CO₃ (190.0 mg, 1.38 mmol, 2.5 equivalent), and KI (91.0 mg, 0.55 mmol, 1.0 equivalent) in CH₃CN (5 mL) was stirred at 90 °C for 16 hours. The reaction solution was diluted with H₂O (10 mL) and extracted with EtOAc (10 mL × 2). The organic layers were combined, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH = 50 / 1 to 20 / 1) to give the target product as a colorless oil (154.8 mg, yield 36.2%). MS: m / z [M+H + = 770.6. 1H NMR (300 MHz, CDCl3) δ 4.18 (dd, J = 11.1, 4.9 Hz, 2H), 4.09 (dd, J = 11.6, 5.7 Hz, 6H), 3.78 (t, J = 5.4 Hz, 2H), 2.62 (s, 2H), 2.45-2.27 (m, 10H), 1.75-1.66 (m, 10H), 1.64-1.54 (m, 5H), 1.49 (dd, J =14.8, 6.9 Hz, 7H), 1.33-1.20 (m, 25H), 0.94-0.83 (m, 15H).

[0535] Example 2. Synthesis of Compound 2

[0536] 2-Butyloctanoic acid-8-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-10-ethyl-13-hydroxy-5-oxane-10-aza-6-oxatridecane-1-yl ester

[0537]

[0538] Compound 2 was prepared from 3-(ethylamino)prop-1-ol according to the procedure of compound 1, with a yield of 40.8%.

[0539] MS: m / z [M+H] + = 756.6. 1 H NMR (300 MHz, CDCl3) δ 4.18 (dd, J = 11.2,5.0 Hz, 2H), 4.10 (dd, J = 10.3, 5.7 Hz, 6H), 3.78 (t, J = 5.3 Hz, 2H), 2.69(s, 3H), 2.49 (s, 2H), 2.43-2.27 (m, 7H), 1.71 (dd, J = 9.0, 5.9 Hz, 9H), 1.64-1.55 (m, 4H), 1.51-1.39 (m, 5H), 1.34-1.21 (m, 25H), 1.08 (t, J = 6.6Hz, 4H), 0.93-0.83 (m, 12H).

[0540] Example 3. Synthesis of Compound 3

[0541] 2-Butyloctanoic acid-8-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-13-hydroxy-10-methyl-5-oxane-10-aza-6-oxatridecane-1-yl ester

[0542]

[0543] Compound 3 was prepared from 3-(methylamino)prop-1-ol following the same procedure as compound 1, with a yield of 46.9%. MS: m / z [M+H] + = 742.6. 1 HNMR (400 MHz-CDCl3) δ 4.17 (dd, J = 11.2, 4.8 Hz, 2H), 4.08 (dd, J = 11.4, 5.7 Hz, 6H), 3.78 (t, J = 5.3 Hz, 2H), 2.57 (t, J = 5.8 Hz, 2H), 2.44-2.22 (m, 11H), 1.81-1.64 (m, 10H), 1.63-1.52 (m, 5H), 1.45 (dd, J =7.4, 5.1 Hz, 5H), 1.30 (dd, J = 18.8, 12.5 Hz, 24H), 0.99-0.74 (m, 12H).

[0544] Example 4. Synthesis of Compound 4

[0545] 2-Butyloctanoic acid-8-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-10-ethyl-12-hydroxy-5-oxane-10-aza-6-oxadodecane-1-yl ester

[0546]

[0547] Compound 4 was prepared from 2-(ethylamino)ethane-1-ol following the procedure used for compound 1, with a yield of 40.2%. MS: m / z [M+H] + = 742.6. 1 HNMR (400 MHz-CDCl3) δ 4.13 (d, J = 31.4 Hz, 8H), 3.55 (s,2H), 2.60 (s, 5H), 2.34 (dd, J = 56.7, 37.9 Hz, 9H), 1.64 (d, J = 41.8 Hz,12H), 1.45 (s, 5H), 1.26 (s, 23H), 1.03 (s, 4H), 0.88 (s, 11H).

[0548] Example 5. Synthesis of Compound 5

[0549] 2-Butyloctanoic acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-19-(3-hydroxypropyl)-8,14-dioxane-19-aza-9,15-dioxaoctadecane-28-yl ester

[0550]

[0551] Intermediate 5-2: ((2-(((3-hydroxypropyl)amino)methyl)propane-1,3-diyl)bis(oxy))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate)

[0552] A solution of intermediates 1-8 (600.0 mg, 0.80 mmol, 1.0 equivalent), 3-aminoprop-1-ol (300.0 mg, 3.99 mmol, 5.0 equivalent), K₂CO₃ (280.0 mg, 2.00 mmol, 2.5 equivalent), and KI (130.0 mg, 0.80 mmol, 1.0 equivalent) in CH₃CN (10 mL) was stirred at 90 °C for 16 hours. The reaction solution was diluted with H₂O (10 mL) and extracted with EtOAc (10 mL × 2). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH = 40 / 1 to 20 / 1) to give the target product (210.0 mg, yield 36.1%) as a colorless oil. MS: m / z [M+H] + =728.6.

[0553] Compound 5: ((2-(((9-(((2-Butyloctanoyl)oxy)nonyl)(3-hydroxypropyl)amino)methyl)propane-1,3-diyl)bis(oxy))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate).

[0554] Intermediate 5-2 (210.0 mg, 0.29 mmol, 1.0 equivalent), 9-bromononyl 2-butyloctanoate (130.0 mg, 0.32 mmol, 1.1 equivalent), potassium carbonate (K₂CO₃, 120.0 mg, 0.87 mmol, 3.0 equivalent), and potassium iodide (KI, 48.0 mg, 0.29 mmol, 1.0 equivalent) were dissolved in acetonitrile (CH₃CN, 5 mL) and reacted at 90 °C for 16 hours with stirring. The reaction solution was diluted with distilled water (H₂O, 10 mL) and extracted with ethyl acetate (EtOAc, 10 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate (Na2SO4), concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 15 / 1) to give the target product (72.0 mg, yield 23.7%) as a colorless oil.

[0555] MS: m / z [M+H] + = 1052.9. 1 H NMR (300 MHz, CDCl3) δ 4.20-4.16 (m, 2H),4.15-4.01 (m, 8H), 3.83-3.66 (m, 2H), 2.68-2.59 (m, 4H), 2.47-2.30 (m, 12H),1.76-1.53 (m, 28H), 1.49-1.22 (m, 42H), 0.94-0.83 (m, 18H).

[0556] Example 6. Synthesis of Compound 6

[0557] 2-Butyloctanoic acid-18-butyl-8-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-10-[3-(diethylamino)propyl]-5,11,17-trioxane-10-aza-6,16-dioxatetracosane-1-yl ester

[0558]

[0559] Intermediate 6-3: 5-chloro-5-oxopentyl-2-butyloctanoate

[0560] Oxaloyl chloride (2.54 g, 19.98 mmol, 3.0 equivalent) was slowly added to a solution of intermediates 1-5 (2.00 g, 6.66 mmol, 1.0 equivalent) and DMF (49.0 mg, 0.67 mmol, 0.1 equivalent) in dichloromethane (DCM, 20 mL). The reaction mixture was stirred at 0 °C for 3 hours under nitrogen (N2) protection, followed by concentration under reduced pressure to give the target product (2.00 g, 94.2% yield) as a colorless oil, which could be used for the next step of the reaction without further purification.

[0561] Intermediate 6-2: ((2-(((3-(diethylamino)propyl)amino)methyl)propane-1,3-diyl)bis(oxy))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate)

[0562] Intermediates 1-9 (1.00 g, 1.34 mmol, 1.0 equivalent), N,N-diethylpropane-1,3-diamine (870.0 mg, 6.70 mmol, 5.0 equivalent), potassium carbonate (K₂CO₃, 560.0 mg, 4.02 mmol, 3.0 equivalent), and potassium iodide (KI, 220.0 mg, 1.34 mmol, 1.0 equivalent) were dissolved in acetonitrile (CH₃CN, 20 mL) and stirred at 90 °C for 16 hours. The reaction mixture was diluted with ethyl acetate (EtOAc, 20 mL × 2), and the organic phases were combined, dried over anhydrous sodium sulfate (Na₂SO₄), and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 10 / 1) to give the target product (700.0 mg, yield 66.9%) as a colorless oil. MS: m / z [M+H] + =783.6.

[0563] Compound 6: ((2-((5-((2-Butyloctanoyl)oxy)-N-(3-(diethylamino)propyl)pentamido)methyl)propane-1,3-diyl)bis(oxy))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate)

[0564] To a solution of intermediate 6-2 (500.0 mg, 0.64 mmol, 1.0 equivalent) and triethylamine (TEA, 190.0 mg, 1.92 mmol, 3.0 equivalent) in dichloromethane (DCM, 2 mL), a solution of 5-chloro-5-oxopentyl-2-butyloctanoate (310.0 mg, 0.96 mmol, 1.5 equivalent) in dichloromethane (DCM, 1 mL) was added. The resulting mixture was stirred at 25 °C for 3 hours. The reaction solution was diluted with distilled water (H2O, 10 mL) and extracted with dichloromethane (DCM, 10 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate (Na2SO4), concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 15 / 1) to give the target product (410.0 mg, yield 60.3%) as a colorless oil.

[0565] MS: m / z [M+H] + = 1065.9. 1 H NMR (400 MHz, CDCl3) δ 4.28-3.95 (m, 11H), 3.46-3.26 (m, 5H), 2.87-2.64 (m, 3H), 2.57 (dd, J = 14.2, 7.0 Hz, 3H), 2.50-2.25 (m, 12H), 1.80-1.52 (m, 22H), 1.44 (dd, J = 24.0, 18.7 Hz, 8H), 1.33-1.20 (m, 34H), 1.04 (t, J = 7.1 Hz, 4H), 0.88 (dt, J = 7.3, 3.6 Hz, 16H).

[0566] Example 7. Synthesis of Compound 7

[0567] 2-Butyloctanoic acid-18-butyl-8-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-10-[4-(diethylamino)butyl]-5,11,17-trioxane-10-aza-6,16-dioxatetracosane-1-yl ester

[0568]

[0569] Compound 7 was prepared from N1,N1-diethylbutane-1,4-diamine according to the procedure of compound 6, with a yield of 51.7%.

[0570] MS: m / z [M+H] + = 1079.9. 1H NMR (400 MHz, CDCl3) δ 4.25-3.95 (m, 10H), 3.44-3.23 (m, 4H), 3.01-2.68 (m, 6H), 2.32 (ddd, J = 22.7, 13.3, 6.0 Hz,10H), 1.61 (ddd, J = 21.7, 19.1, 4.9 Hz, 22H), 1.51-1.37 (m, 8H), 1.21 (dt, J= 14.4, 9.0 Hz, 40H), 1.00 – 0.74 (m, 18H).

[0571] Example 8. Synthesis of Compound 8

[0572] 2-Butyloctanoic acid-18-butyl-8-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-10-[3-(dimethylamino)propyl]-5,11,17-trioxane-10-aza-6,16-dioxatetracosane-1-yl ester

[0573]

[0574] Compound 8 was prepared from N1,N1-dimethylpropane-1,3-diamine using the same procedure as compound 6, with a yield of 51.2%. MS: m / z [M+H] + = 1037.8. 1 H NMR (300 MHz, CDCl3) δ 4.07 (d, J = 12.5 Hz, 10H), 3.4-3.26 (m, 4H), 2.55-2.16 (m, 17H), 1.85-1.52 (m, 20H), 1.43 (d, J = 12.4Hz, 7H), 1.27 (d, J = 12.5 Hz, 36H), 0.88 (t, J = 6.6 Hz, 18H).

[0575] Example 9. Synthesis of Compound 9

[0576] 2-Butyloctanoic acid-18-butyl-8-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-10-[2-(diethylamino)ethyl]-5,11,17-trioxane-10-aza-6,16-dioxatetracosane-1-yl ester

[0577]

[0578] Compound 9 was prepared from N1,N1-diethylethane-1,2-diamine according to the procedure for compound 6, with a yield of 46.7%. MS: m / z [M+H] + = 1051.8. 1 H NMR (300 MHz, CDCl3) δ 4.39-3.87 (m, 10H), 3.54-3.24(m, 4H), 2.54 (d, J = 6.9 Hz, 4H), 2.46-2.26 (m, 8H), 1.76-1.53 ​​(m, 16H),1.45 (s, 8H), 1.26 (s, 38H), 1.03 (t, J = 7.0 Hz, 6H), 0.88 (t, J = 6.4 Hz, 20H).

[0579] Example 10. Synthesis of Compound 10

[0580] 2-Butyloctanoic acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-19-[3-(diethylamino)propyl]-8,14-dioxane-19-aza-9,15-dioxaoctadecane-28-yl ester

[0581]

[0582] Intermediate 6-2 (500.0 mg, 0.64 mmol, 1.0 equivalent), intermediate 5-3 (390.0 mg, 0.96 mmol, 1.5 equivalent), potassium carbonate (K₂CO₃, 270.0 mg, 1.92 mmol, 3.0 equivalent), and potassium iodide (KI, 110.0 mg, 0.64 mmol, 1.0 equivalent) were dissolved in acetonitrile (CH₃CN, 10 mL) and stirred at 90 °C for 16 hours. The reaction solution was diluted with distilled water (H₂O, 20 mL) and extracted with ethyl acetate (EtOAc, 20 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate (Na₂SO₄), concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 20 / 1) to give the target product (132.8 mg, yield 18.8%) as a colorless oil. MS: m / z [M+H] + =1107.9. 1H NMR (300 MHz, CDCl3) δ 4.15-4.01 (m, 10 H), 3.44-3.20 (m, 4H), 2.71-2.50 (m, 6H), 2.39-2.23 (m, 12H), 2.02-1.40 (m, 28H), 1.38-1.22 (m, 48H), 0.92-0.75 (m, 18H).

[0583] Example 11. Synthesis of Compound 11

[0584] 2-Butyloctanoic acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-19-[3-(diethylamino)propyl]-8,14,20-trioxane-19-aza-9,15-dioxahexadecane-26-yl ester

[0585]

[0586] Compound 11 was prepared according to the procedure for compound 6, using 7-chloro-7-oxohepyl-2-butyloctanoate as the starting material (synthesized according to intermediates 6-3). Yield: 31.4%. MS: m / z [M+H] + = 1093.9. 1 H NMR (400MHz, CDCl3) δ 4.16-4.02 (m, 10H), 3.56-3.14 (m, 2H), 2.53 (q, J = 7.1 Hz, 3H), 2.42-2.31 (m, 10H), 2.03 (d, J = 48.7 Hz, 1H), 1.70 (d, J = 6.5 Hz, 9H), 1.66-1.57 (m, 9H), 1.46 (dd, J = 13.0, 5.3 Hz, 6H), 1.40-1.36 (m, 4H), 1.33-1.23 (m, 40H), 1.03 (t, J = 7.2 Hz, 6H), 0.96-0.83 (m, 20H).

[0587] Example 12. Synthesis of Compound 12

[0588] 2-Butyloctanoic acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-19-[3-(diethylamino)propyl]-8,14,20-trioxane-19-aza-9,15-dioxaoctadecane-28-yl ester

[0589]

[0590] Compound 12 was prepared according to the procedure for compound 6, using 9-chloro-9-oxononyl-2-butyloctanoate as the starting material (which was synthesized according to the steps of intermediates 6-3). Yield: 41.2%.

[0591] MS: m / z [M+H] + = 1121.9. 1 H NMR (400 MHz, CDCl3) δ 4.15-4.04 (m, 10H), 3.61-3.18 (m, 2H), 2.53 (q, J = 7.1 Hz, 4H), 2.43-2.31 (m, 10H), 1.90 (s,1H), 1.70 (d, J = 4.2 Hz, 10H), 1.61 (dd, J = 14.0, 7.5 Hz, 9H), 1.50-1.42(m, 6H), 1.35-1.24 (m, 47H), 1.03 (t, J = 7.1 Hz, 6H), 0.92-0.88 (m, 19H).

[0592] Example 13. Synthesis of Compound 13

[0593] 2-Butyloctanoic acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-19-[3-(diethylamino)propyl]-8,14-dioxane-19-aza-9,15-dioxatetracosane-24-yl ester

[0594]

[0595] Compound 13 was prepared following the procedure for compound 10, using 5-bromopentyl-2-butyloctanoate as the starting material. The yield was 44.4%.

[0596] MS: m / z [M+H] + = 1051.9. 1H NMR (400 MHz, CDCl3) δ 4.22-3.99 (m, 10H), 2.81-2.50 (m, 7H), 2.47-2.26 (m, 13H), 2.23-2.07 (m, 2H), 1.78-1.52 (m, 18H), 1.46 (dd, J = 14.6, 6.9 Hz, 8H), 1.38-1.19 (m, 37H), 1.11 (t, J = 6.8 Hz, 6H), 0.89 (dd, J = 9.4, 4.4 Hz, 17H).

[0597] Example 14. Synthesis of Compound 14

[0598] 2-Butyloctanoic acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-19-[3-(diethylamino)propyl]-8,14-dioxane-19-aza-9,15-dioxatetracosane-24-yl ester

[0599]

[0600] Compound 14 was prepared following the procedure for compound 10, using 7-bromoheptyl-2-butyloctanoate as the starting material. Yield: 23.6%. MS: m / z [M+H] + = 1079.9. 1 H NMR (300 MHz, CDCl3) δ 4.17-3.96 (m, 10 H), 3.44-3.15 (m, 4H), 2.77-2.54 (m, 6H), 2.40-2.30 (m, 8H), 2.28-2.19 (m, 4H), 2.10-1.73 (m, 20H), 1.70-1.40 (m, 8H), 1.40-1.18 (m, 44H), 0.92-0.75 (m, 18H).

[0601] Example 15. Synthesis of Compound 15

[0602] 6-[7-Butyl-17-(10-Butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-23-ethyl-8,14-dioxane-19,23-diaza-9,15-dioxapentanodecane-19-yl]-6-oxanehexanoic acid-(2Z)-non-2-en-1-yl ester

[0603]

[0604] Intermediate 15-3: (Z)-6-(2-nonen-1-yloxy)-6-oxohexanoic acid

[0605] 15-1 (6.16 g, 42.18 mmol, 3.0 equivalent), N,N-diisopropylethylamine (DIEA, 5.45 g, 42.18 mmol, 3.0 equivalent), 4-dimethylaminopyridine (DMAP, 1.72 g, 14.06 mmol, 1.0 equivalent), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 5.39 g, 28.12 mmol, 2.0 equivalent) were dissolved in dichloromethane (DCM, 20 mL), stirred at 25 °C for 0.5 hours, and then 15-2 (2.00 g, 14.06 mmol, 1.0 equivalent) was added. The resulting solution was stirred at 25°C for 2.5 hours, extracted with dichloromethane (DCM, 20 mL × 3), the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate (Na2SO4), concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: PE / EtOAc = 10 / 1 to 1 / 1) to give the target product (2.90 g, yield 76.3%) as a colorless oil.

[0606] Intermediate 15-4: (Z)-6-chloro-6-oxohexanoic acid-2-nonen-1-yl ester

[0607] Intermediate 15-3 (500.0 mg, 1.85 mmol, 1.0 equivalent), oxalyl chloride (700.0 mg, 5.55 mmol, 3.0 equivalent), and N,N-dimethylformamide (DMF, 14.0 mg, 0.19 mmol, 0.1 equivalent) were dissolved in dichloromethane (DCM, 20 mL) and stirred at 0 °C for 3 hours. The reaction solution was concentrated under reduced pressure to give a crude product (490.0 mg, 91.7% yield) as a colorless oil, which could be used for the next step of the reaction without further purification.

[0608] Compound 15: (Z)-((2-((N-(3-(diethylamino)propyl)-6-(2-nonen-1-yloxy)-6-oxohexamido)methyl)propane-1,3-diyl)bis(oxo))bis(5-oxopentane-5,1-diyl)bis(2-butyloctanoate)

[0609] Compound 15 was prepared from intermediate 15-4 using the same procedure as compound 6, with a yield of 45.0%. MS: m / z [M+H] + = 1035.8. 1H NMR (400 MHz, CDCl3) δ 5.65 (dd, J = 18.3, 7.6 Hz, 1H), 5.56-5.48 (m, 1H), 4.63 (d, J = 6.8 Hz, 2H), 4.22-3.98 (m, 8H), 3.44-3.28 (m,4H), 2.66-2.24 (m, 17H), 2.10 (dd, J = 14.3, 7.3 Hz, 2H), 1.86 (s, 2H), 1.78-1.55 (m, 17H), 1.52-1.12 (m, 36H), 1.01 (t, J = 7.0 Hz, 6H), 0.88 (t, J = 6.8Hz, 14H).

[0610] Example 16. Preparation, characterization, and in vivo editing experiments of lipid nanoparticles.

[0611] 1. Animal Experiment Design

[0612] mRNA and sgRNA delivery experiments of the PCSK9-targeting base editor ABE8e

[0613] Cholesterol in the blood is mainly synthesized by the liver, which is also the main organ for breaking down excess cholesterol. On the surface of the liver, there is a low-density lipoprotein (LDL) receptor (LDLR). This receptor binds to cholesterol circulating back to the liver, breaking it down into bile acids, which are then excreted through the intestines. PCSK9 is a liver-synthesized protease that binds to the LDL receptor, promoting its entry into hepatocytes. This leads to the degradation of the LDL receptor by lysosomes, reducing its number. Therefore, inhibiting the activity of PCSK9 increases the number of LDLR receptors, thereby enhancing the uptake and breakdown of cholesterol. Basic and clinical research has shown that the PCSK9 gene is an effective target for treating hyperlipidemia and atherosclerosis. Figure 1 This study illustrates the changes in the number of LDL receptors and the resulting alterations in cholesterol metabolism caused by editing specific sites in the PCSK9 gene.

[0614] Strategies for PCSK9 gene editing delivery in mouse liver cells, such as Figure 1 As shown, the main process is as follows: The prepared lipid nanoparticles are used to target and deliver the mRNA and sgRNA encoding ABE8e to mouse hepatocytes via intravenous injection. Under the action of ABE8e and sgRNA, mutations are introduced into the PCSK9 gene, and the A to G mutation is achieved at a specific site. The editing efficiency is calculated by sequencing.

[0615] The specific experimental design is as follows:

[0616] 1.1 Selection of suitable mutation sites and editing design

[0617] The single-base editor ABE8e achieves precise A-to-G base substitution without requiring a donor template or causing DSB (Dissociative Identity Deficit Hyperbacterial Syndrome). Based on this, the first exon of the PCSK9 gene was selected as the screening mutation site. The mRNA and sgRNA encoding the single-base editing tool ABE8e were co-delivered to the animal via lipid nanoparticles. The mRNA encoding ABE8e was translated into protein in the cytoplasm, formed a complex with the sgRNA, and entered the nucleus. Guided by the sgRNA, the base editor ABE8e targeted the splicing donor site in the first exon of the PCSK9 gene, deaminated adenine (A) on the template strand of the first exon to convert it to inosine (I). I is read and replicated at the DNA level as G, ultimately achieving the A-to-G substitution, thereby disrupting the splicing donor site and causing premature termination of the PCSK9 gene reading frame.

[0618] 1.2 Preparation of mRNA and sgRNA of base editor ABE8e

[0619] The sequences of the first exon and the first intron of the mouse PCSK9 gene (NCBI Gene ID: 100102) were selected as the target region to determine the target sequence PCSK9-sgRNA for single-base editing of the PCSK9 gene.

[0620] By analyzing the sequence across the first exon and intron of the PCSK9 gene, a target region sgRNA was designed: PCSK9-sgRNA (synthesized by Nanjing Genscript Biotech Co., Ltd.). The PCSK9-sgRNA sequence is as follows:

[0621] PCSK9-sgRNA: 5'-cccataccttggagcaacgg-3' (SEQ ID NO: 1);

[0622] sgRNAs were designed and oligonucleotides were synthesized based on the target sequence. The sgRNA sequences used are shown in SEQ ID NO:1. A CACC sequence was added to the 5' end of the upstream sequence of each sgRNA, and an AAAC sequence was added to the 5' end of the downstream sequence. After synthesis, the upstream and downstream sequences were annealed using a preset program (95℃, 5 min; 95℃-85℃ at -2℃ / s; 85℃-25℃ at -0.1℃ / s; held at 4℃). The annealed products were then ligated into the lenti U6-sgRNA / EF1a-mCherry vector (Addgene, Plasmid, #114199) linearized with BbsI (NEB, R3539S).

[0623] The system used in the construction of the sgRNA plasmid is as follows:

[0624] The linearization system of lenti U6-sgRNA / EF1a-mCherry vector is as follows: 3 μg vector; 6 μL buffer (NEB: R0539L); 2 μL BbsI; ddH2O to make up to 60 μL, digested overnight at 37℃.

[0625] The ligation system for the annealed sgRNA product and the linearized vector was as follows: 1 μL of T4 ligase buffer (NEB: M0202L), 20 ng of linearized vector, 5 μL of annealed oligo fragment (10 μM), 0.5 μL of T4 ligase (NEB: M0202L), and ddH2O to a final volume of 10 μL. The mixture was incubated overnight at 16°C.

[0626] The ligation vector was transformed into *E. coli* DH5α competent cells (Weidi Bio, DL1001). The specific procedure is as follows: DH5α competent cells were removed from -80℃ and immediately placed on ice. After 5 minutes, once the bacterial block had thawed, the ligation product was added, and the mixture was gently stirred by tapping the bottom of the centrifuge tube. The cells were then incubated on ice for 25 minutes. A heat shock was performed at 42℃ for 45 seconds, followed by immediate return to ice and incubation for 2 minutes. 700 μl of antibiotic-free sterile LB medium was added to the centrifuge tube, mixed, and then incubated at 37℃, 200 rpm for 60 minutes. The cells were harvested by centrifugation at 5000 rpm for one minute. Approximately 100 μl of the supernatant was collected, gently resuspended by pipetting, and spread onto LB medium containing Amp antibiotics. The plates were inverted and incubated overnight at 37℃. Single colonies were picked, and after sequencing confirmation, positive clones were shaken and plasmids (TIANGEN: DP120-01) were extracted and their concentration determined. The plasmids were then stored at -20℃ for later use.

[0627] The base editor ABE8e used in this experiment is the highly efficient base editor ABE8e evolved by David R. Liu's team (Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol. 2020 Jul;38(7):883-891. doi: 10.1038 / s41587-020-0453-z. Epub 2020 Mar 16. Erratum in: Nat Biotechnol. 2020 May 20; PMID:32433547; PMCID: PMC7357821). The plasmid ABE8e (Plasmid#138489) was purchased from Addgene and the ABE8e mRNA was expressed and purified in the laboratory for later use.

[0628] 2. Lipid nanoparticles were prepared by ionizable lipid MC3 or the compounds disclosed herein / DSPC / cholesterol / PEG-lipid at a molar ratio of 50:10:38.5:1.5.

[0629] 2.1 Dilinylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, commonly abbreviated as MC3) and compounds 1-16 of this disclosure are dissolved in anhydrous ethanol in the above molar ratio with DSPC, cholesterol, and PEG-DMG, respectively.

[0630] 2.2 Ethanol solutions of different lipid carriers were mixed with mRNA buffer at a 1:3 (volume / volume) ratio (total lipid to mRNA mass ratio (w / w) was 40:1, and ABE8e mRNA:sgRNA mass ratio (w / w) was 3:2). Nucleic acid lipid nanoparticles 1-16 were obtained by passing the mixture through a microfluidic nanomedicine manufacturing system (NanoAssemblr Ignite, Canada) at a flow rate of 12 ml / min. The obtained nucleic acid lipid nanoparticles were immediately diluted 40-fold in 1×DPBS buffer. The diluted nucleic acid lipid nanoparticle solution was concentrated to the target volume using an ultracentrifuge tube. The diluted solution was then used for DLS particle size measurement and encapsulation efficiency detection.

[0631] 2.3 The particle size and polydispersity index of lipid nanoparticles were determined by dynamic light scattering in 173° backscatter detection mode using a Malvern Zetasizer Nano ZS (Malvern UK). The encapsulation efficiency of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA Quantification Kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions. The test results are shown in Table 2.

[0632] Table 2 Characterization of nanolipid particles

[0633]

[0634] 3. Evaluation of in vivo editing experiments

[0635] 3.1 The lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, abbreviated as MC3) and the compounds disclosed herein (see Table 2), encapsulating the mRNA and sgRNA of the base editor ABE8e prepared in step 2, were administered via tail vein injection to 6-7 week old female C57BL / 6 mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.). The lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, abbreviated as MC3) encapsulating the mRNA and sgRNA of the base editor ABE8e were administered to mice of similar age and sex as a positive control. Additionally, PBS buffer was administered via tail vein injection to mice of similar age and sex as a negative control.

[0636] 3.2 Editing Efficiency Test

[0637] One week after administration to mice, the editing efficiency was tested. The mice were sacrificed, liver tissue was collected, the genome was extracted after lysis, and efficiency was analyzed by deep sequencing.

[0638] The deep sequencing steps are as follows:

[0639] (1) Design primers based on the location of the target gene, see Table 3 for details.

[0640] Table 3. Primer design targeting the PCSK9 gene

[0641]

[0642] (2) Editing efficiency test.

[0643] The PCR program was as follows: 94℃, 2 min; 98℃, 10 s; 60℃, 30 s; 68℃, 20 s; 34 cycles; 68℃, 5 min. After PCR, gel electrophoresis was used to verify the amplification of the product by selecting a single band of appropriate size. The obtained PCR product was then sent to Nanjing GenScript Biotech Co., Ltd. for sequencing.

[0644] (3) The deep sequencing results were analyzed using Crispresso software to perform specific site analysis and calculate the editing efficiency. The calculation results are shown in Table 4. The editing efficiency corresponding to each lipid nanoparticle can be found in Table 4. Figure 2 .

[0645] Table 4. Evaluation of In-Vivo Editing Efficiency

[0646]

[0647] As shown in Table 4, the ionizable lipid compounds used in this disclosure can effectively deliver drugs such as nucleic acid molecules and small molecule compounds; and by comparison, the lipid nanoparticles of this disclosure have a better particle size distribution, higher encapsulation efficiency, and significantly better delivery effect than the comparative lipid nanoparticles, which can meet the needs of in vivo delivery.

[0648] The descriptions of the exemplary embodiments presented above are merely illustrative of the technical solutions of this disclosure and are not intended to be exhaustive or to limit this disclosure to the precise forms described. Obviously, those skilled in the art can make many changes and variations based on the above teachings. The exemplary embodiments were chosen and described to explain the specific principles of this disclosure and its practical applications, thereby enabling others skilled in the art to understand, implement, and utilize the various exemplary embodiments of this disclosure and their various alternatives and modifications. The scope of protection of this disclosure is intended to be defined by the claimed scope and its equivalents.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, said compound having the following structure: (I) in, R1 is selected from -OH and R a (R b )N-, where R a and R b Each independently is hydrogen, C1-C 10 Alkyl or C1-C 10 Halogenated alkyl groups; R2 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 acetylin R3 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, or R c -(CH2)n-, where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, and more preferably a positive integer from 1 to 10; R c Selected from the structure shown below: , , , , and ,in, Represents a connection key; L1, L2, L3, L4, and L5 do not exist, or each can be independently selected from the following groups: 、 、 、 、 ； X does not exist, or is -CH- or N; R4 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R5 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R6 is C1-C 30 Alkyl, C2-C 30 alkenyl, C2-C 30 alkynyl group; R7 is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group; R8 and R9 do not exist, or they are each independently C1-C. 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S; R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h It can be O or S.

2. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, wherein the compound of formula I has the structure of formula I-1: (I-1) in, R1-R2, R6-R 11 The definitions of L2-L5 are as described above; R3 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group.

3. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, wherein the compound of formula I has the structure of formulas I-2 as follows: （I-2） in, R1-R2, R6-R7, R 10 -R 11 The definitions of L1-L5 are as described above; R3 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group, or R c -(CH2) n - where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, R c The definition is as described above; R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group.

4. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, wherein the compound of formula I has the structure of formulas I-3 as follows: （I-3） in, R1-R3, R6, R7, R 10 R 11 The definitions of L1-L5 are as described above; R4 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group.

5. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, wherein the compound of formula I has the structure of formulas I-4 as follows: （I-4） in, R1-R7, R 10 -R 11 The definitions of L1-L3 and L5 are as described above.

6. A compound of formula II or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, said compound having the following structure: (II); in, R1 is selected from -OH and R a (R b )N-, where R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; R3 is C1-C 15 Alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl group, or R c -(CH2) n - where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, and even more preferably a positive integer from 1 to 6; R c Selected from the following structures: , , , , and ,in Indicates a connection key; L1, L2, L3, L4, and L5 do not exist, or each can be independently selected from the following groups: 、 、 、 、 、 ； R4 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R5 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S; R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S; r is 2 or 3; u is a positive integer from 1 to 10; t is a positive integer from 1 to 10; Furthermore, when R1 is selected from R a (R b When N-, r is 2.

7. A compound of formula III or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, said compound having the following structure: (III) in, R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; R c Selected from the following structures: , , , , and ,in Indicates a connection key; s is a positive integer from 1 to 10; L1, L2, L3, L4, and L5 do not exist, or each can be independently selected from the following groups: 、 、 、 、 、 ； R4 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R5 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S; R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S; r is a positive integer of at least 3; u is a positive integer from 1 to 10; t is a positive integer from 1 to 10.

8. A compound of formula IV or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, said compound having the following structure: (IV) in, R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; R3 is C1-C 15 Alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl group, or R c -(CH2) n - where n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, and more preferably a positive integer from 1 to 10; R c Selected from the following structures: , , , , and ,in Indicates a connection key; L1, L2, L3, L4, and L5 do not exist, or each can be independently selected from the following groups: 、 、 、 、 、 ； R4 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R5 does not exist, or it is C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R8 and R9 are each independently C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S; R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 Alkyne group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S; r is a positive integer of at least 3; u is a positive integer from 1 to 10; t is a positive integer from 1 to 10.

9. A compound of formula V or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof, said compound having the following structure: (V) in, R a and R b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; R c Selected from the following structures: , , , , and ,in Indicates a connection key; L1, L2, L3, and L5 do not exist, or each can be independently selected from the following groups: 、 、 、 、 、 ； R4 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R5 is C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl group; R6 is C1-C 30 Alkyl, C2-C 30 alkenyl, C2-C 30 alkynyl group; R 10 R 11 Each independently is C1-C 14 Alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl group, or -R h -C1-C 14 Alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl group, wherein R h For O or S; r is a positive integer of at least 3; t is a positive integer from 1 to 10.

10. A compound of formula I, II, III, IV or V according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, wherein the compound is selected from Table 1: Table 1 。 11. The compound according to claim 10, wherein the compound has the following structure: 。 12. A lipid carrier comprising a compound of formula I, II, III, IV or V as described in any one of claims 1-11, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof.

13. A lipid nanoparticle (LNP) comprising a compound of formula I, II, III, IV or V as described in any one of claims 1-11, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof.

14. A lipid nanoparticle composition comprising the lipid carrier of claim 12 or the lipid nanoparticle of claim 13, and a bioactive substance encapsulated in the lipid carrier or the lipid nanoparticle.

15. A pharmaceutical composition comprising a lipid carrier of claim 12 or a lipid nanoparticle of claim 13, a bioactive substance encapsulated in the lipid carrier or the lipid nanoparticle, and a pharmaceutically acceptable excipient, carrier, or diluent.

16. A pharmaceutical formulation comprising a lipid carrier of claim 12 or lipid nanoparticles of claim 13, a bioactive substance encapsulated in the lipid carrier or lipid nanoparticles, and a pharmaceutically acceptable excipient, carrier, or diluent; or, the pharmaceutical formulation comprising a lipid nanoparticle composition of claim 14, and a pharmaceutically acceptable excipient, carrier, or diluent.

17. Use of any compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, according to any one of claims 1-11, in the preparation of lipid nanoparticles.

18. Use of any compound of formula I, II, III, IV or V as described in any one of claims 1-11, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, or a lipid carrier as described in claim 12, or a lipid nanoparticle as described in claim 13, or a lipid nanoparticle composition as described in claim 14, or a pharmaceutical composition as described in claim 15, or a pharmaceutical formulation as described in claim 16, in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs.

19. A method for preparing the lipid nanoparticle composition of claim 14, comprising: (a) A compound of formula I, II, III, IV or V or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor of any one of claims 1-11 is mixed with an optional auxiliary lipid and an organic solvent to obtain a lipid organic phase; (b) The bioactive substance is mixed with an aqueous solvent to obtain an aqueous phase containing the bioactive substance; (c) The lipid organic phase from step (a) is mixed with the aqueous phase from step (b) to obtain the lipid nanoparticle composition.

20. Use of any compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, as claimed in any one of claims 1-11, in the preparation of a drug delivery system.

21. Use of any compound of formula I, II, III, IV or V of any one of claims 1-11, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, or the lipid carrier of claim 12, or the lipid nanoparticle of claim 13, or the lipid nanoparticle composition of claim 14, or the pharmaceutical composition of claim 15, or the pharmaceutical formulation of claim 16, in the preparation of a medicament for the treatment and / or prevention of disease.

22. The use according to claim 21, wherein the disease is selected from the group consisting of metabolic diseases, hereditary diseases, cancer, cardiovascular diseases, infectious diseases, and combinations thereof; preferably, the metabolic disease includes familial hypercholesterolemia (FH), the hereditary disease includes transthyretin amyloidosis (ATTR), primary hyperoxaluria type 1 (PH1), and hereditary angioedema (HAE), and the infectious disease includes hepatitis B.

23. A method of delivering a therapeutic or preventative agent to the cells, tissues, or organs of a subject, the method comprising contacting the subject's cells, tissues, or organs with the lipid nanoparticle composition of claim 14, the pharmaceutical composition of claim 15, or the pharmaceutical formulation of claim 16.

24. A method for generating a target protein or target polypeptide in subject cells, the method comprising contacting the subject cells with the lipid nanoparticle composition of claim 14, the pharmaceutical composition of claim 15, or the pharmaceutical formulation of claim 16.

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