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

Abstract

A lipid compound for delivering a therapeutic agent, and a method of preparing and using the same. The lipid compound is a compound having the 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-conjugated lipids, to form a lipid nanoparticle for delivering a therapeutic agent (e.g., a nucleic acid molecule) for therapeutic or prophylactic purposes (e.g., vaccination), enriching the repertoire of ionizable lipid compounds.

Description

Lipid compound for delivering therapeutic agent and preparation method and application thereof Technical Field

[0001] The present invention relates to the field of drug delivery, and in particular to a lipid compound for delivering a therapeutic agent (such as a nucleic acid molecule), a lipid carrier comprising the same, a nucleic acid lipid nanoparticle composition and a pharmaceutical preparation, as well as their preparation methods and related applications. Background Art

[0002] Gene therapy technology is a hot topic in modern biomedical research. Nucleic acid drugs can be used to prevent and treat cancer, bacterial and viral infections, and diseases with genetic etiologies. Because nucleic acid drugs are easily degraded and difficult to enter cells, they typically require encapsulation with a vector for delivery to target cells. Therefore, the development of 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 delivery. In 2018, the FDA approved the LNP-delivered drug patisiran (onpattro) for the treatment of hereditary transthyretin amyloidosis. Since then, research on the use of LNP technology to deliver nucleic acid drugs has exploded. In particular, at the end of 2020, the FDA approved the COVID-19 vaccines from Moderna and BioNtech & Pfizer, respectively. Both vaccines utilize LNP technology to deliver mRNA drugs, thereby achieving protection against the SARS-CoV-2 virus.

[0004] LNPs are typically composed of four lipid compounds: ionizable lipids, neutral lipids, steroids, and polymer-bound lipids. The choice of ionizable lipids has the greatest impact on LNPs. Current nucleic acid therapeutics still face several challenges, including delivery efficiency, low cell permeability, and high sensitivity to degradation of certain nucleic acid molecules, including RNA. Therefore, 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 preventive purposes.

[0005] Summary of the Invention

[0006] One object of the present invention is to provide a lipid compound or a pharmaceutically acceptable form thereof (e.g., salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs, etc.). The lipid compound can be used together 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 also mRNA) to improve the delivery efficiency of nucleic acid drugs in the body. Lipid compounds with specific structures can be selected as lipid carriers according to the organs where the nucleic acid drugs need to be enriched.

[0007] Another object of the present invention is to provide a method for preparing the lipid compound or a pharmaceutically acceptable form thereof.

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

[0009] Another object of the present invention is to provide a nucleic acid lipid nanoparticle composition comprising the above-mentioned compound or a pharmaceutically acceptable form thereof or the above-mentioned lipid carrier.

[0010] Another object of the present invention is to provide a pharmaceutical preparation comprising the above-mentioned compound or a pharmaceutically acceptable form thereof, or the above-mentioned lipid carrier, or the above-mentioned nucleic acid lipid nanoparticle composition.

[0011] Another object of the present invention is to provide the use of the above-mentioned compound or its pharmaceutically acceptable form or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0012] Another object of the present invention is to provide a method for delivering a nucleic acid drug in vivo, which comprises administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.

[0013] <First Aspect>

[0014] The present invention provides a compound of formula I or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug,

[0015] Where,

[0016] G 1 C 1-10 alkylene;

[0017] R 1 、R 2 Each independently is C 1-6 alkyl;

[0018] Optionally, R 1 、R 2 Together with the connected N, it forms a 3-8 membered heterocyclic group, or, R 1 、R 2 Any one of G 1 Any carbon atom in is directly connected to form a 3-8 membered heterocyclic group;

[0019] L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 Each is independently selected from an ester group, an amide group, a carbonate group, a carbamate group, a mercaptoformate group, a urea group, a phosphate group, or none;

[0020] R 3 、R 5 、R 7 、R 9 Each independently selected from C 1-20 Straight chain alkylene, C 3-20 Branched alkylene, C 1-20 Straight chain heteroatom-containing alkylene, C 3-20 Branched chain containing heteroatom alkylene, C 2-20 Straight chain alkenylene, C 3-20 Branched alkenylene, C 2-20 Straight chain heteroatom-containing alkenylene, C 3-20 Branched chain containing heteroatom alkenylene, C 2-20 Straight chain alkynylene, C 3-20 Branched chain alkynylene, C 2-20 Straight chain heteroatom-containing alkynylene, C 3-20 The branched chain contains heteroatom alkynylene groups or none;

[0021] R 4 、R 6 、R 8 、R 10 Each independently selected from C 1-20 Straight chain alkyl, C 2-20 Straight chain alkenyl, C 2-20 Straight chain alkynyl, C 1-20 Straight chain heteroatom-containing alkyl, C 3-20 Branched alkyl, C 3-20 Branched chain containing heteroatom alkyl, C 3-10 Cycloalkyl, C 4-10 Bridged cycloalkyl or none.

[0022] According to some specific embodiments of the present invention, in the compound of formula I of the present invention or a pharmaceutically acceptable form thereof, the heteroatom is selected from one or more of O, N, and S.

[0023] According to some specific embodiments of the present invention, in the compound of formula I of the present invention or its pharmaceutically acceptable form, 0 to 3 H in the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene or heteroatom-containing groups are optionally further replaced by halogen, hydroxyl, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 1-12 Alkoxy, halogen-substituted C 1-12 Alkyl or hydroxy substituted C 1-12 Alkyl substituted.

[0024] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, G 1 for wherein m is selected from an integer of 1-6 (1, 2, 3, 4, 5 or 6).

[0025] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, G 1 The group corresponding to any one of Compounds 1 to 35.

[0026] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, R 1 、R 2 are each independently methyl, ethyl or propyl; or, R 1 、R 2 Together with the connected nitrogen, it forms a 5-membered or 6-membered heterocyclic group.

[0027] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, R 1 、R 2 They correspond to the groups or values ​​shown in any one of Compounds 1 to 35 respectively.

[0028] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 Each independently selected from Or none. The “-” next to O in the formula represents an anion.

[0029] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 They correspond to the groups or values ​​shown in any one of Compounds 1 to 35 respectively.

[0030] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form:

[0031] R 3 、R 5 、R 7 、R 9 Each independently

[0032] wherein X is O, S, Se, SS, Se-Se or none;

[0033] R 11 、R 12 、R 13 、R 14 Each is independently H or a C1-C8 straight-chain alkyl group;

[0034] m and o are each independently an integer selected from 1 to 10. That is, m can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; o can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0035] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, R 3 、R 5 、R 7 、R 9 They correspond to the groups or values ​​shown in any one of Compounds 1 to 35 respectively.

[0036] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form:

[0037] R 4 、R 6 、R 8 、R 10 Each independently

[0038] Where Y is or none;

[0039] R 15 、R 16 、R 17 、R 18 、R 19 Each independently represents H, C1-C 10 Straight chain alkyl, C 3-10 Cycloalkyl or C 4-10 bridged cycloalkyl;

[0040] p and q are each independently an integer selected from 0 to 10. That is, p can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; q can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0041] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, R 4 、R 6 、R 8 、R 10 They correspond to the groups or values ​​shown in any one of Compounds 1 to 35 respectively.

[0042] According to some specific embodiments of the present invention, in Formula I, G 1 、R 1 、R 2 , L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 、R 3 、R 5 、R 7 、R 9 、R 4 、R 6 、R 8 、R 10 They correspond to the groups or values ​​shown in any one of Compounds 1 to 35 respectively.

[0043] According to some specific embodiments of the present invention, in the compound of formula I of the present invention or a pharmaceutically acceptable form thereof, the pharmaceutically acceptable form is selected from a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug.

[0044] According to a specific embodiment of the present invention, the compound in Formula I is selected from one or more of Compounds 1 to 35 shown in Table 1.

[0045] The present invention also provides a method for preparing the compound of formula I or a pharmaceutically acceptable form thereof. Based on the chemical structure of the compound of formula I or a pharmaceutically acceptable form thereof, a synthetic route can be designed to prepare the compound of formula I or a pharmaceutically acceptable form thereof with reference to methods known in the art.

[0046] Table 1

[0047] <Second Aspect>

[0048] The present invention also provides the use of the compound or a pharmaceutically acceptable form thereof in the preparation of liposome nanocarriers.

[0049] <Third Aspect>

[0050] The present invention provides a lipid carrier comprising the compound according to the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug. This lipid carrier has high encapsulation efficiency for nucleic acid drugs, greatly improving the delivery efficiency of nucleic acid drugs in vivo.

[0051] In some embodiments, the lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises the compound according to the <First Aspect> or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, and optionally other ionizable lipids, and the second lipid compound comprises one or a combination of two or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids.

[0052] In some embodiments, the first lipid compound is any one of the compounds described above or 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 any one of the compounds described above 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.

[0054] In some embodiments, the other cationic or ionizable lipid compounds include 1,2-dilinoleyloxy-N,N-dimethylaminopropane DLinDMA, 1,2-dioleyloxy-N,N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane DLin-KC2-DMA, 1,2-dioleoyl-3-trimethylammonium-propane DOT One or a combination of two or more of AP, 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)di-dodecan-2-ol C12-200, 3β[N-N'N'-dimethylaminoethane)-carbamoyl]cholesterol DC-Chol and N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylamine chloride DOTMA.

[0055] In some embodiments, the anionic lipid comprises one or a combination of two or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol DOPG, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine DOPS, and dimyristoylphosphatidylglycerol.

[0056] In some embodiments, the neutral lipids include at least one of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine DOPE, 1,2-distearoyl-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, distearoylphosphatidylethanolamine DSPE, and 1-stearoyl-2-oleoylphosphatidylethanolamine SOPE, or lipids modified with anionic or cationic modifying groups. The anionic or cationic modifying groups are not limited.

[0057] In some embodiments, the steroid comprises one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, coproposterol, and corticosteroids.

[0058] In some embodiments, the polymer-bound lipids include 1,2-dimyristoyl-sn-glyceromethoxy-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-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]PEG-DSPE, PEGylated phosphatidylethanolamine PEG-PE, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, Tween-20, Tween-80, 1,2-dipalmityl-sn-glycero-methoxypolyethylene glycol PEG-DPG, 4-O-(2',3'-di(tetradecanoyl)-1,2-di-tetradecanoyl)-1,2-di-di-tetradecanoyl-1,2-di ... One or a combination of two or more of PEG-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, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20-65):(0-20):(5-25):(25-55):(0.3-15); illustratively, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the 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 any one of the above compounds or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (1-10):(0-10); illustratively, 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 lipids, neutral lipids, steroids and polymer-bound lipids 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 pharmaceutically acceptable forms thereof such as salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (3-4):(0-5).

[0061] <Fourth Aspect>

[0062] The present invention provides a nucleic acid-lipid nanoparticle composition comprising the compound according to 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 according to the third aspect, and a therapeutic and / or prophylactic agent. The therapeutic and / or prophylactic agent is, for example, a nucleic acid drug.

[0063] In some embodiments, the nucleic acid drug comprises RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA. In some embodiments, the antisense nucleic acid is an antisense oligonucleotide. In some embodiments, the RNA comprises one or more of mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomiR, microRNA inhibitor, microRNA activator or shRNA. In some embodiments, the mRNA comprises one or more functional nucleotide analogs, including pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.

[0064] In some embodiments, the mRNA encodes at least one antigen. In some embodiments, the antigen is a pathogenic antigen. In some embodiments, the antigen is a tumor-associated antigen.

[0065] In some embodiments, the DNA comprises a plasmid.

[0066] In some embodiments, the RNA component comprises mRNA encoding an RNA-guided nuclease or encoding a base editor, and / or gRNA.

[0067] In some embodiments, the mRNA includes a sequence encoding an RNA-guided DNA binder, more specifically, an mRNA comprising a Cas protein.

[0068] In some embodiments, the nucleic acid drug comprises a guide RNA, specifically, the guide RNA comprises a gRNA nucleic acid.

[0069] In some embodiments, the nucleic acid drug includes mRNA and gRNA of the Cas protein.

[0070] In some embodiments, the nuclease is selected from Cas9, Cas12, Cas13, IscB, TnpB, IsrB, and homologs thereof.

[0071] In some embodiments, the RNA comprises modified nucleotides.

[0072] In some embodiments, the gRNA is modified.

[0073] In some embodiments, the mass ratio of the nucleic acid drug to any of the compounds or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs thereof is 1:(3-40).

[0074] In some embodiments, the mass ratio of the nucleic acid drug to the lipid carrier is 1:(3-40).

[0075] Illustratively, the above mass ratio is 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, etc.

[0076] <Fifth Aspect>

[0077] The present invention provides a pharmaceutical composition comprising a compound according to the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or a lipid carrier according to the third aspect, or a nucleic acid lipid nanoparticle composition according to the fourth aspect, and a pharmaceutically acceptable excipient, carrier or diluent.

[0078] <Sixth Aspect>

[0079] The present invention provides a pharmaceutical preparation comprising any one of the above-mentioned compounds or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or the above-mentioned lipid carrier, or the above-mentioned nucleic acid lipid nanoparticle composition, and a pharmaceutically acceptable excipient, carrier or diluent.

[0080] In some embodiments, the particle size of the pharmaceutical preparation is 30 to 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 efficiency of the nucleic acid drug in the pharmaceutical formulation is greater than 50%. Exemplarily, the encapsulation efficiency can be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, etc.

[0082] <Seventh Aspect>

[0083] The present invention also provides the use of the above-mentioned compound or its pharmaceutically acceptable form or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0084] The present invention also provides a method for delivering a nucleic acid drug in vivo, comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.

[0085] The present invention also provides a method for delivering a therapeutic agent and / or prophylactic agent to cells of a subject, the method comprising administering to the subject the compound of the present invention or its pharmaceutically acceptable form, the lipid carrier, the nucleic acid lipid nanoparticle composition, or the pharmaceutical preparation, wherein the administration comprises contacting the cells of the subject with the compound or its pharmaceutically acceptable form, the lipid carrier, the nucleic acid lipid nanoparticle composition, or the pharmaceutical preparation, thereby delivering the therapeutic agent and / or prophylactic agent to the cells of the subject.

[0086] The present invention also provides a method for producing a target protein or target polypeptide in a subject cell, the method comprising contacting the subject cell with the nucleic acid lipid nanoparticle composition, wherein the therapeutic agent or preventive agent is mRNA, and wherein the mRNA encodes the target protein or target polypeptide, whereby the mRNA can be translated in the cell to produce the target protein or target polypeptide.

[0087] The present invention also provides a method for preventing, improving or treating a disease or condition in a subject in need thereof, comprising administering to the subject the compound of the present invention or a pharmaceutically acceptable form thereof, the lipid carrier, the nucleic acid lipid nanoparticle composition, or the pharmaceutical preparation.

[0088] In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is used to treat or prevent a disease or disorder in a subject in need thereof.

[0089] In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is administered, for example, via an enteral or parenteral route of administration. In some embodiments, a dose of about 0.001 mg / kg to about 10 mg / kg of the nucleic acid lipid nanoparticle composition or pharmaceutical formulation is administered to the subject.

[0090] In some embodiments, the subject is a mammal or a human, preferably, the subject is a human.

[0091] In some specific embodiments, the disease or condition is selected from metabolic diseases, hereditary diseases, cancer, cardiovascular diseases and infectious diseases; preferably, the metabolic diseases include familial hypercholesterolemia (FH), the hereditary diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluria (PH1) and hereditary angioedema (HAE), and the infectious diseases include hepatitis B (HEPATITIS B).

[0092] The present invention provides a series of novel compounds of Formula I. These compounds can be used as ionizable lipids and, together with other lipid compounds, prepare lipid carriers. These lipid carriers have controllable particle size, uniform distribution, and high encapsulation efficiency. The synthesis method is simple, high in yield, and can be rapidly synthesized at low cost. The compounds of the present invention can be used to deliver nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, or protein drugs, enriching the variety of ionizable lipid compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 is a schematic diagram of the delivery strategy for detecting the efficiency of PCSK9 gene editing in mouse liver cells in the present invention.

[0094] Figure 2 shows the PCSK9 gene editing efficiency of base editors encapsulated by different lipid compounds in mouse liver cells. DETAILED DESCRIPTION

[0095] For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0096] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0097] In this specification, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 1%.

[0098] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0099] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0100] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0101] Before the present invention is further described, it is to be understood that the present invention is not limited to the particular embodiments described herein; it is to be understood that the terminology used herein is for the purpose of describing only and is not intended to be limiting of the particular embodiments.

[0102] [Definition of terms]

[0103] Unless otherwise stated, the following terms have the following meanings:

[0104] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is substantially non-toxic to living organisms. Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by reacting a compound of the present invention with a pharmaceutically acceptable inorganic / organic acid or inorganic / organic base. Such salts are also referred to 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.; common organic bases include (but are not limited to) diethylamine, triethylamine, ethambutol, etc.

[0105] The term "stereoisomer" (or "optical isomer") refers to a stable isomer that possesses at least one chiral element (including a chiral center, chiral axis, chiral plane, etc.) resulting in a perpendicular asymmetric plane, thereby rotating plane-polarized light. Because the compounds of the present invention contain asymmetric centers and other chemical structures that may lead to stereoisomerism, the present invention also encompasses these stereoisomers and mixtures thereof. Because the compounds of the present invention and their salts contain asymmetric carbon atoms, they can exist as single stereoisomers, racemates, enantiomers, and mixtures of 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 in a single stereoisomer (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). Individual stereoisomers of a compound are synthesized from optically active starting materials containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, for example, by conversion to a mixture of diastereomers followed by separation or recrystallization, chromatography, use of a chiral resolving agent, or direct separation of the enantiomers on a chiral chromatographic column. Starting compounds of specific stereochemistry are either commercially available or prepared as described herein and resolved by methods well known in the art.

[0106] The term "tautomer" (or "tautomeric form") refers to structural isomers of different energies that are interconvertible via a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or prototropic tautomers) include, but are not limited to, interconversions via proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-iminoalcohol isomerization, and the like. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.

[0107] The term "solvate" 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, ethanolates, acetonides, etc.

[0108] The term "chelate" refers to a complex having a cyclic structure, which is obtained by the chelation of two or more ligands with the same metal ion to form a chelate ring.

[0109] The term "non-covalent complex" is formed by the interaction of a compound with another molecule, wherein no covalent bond is formed between the compound and the molecule. For example, complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

[0110] The term "prodrug" refers to a derivative compound that, upon application to a patient, is capable of providing, directly or indirectly, a compound of the invention. Particularly preferred derivative compounds or prodrugs are compounds that, when administered to a patient, can increase the bioavailability of the compound of the invention (e.g., more readily absorbed into the bloodstream) or compounds that facilitate delivery of the parent compound to the site of action (e.g., the lymphatic system). Unless otherwise indicated, all prodrug forms of the compounds of the invention are within the scope of the invention, and various prodrug forms are well known in the art.

[0111] The term "each independently" means that at least two groups (or ring systems) present in a structure with the same or similar value ranges may have the same or different meanings in specific circumstances. For example, if substituent X and substituent Y are each independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl. Similarly, when substituent Y is hydrogen, substituent X may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl.

[0112] The terms "including" and "comprising" are used in their open, non-limiting sense.

[0113] The term "alkyl" refers to a monovalent straight or branched alkane group consisting only of carbon and hydrogen atoms, containing no unsaturation, and connected to other moieties 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 or branched chain hydrocarbon group containing 1 to 30 carbon atoms.

[0114] The term "alkylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing no saturation, and connected to other fragments by two single bonds, including (but not limited to) methylene, 1,1-ethylene and 1,2-ethylene. For example, "C 1-30 "Alkylene" refers to a saturated divalent straight or branched chain alkyl group containing from 1 to 30 carbon atoms.

[0115] The term "cycloalkyl" refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting only of carbon atoms and hydrogen atoms. Cycloalkyl groups may include fused, bridged or spirocyclic ring systems. For example, the term "C 3-6"Cycloalkyl" refers to a cycloalkyl group having 3 to 6 carbon atoms. For example, the cycloalkyl group may be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or bicyclo[2.2.1]heptyl, etc.

[0116] 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 cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. For example, "C 3-30 The "cycloalkylene group" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group containing 3 to 30 carbon atoms.

[0117] The term "branched alkyl" refers to an alkane radical that is attached to a parent molecule and forms at least two branched structures. For example

[0118] The term "alkenyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by a single bond, including (but not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl and isobutenyl groups. For example, "C 2-30 "Alkenyl" refers to a monovalent straight or branched chain hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

[0119] The term "alkenylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by two single bonds, including (but not limited to) vinylene, etc. For example, "C 2-30 "Alkenylene" refers to a divalent straight or branched chain hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

[0120] The term "alkynyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by a single bond, including (but not limited to) ethynyl, propynyl, butynyl and pentynyl groups. For example, "C 2-30 "Alkynyl" refers to a monovalent straight or branched chain hydrocarbon radical containing from 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.

[0121] The term "alkynylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by two single bonds, including (but not limited to) ethynylene. 2-30 "Alkyne" refers to a divalent straight or branched chain hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.

[0122] The term "cycloalkenyl" refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) non-aromatic hydrocarbon group consisting solely of carbon and hydrogen atoms. Cycloalkenyl groups may include cyclic, bridged, or spirocyclic ring systems. Examples include cyclopropenyl and cyclobutenyl.

[0123] The term "cycloalkenylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group as defined above, including but not limited to cyclopropenylene and cyclobutenylene. For example, "C 3-30 The "cycloalkenylene group" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group containing 3 to 30 carbon atoms.

[0124] The term "branched alkenyl" refers to an alkene radical attached to a parent molecule and forming at least two branches. For example

[0125] The term "ester group" is understood to be a monovalent or divalent (depending on the position of these groups in formula I) ester group, which is connected to the other moieties in formula I by one or two single bonds, respectively. In some embodiments, "ester group" specifically refers to a group containing the formula -CO-O- or -O-CO-, such as -(CnH2n)x-CO-O-, -(CnH2n)x-CO-O-alkyl or -(CnH2n)x-CO-O-alkylene, wherein n can be an integer selected from 0-10 and x is 0, 1, 2 or 3.

[0126] Similarly, in the present invention, the amide group, carbonate group, carbamate group, mercaptoformate group, urea group, and phosphate group refer to the corresponding monovalent or divalent (depending on the position of these groups in Formula I) groups of amide, carbonate, carbamate, mercaptoformate, urea, and phosphate, which are connected to other fragments in Formula I through one or two single bonds, respectively.

[0127] The term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, for example, fused, bridged or spiro) non-aromatic group, the ring atoms of which are composed of carbon atoms 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 1-4Alkyl. If the valence bond requirements are met, the heterocyclic group can be attached to the rest of the molecule through any one of the ring atoms. For example, the term "3-8 membered heterocyclic group" as used in the present invention refers to a heterocyclic group having 3 to 8 ring atoms. For example, the heterocyclic group can be an oxiranyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl or trithianyl.

[0128] The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π electron system. For example, the term "C 6-10 The term "aryl" refers to an aromatic group having 6 to 10 carbon atoms. For example, the aromatic group may be phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthenyl, azulenyl, fluorenyl, indenyl, pyrenyl, and the like.

[0129] The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated π electron system, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O and S. If the valence bond requirements are met, the heteroaryl group can be connected to the rest of the molecule through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" as used in the present invention refers to a heteroaryl group having 5 to 10 ring atoms. For example, the heteroaryl group can be thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and benzo derivatives thereof, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, etc.

[0130] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). The term "hydroxy" refers to -OH. The term "cyano" refers to -CN. The term "amino" refers to -NH2. The term "nitro" refers to -NO2. The term "oxo" refers to (=O).

[0131] [Preparation method]

[0132] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0133] In the present invention, "appropriate amount" means that the amount of the added solvent or the amount of the drug can be adjusted in a wide range and has little effect on the synthesis result, and is not specifically limited.

[0134] In the following examples, all solvents and drugs used were of analytical or chemical purity; all solvents were redistilled before use; and all anhydrous solvents were treated according to standard methods or literature methods.

[0135] Example

[0136] Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Reagents or instruments used without specifying the manufacturer are conventional products that can be obtained commercially. It will be appreciated by those skilled in the art that the examples describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention. The technical features involved in the various embodiments of the present invention may 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.

[0137] Example 1: 6-({4-[3-(diethylamino)propyl]-2,6-bis(3,8-dioxyylidene-2,9-dioxahexadecan-1-yl)-9,14-dioxyylidene-4-aza-8,15-dioxadocosan-1-yl}oxy)-6-oxyylidenehexanoate

[0138] Step 1: Synthesis of compound 1-2

[0139] To 100 mL of dichloromethane at 0°C were added (2,2-dimethyl-1,3-dioxan-5-yl)methanol (10.00 g, 68.41 mmol, 1.0 eq), triethylamine (20.77 g, 205.23 mmol, 3.0 eq), and methanesulfonic anhydride (23.83 g, 136.82 mmol, 2.0 eq). The mixture was allowed to react for 0.5 hours and then returned to room temperature for 2.5 hours. The product was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to afford (2,2-dimethyl-1,3-dioxan-5-yl)methyl methanesulfonate (13.70 g, 89.30% yield).

[0140] Step 2: Synthesis of Compounds 1-5

[0141] Adipic acid (12.58 g, 86.06 mmol, 2.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16.50 g, 86.06 mmol, 2.0 eq), N,N-diisopropylethylamine (16.68 g, 129.09 mmol, 3.0 eq), and 4-dimethylaminopyridine (2.63 g, 21.52 mmol, 0.5 eq) were added to 10 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, n-heptanol (5.0 g, 43.03 mmol, 1.0 eq) was added. The reaction mixture was allowed to react at room temperature for another 2.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain 6-(heptyloxy)-6-oxopropanoic acid (4.80 g, 45.7% yield).

[0142] Step 3: Synthesis of Compound 1-3

[0143] At room temperature, the compound methanesulfonic acid-(2,2-dimethyl-1,3-dioxane-5-yl)methyl ester (12.92g, 57.60mmol, 3.0eq), 3-(diethylamino)propan-1-amine (2.50g, 19.20mmol, 1.0eq), potassium carbonate (7.96g, 57.60mmol, 3.0eq), potassium iodide (3.19g, 19.20mmol, 1.0eq) were added to 100 ml of acetonitrile, protected by nitrogen, heated to 90 degrees Celsius, and reacted for 24 hours. The reaction solution was concentrated, diluted with water, extracted three times with dichloromethane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 1-(2,2-dimethyl-1,3-dioxane-5-yl)-2-[(2,2-dimethyl-1,3-dioxane-5-yl)methyl]-6-ethyl-2,6-diazaoctane (2.40 g, yield 32.3%).

[0144] Step 4: Synthesis of Compounds 1-4

[0145] Compound 1-(2,2-dimethyl-1,3-dioxane-5-yl)-2-[(2,2-dimethyl-1,3-dioxane-5-yl)methyl]-6-ethyl-2,6-diazaoctane (2.40 g, 6.21 mmol, 1.0 eq) and p-toluenesulfonic acid (3.21 mg, 18.63 mmol, 3.0 eq) were added to 30 ml of methanol and reacted at room temperature for two hours. The solvent was removed by concentration under reduced pressure to obtain compound 2-{6-ethyl-2-[3-hydroxy-2-(hydroxymethyl)propyl]-2,6-diazaoctan-1-yl}propane-1,3-diol (1.80 g, yield 94.6%).

[0146] Step 5: Synthesis of Compound 1

[0147] Compound 6-(heptyloxy)-6-oxohexanoic acid (1.92 g, 7.84 mmol, 8.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.50 g, 7.84 mmol, 8.0 eq), N,N-diisopropylethylamine (1.27 g, 9.80 mmol, 10.0 eq), and 4-dimethylaminopyridine (0.12 g, 0.98 mmol, 1.0 eq) were added to 10 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, compound 1-4 (300.0 mg, 0.98 mmol, 1.0 eq) was added. The reaction mixture was allowed to react at room temperature for 15.5 hours, then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the compound 6-({4-[3-(diethylamino)propyl]-2,6-bis(3,8-dioxyidene-2,9-dioxahexadecan-1-yl)-9,14-dioxyidene-4-aza-8,15-dioxadocosan-1-yl}oxy)-6-oxyidene hexanoic acid heptyl ester (296.9 mg, 25.0% yield). MS: m / z [M+H] + =1212.7. 1 H NMR(400MHz, CDCl3) δ4.13(dd,J=10.9,4.1Hz,4H),4.09-3.91(m,10H),2.50(dd,J=14.0,6.9Hz,5H),2.36(dd,J=20.8,13.5H z, 20H), 2.13 (s, 3H), 1.60 (dd, J = 24.6, 18.0Hz, 26H), 1.29 (d, J = 10.3Hz, 34H), 1.00 (t, J = 7.0Hz, 6H), 0.87 (d, J = 6.9Hz, 14H).

[0148] Example 2: 6-{[(20Z)-4-[3-(diethylamino)propyl]-2,6-bis[(14Z)-3,8-dioxyylidene-2,9-dioxaheptadecan-14-en-1-yl]-9,14-dioxyylidene-4-aza-8,15-dioxatricos-20-en-1-yl]oxy}-6-oxyylidenehexanoic acid (5Z)-oct-5-en-1-yl ester

[0149] Example 2 was synthesized using the method of Example 1, except that acid 1-5 in Example 1 was replaced with 6-{[(5Z)-oct-5-enyl]oxy}-6-oxyidenehexanoic acid. 155.3 mg, 12.6% yield. MS: m / z [M+H] + =1230.7. 1H NMR (400MHz, CDCl3) δ5.35(d,J=23.3Hz,8H),4.08(dd,J=18.2,11.5Hz,14H),2.51(d,J=7.1Hz,4H),2.37(dd,J=20.8,14.0Hz ,20H),2.05(dt,J=14.3,7.2Hz,16H),1.64(d,J=14.0Hz,26H),1.47-1.35(m,8H),1.26(s,6H),0.98(dt,J=15.0,7.3Hz,20H).

[0150] Example 3: 6-{[(17Z)-4-[3-(diethylamino)propyl]-2,6-bis[(11Z)-3,8-dioxyylidene-2,9-dioxaoctadec-11-en-1-yl]-9,14-dioxyylidene-4-aza-8,15-dioxa-tetracos-17-en-1-yl]oxy}-6-oxyylidenehexanoic acid-(2Z)-non-2-en-1-yl ester

[0151] Example 3 was synthesized using the method of Example 1, except that acid 1-5 in Example 1 was replaced with 6-{[(2Z)-non-2-enyl]oxy}-6-oxyylidenehexanoic acid. 292.3 mg, 22.7% yield. MS: m / z [M+H] + =1316.8. 1 H NMR (400MHz, CDCl3) δ5.71-5.42(m,8H),4.62(d,J=6.6Hz,8H),4.20-3.97(m,8H),2.42(ddd,J=22.3,14.5,5.4Hz,26H),2.10(dd ,J=13.7,6.7Hz,10H),1.81(s,4H),1.65(s,14H),1.54(s,4H),1.41-1.20(m,30H),1.00(t,J=7.1Hz,6H),0.88(d,J=6.9Hz,12H).

[0152] Example 4: 2-Butyloctanoate-22-butyl-8,12-bis(10-butyl-3,9-dioxyidene-2,8-dioxahexadecan-1-yl)-10-[3-(diethylamino)propyl]-5,15,21-trioxyidene-10-aza-6,14,20-trioxaoctacosan-1-yl ester

[0153] Example 4 was synthesized using the method of Example 1, except that acid 1-5 in Example 1 was replaced with 5-[(2-butyl-1-oxyoctylene)oxy]pentanoic acid. 185.5 mg, 34.1% yield. MS: m / z [M+H] + =1436.13. 1 H NMR (400MHz, CDCl3) δ4.15-4.01(m,16H),2.52-2.47(m,5H),2.44-2.26(m,20H),2.18-2.10(m,2H),1.80-1.74(m,2H), 1.72-1.63(m,15H),1.59-1.50(m,11H),1.49-1.36(m,9H),1.34-1.16(m,44H),1.01-0.98(m,6H),0.89-0.85(m,24H).

[0154] Example 5: Octanoic acid-10-[3-(diethylamino)propyl]-8,12-bis(3,9-dioxyylidene-2,8-dioxahexadecan-1-yl)-5,15,21-trioxyylidene-10-aza-6,14,20-trioxaoctacosan-1-yl ester

[0155] Example 5 was synthesized using the method of Example 1, except that acid 1-5 in Example 1 was replaced with 5-[(1-oxyoctylene)oxy]pentanoic acid. 98.6 mg, 30.3% yield. MS: m / z [M+H] + =1211.88. 1 H NMR (400MHz, CDCl3) δ4.15-4.01(m,16H),2.52-2.47(m,4H),2.43-2.26(m,15H),2.16-2. 10(m,2H),1.72-1.54(m,29H),1.35-1.22(m,31H),1.02-0.98(m,6H),0.88-0.85(m,12H).

[0156] Example 6. Preparation, Characterization, and In Vivo Editing Experimental Evaluation of Lipid Nanoparticles

[0157] 1. Animal Experiment Design

[0158] mRNA and sgRNA delivery experiments of the base editor ABE8e targeting PCSK9

[0159] Cholesterol in the blood is primarily synthesized by the liver, which is also the primary 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 for excretion through the intestines. PCSK9 is a protease synthesized by the liver that binds to the LDL receptor, promoting its entry into hepatocytes. This leads to its degradation in the lysosomes, reducing its number. Therefore, inhibiting the activity of the enzyme PCSK9 can increase the number of LDLRs, thereby enhancing cholesterol uptake and breakdown. Basic and clinical studies have shown that the PCSK9 gene is an effective target for the treatment of hyperlipidemia and atherosclerosis. Figure 1 shows the changes in LDL receptor number and the resulting changes in cholesterol metabolism caused by editing specific sites in the PCSK9 gene before and after editing.

[0160] The strategy for delivering PCSK9 gene editing to mouse liver cells is shown in Figure 1. The main process is as follows: mRNA and sgRNA encoding ABE8e are delivered to mouse liver cells via intravenous injection using the prepared lipid nanoparticles. Under the action of ABE8e and sgRNA, mutations are introduced into the PCSK9 gene, and base mutations from A to G are achieved at specific sites. The editing efficiency is calculated by sequencing.

[0161] The specific experimental design is as follows:

[0162] 1.1 Selection of appropriate mutation sites and editing design

[0163] The single-base editor ABE8e achieves precise A-to-G base substitutions without the need for a donor template and without causing DSBs. Based on this, the first exon of the PCSK9 gene was selected as a mutation site for screening. The mRNA encoding the single-base editor ABE8e and sgRNA were co-delivered into animals via lipid nanoparticles. The mRNA encoding the base editor ABE8e is translated into protein in the cytoplasm, forming a complex with the sgRNA before entering the cell nucleus. Under the guidance of the sgRNA, the base editor ABE8e targets the splice donor site in the first exon of the PCSK9 gene, deaminating the adenine (A) on the first exon template chain to inosine (I). I is read and replicated as G at the DNA level, ultimately achieving the A-to-G substitution, thereby destroying the splice donor site and prematurely terminating the PCSK9 gene reading frame.

[0164] 1.2 Preparation of mRNA and sgRNA for base editor ABE8e

[0165] The sequences of the first exon and the first intron of the mouse PCSK9 gene (NCBI Gene ID: 100102) were selected as the targeting region, and the target sequence PCSK9-sgRNA for single-base editing of the PCSK9 gene was determined.

[0166] By analyzing the sequence spanning the first exon and intron of the PCSK9 gene, an sgRNA targeting the region was designed: PCSK9-sgRNA (synthesized by Nanjing GenScript). The PCSK9-sgRNA sequence is:

[0167] PCSK9-sgRNA: 5'-CCCATACCTTGGAGCAACGG-3' (SEQ ID NO: 1);

[0168] sgRNAs were designed and oligonucleotides 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 pre-set protocol (95°C, 5 min; 95°C-85°C at -2°C / s; 85°C-25°C at -0.1°C / s; hold at 4°C). The annealed products were ligated into the lenti U6-sgRNA / EF1a-mCherry vector (Addgene, Plasmid, #114199) linearized with BbsI (NEB, R3539S).

[0169] Among them, the system used in the construction of sgRNA plasmid is as follows:

[0170] The linearization system of lenti U6-sgRNA / EF1a-mCherry vector is as follows: 3 μg of vector; 6 μL of buffer (NEB: R0539L); 2 μL of BbsI; fill to 60 μL with ddH2O, and digest at 37°C overnight.

[0171] The sgRNA annealing product and linearized vector ligation system is as follows: T4 ligase buffer (NEB: M0202L) 1 μL, linearized vector 20 ng, annealed oligo fragment (10 μM) 5 μL, T4 ligase (NEB: M0202L) 0.5 μL, ddH2O is added to 10 μL, and ligation is carried out at 16°C overnight.

[0172] The ligated vector was transformed into Escherichia coli DH5α competent cells (Weidi Biotech, DL1001). The specific process is as follows: Remove the DH5α competent cells from -80°C and quickly place them on ice. After 5 minutes, allow the bacterial mass to thaw. Add the ligated product and gently mix by fingering the bottom of the centrifuge tube. Let it rest on ice for 25 minutes. Heat shock the tube in a 42°C water bath for 45 seconds, quickly return it to ice, and let it rest for 2 minutes. Add 700 μl of sterile LB medium without antibiotics to the centrifuge tube, mix thoroughly, and then recover at 37°C, 200 rpm, for 60 minutes. Harvest the cells by centrifugation at 5000 rpm for one minute. Retain approximately 100 μl of the supernatant, gently pipette to resuspend the bacterial mass, and spread it onto LB medium supplemented with Amp antibiotics. Incubate the plate upside down at 37°C in a humidified incubator overnight. Single colonies were picked, and after sequencing confirmation, positive clones were shaken out and the plasmid (TIANGEN: DP120-01) was extracted and the concentration was determined. The cells were then stored in a -20°C refrigerator until further use.

[0173] The base editor ABE8e used in this experiment is an 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). Plasmid ABE8e (Plasmid #138489) was purchased from Addgene, and ABE8e mRNA was expressed and purified in the laboratory for future use.

[0174] 2. Lipid nanoparticles were prepared by mixing ionizable lipid or the compound of the present invention / DSPC / cholesterol / PEG-lipid at a molar ratio of 50:10:38.5:1.5.

[0175] 2.1 Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated as MC3) and Compound 1 to Compound 10 of the present invention were dissolved in anhydrous ethanol with DSPC, cholesterol, and PEG-DMG according to the above molar ratios.

[0176] 2.2 Ethanol solutions of different lipid carriers were mixed with mRNA buffer at a 1:3 (vol / vol) ratio (the total lipid to mRNA mass ratio was 40:1, and the sgRNA:ABE8e mRNA (w / w) ratio was 1:1). Nucleic acid lipid nanoparticles 1-10 were prepared using a microfluidic nanomedicine manufacturing system (NanoAssemblr Ignite, Canada) at a flow rate of 12 ml / min. The resulting nucleic acid lipid nanoparticles were immediately diluted 40 times the volume into 1× DPBS buffer. The diluted nucleic acid lipid nanoparticle solution was concentrated to the desired volume by ultracentrifugation. The diluted solution was then used for DLS particle size measurement and encapsulation efficiency testing.

[0177] 2.3 The particle size and polydispersity index of the lipid nanoparticles were measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK) in 173° backscatter detection mode. 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.

[0178] Table 2 Characterization of nanolipid particles

[0179] 3 In vivo editing experimental evaluation

[0180] 3.1 Lipid nanoparticles containing the compound of the present invention (see Table 2) encapsulating the mRNA and sgRNA encoding the base editor ABE8e were systemically administered to 6-7 week old C57BL / 6 female mice (purchased from Jiangsu Jicui Pharmaceutical Kang Co., Ltd.) at a dose of 0.2 mg / kg via tail vein injection. Lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, abbreviated as MC3) encapsulating the mRNA and sgRNA of the base editor ABE8e were similarly administered to mice of the same age and sex as a positive control. In addition, PBS buffer was also injected into the tail vein of mice of the same age and sex in a similar manner as a negative control.

[0181] 3.2 Editing efficiency detection

[0182] The editing efficiency was tested one week after the mice were given the drug. The liver tissue was taken after the mice were killed, and the genome was extracted after lysis, and the efficiency was analyzed by deep sequencing.

[0183] The deep sequencing steps are as follows:

[0184] (1) Design primers according to the target gene location, see Table 3 for details.

[0185] Table 3 Design of primers targeting PCSK9 gene

[0186] (2) Editing efficiency detection.

[0187] The PCR program was as follows: 94°C for 2 minutes; 34 cycles of 98°C for 10 seconds, 60°C for 30 seconds, and 68°C for 20 seconds; and 68°C for 5 minutes. After PCR, gel electrophoresis was performed to confirm the amplification product by selecting a single band of appropriate size. The resulting PCR product was then sent to Nanjing GenScript for sequencing.

[0188] (3) The deep sequencing results were analyzed using Crispresso software to perform site-specific analysis and calculate the editing efficiency. The calculation results are shown in Table 4, and the editing efficiency corresponding to each lipid nanoparticle can be seen in Figure 2.

[0189] Table 4 Evaluation of in vivo editing efficiency

[0190] As shown in Table 4, the ionizable lipid compound used in the present invention can effectively deliver drugs such as nucleic acid molecules and small molecule compounds; and by comparison, the lipid nanoparticles of the present invention have a better particle size distribution, a high encapsulation efficiency, and a delivery effect that is significantly better than that of the comparative lipid nanoparticles, and can meet the needs of in vivo delivery.

[0191] The descriptions presented in the above exemplary embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to be exhaustive or to limit the present invention to the precise forms described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described to explain the specific principles of the present invention and its practical applications, so that other persons skilled in the art can easily understand, implement and utilize the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be defined by the scope of the claims and their equivalents.

Claims

1. A compound of formula I or a pharmaceutically acceptable form thereof, In the formula, G 1 C 1-10 Alkylene; R 1 , R 2 Each independently is C 1-6 alkyl; Optionally, R 1 , R 2 Together with the connected N, it forms a 3-8 membered heterocyclic group, or, R 1 , R 2 Any one of them and G 1 Any carbon atom in is directly connected to form a 3-8 membered heterocyclic group; L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 Each is independently selected from an ester group, an amide group, a carbonate group, a carbamate group, a mercaptoformate group, a urea group, a phosphate group or none; R 3 , R 5 , R 7 , R 9 Each independently selected from C 1-20 Straight chain alkylene, C 3-20 Branched alkylene, C 1-20 Straight chain heteroatom-containing alkylene, C 3-20 Branched chain containing heteroatom alkylene, C 2-20 Straight chain alkenylene, C 3-20 Branched alkenylene, C 2-20 Straight chain heteroatom-containing alkenylene, C 3-20 Branched chain containing heteroatom alkenylene, C 2-20 Straight chain alkynylene, C 3-20 Branched chain alkynylene, C 2-20 Straight chain heteroatom-containing alkynylene, C 3-20 The branched chain contains heteroatom alkynylene or none; R 4 , R 6 , R 8 , R 10 Each independently selected from C 1-20 Straight chain alkyl, C 2-20 Straight chain alkenyl, C 2-20 Straight chain alkynyl, C 1-20 Straight chain heteroatom-containing alkyl, C 3-20 Branched alkyl, C 3-20 Branched chain containing heteroatom alkyl, C 3-10 Cycloalkyl, C 4-10 Bridged cycloalkyl or none.

2. The compound according to claim 1 or a pharmaceutically acceptable form thereof, in: G 1 for wherein m is selected from an integer of 1-6.

3. A compound according to any one of claims 1 to 2 or a pharmaceutically acceptable form thereof, in: R 1 , R 2 are each independently methyl, ethyl or propyl; or, R 1 , R 2 Together with the connected nitrogen, it forms a 5-membered or 6-membered heterocyclic group.

4. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable form thereof, in: L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 Each independently selected from Or none.

5. A compound according to any one of claims 1 to 4 or a pharmaceutically acceptable form thereof, in: R 3 , R 5 , R 7 , R 9 Each independently wherein X is O, S, Se, SS, Se-Se or none; R 11 , R 12 , R 13 , R 14 Each independently is H or C 1 -C 8 A straight chain alkyl group; m and o are each independently an integer selected from 1-10.

6. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable form thereof, wherein R 4 , R 6 , R 8 , R 10 Each independently Where Y is or none; R 15 , R 16 , R 17 , R 18 , R 19 Each independently is H, C 1 -C 10 Straight chain alkyl, C 3-10 Cycloalkyl or C 4-10 bridged cycloalkyl; p and q are each independently an integer selected from 0-10.

7. A compound according to any one of claims 1 to 6 or a pharmaceutically acceptable form thereof, in, The compound is selected from one or more of Compound 1 to Compound 35; preferably, the pharmaceutically acceptable form is selected from a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug.

8. Use of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof in the preparation of liposome nanocarriers.

9. A lipid carrier comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof; Preferably, the lipid carrier comprises a first lipid compound and a second lipid compound, in, The first lipid compound comprises a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof and optionally other lipid compounds, and the second lipid compound comprises one or a combination of two or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids.

10. A nucleic acid lipid nanoparticle composition comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof or a lipid carrier according to claim 9, and a therapeutic agent and / or a preventive agent.

11. The nucleic acid lipid nanoparticle composition of claim 10, wherein the therapeutic agent and / or prophylactic agent comprises RNA, DNA, antisense nucleic acid, aptamer, nuclease, immunostimulatory nucleic acid or PNA component; Preferably, the antisense nucleic acid is an antisense oligonucleic acid; Preferably, the RNA comprises one or more of mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator or shRNA; Preferably, the DNA comprises a plasmid; Preferably, the RNA component comprises mRNA encoding an RNA-guided nuclease or encoding a base editor, and / or gRNA; Preferably, the nuclease is selected from Cas9, Cas12, Cas13, IscB, TnpB, IsrB and homologs thereof; Preferably, the RNA component comprises modified nucleotides.

12. A pharmaceutical preparation comprising: a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or a lipid carrier according to claim 9, or a nucleic acid lipid nanoparticle composition according to claim 10 or 11, and a pharmaceutically acceptable excipient, carrier or diluent.

13. A method for delivering a therapeutic and / or prophylactic agent to a subject's cells, the method comprising administering to the subject a compound according to any one of claims 1-7 or a pharmaceutically acceptable form thereof, or a lipid carrier according to claim 9, or a nucleic acid lipid nanoparticle composition according to claim 10 or 11, or a pharmaceutical preparation according to claim 12, wherein the administration comprises contacting the subject's cells with the compound or a pharmaceutically acceptable form thereof, or the lipid carrier, or the nucleic acid lipid nanoparticle composition, or the pharmaceutical preparation, thereby delivering the therapeutic and / or prophylactic agent to the subject's cells.

14. A method for producing a target protein or target polypeptide in a subject cell, It is characterized in that The method comprises contacting the subject's cells with the composition of claim 10, wherein the therapeutic agent or prophylactic agent is mRNA, and wherein the mRNA encodes a protein or polypeptide of interest, whereby the mRNA can be translated in the cell to produce a protein or polypeptide of interest.

15. Use of a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or a lipid carrier according to claim 9, or a nucleic acid lipid nanoparticle composition according to claim 10 or 11, or a pharmaceutical preparation according to claim 12 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

16. The use according to claim 15, wherein the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation is used for treating or preventing a disease or disorder in a subject in need thereof.

17. The use according to claim 16, wherein the subject is a mammal or a human, preferably, the subject is a human.

18. The use according to claim 16 or 17, wherein the disease or condition is selected from metabolic diseases, hereditary diseases, cancer, cardiovascular diseases and infectious diseases; preferably, the metabolic diseases include familial hypercholesterolemia (FH), the hereditary diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluria (PH1) and hereditary angioedema (HAE), and the infectious diseases include hepatitis B.

19. A method for preventing, ameliorating or treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a compound as described in any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or a lipid carrier as described in claim 9, or a nucleic acid lipid nanoparticle composition as described in claim 10 or 11, or a pharmaceutical formulation as described in claim 12.

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