Lipid compounds for delivering therapeutic agents, and methods of making and using the same

Abstract

This invention provides a lipid compound for delivering therapeutic agents, a method for its preparation, and its applications. Specifically, this invention provides a lipid compound having structural formula I or a pharmaceutically acceptable form thereof. The lipid compound can be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer-bound lipids, to form lipid nanoparticles for delivering therapeutic agents, such as nucleic acid molecules, for therapeutic or preventative purposes, such as vaccination, thus enriching the variety of ionizable lipid compounds.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery, specifically relating to a lipid compound for delivering therapeutic agents, its preparation method and application, and more particularly to a lipid compound for delivering therapeutic agents (e.g., nucleic acid molecules), a lipid carrier containing the compound, a nucleic acid lipid nanoparticle composition and a pharmaceutical formulation, as well as their preparation methods and related applications. Background Technology

[0002] Gene therapy is a hot research topic in modern biomedicine. Nucleic acid drugs can be used to prevent and treat cancer, bacterial and viral infections, and diseases with genetic causes. Because nucleic acid drugs are easily degraded and have difficulty entering 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, and since then, research on the delivery of nucleic acid drugs using LNP technology has seen explosive growth. In particular, at the end of 2020, the FDA approved Moderna's and BioNTech & Pfizer's COVID-19 vaccines, both of which used LNP technology to deliver mRNA drugs, thus achieving prevention against the SARS-CoV-2 virus.

[0004] Lipid-dependent nucleic acid (LNP) molecules typically consist of four types of lipid compounds: ionized lipids, neutral lipids, steroids, and polymer-bound lipids. The choice of ionized lipids has the greatest impact on LNP efficacy. Current nucleic acid therapeutics still face several challenges, primarily including delivery efficiency, low cell penetration, and high sensitivity to degradation of certain nucleic acid molecules, including RNA. Therefore, there is still a need to develop novel 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] To address the shortcomings of existing technologies, one objective of this 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), wherein the lipid compound can be co-prepared 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. A 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.

[0006] In one embodiment, this document provides a compound of formula (I) or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof:

[0007]

[0008] In formula (I),

[0009] G 1 C 1-10 Alkylene; G 1 Preferred Where m is selected from integers from 1 to 6;

[0010] R 1 R 2 Each independently is C 1-6 Alkyl; preferably, R 1 R 2 Together with the attached N, they form a 3-8 membered heterocyclic group, or, R 1 R 2 Any of the two G 1 Any carbon atom in R is directly linked to form a 3-8 membered heterocyclic group; more preferably, R 1 R 2 Each is independently methyl, ethyl, or propyl; or, R 1 R 2 Together with the attached N, they form 4-, 5-, or 6-membered heterocyclic groups;

[0011] L 1 L 2 L 3 L 4 L 5 Each is independently selected from esters, amides, carbonates, carbamates, mercaptocarbamates, ureas, phosphates, or none; preferably, L 1 ~L 5 Each independently selected Or none;

[0012] R 3 R5 R 7 Each is independently selected from straight-chain alkanes, branched alkanes, straight-chain alkyl groups containing heteroatoms, branched alkyl groups containing heteroatoms, or none; preferably, R 3 R 5 R 7 Each independently Where X is selected from O, S, Se, SS, Se-Se, or none; R 11 ~R 14 It is a straight-chain alkane of H or C1-C8; n and o are integers selected from 1 to 10;

[0013] R 4 R 6 R 8 Each is independently selected from straight-chain alkyl, straight-chain alkenyl, straight-chain alkynyl, branched alkyl, cycloalkyl, bridged cycloalkyl, or none; preferably, R 4 R 6 R 8 Each independently Where Y is, Or none; R 15 ~R 19 For H or C1-C 10 Straight-chain alkanes, cycloalkanes, or bridged cycloalkanes; p and q are integers from 0 to 10.

[0014] In one embodiment, this document also provides a lipid carrier comprising the above-described lipid compound or a pharmaceutically acceptable form thereof.

[0015] In one embodiment, this document also provides a nucleic acid lipid nanoparticle composition comprising the above-described lipid compound or a pharmaceutically acceptable form thereof, or the above-described lipid carrier.

[0016] In one embodiment, this document also provides pharmaceutical formulations comprising the above-described lipid compound or a pharmaceutically acceptable form thereof, or the above-described lipid carrier, or the above-described nucleic acid lipid nanoparticle composition.

[0017] In one embodiment, this document also provides the use of the above-described lipid compound or its pharmaceutically acceptable form, or the above-described lipid carrier, or the above-described nucleic acid lipid nanoparticle composition, or the above-described pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs.

[0018] In one embodiment, this document also provides 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 formulation to a subject in need.

[0019] Solution for solving the problem

[0020] <First Aspect>

[0021] This invention provides a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug thereof:

[0022]

[0023] In equation (I),

[0024] G 1 C 1-10 Alkylene;

[0025] R 1 R 2 Each independently is C 1-6 alkyl;

[0026] Optional, R 1 R 2 Together with the attached N, they form a 3-8 membered heterocyclic group, or, R 1 R 2 Any of the two G 1 Any carbon atom in the group can be directly linked to form a 3-8 membered heterocyclic group;

[0027] L 1 L 2 L 3 L 4 L 5 Each group is independently selected from esters, amides, carbonates, carbamates, mercaptocarbamates, ureas, phosphates, or none;

[0028] R 3 R 5 R 7 Each is independently selected from straight-chain alkanes, branched alkanes, straight-chain alkyl groups containing heteroatoms, branched alkyl groups containing heteroatoms, or none;

[0029] R 4 R 6 R 8 Each is independently selected from straight-chain alkyl, straight-chain alkenyl, straight-chain alkynyl, branched alkyl, cycloalkyl, bridged cycloalkyl, or none.

[0030] In one implementation, G 1 for Where m is selected from integers from 1 to 6.

[0031] In one implementation, R 1 R 2 Each is independently methyl, ethyl, or propyl; or, R 1 R 2Together with the attached N, they form 4-, 5-, or 6-membered heterocyclic groups.

[0032] In one implementation, L 1 ~L 5 Each independently selected Or none.

[0033] In one implementation, R 3 R 5 R 7 Each independently

[0034] Where X is O, S, Se, SS, Se-Se or none;

[0035] R 11 ~R 14 It is a straight-chain alkane, either H or C1-C8;

[0036] n and o are integers selected from 1 to 10.

[0037] In one implementation, R 4 R 6 R 8 Each independently

[0038] Where Y is, Or none;

[0039] R 15 ~R 19 For H or C1-C 10 Straight-chain alkanes, cycloalkanes, or bridged cycloalkanes;

[0040] p and q are integers selected from 0 to 10.

[0041] In one embodiment, the lipid compound is selected from any one or a combination of at least two of the structural compounds shown in Table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0042] Table 1

[0043]

[0044]

[0045] In one embodiment, the pharmaceutically acceptable form is selected from pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes, or prodrugs.

[0046] <Second aspect>

[0047] The present invention provides the use of the lipid compound described in the first aspect or in a pharmaceutically acceptable form thereof in the preparation of liposome nanocarriers.

[0048] <Third aspect>

[0049] The present invention provides a lipid carrier comprising the lipid compound described in the first aspect or in a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug.

[0050] This type of lipid carrier has high encapsulation efficiency for nucleic acid drugs, which greatly improves the delivery efficiency of nucleic acid drugs in vivo.

[0051] In one embodiment, the lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises the lipid compound described in the first aspect or in a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug, and optionally an ionizable lipid, and the second lipid compound comprises any one or a combination of at least two of anionic lipids, neutral lipids, steroid-bound lipids, or polymer-bound lipids.

[0052] In some embodiments, the first lipid compound is the lipid compound described in the first aspect or in a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug.

[0053] In some embodiments, the first lipid compound is the lipid compound described in the first aspect or in a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, and a combination of ionizable lipids.

[0054] In one embodiment, the ionizable lipid is selected from: 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]-dioxolane DLin-KC2-DMA, and 1,2-dioleoyl-3-trimethylammonium-propane DOTAP. , any one or a combination of at least two of the following: 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 or N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylamine chloride DOTMA.

[0055] In some embodiments, the anionic lipid is selected from any one or a combination of at least two of the following: phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycerol-3-phosphatidylserine (DOPS), and dimyroylphosphatidylglycerol.

[0056] In some embodiments, the neutral lipid is selected from any one of: 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-distearyl-sn-glycerol-3-phosphatidylcholine (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DPPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), and 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or lipids modified with anionic or cationic modifying groups. There are no restrictions on whether the modifying groups are anionic or cationic.

[0057] In some embodiments, the steroid is selected from any one or a combination of at least two of the following: cholesterol, nonsteroidal, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, coccosterol, or corticosteroids.

[0058] In some embodiments, the lipid bound to the polymer is selected from: 1,2-dimyristoyl-sn-glycerol methoxy-polyethylene glycol (PEG-DMG), dimyristoylglycerol-polyethylene glycol (PEG-c-DMG), polyethylene glycol-dimyristoylglycerol (PEG-C14), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), 1,2-distearate-sn-glycero-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEGylated phosphatidylethanolamine (PEG-PE), PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and Tween-2-. 0. One or more combinations of 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).

[0059] 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 (20–65):(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.; wherein, in the first lipid compound, the molar ratio of the lipid compound or its pharmaceutically acceptable form to the ionizable lipid 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.

[0060] 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 the lipid compound or its pharmaceutically acceptable form to the ionizable lipid is (3-4):(0-5).

[0061] <Fourth Aspect>

[0062] The present invention provides a nucleic acid lipid nanoparticle composition comprising a lipid compound as described in the first aspect or in a pharmaceutically acceptable form thereof, or a lipid carrier as described in the third aspect, and a therapeutic or preventative agent.

[0063] In one embodiment, the therapeutic or preventative agent comprises any one or a combination of at least two of RNA, DNA, antisense nucleic acid, aptamer, nuclease, immunostimulatory nucleic acid, or peptide nucleic acid.

[0064] In one implementation, the antisense nucleic acid is an antisense oligonucleotide.

[0065] In one embodiment, the RNA is any one or a combination of at least two of mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA.

[0066] In one implementation, the RNA is modified RNA.

[0067] In one embodiment, the mRNA includes a sequence encoding an RNA-directed DNA binder, and more specifically, includes the mRNA of a Cas protein.

[0068] In one embodiment, the DNA includes a plasmid.

[0069] In one embodiment, the nuclease is selected from any one or a combination of at least two of Cas9, Cas12, Cas13, IscB, TnpB, IsrB and their homologs.

[0070] In some embodiments, the mass ratio of the therapeutic agent or preventative agent to the compound or its pharmaceutically acceptable form is 1:(3 to 40). Exemplary examples include mass ratios of 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, 1:40, etc.

[0071] In some embodiments, the mass ratio of the therapeutic agent or preventative agent to the lipid carrier is 1:(3-40). Exemplary examples include 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, 1:40, etc.

[0072] <Fifth Aspect>

[0073] The present invention provides a pharmaceutical composition comprising the lipid compound of the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or the lipid carrier of the third aspect, or the nucleic acid lipid nanoparticle composition of the fourth aspect, and a pharmaceutically acceptable excipient.

[0074] In some embodiments, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, pH regulators, antioxidants, or buffers.

[0075] <Sixth Aspect>

[0076] The present invention provides a pharmaceutical formulation comprising the lipid compound of the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or the lipid carrier of the third aspect or the nucleic acid lipid nanoparticle composition of the fourth aspect, and pharmaceutically acceptable excipients.

[0077] In one embodiment, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, pH regulators, antioxidants, or buffers.

[0078] In some embodiments, the dosage form of the pharmaceutical preparation is selected from any one or a combination of at least two of the following: tablets, capsules, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, or sprays.

[0079] In some embodiments, the injectable includes an injection solution, a sterile powder for injection, and a concentrated solution for injection.

[0080] In some embodiments, the particle size of the pharmaceutical preparation 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.

[0081] In some embodiments, the encapsulation rate of the therapeutic or preventative agent 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.

[0082] <Seventh Aspect>

[0083] The present invention provides the use of the lipid compound of the first aspect or a pharmaceutically acceptable form thereof, the lipid carrier of the third aspect, the nucleic acid lipid nanoparticle composition of the fourth aspect, the pharmaceutical composition of the fifth aspect, or the pharmaceutical preparation of the sixth aspect in the preparation of nucleic acid drugs, gene vaccines, peptide or protein drugs.

[0084] In one embodiment, the nucleic acid lipid nanoparticle composition or the above-described pharmaceutical preparation is used to treat or prevent diseases or conditions in a subject in need.

[0085] In one implementation, the subject is a mammal.

[0086] In one implementation, the mammal is a human.

[0087] In one implementation, the disease or condition is selected from metabolic diseases, genetic diseases, cancer, cardiovascular diseases, or infectious diseases.

[0088] In one embodiment, the metabolic disease includes familial hypercholesterolemia, the hereditary disease includes transthyretin amyloidosis, primary hyperoxaluria, or hereditary angioedema, and the infectious disease includes hepatitis B.

[0089] <Eighth Aspect>

[0090] The present invention provides a method for delivering a therapeutic or preventive agent to subject cells, the method comprising: administering to the subject a nucleic acid lipid nanoparticle composition according to the fourth aspect or a pharmaceutical preparation according to the sixth aspect, the administration comprising contacting the subject cells with the lipid nanoparticle composition or pharmaceutical preparation, thereby delivering the therapeutic and / or preventive agent to the subject cells.

[0091] In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation is administered via one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.

[0092] In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is administered, for example, via an enteric or parenteral route.

[0093] In some embodiments, the nucleic acid lipid nanoparticle composition or pharmaceutical formulation is administered to the subject at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0094] <Ninth Aspect>

[0095] The present invention provides a method for generating a target protein or target peptide in subject cells, the method comprising: contacting the subject cells with a nucleic acid lipid nanoparticle composition according to a fourth aspect, wherein the therapeutic agent or preventive agent is mRNA, and wherein the mRNA encodes a target protein or peptide, thereby enabling the mRNA to be translated in the cells to generate the target protein or target peptide.

[0096] <Tenth Aspect>

[0097] The present invention provides a method for preventing, improving or treating a disease or condition in a subject in need, the method comprising administering to the subject the nucleic acid lipid nanoparticle composition of the fourth aspect or the pharmaceutical preparation of the sixth aspect.

[0098] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0099] Compared with the prior art, the present invention has the following beneficial effects:

[0100] This invention provides a series of novel Formula I compounds that can be used as ionizable lipids to prepare lipid carriers in combination with other lipid compounds. These carriers exhibit controllable particle size, uniform distribution, and high encapsulation efficiency. The synthesis methods are simple, yield-efficient, and can be rapidly synthesized at low cost. The compounds of this invention can be used to deliver nucleic acid drugs, gene vaccines, small molecule drugs, peptides, or protein drugs, enriching the variety of ionizable lipid compounds. Attached Figure Description

[0101] Figure 1 This is a schematic diagram of the delivery strategy for detecting the PCSK9 gene editing efficiency in mouse liver cells in this invention.

[0102] Figure 2 The efficiency of PCSK9 gene editing in mouse liver cells using base editors encapsulated with different lipid compounds. Detailed Implementation

[0103] To facilitate understanding of this invention, 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.

[0104] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0105] In this specification, the term "substantially" is used to indicate that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0106] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0107] 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.

[0108] In this specification, references to "some embodiments," "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that 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.

[0109] 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.

[0110] [Terminology Definition]

[0111] Unless otherwise stated, the following terms have the following meanings:

[0112] 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 the reaction of 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.

[0113] 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 treatment, using chiral resolving reagents, or by direct separation of the enantiomers on a chiral chromatographic column. Starting compounds with specific stereochemistry are either commercially available or prepared according to the methods described herein and then resolved using methods well known in the art.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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).

[0118] The term "prodrug" refers to a derived compound that, when administered 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.

[0119] 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.

[0120] The terms “contain” and “include” are used in their open, non-restrictive sense.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] The term "branched alkyl" refers to an alkane radical that is attached to a parent molecule and forms at least two branches.

[0126] For example

[0127] 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<).

[0128] 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<).

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] The term "branched alkenyl" refers to an alkene radical that is linked to a parent molecule and forms at least two branches. For example...

[0134] 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 consist of 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, dioxadiazopentenyl, pyrrolyl, pyrrolidone, imidazoalkyl, pyrazolyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithiaalkyl, or trithiaalkyl.

[0135] 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 groups can be phenyl, naphthyl, anthracene, phenanthryl, acenaphthene, azulene, fluorenyl, indene, pyrene, etc.

[0136] The term "heteroaryl" refers to an aromatic group consisting of a monocyclic or fused polycyclic ring with a conjugated π-electron system, the ring atoms of which consist of a carbon atom and at least one heteroatom selected from N, O, and S. If valence requirements are met, the heteroaryl group can be linked 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, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl and their benzo[a] derivatives, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purine, etc.

[0137] The term "bridged cycloalkyl" refers to a fully carbon polycyclic system in which two non-directly bonded carbon atoms are shared between rings. The rings may contain one or more double bonds and have 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., 5 to 20-membered bridged cycloalkyl). The bridged cycloalkyl is preferably a bridged cycloalkyl having 6 to 14 carbon atoms (i.e., 6 to 14-membered bridged cycloalkyl), more preferably a bridged cycloalkyl having 7 to 10 carbon atoms (i.e., 7 to 10-membered bridged cycloalkyl). The bridged cycloalkyl includes bicyclic bridged cycloalkyl and polycyclic bridged cycloalkyl (e.g., tricyclic bridged cycloalkyl, tetracyclic bridged cycloalkyl, etc.), preferably bicyclic or tricyclic bridged cycloalkyl. Non-limiting examples include:

[0138]

[0139] The cycloalkyl ring may be fused to an aryl, heteroaryl, or heterocycloalkyl ring, wherein the ring connected to the parent structure is a cycloalkyl group, and non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0140] The term "heteroatom-containing alkyl" refers to an alkyl group having at least one heteroatom with one connection point, the heteroatom being selected from oxygen, nitrogen, sulfur, phosphorus or halogen atoms.

[0141] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0142] The term "hydroxyl group" refers to -OH.

[0143] The term "cyano" refers to -CN.

[0144] The term "amino" refers to -NH2.

[0145] The term "nitro" refers to -NO2.

[0146] The term "oxo group" refers to (=O).

[0147] Unless otherwise stated, the descriptions provided herein apply to all formulas provided herein (e.g., formula (I), including their subformulas) to the extent to which they apply.

[0148] [Preparation Method]

[0149] 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.

[0150] 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.

[0151] 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.

[0152] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely illustrative of the invention and should not be considered as specific limitations thereof. Those skilled in the art will recognize that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the invention. The technical features involved in the various embodiments of the present 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.

[0153] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0154] Example 1 Compound 1

[0155] Synthesis of 3-(diethylamino)propionic acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-8,14,19-trioxane-20-aza-9,15-dioxane-nonadecan-18-yl ester

[0156]

[0157] The chemical structure of compound 1 is shown in formula (II).

[0158]

[0159] Specifically, the steps include the following:

[0160] Step 1: Synthesis of compounds 1-4

[0161] In a 100 mL round-bottom flask, 2,2-dimethyl-1,3-dioxane-5-carboxaldehyde (282.6 mg, 1.96 mmol, 1.0 eq), 3-(diethylamino)propionate (356.0 mg, 1.96 mmol, 1.0 eq), 1-isocyanonane (300.0 mg, 1.96 mmol, 1.0 eq), triethylamine (200.0 mg, 1.96 mmol, 1.0 eq), and 10 mL of dichloromethane were added. After reacting at room temperature for 16 hours, the solvent was removed by concentration under reduced pressure, and column chromatography yielded 1-isocyanoctane (350.0 mg, yield 40.4%). MS: m / z [M+H] + =443.3.

[0162] Step 2: Synthesis of compounds 1-5

[0163] In a 100 mL round-bottom flask, compounds 1-4 (330.0 mg, 0.75 mmol, 1.0 eq), p-toluenesulfonic acid (260.0 mg, 1.50 mmol, 2.0 eq), and methanol (10 mL) were added sequentially. The mixture was reacted at room temperature for 3 hours. Then, 20 mL of saturated sodium bicarbonate solution was added to the reaction mixture, and the mixture was extracted five times with 20 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to give compound 3-(diethylamino)propionic acid-4-hydroxy-3-(hydroxymethyl)-1-(nonylamino)-1-oxoethylenebutyl-2-yl ester (200 mg, yield 66.6%). MS: m / z [M+H] + =403.3.

[0164] Step 3: Synthesis of Compound 1

[0165] Add 5-[(2-butyl-1-oxoylideneoctyl)oxy]valeric acid (448.0 mg, 1.49 mmol, 3.0 eq), 4-dimethylpyridine (60.7 mg, 0.50 mmol, 1.0 eq), N,N-diisopropylethylamine (385.2 mg, 2.98 mmol, 6.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (285.7 mg, 1.49 mmol, 3.0 eq), and 15 mL of dichloromethane to a 100 mL round-bottom flask in sequence. After stirring at room temperature for half an hour, add compounds 1-5 (200.0 mg, 0.50 mmol, 1.0 eq). After reacting at room temperature for 16 hours, the solvent was removed by concentration under reduced pressure. The solution was diluted with 100 mL of water and extracted three times with 100 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to give 3-(diethylamino)propionic acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-8,14,19-trioxane-20-aza-9,15-dioxane-nonacol-18-yl ester (200.0 mg, yield 41.6%). MS: m / z [M+H] + =967.8.

[0166] 1 H NMR (300MHz, CDCl3) δ6.40-6.20(m,1H),5.31-5.22(m,1H),4.19-4.07(m,7H),3.29-3.19(m,1H),2.82-2.72(m,2H),2.60-2.43(m,7H) ,2.36-2.27(m,4H),1.76-1.64(m,9H),163-1.53(m,5H),1.51-1.38(m,8H),1.33-1.19(m,35H),1.05-1.01(m,6H),0.89-0.85(m,15H).

[0167] Example 2 Compound 2

[0168] Synthesis of 4-(diethylamino)butyric acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-8,14,19-trioxane-20-aza-9,15-dioxane-nonadecan-18-yl ester

[0169] The chemical structural formula of compound 2 is shown in Formula III. The preparation steps are the same as in Example 1, except that compound 1-1 is replaced with compound 4-(diethylamino)butyric acid for synthesis.

[0170]

[0171] Yield: 56.6%. MS: m / z [M+H] + =981.8.

[0172] 1 H NMR (400MHz, CDCl3) δ6.19-6.15(m,1H),5.32-5.31(m,1H),4.18-4.15(m ,2H),4.11-4.07(m,6H),3.31-3.20(m,2H),2.80-2.74(m,1H),2.54-2.4 1(m,8H),2.33-2.27(m,6H),1.77-1.64(m,12H),1.61-1.55(m,5H),1.50 -1.38(m,7H),1.30-1.20(m,32H),1.02-0.99(m,6H),0.89-0.85(m,15H).

[0173] Example 3 Compound 3

[0174] Synthesis of (diethylamino)acetic acid-7-butyl-17-(10-butyl-3,9-dioxane-2,8-dioxahexadecane-1-yl)-8,14,19-trioxane-20-aza-9,15-dioxanonadecan-18-yl ester

[0175] The chemical structure of compound 3 is shown in Formula IV. The preparation steps are the same as in Example 1, except that compound 1-1 is replaced with compound N,N-diethylglycine for reaction synthesis.

[0176]

[0177] Yield was 47.9%. MS: m / z [M+H] + =953.7.

[0178] 1H NMR (300MHz, CDCl3) δ6.22-6.19(m,1H),6.16-6.13(m,1H),5.32-5.29(m,2H),4.25-4.15(m ,3H),4.12-4.07(m,5H),3.31-3.30(m,2H),3.29-3.19(m,2H),2.80-2.76(m,1H),2.66-2.6 1(m,4H),2.46-2.43(m,2H),2.37-2.27(m,4H),1.84-1.77(m,2H),1.74-1.64(m,8H),1.62- 1.53(m,4H),1.50-1.39(m,8H),1.32-1.22(m,31H),1.06-1.03(m,6H),0.88-0.85(m,15H).

[0179] Example 4 Compound 4

[0180] Synthesis of 3-(diethylamino)propionic acid-7-butyl-17-({[(10Z,12Z)-1-oxylidene-octadec-9,12-dienyl]oxy}methyl)-8,14,19-trioxylidene-20-aza-9,15-dioxane-18-yl ester)

[0181] The chemical structure of compound 4 is shown in Formula V. The preparation steps are the same as in Example 1, except that one of the compounds 1-6 is replaced by a linoleic acid condensation reaction for synthesis.

[0182]

[0183] The yield was 35.1%. MS: m / z [M+H] + =947.8.

[0184] 1 H NMR (400MHz, CDCl3) δ6.23-6.18(m,1H),6.18-6.13(m,1H),5.50-5.20(m,6H),4.25-4.07(m,6H), 3.38-3.15(m,4H),2.88-2.45(m,6H),2.37-1.74(m,16H),1.68-1.25(m,44H),1.05-0.90(m,18H).

[0185] Example 5 Compound 5

[0186] Synthesis of 3-(diethylamino)propionic acid-16-({[(10Z,12Z)-1-oxoylidene-18-9,12-dienyl]oxy}methyl)-10-(octyloxy)-13,18-dioxoylidene-19-aza-9,14-dioxaoctadecane-17-yl ester)

[0187] The chemical structure of compound 5 is shown in Formula VI. The preparation steps are the same as in Example 1, except that compounds 1-6 are replaced by the condensation reaction of linoleic acid and 4,4-bis(octyloxy)butyric acid.

[0188]

[0189] Yield was 37.9%. MS: m / z [M+H] + =991.9.

[0190] 1 H NMR (400MHz, CDCl3) δ6.25-6.20(m,1H),6.19-6.15(m,1H),5.86-5.45(m,3H),4.44-4.10(m,8H),3.40-3.27(m,4H), 3.27-3.03(m,4H),2.95-2.68(m,4H),2.63-2.48(m,4H),2.40-1.75(m,16H),1.68-1.25(m,47H),1.02-0.89(m,18H).

[0191] Example 6 Compound 6

[0192] Synthesis of 5-[(2-butyl-1-oxoylidecyl)oxy]valerate-2-(10-butyl-3,9-dioxoylidecyl-2,8-dioxahexadecane-1-yl)-8-ethyl-3-({[(10Z,12Z)-octadecyl-9,12-dienyl]amino}carbonyl)-5-oxoylidecyl-8-aza-4-oxadecyl-1-yl ester

[0193] The chemical structural formula of compound 6 is shown in formula VII.

[0194]

[0195] Intermediate 6-5: (6Z,10Z)-18-isocyanooctadec-6,9-diene.

[0196]

[0197] Specifically, the steps include the following:

[0198] Step 1: Synthesis of Compound 6-2

[0199] 6-1 (10.00 g, 35.66 mmol, 1.0 eq) and N,N-dimethylformamide (0.26 g, 3.57 mmol, 0.1 eq) were added to a round-bottom flask containing 100 mL of dichloromethane. Then, oxalyl chloride (6.79 g, 53.49 mmol, 1.5 eq) was slowly added dropwise. The mixture was stirred at 0 °C for 3 hours. The reaction solution was then concentrated under reduced pressure to obtain the crude product. In a 500 mL round-bottom flask, ammonia (7.04 g, 50.19 mmol, 1.5 eq), triethylamine (10.16 g, 100.38 mmol, 3.0 eq), and 100 mL of dichloromethane were added. The crude product diluted with dichloromethane was slowly added dropwise at 0 °C. The mixture was stirred at 0 °C for 3 hours. 80 mL of water was added to the reaction mixture, and the mixture was extracted three times with 100 mL of dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography to give (10Z,12Z)-octadecyl-9,12-dieneamide (7.5 g, 80.2% yield). MS: m / z [M+H] + =280.3.

[0200] Step 2: Synthesis of Compound 6-3

[0201] 6-2 (7.50 g, 26.84 mmol, 1.0 eq) was added to a round-bottom flask containing 60 mL of tetrahydrofuran. Lithium aluminum hydride (4.07 g, 107.35 mmol, 4.0 eq) was slowly added at 0°C, and the mixture was stirred for 30 minutes. The reaction mixture was then stirred at 65°C for 12 hours. The reaction mixture was cooled to 0°C, and 4.1 mL of water was carefully added dropwise, followed by 4.1 mL of 15% sodium hydroxide aqueous solution, and then 12.3 mL of water. The mixture was heated to room temperature and stirred for 1 hour. Some tetrahydrofuran was added for dilution, and a small amount of anhydrous sodium sulfate was added. The mixture was stirred and dried for 15 minutes, then filtered and concentrated to give the compound (10Z,12Z)-octadecyl-9,12-diene-1-amine (6.00 g, yield 84.3%). MS: m / z [M+H] + =266.3.

[0202] Step 3: Synthesis of Compound 6-4

[0203] Intermediate 6-3 (6.00 g, 22.60 mmol, 1.0 eq) was added to 60 mL of ethyl formate, heated to 65°C and refluxed for 12 hours. The mixture was then concentrated under reduced pressure and subjected to column chromatography to give N-[(6Z,10Z)-octadecyl-6,9-dien-18-yl]methaneamide (5.50 g, yield 82.92%). MS: m / z [M+H] + =294.3.

[0204] Step 4: Synthesis of Compounds 6-5

[0205] Compound 6-4 (5.50 g, 18.74 mmol, 1.0 eq) and triethylamine (9.48 g, 93.70 mmol, 5.0 eq) were added to 40 mL of tetrahydrofuran. Phosphorus oxychloride (4.31 g, 28.11 mmol, 1.5 eq) was then slowly added dropwise at 0°C. After the addition was complete, the temperature was raised to 25°C, and the reaction was allowed to proceed for 2 hours. The mixture was concentrated under reduced pressure, and column chromatography was used to obtain compound (6Z,10Z)-18-isocyanooctadecyl-6,9-diene (4.30 g, 83.30% yield). MS: m / z [M+H] + =276.3.

[0206] Example 6 was synthesized by replacing compounds 1-2 with intermediate 6-5 using the method employed in Example 1. Intermediate 6-5 is (6Z,10Z)-18-isocyanooctadecyl-6,9-diene. The yield was 48.9%. MS: m / z [M+H] + =1089.9.

[0207] 1 H NMR (300MHz, CDCl3) δ6.47-6.42(m,1H),6.28-6.23(m,1H),5.39-5.32(m,4H),4.22-4. 18(m,3H),4.15-4.07(m,5H),3.28-3.25(m,1H),2.83-2.77(m,5H),2.62-2.56(m,4H), 2.52-2.44(m,4H),2.40-2.29(m,4H),2.09-2.04(m,4H),1.76-1.66(m,10H),1.63-1.5 8(m,4H),1.52-1.42(m,6H),1.36-1.23(m,37H),1.08-1.05(m,6H),0.94-0.86(m,15H).

[0208] Example 7 Compound 7

[0209] Synthesis of 10-[22-methyl-10-(octyloxy)-13,19-dioxane-18,22-diaza-9,14-dioxane-18-yl]-11-(octylamino)-11-oxane-undecanoic acid-2-butyloctyl ester

[0210] The chemical structure of compound 7 is shown in Formula VIII. The preparation steps are the same as in Example 1, except that compounds 1-2 are replaced with intermediate 8-isocyanoctanoic acid-2-butyloctyl ester for reaction synthesis.

[0211]

[0212] The yield was 25.5%. MS: m / z [M+H] + =1151.9.

[0213] 1 H NMR (300MHz, CDCl3) δ6.54-6.48(m,1H),6.34-6.28(m,1H),5.33-5.24(m,1H),4.21-4. 20(m,2H),4.14-4.06(m,5H),3.99-3.97(m,2H),3.31-3.20(m,2H),2.88-2.83(m,1H), 2.81-2.73(m,1H),2.66-2.57(m,3H),2.55-2.44(m,3H),2.38-2.29(m,6H),1.82-1.56 (m,18H),1.53-1.41(m,6H),1.33-1.21(m,45H),1.09-1.06(m,6H),0.94-0.85(m,18H).

[0214] Example 8: Preparation, characterization, and in vivo editing evaluation of lipid nanoparticles

[0215] 1. Animal Experiment Design

[0216] mRNA and sgRNA delivery experiments of the PCSK9-targeting base editor ABE8e

[0217] 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). The LDL 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 studies have 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.

[0218] Strategies for PCSK9 gene editing delivery in mouse liver cells, such as Figure 1As 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.

[0219] The specific experimental design is as follows:

[0220] 1.1 Selection of suitable mutation sites and editing design

[0221] 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.

[0222] 1.2 Preparation of mRNA and sgRNA of base editor ABE8e

[0223] 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.

[0224] 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:

[0225] PCSK9-sgRNA: 5'-cccataccttggagcaacgg-3' (SEQ ID NO: 1);

[0226] 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 minutes; 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).

[0227] The system used in the construction of the sgRNA plasmid is as follows:

[0228] 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℃.

[0229] 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 oligonucleotide 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.

[0230] 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 collected 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.

[0231] 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 anadenine 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 Mar16. 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.

[0232] 2. Lipid nanoparticles were prepared by using ionizable lipids or the compounds of this invention / DSPC / cholesterol / PEG-lipids in a molar ratio of 50:10:38.5:1.5.

[0233] 2.1 Dilinylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated as MC3) and compounds 1-7 of the present invention were dissolved in anhydrous ethanol with DSPC, cholesterol and PEG-DMG in the above molar ratio.

[0234] 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 of 40:1, sgRNA:ABE8e mRNA (w / w) ratio of 1:1), and the mixture was flowed at 12 mL / min through a microfluidic nanomedicine manufacturing system (NanoAssemblr Ignite, Canada) to obtain nucleic acid lipid nanoparticles 1-8. 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.

[0235] 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 (ThermoFisherScientific, UK) according to the manufacturer's instructions. The test results for the characterization of the lipid nanoparticles are shown in Table 2.

[0236] Table 2

[0237] 1 MC3 76.5 0.16 92.3 2 Compound 1 72.5 0.01 89.5 3 Compound 2 73.5 0.06 97.9 4 Compound 3 74.3 0.06 76.9 5 Compound 4 70.7 0.01 95.5 6 Compound 5 66.4 0.02 95.3 7 Compound 6 71.6 0.01 93.5 8 Compound 7 63.9 0.01 94.6

[0238] 3. In vivo editing experiment evaluation

[0239] 3.1 Lipid nanoparticles containing the compounds of this invention (see Table 2) and encapsulating the mRNA and sgRNA encoding the base editor ABE8e were administered systemically via tail vein injection to 6-7 week old female C57BL / 6 mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.). Lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, abbreviated as MC3) and encapsulating the mRNA and sgRNA of the base editor ABE8e were administered in a similar manner to age- and sex-matched mice as positive controls. Additionally, PBS buffer was administered via tail vein injection to age- and sex-matched mice in a similar manner as negative controls.

[0240] 3.2 Editing Efficiency Test

[0241] 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.

[0242] The deep sequencing steps are as follows:

[0243] (1) Primers were designed based on the location of the target gene. See Table 3 for primer design targeting the PCSK9 gene.

[0244] Table 3

[0245] PCSK9-F2 5'-accagacggctagatgagca-3'(SEQ ID NO:2) PCSK9-R2 5'-cccaggacgaggatggagatta-3'(SEQ ID NO:3)

[0246] (2) Editing efficiency test.

[0247] The PCR program was as follows: 94℃ for 2 minutes; 98℃ for 10 seconds, 60℃ for 30 seconds, 68℃ for 20 seconds, 34 cycles; 68℃ for 5 minutes. After PCR, the results were verified by gel electrophoresis. A single band of appropriate size was selected to confirm the correct amplification product. The obtained PCR products were then sent to Nanjing GenScript Biotech Co., Ltd. for sequencing.

[0248] (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 Table 4 shows the in vivo editing efficiency assessment.

[0249] Table 4

[0250]

[0251]

[0252] As shown in Table 4, the ionizable lipid compounds used in this invention can effectively deliver drugs such as nucleic acid molecules and small molecule compounds; and by comparison, the lipid nanoparticles of this invention 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.

[0253] The descriptions of the exemplary embodiments presented above are merely illustrative of the technical solutions of the present invention and are not intended to be exhaustive, nor are they intended to limit the invention 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 the invention and its practical applications, thereby enabling other those skilled in the art to understand, implement, and utilize the various exemplary embodiments of the invention and their various alternatives and modifications. The scope of protection of the present invention is intended to be defined by the claimed scope and its equivalents.

[0254] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A lipid compound, characterized in that, The lipid compound is selected from any one of the following structural compounds. 。 2. The application of the lipid compound of claim 1 in the preparation of liposome nanocarriers.

3. A lipid carrier, characterized in that, The lipid carrier comprises the lipid compound of claim 1.

4. The lipid carrier according to claim 3, characterized in that, The lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises the lipid compound of claim 1, and optionally an ionizable lipid, and the second lipid compound comprises any one or a combination of at least two of anionic lipids, neutral lipids, steroid-bound lipids, or polymer-bound lipids.

5. The lipid carrier according to claim 4, characterized in that, The ionizable lipids are selected from any one or a combination of at least two of the following: DLinDMA, DODMA, DLin-MC2-MPZ, DLin-KC2-DMA, DOTAP, C12-200, and DOTMA.

6. A nucleic acid lipid nanoparticle composition, characterized in that, The nucleic acid lipid nanoparticle composition comprises the lipid compound of claim 1, or the lipid carrier of any one of claims 3-5, and a therapeutic or preventative agent.

7. The nucleic acid lipid nanoparticle composition according to claim 6, characterized in that, The therapeutic or preventive agent is any one or a combination of at least two of RNA, DNA, antisense nucleic acid, aptamer, nuclease, immunostimulatory nucleic acid, or peptide nucleic acid.

8. The nucleic acid lipid nanoparticle composition according to claim 7, characterized in that, The antisense nucleic acid is an antisense oligonucleotide.

9. The nucleic acid lipid nanoparticle composition according to claim 7, characterized in that, The RNA is any one or a combination of at least two of the following: mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, or shRNA.

10. The nucleic acid lipid nanoparticle composition according to claim 7, characterized in that, The RNA in question is modified RNA.

11. The nucleic acid lipid nanoparticle composition according to claim 9, characterized in that, The mRNA is a sequence encoding an RNA-directed DNA binder.

12. The nucleic acid lipid nanoparticle composition according to claim 7, characterized in that, The DNA is a plasmid.

13. The nucleic acid lipid nanoparticle composition according to claim 7, characterized in that, The nuclease is selected from any one or a combination of at least two of Cas9, Cas12, Cas13, IscB, TnpB, and IsrB.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nucleic acid lipid nanoparticle composition of any one of claims 6-13, and pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, antioxidants or buffers.

15. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises the nucleic acid lipid nanoparticle composition according to any one of claims 6-13, and pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, antioxidants or buffers.

16. The pharmaceutical preparation according to claim 15, characterized in that, The dosage form of the pharmaceutical preparation is selected from any one or a combination of at least two of the following: tablets, capsules, pills, granules, solutions, suspensions, suppositories, inhalers, or sprays.

17. Use of the nucleic acid lipid nanoparticle composition of any one of claims 6-13, the pharmaceutical composition of claim 14, or the pharmaceutical formulation of claim 15 or 16 in the preparation of gene vaccines, peptide or protein drugs.

18. The use according to claim 17, characterized in that, The nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation is used to treat or prevent diseases or conditions in subjects in need.

19. The use according to claim 18, characterized in that, The subjects were mammals.

20. The use according to claim 19, characterized in that, The mammal in question is a human.

21. The use according to claim 18, characterized in that, The disease or condition is selected from metabolic diseases, genetic diseases, cancer, cardiovascular diseases, or infectious diseases.

22. The use according to claim 21, characterized in that, The metabolic disease is familial hypercholesterolemia, the hereditary disease includes transthyretin amyloidosis, primary hyperoxaluria, or hereditary angioedema, and the infectious disease includes hepatitis B.

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