Quinolinyl-containing cationic lipid compound, nano-composition thereof and use thereof

Abstract

A quinolinyl-containing cationic lipid compound, a nano-composition thereof and the use thereof. Disclosed in the present application are a quinolinyl-containing cationic lipid compound having a structure shown as formula I, and a pharmaceutically acceptable salt or stereoisomer thereof. Also disclosed in the present application is a nano-composition comprising the compound or the salt or isomer thereof. The nano-composition disclosed in the present application can achieve efficient organ-selective delivery of mRNAs, and has low cytotoxicity and in vivo toxicity and high biocompatibility, and thus has great application prospects in the delivery of nucleic acid drugs, especially the delivery of mRNA drugs.

Description

A quinoline-containing cationic lipid compound, its nanocomposition, and its applications. Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a quinoline-containing cationic lipid compound, its nanocomposition, and its applications. Background Technology

[0002] Gene therapy improves diseases caused by gene defects by regulating the expression or silencing of specific genes, demonstrating great potential in treating hereditary diseases and many other conditions. Current technologies primarily utilize nucleic acid drugs to regulate genes, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), and messenger RNA (mRNA). siRNA utilizes RNA interference (RNAi) to specifically degrade target mRNA, blocking the synthesis of specific proteins. The mRNA is then introduced into host cells to express pathogens or tumor-associated antigens, thereby inducing an immune response and achieving disease treatment. However, nucleic acid drugs still face several challenges in clinical application. First, nucleic acid drugs are easily degraded by nucleases in vivo, exhibiting poor stability, and their negative charge makes it difficult to effectively penetrate cell membranes, limiting their entry into cells to exert their effects. Second, nucleic acid drugs may trigger off-target effects and immune responses, leading to potential toxic side effects. Therefore, there is an urgent need to develop effective delivery vectors to protect nucleic acid drugs from degradation, improve their intracellular delivery efficiency, and reduce toxic side effects.

[0003] Existing delivery vectors for nucleic acid drugs are mainly divided into viral vectors and non-viral vectors. Although viral vectors have high delivery efficiency, their inherent immunogenicity poses safety risks. In contrast, lipid nanoparticles (LNPs) among non-viral vectors are widely used for nucleic acid drug delivery. LNPs typically consist of four components: cationic lipids, phospholipids, cholesterol, and polyethylene glycol lipids. Among them, cationic lipids are the key component for nucleic acid drug delivery. The structure of cationic lipids typically consists of three parts: a positively charged head, a hydrophobic tail, and an intermediate linker. The positively charged head can bind to nucleic acid molecules (which are negatively charged), promoting efficient encapsulation and intracellular delivery of nucleic acids. The intermediate linker usually affects the stability and degradability of the lipid and is used to connect the head and tail. The hydrophobic tail usually consists of one or more fatty acid chains, which determine the physicochemical properties of the lipid.

[0004] Currently, several cationic lipids have been approved by the FDA for the preparation of LNPs, including DLin-MC3-DMA (for...). ), SM-102 (for ) and ALC-0315 (for Although existing technologies have reported the structures and applications of various cationic lipids, further optimization of their structures is needed to improve their delivery efficiency and safety, as well as their in vivo distribution. However, most systemically administered LNPs primarily target the liver, which presents significant limitations in the treatment of regional diseases and immunotherapy.

[0005] Therefore, it is crucial to research new LNPs to enable more organ-targeting delivery systems. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a quinoline-containing cationic lipid compound, its nanocomposition, and its application.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] Technical Topic 1

[0009] A cationic lipid compound containing quinoline, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, having the structure shown in Formula I:

[0010] Among them, R b Selected from H, C1-C40 alkyl, -C(O)-C1-C40 alkyl;

[0011] R a1 R a2 Independently, they are H, F, Cl, Br, and I;

[0012] R c1 R c2 Independently H, C1-C8 alkyl;

[0013] A is selected from -CH2-, -C(O)-, C1-C10 alkylene-NHC(O)-;

[0014] R d Selected from

[0015] R 1 Selected from C1-C40 alkyl, C2-C40 alkenyl, or C2-C40 alkynyl;

[0016] R 2 Selected from C1-C40 alkyl, C2-C40 alkenyl, or C2-C40 alkynyl;

[0017] R 3 It is -C1-C10 alkylene-M-C1-C40 alkyl;

[0018] R 4It is -C1-C10 alkylene-M-C1-C40 alkyl;

[0019] L stands for -SS-;

[0020] M is -C(O)-O- or -OC(O)-;

[0021] R 3 R 4 The C1-C40 alkyl group attached to M is independently a single-chain alkyl group or a double-chain alkyl group;

[0022] m 1 m 2 n can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 independently;

[0023] o can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0024] As a further improvement to the present invention, its structure is shown in Formula II:

[0025] Among them, R b Selected from H, C1-C10 alkyl, -C(O)-C1-C10 alkyl;

[0026] R a1 R a2 Independently, they are H, F, Cl, Br, and I;

[0027] R c1 R c2 Independently, it is H or a C1-C5 alkyl group;

[0028] A is selected from -CH2-, -C(O)-, C1-C6 alkylene-NHC(O)-;

[0029] R d Selected from

[0030] R 1 Selected from C1-C30 alkyl, C2-C30 alkenyl, or C2-C30 alkynyl;

[0031] R 2 Selected from C1-C30 alkyl, C2-C30 alkenyl, or C2-C30 alkynyl;

[0032] R 3 It is -C1-C10 alkylene-M-C1-C30 alkyl;

[0033] R 4 It is -C1-C10 alkylene-M-C1-C30 alkyl;

[0034] L stands for -SS-;

[0035] M is -C(O)-O- or -OC(O)-;

[0036] R 3 R 4 The C1-C30 alkyl group attached to M is independently a single-chain alkyl group or a double-chain alkyl group;

[0037] m 1 m 2 n can be 1, 2, 3, 4, 5, 6, 7 or 8 independently;

[0038] o can be selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0039] As a further improvement to the present invention, its structure is shown in Formula II:

[0040] R b Selected from H, C1-C5 alkyl, -C(O)-C1-C5 alkyl;

[0041] R a1 R a2 Independently, they are H, F, Cl, Br, and I;

[0042] R c1 R c2 Independently, it is H or a C1-C3 alkyl group;

[0043] A is selected from -CH2-, -C(O)-, and -(CH2). q NHC(O)-;

[0044] R d Selected from

[0045] R 1 Selected from C6-C24 alkyl, C6-C24 alkenyl, or C6-C24 alkynyl;

[0046] R 2 Selected from C6-C24 alkyl, C4-C24 alkenyl, or C6-C24 alkynyl;

[0047] R 3 It is -C1-C10 alkylene-M-C1-C30 alkyl;

[0048] R 4 It is -C1-C10 alkylene-M-C1-C30 alkyl;

[0049] L stands for -SS-;

[0050] M is -C(O)-O- or -OC(O)-;

[0051] R 3 R 4 The C1-C30 alkyl group attached to M is independently a single-chain alkyl group or a double-chain alkyl group;

[0052] m 1 m 2 n and q are independently 1, 2, 3, 4, 5 or 6;

[0053] o can be selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0054] As a further improvement of the present invention, the structure of the quinoline-containing cationic lipid compound is as follows:

[0055] Technical Theme Two

[0056] A lipid nanocomposition comprising a lipid component, said lipid component comprising a compound or a pharmaceutically acceptable salt thereof, as described in any one of the technical subjects.

[0057] As a further improvement of the present invention, the lipid component further comprises neutral phospholipids, cholesterol, and PEG lipids.

[0058] The neutral phospholipids are selected from one or more of the following compounds: distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), soybean lecithin (SL), egg yolk lecithin (EPC), hydrogenated soybean lecithin (HSPC), hydrogenated egg yolk lecithin (HEPC), phosphatidic acid, and sphingomyelin;

[0059] The PEG lipid is selected from one or more of the following compounds: PEG-modified distearylphosphatidylethanolamine (DSPE-PEG), PEG-modified dimyristoylglycerol (DMG-PEG), PEG-modified dipalmitoylphosphatidylethanolamine (DPPE-PEG), PEG-modified dimyristoylphosphatidylethanolamine (DMPE-PEG), PEG-modified dilauroylphosphatidylethanolamine (DLPE-PEG), PEG-modified ceramide (Ceramide-PEG), and mPEG-modified bis(tetradecyl)acetamide (mPEG-DTA).

[0060] As a further improvement of the present invention, the molecular weight of the PEG or mPEG is 1000-5000 Da.

[0061] As a further improvement of the present invention, the PEG lipid is DMG-PEG 2000.

[0062] As a further improvement of the present invention, the nanocomposition further includes a therapeutic agent and / or a preventive agent, wherein the therapeutic agent and / or preventive agent is a nucleic acid;

[0063] As a further improvement of the present invention, the nucleic acid is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA, snRNA, tRNA, rRNA, circRNA, saRNA, antisense oligonucleotide, lncRNA, ssDNA, mRNA, nucleic acid aptamers or ribozymes.

[0064] As a further improvement of the present invention, the nucleic acid is mRNA.

[0065] Technical Theme 3

[0066] A pharmaceutical composition comprising the lipid nanocomposition described in Technical Subject 2 and a pharmaceutically acceptable carrier.

[0067] Technical Theme 4

[0068] The use of the compound described in Technical Subject 1 in the preparation of a medicament for targeting the liver and spleen to treat infectious diseases, genetic defects, autoimmune diseases or tumors.

[0069] The beneficial effects of adopting the above technical solution are as follows:

[0070] The cationic lipids provided by this invention are positively charged in an acidic environment. This property allows them to bind tightly to nucleic acid drugs, improving their stability and delivery efficiency. The nanocomposition obtained by encapsulating nucleic acid drugs with cationic lipids has high encapsulation efficiency and good stability.

[0071] The nanocomposition disclosed in this application does not require the introduction of targeting ligands. It can achieve targeted delivery of nucleic acid drugs simply by screening compounds and formulations, preferentially targeting the spleen, which is beneficial for immune activation and disease treatment. Moreover, its preparation process is controllable, highly reproducible, suitable for the delivery of different types and chain lengths of nucleic acid drugs, and suitable for large-scale production. Attached Figure Description

[0072] Figure 1 shows the mRNA encapsulation efficiency of the lipid nanoparticles in the examples;

[0073] Figure 2 shows the stability test results of lipid nanoparticles in the examples;

[0074] Figure 3 shows the effect of lipid nanoparticles on the in vitro survival rate of DC2.4 cells in the examples. The 3-0 and DLin-MC3-MDA are lipid nanoparticles based on lipid 3-0 or DLin-MC3-MDA with EGFP mRNA as the reporter gene.

[0075] Figure 4 shows the effect of lipid nanoparticles on the in vitro survival rate of 293T cells in the examples. The 3-0 and DLin-MC3-MDA are lipid nanoparticles based on lipid 3-0 or DLin-MC3-MDA with EGFP mRNA as the reporter gene.

[0076] Figure 5 shows the expression results of lipid nanoparticles in mice in the example. The 3-0 and DLin-MC3-MDA are lipid nanoparticles based on lipid 3-0 or DLin-MC3-MDA, with Luc mRNA as the reporter gene.

[0077] Figure 6 shows the expression results of lipid nanoparticles in mouse organs in the example. The 3-0 and DLin-MC3-MDA are lipid nanoparticles based on lipid 3-0 or DLin-MC3-MDA, with Luc mRNA as the reporter gene.

[0078] Figure 7 shows the quantitative results of bioluminescence in ex vivo tissues after administration of lipid nanoparticles in the example. The 2-0, 3-0, 5-0 and DLin-MC3-MDA are lipid nanoparticles based on lipid 2-0, lipid 3-0, lipid 5-0 or DLin-MC3-MDA, with Luc mRNA as the reporter gene.

[0079] Figure 8 shows the changes in animal body weight after administration of lipid nanoparticles in the example. The 3-0 and DLin-MC3-MDA are lipid nanoparticles based on lipid 3-0 or DLin-MC3-MDA, with OVA mRNA as the reporter gene.

[0080] Figure 9 shows the blood biochemical assay results after in vivo administration of lipid nanoparticles in the example. The 3-0 and DLin-MC3-MDA are lipid nanoparticles based on lipid 3-0 or DLin-MC3-MDA with OVA mRNA as the reporter gene. ALT is alanine aminotransferase, AST is aspartate aminotransferase, BUN / UREA is blood urea nitrogen / urea, CR (CREA) is creatinine, and UA is uric acid. Detailed Implementation

[0081] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0082] Terms and Definitions

[0083] It should be noted that the structural and chemical formula descriptions in the embodiments or implementations of this invention are intended to cover all alternative, modified, and equivalent technical solutions, all of which should be considered within the scope of this invention. Those skilled in the art should understand that many similar or equivalent methods and materials can be used to implement this invention; therefore, this invention is not limited to the specific methods and materials described. In the event of any discrepancies or contradictions between the cited documents, patents, or similar materials and this application (including but not limited to terminology definitions, terminology application, and described techniques), the content of this invention shall prevail.

[0084] In this document, the minimum and maximum carbon atom content in hydrocarbon groups are indicated by prefixes, for example, the prefix Ca-Cb indicates the presence of "a" to "b" carbon atoms. Exemplarily, "C1-Cn" refers to a straight or branched saturated / unsaturated carbon chain containing 1, 2, 3, 4, 5, ..., or n carbon atoms; further understood, "C1-Cn" should be interpreted as any subrange included, such as C1-C40, C2-C40, C6-C30, C1-C10, C1-C8, C1-C6, C1-C5, C1-C3, etc.

[0085] As used herein, “alkyl” means a fully saturated (without double or triple bonds) straight-chain or branched hydrocarbon chain group. An alkyl group can have 1 to 40 carbon atoms (wherever it appears herein, a numerical range of “1 to 40” refers to each integer within the given range; for example, “1 to 40 carbon atoms” means that an alkyl group can consist of 1, 2, 3, etc., up to and including 40 carbon atoms, although this definition also covers the occurrence of the term “alkyl” without specifying a numerical range). An alkyl group can also be a medium-sized alkyl group having 1 to 10 carbon atoms, such as “C1-6”. An alkyl group can also be a lower alkyl group having 1 to 4 carbon atoms. The alkyl group of a compound can be specified as “C1-C4 alkyl”, “C1-4 alkyl”, or similar names. By way of example only, "C1-C4 alkyl" or "C1-4 alkyl" indicates that there are one to four carbon atoms in the alkyl chain, meaning the alkyl chain is selected from the group consisting of: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and hexyl.

[0086] As used herein, “alkenyl” refers to an alkyl group containing one or more double bonds in a straight-chain or branched hydrocarbon chain. If more than one double bond is present, the double bonds may be concatenated or unconcatenated. Alkenyl groups can have 2 to 40 carbon atoms (wherever it appears herein, a numerical range of “2 to 40” refers to each integer within the given range; for example, “2 to 40 carbon atoms” means that an alkenyl group can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to and including 40 carbon atoms). In alkenyl groups, stereochemically unspecified C=C double bonds (e.g., -CH=CHCH3) can be (E)- or (Z)- double bonds.

[0087] As used herein, “alkynyl” refers to an alkyl group containing one or more triple bonds in a straight or branched hydrocarbon chain. An alkynyl group can have 2 to 40 carbon atoms (whenever it appears herein, for example, “2 to 40” means every integer within a given range; for example, “2 to 40 carbon atoms” means that an alkynyl group can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to and including 40 carbon atoms).

[0088] The term "treatment" generally refers to the use of medication to improve, alleviate, or cure a disease state, meaning partial or complete relief, improvement, delay of its onset, inhibition of its progression, reduction of its severity, and / or reduction of the incidence of one or more of its symptoms or features. It can also refer to a specific infection, disease, symptom, and / or condition. For example, "treatment" of cancer can refer to inhibiting tumor survival, growth, and / or spread. To reduce risk, treatment can be administered to subjects who do not exhibit disease, symptom, and / or condition and / or to subjects who only exhibit early signs of disease, symptom, and / or condition. It can also refer to the pathological development of a disease, symptom, and / or condition.

[0089] The terms "therapeutic agent" or "preventive agent" refer to any pharmaceutical agent that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect and / or induces the desired biological and / or pharmacological action. Therapeutic agents are also referred to as "active agents" or "active components." In this invention, therapeutic agents can be nucleic acids, small molecule compounds, macromolecule compounds, peptides, immunomodulators, antigens or fragments thereof, vaccines, antitumor drugs, antibiotics, or mixtures thereof.

[0090] In this invention, the term "pharmaceutical acceptable" means that a substance or composition, when exposed to mammals, will not produce unreasonable toxicity, irritation, allergic reactions, or other adverse side effects.

[0091] In this invention, the term "pharmaceutically acceptable salt" refers to the organic and inorganic salts of the compounds of this invention. These salts not only retain the pharmacological activity of the compounds but also possess favorable pharmaceutical properties, such as enhanced solubility, stability, or bioavailability. These salts include, but are not limited to, organic acid salts such as acetates, citrates, fumarates, maleates, oxalates, malates, citrates, succinates, tartrates, lactates, camphor sulfonates, benzene sulfonates, p-toluene sulfonates, methanesulfonates, trifluoroacetates, trifluoromethanesulfonates, etc.; and inorganic acid salts such as hydrohalides, sulfates, phosphates, nitrates, etc. Furthermore, the active pharmaceutical ingredient can also form salts with amino acids such as glutamic acid or aspartic acid, i.e., glutamate or aspartate.

[0092] As used herein, the phrase “pharmaceuticalally acceptable excipient” means any component other than the compounds described herein (e.g., a medium capable of suspending, complexing, or dissolving an active compound) and is substantially non-toxic and non-inflammatory to patients. Excipients may include, for example: anti-adhesives, antioxidants, adhesives, coatings, anti-compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, flow enhancers (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydrating agents. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (diacid), calcium stearate, croscarmellose, croscarmellose, citric acid, croscarmellose, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, corn starch, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0093] In this invention, the compounds and their stereoisomers described are all within the scope of protection, including but not limited to enantiomers, diastereomers, and cis-trans isomers. Therefore, the compounds protected by this invention include not only all possible single stereoisomers, but also their optically active mixtures and racemates. Furthermore, those skilled in the art will understand that different stereoisomers may exhibit significant differences in biological activity, pharmacodynamics, and toxicity; therefore, selecting specific stereoisomers or combinations thereof may optimize therapeutic effects. Stereoisomers can be obtained by known chemical or physical methods, such as chiral catalysis, chiral synthesis, or chiral resolution by chromatographic or chemical methods. This invention also includes stereoisomers obtained by these methods and their pharmaceutically acceptable salts.

[0094] The term "cationic lipid" as used in this invention refers to lipids that exhibit a positive charge under specific pH conditions and can bind to negatively charged nucleic acid molecules through electrostatic interactions.

[0095] As used herein, a “lipid nanocomposition” is a composition comprising one or more lipids. Lipid nanocompositions typically have particle sizes on the order of micrometers or smaller and may comprise a lipid bilayer. Lipid nanocompositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, a lipid nanocomposition may be a liposome having a lipid bilayer with a diameter of 500 nm or smaller.

[0096] As used herein, "PEG lipid" or "PEGylated lipid" refers to lipids containing polyethylene glycol.

[0097] As used herein, "phospholipid" is a lipid comprising a phosphate ester moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipids may contain one or more (e.g., double or triple) bonds (e.g., one or more unsaturated bonds). Certain phospholipids can facilitate fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). Fusion of phospholipids with membranes can allow one or more elements of a lipid-containing composition to cross the membrane, thereby allowing, for example, the delivery of one or more elements to the cell.

[0098] As used herein, “encapsulation ratio” refers to the amount of therapeutic and / or preventive agents that are part of the lipid nanocomposition, relative to the total amount of therapeutic or preventive agents used in the preparation of the lipid nanocomposition. For example, if 97 mg of therapeutic and / or preventive agents are encapsulated in the lipid nanocomposition out of a total of 100 mg initially provided to the composition, the encapsulation ratio can be 97%. As used herein, “encapsulation” can mean complete, substantial, or partial encapsulation, closure, enclosure, or sealing.

[0099] As used herein, in the context of lipid nanocomposites, “particle size” or “average particle size” refers to the average diameter of the lipid nanocomposites.

[0100] The term "nucleic acid" refers to biological macromolecules composed of nucleotide units, primarily of two types: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They play crucial roles in the storage, transmission, and expression of genetic information. Nucleic acids include: messenger RNA (mRNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), small nuclear RNA (snRNA), transfer RNA (tRNA), ribosomes (rRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), antisense oligonucleotides (ASO), microRNA (miRNA), long non-coding RNA (lncRNA), small hairpin RNA (shRNA), single-stranded DNA (ssDNA), nucleic acid aptamers, and ribozymes. In some therapeutic regimens, the RNA used is mRNA.

[0101] In some implementations, the therapeutic or preventative agent is mRNA, and OVA (ovalbumin) is used as a model antigen to stimulate a specific immune response against melanoma.

[0102] In one embodiment, the lipid nanoparticles are administered via intravenous injection.

[0103] In one embodiment, the lipid nanoparticles are used to target the spleen.

[0104] The term "targeting" generally refers to the ability of a delivery system to deliver a drug to a predetermined target area to enhance therapeutic or diagnostic efficacy. In one implementation, "targeting the spleen" means that the drug exhibits significant enrichment in the spleen compared to other tissues, but this does not preclude distribution of the drug in other sites.

[0105] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than events that occur within a living organism (e.g., an animal, plant, or microorganism).

[0106] As used herein, the term "in vivo" refers to events that occur within an organism (such as an animal, plant, or microorganism, or its cells or tissues).

[0107] As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or its cells or tissues). Ex vivo events can occur in environments with minimal alteration from the natural (e.g., internal) environment.

[0108] In the embodiments of this patent, all reagents and instruments used in the experiments are conventional items in the art, and their functions and uses are known. The technical means involved are conventional operating methods commonly mastered by those skilled in the art, and experiments without special instructions are all conducted under normal temperature and pressure conditions. For operating steps without specified special requirements, the standard procedures in the product instructions of the reagents or instruments are followed to ensure the repeatability and reliability of the results.

[0109] DLin-MC3-DMA was purchased from Xiamen Sinobond Biotechnology Co., Ltd.

[0110] EGFP mRNA, Luc mRNA, OVA mRNA, and Cas9 mRNA were all purchased from APE×BIO.

[0111] Example 1: Synthesis of quinoline head-group compound A1:

[0112] At room temperature, N-Boc-N-methylethylenediamine (5 g, 28.7 mmol) and 4,7-dichloroquinoline (6.8 g, 34.4 mmol) were added to a 500 mL reaction flask, and then transferred to a preheated oil bath at 130 °C with stirring for 5 h. TLC showed that the reaction was complete. The reaction solution was cooled to room temperature, and the crude product was purified by silica gel column chromatography (eluent: DCM:MeOH = 20:1) to give a yellow-brown solid compound 3 (8.6 g, 89.2%).

[0113] Compound 3 (5 g, 14.9 mmol) was added to a 200 mL reaction flask at 0 °C, dissolved in dichloromethane (60 mL), and then trifluoroacetic acid (20 mL) was added dropwise. The mixture was stirred at room temperature for 5 h, and TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and deionized water (200 mL) was added to the concentrated system. The pH was adjusted to 10-12 with 1 N NaOH, and the mixture was extracted with dichloromethane (100 mL × 2). The organic layer was separated, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and the organic layer was concentrated under reduced pressure to obtain a pale yellow solid compound MAQ (3.2 g, 91.2%). ESI-HRMS: C 12 H 15 ClN3 + m / z[M+H] + The calculated value is 236.09, and the measured value is 236.09.

[0114] At room temperature, MAQ (500 mg, 2.1 mmol) and methyl 4-oxobutyrate (492 mg, 4.2 mmol) were added to a 50 mL reaction flask, dissolved in methanol (10 mL), and then one drop of acetic acid was added. After stirring for 0.5 h, sodium cyanoborohydride (255 mg, 2.7 mmol) was slowly added. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and then deionized water (50 mL) and dichloromethane (50 mL × 2) were added to the concentrated system for extraction. The organic layer was separated, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the crude product obtained by concentrated organic layer under reduced pressure was purified by silica gel column chromatography (eluent: MeOH:DCM = 30:1) to give a light yellow solid compound 4 (564 mg, 79.2%).

[0115] Compound 4 (564 mg, 1.7 mmol) was added to a 50 mL reaction flask at room temperature, dissolved in methanol (10 mL), followed by 1 N NaOH (10 mL). After stirring for 1 h, TLC showed the reaction was complete. MeOH was removed by vacuum concentration, and the pH was adjusted to 2-4 with 1 N HCl. Filtering yielded a brown solid compound A1 (512 mg, 94.8%). ESI-HRMS: C 16 H 19 ClN3O2 - m / z[M+H] - The calculated value is 320.12, and the measured value is 320.12.

[0116] Example 2: Synthesis of quinoline head-group compound A2

[0117] At room temperature, MAQ (37 mg, 158 μmol), monomethyl succinate (25 mg, 190 μmol), tetramethylchlorourea hexafluorophosphate (TCFH, 54 mg, 190 μmol), and N-methylimidazole (NMI, 39 mg, 474 μmol) prepared in Example 1 were added to a 50 mL reaction flask, dissolved in DMF (5 mL), and stirred for 16 h. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and then deionized water (20 mL) and dichloromethane (20 mL × 2) were added to the concentrated system for extraction. The organic layer was separated, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the crude product obtained by concentrated organic layer under reduced pressure was purified by silica gel column chromatography (eluent: MeOH:DCM = 1:10) to give a light yellow oily compound 6 (41 mg, 74.2%).

[0118] Compound 6 (41 mg, 117.2 μmol) was added to a 50 mL reaction flask at room temperature, dissolved in methanol (10 mL), followed by 1 N NaOH (10 mL). After stirring for 1 h, TLC showed the reaction was complete. MeOH was removed by vacuum concentration, and the pH was adjusted to 2-4 with 1 N HCl. Filtering yielded a brown solid compound A2 (32 mg, 81.3%). ESI-HRMS: C 16 H 17 ClN3O3 - m / z[M+H] - The calculated value is 334.10, and the measured value is 334.11.

[0119] Example 3: Synthesis of quinoline head-group compound A3

[0120] At room temperature, MAQ (2 g, 8.5 mmol), N-Boc-2-bromoethylamine (3.8 g, 17 mmol), and potassium carbonate (2.33 g, 17 mmol) prepared in Example 1 were added to a 200 mL reaction flask, DMF (50 mL) was added, and the mixture was then transferred to a preheated oil bath at 70 °C and stirred for 16 h. TLC detection showed that the reaction was complete. The reaction solution was transferred to room temperature and cooled. The crude product obtained by vacuum distillation was purified by silica gel column chromatography (eluent: DCM:MeOH = 20:1) to obtain a yellow-brown solid compound 7 (2.1 g, 65.3%).

[0121] Compound 7 (2.1 g, 5.5 mmol) was added to a 200 mL reaction flask at 0 °C, dissolved in dichloromethane (30 mL), and then trifluoroacetic acid (10 mL) was added dropwise. The mixture was then stirred at room temperature for 3 h. TLC analysis showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and deionized water (100 mL) was added to the concentrated system. The pH was adjusted to 10-12 with 1 N NaOH, and the mixture was extracted with dichloromethane (100 mL × 2). The organic layer was separated, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and the organic layer was concentrated under reduced pressure to obtain a pale yellow solid compound 8 (1.2 g, 77.7%). ESI-HRMS: C 14 H 20 ClN4 + m / z[M+H] + The calculated value is 279.2371, and the measured value is 279.2368.

[0122] At room temperature, compound 8 (44.5 mg, 158 μmol), monomethyl succinate (25 mg, 190 μmol), TCFH (54 mg, 190 μmol), and NMI (39 mg, 474 μmol) were added to a 50 mL reaction flask, dissolved in DMF (5 mL), and stirred for 16 h. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and then deionized water (20 mL) and dichloromethane (20 mL × 2) were added to the concentrated system for extraction. The organic layer was separated, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the crude product obtained by concentrated organic layer under reduced pressure was purified by silica gel column chromatography (eluent: MeOH:DCM = 1:10) to give a light yellow oily compound 9 (51 mg, 82.2%).

[0123] Compound 9 (51 mg, 128.8 μmol) was added to a 50 mL reaction flask at room temperature, dissolved in methanol (10 mL), followed by 1 N NaOH (10 mL). After stirring for 1 h, TLC showed the reaction was complete. MeOH was removed by vacuum concentration, and the pH was adjusted to 2-4 with 1 N HCl. Filtering yielded a brown solid compound A3 (42 mg, 85.4%). ESI-HRMS: C 18 H 22 ClN4O3 - m / z[M+H] - The calculated value is 377.14, and the measured value is 377.14.

[0124] Example 4: Synthesis of quinoline head-group compound A4

[0125] The synthetic route for compound A4 was similar to that in Example 2, yielding a brown solid compound A4 (69 mg, 63.2%). ESI-HRMS: C 26 H 24 Cl2N5O3 - m / z[M+H] - The calculated value is 524.13, and the measured value is 524.13.

[0126] Example 5: Synthesis of quinoline head-group compound A5

[0127] The synthetic route for compound A5 was similar to that in Example 3, yielding a brown solid, compound A5 (76 mg, 68.9%). ESI-HRMS: C 28 H 29 Cl2N6O3 - m / z[M+H] - The calculated value is 567.17, and the measured value is 567.17.

[0128] Example 6 Synthesis of Al2

[0129] At room temperature, compound 16 (1.0 g, 8.05 mmol) and 2,2'-dithiodipyridine (5.32 g, 24.1 mmol) were added to a 100 mL reaction flask, and MeOH (50 mL) was added to dissolve them. After stirring for 16 h, the reaction was stopped by TLC. The crude product obtained by concentrating the reaction solution under reduced pressure was purified by silica gel column chromatography (eluent: MeOH:DCM = 1:10) to give compound 17 (1.86 g, 82.7%) as a white solid.

[0130] Compound 18 (1.0 g, 3.75 mmol), triethylamine (1.14 g, 11.26 mmol), 4-dimethylaminopyridine (230 mg, 1.88 mmol), and p-toluenesulfonyl chloride (1.07 g, 5.63 mmol) were added to a 100 mL reaction flask at 0 °C and under a nitrogen atmosphere. After dissolving in DCM (20 mL), the mixture was moved to room temperature and stirred for 6 h. TLC analysis showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and deionized water (200 mL) was added to the concentrated system. The mixture was then extracted with ethyl acetate (100 mL × 2). The organic layer was separated, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and the organic layer was concentrated under reduced pressure to obtain a light yellow oily compound 19 (1.32 g, 83.6%).

[0131] At room temperature, compound 19 (1.0 g, 2.38 mmol) and potassium thioacetate (542 mg, 4.75 mmol) were added to a 50 mL reaction flask, dissolved in DMF (20 mL), and heated to 80 °C with stirring for 5 h. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and then deionized water (100 mL) and ethyl acetate (100 mL × 2) were added to the concentrated system for extraction. The organic layer was separated, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and the organic layer was concentrated by vacuum distillation to obtain a light yellow oily compound 20 (688 mg, 89.2%). Compound 20 (500 mg, 1.54 mmol) and lithium aluminum hydride (64.3 mg, 1.69 mmol) were added to a 50 mL reaction flask at 0 °C under a nitrogen atmosphere. After dissolving in tetrahydrofuran (20 mL), the mixture was moved to room temperature and stirred for 6 h. TLC analysis showed that the reaction was complete. The reaction solution was concentrated under reduced pressure. Deionized water (200 mL) was added to the concentrated system, and the mixture was extracted with ethyl acetate (100 mL × 2). The organic layer was separated, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and the organic layer was concentrated under reduced pressure to obtain a light yellow oily compound 21 (323 mg, 74.2%).

[0132] At room temperature, compound 21 (300 mg, 1.06 mmol), triethylamine (130 mg, 1.27 mmol), and compound 17 (118 mg, 0.4 mmol) were added to a 50 mL reaction flask, dissolved in 20 mL of DCM, and stirred for 16 h. TLC analysis showed the reaction was complete. The crude product obtained by concentrating the reaction solution under reduced pressure was purified by silica gel column chromatography (eluent: MeOH:DCM = 1:10) to give a pale yellow oily compound Al2 (220 mg, 75.6%). ESI-HRMS: C 39 H 73 OS4 + m / z[M+H] + The calculated value is 685.4539, and the measured value is 685.4543.

[0133] Example 7 Synthesis of A6

[0134] The synthetic route for compound A6 was similar to that for compound A1, yielding a brown solid, compound A6 (38 mg, 76.2%). ESI-HRMS: C 16 H 17 N3O3 - m / z[M+H] - The calculated value is 300.14, and the measured value is 300.14.

[0135] Example 8 Synthetic route and characterization of A7

[0136] The synthetic route for compound A7 was similar to that for compound A4, yielding a brown solid, compound A7 (86 mg, 67.5%). ESI-HRMS: C 16 H 17 N3O3 - m / z[M+H] - The calculated value is 458.22, and the measured value is 458.21.

[0137] Example 9: Synthetic route and characterization of cationic lipid 1-0

[0138] At 0 °C, compound DLin-MC3-DMA (1 g, 1.56 mmol) was added to a 250 mL reaction flask, dissolved in tetrahydrofuran (60 mL), and then a tetrahydrofuran solution of triethyl borohydride (1 mol / L, 8 mL, 7.79 mmol) was added dropwise. The mixture was then stirred at room temperature for 8 h. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and deionized water (200 mL) was added to the concentrated system. The mixture was then extracted with anhydrous diethyl ether (100 mL × 2) to separate the organic layer. The organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting light yellow oily compound Al1 (784 mg, 95.1%) was directly used for the next reaction.

[0139] At room temperature, compounds A1 (100 mg, 310.8 μmol), Al1 (198 mg, 373 μmol), TCFH (165 mg, 600 μmol), and NMI (83 mg, 900 μmol) were added to a 50 mL reaction flask, dissolved in DMF (5 mL), and stirred for 16 h. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and then deionized water (20 mL) and dichloromethane (20 mL × 2) were added to the concentrated system for extraction. The organic layer was separated, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the crude product obtained by concentrated organic layer under reduced pressure was purified by silica gel column chromatography (eluent: MeOH:DCM = 1:10) to give a light yellow oily compound 1-0 (102 mg, 39.4%). 1 H NMR(600MHz,Chloroform-d)δ8.45(d,J=5.3Hz,1H),7.88(d,J=2.1Hz,1H),7.71(d,J=8.9Hz,1H),7.29(dt,J =8.9,1.7Hz,1H),6.29(d,J=5.3Hz,1H),5.93(t,J=4.4Hz,1H),5.34–5.22(m,8H),4.80(p,J=6.3Hz,1H),3.22 (q,J=5.2Hz,2H),2.69(q,J=6.7,6.2Hz,6H),2.42(t,J=6.9Hz,2H),2.33(t,J=7.1Hz,2H),2.19(s,3H),1.97( p,J=6.9Hz,8H),1.80(p,J=7.1Hz,2H),1.41(q,J=6.9,6.5Hz,4H),1.31–1.13(m,36H),0.81(t,J=6.9Hz,6H). 13C NMR (151MHz, CDCl3) δ173.36,151.28,150.32,135.28,130.22,130.13,128.00,127 .93,125.49,121.66,117.23,99.07,74.66,56.61,55.31,41.08,39.80,34.13,32. 31,31.94,31.92,31.54,31.44,30.58,30.20,29.71,29.68,29.63,29.55,29.50,2 9.36,29.30,29.17,28.96,27.24,27.21,25.64,25.40,22.69,22.58,19.19,13.74. ESI-HRMS:C 53 H 87 ClN3O2 + m / z[M+H] + The calculated value is 832.6481, and the measured value is 832.6436.

[0140] Example 10: Synthetic route and characterization of cationic lipid 2-0

[0141] The synthetic route of compound 2-0 was similar to that of Example 9, yielding a pale yellow oily compound 2-0 (98 mg, 37.5%). 1 H NMR(600MHz,Chloroform-d)δ8.43(dt,J=5.2,2.3Hz,1H),7.89(s,1H),7.67(d,J=8.9Hz,1H),7. 30(d,J=8.9Hz,1H),7.04–6.88(m,1H),6.22(d,J=5.6Hz,1H),5.34–5.21(m,8H),4.78(p,J=6.3Hz ,1H),3.76(t,J=5.1Hz,2H),3.37(q,J=4.5Hz,2H),3.06(s,3H),2.70(d,J=7.0Hz,4H),2.62(t,J =6.8Hz,4H),1.99–1.95(m,8H),1.44(d,J=6.1Hz,4H),1.32–1.11(m,36H),0.81(t,J=6.9Hz,6H). 13C NMR (151MHz, CDCl3) δ174.48,172.72,151.21,150.52,148.25,135.26,130.21,130.15,127.97,127.94,127.75,125.61,122.23,117.07,97.95 ,74.97,47.57,43.15,36.32,34.03,31.53,29.71,29.67,29.58,29.55 ,29.52,29.36,29.32,28.42,27.24,27.21,25.64,25.32,22.58,14.08. ESI-HRMS:C 53 H 85 ClN3O3 + m / z[M+H] + The calculated value is 846.6274, and the measured value is 846.6243.

[0142] Example 11 Synthetic route and characterization of cationic lipid 3-0

[0143] The synthetic route of compound 3-0 is similar to that of Example 9, yielding a pale yellow oily compound 3-0 (76 mg, 31.8%). 1 H NMR(600MHz,Chloroform-d)δ8.31(d,J=6.0Hz,1H),8.03(d,J=9.0Hz,1H),7.77(d,J=2.1Hz,1H),7.34(dd,J=9.0,2.1Hz ,1H),6.40(d,J=6.1Hz,1H),6.32(t,J=5.7Hz,1H),5.28(dddd,J=24.8,18.0,11.0,7.2Hz,8H),4.74(p,J=6.2Hz,1H),3.3 5(dd,J=7.6,5.3Hz,4H),2.75(dd,J=9.2,3.8Hz,2H),2.69(t,J=6.9Hz,4H),2.59(t,J=6.7Hz,2H),2.52(t,J=5.9Hz,2H) ,2.41(t,J=6.7Hz,2H),2.23(s,3H),1.97(p,J=6.8Hz,8H),1.45–1.39(m,4H),1.31–1.09(m,36H),0.81(t,J=6.9Hz,6H). 13C NMR (151MHz, CDCl3) δ173.16,172.07,150.47,135.39,130.22,130.14,127.99,12 7.94,127.73,125.44,121.97,117.16,98.91,75.33,57.08,55.18,41.66,40.20, 37.04,34.03,31.93,31.53,31.29,30.20,29.99,29.71,29.68,29.61,29.57,29. 52,29.36,29.33,29.31,27.25,27.21,25.64,25.35,22.70,22.58,14.13,14.09. ESI-HRMS:C 55 H 90 ClN4O3 + m / z[M+H] + The calculated value is 889.6696, and the measured value is 889.6639.

[0144] Example 12 Synthetic route and characterization of cationic lipid 4-0

[0145] The synthetic route of compound 4-0 was similar to that of Example 9, yielding a pale yellow oily compound 4-0 (76 mg, 31.8%). 1 H NMR(600MHz,Chloroform-d)δ8.18(s,2H),7.93(d,J=8.9Hz,2H),7.69(s,2H),6.82(s,2H),6.75(d,J=8.9Hz,2 H),6.10(t,J=5.1Hz,2H),5.28(tdd,J=17.9,10.8,7.3Hz,8H),4.73(p,J=6.3Hz,1H),3.40(q,J=5.9,5.1Hz,2H) ,3.25(s,4H),2.86–2.77(m,4H),2.69(t,J=7.0Hz,4H),2.65(t,J=5.1Hz,2H),2.46(t,J=6.8Hz,2H),2.24(dd,J =8.3, 4.6Hz, 2H), 1.96 (p, J = 7.5Hz, 8H), 1.43 (dq, J = 13.4, 8.2, 7.1Hz, 4H), 1.23 (m, 36H), 0.81 (t, J = 6.9Hz, 6H). 13C NMR (151MHz, CDCl3) δ173.09,173.05,150.46,135.48,130.23,130.15,127.98,127.94,125.39,122.36,116.99,98.82,75.52,56.10,51. 67,40.70,37.46,34.02,31.53,31.00,29.68,29.57,29.55,29.54,2 9.52,29.36,29.32,27.25,27.21,25.64,25.37,22.58,14.13,14.09. ESI-HRMS:C 65 H 97 Cl2N6O3 + m / z[M+H] + The calculated value is 1079.6994, and the measured value is 1079.6950.

[0146] Example 13 Synthetic route and characterization of cationic lipid 5-0

[0147] The synthetic route of compound 5-0 was similar to that of Example 9, yielding a pale yellow oily compound 5-0 (126 mg, 68.0%). 1 H NMR(600MHz,Chloroform-d)δ8.34(d,J=5.4Hz,1H),8.13(d,J=5.8Hz,1H),7.73(m,3H),7.68(d,J=9.0Hz,1H) ,7.24(dd,J=8.8,2.0Hz,1H),7.01(d,J=8.9Hz,1H),6.34(d,J=5.6Hz,1H),6.11(d,J=6.0Hz,1H),5.33–5.20( m,8H),4.76(p,J=6.2Hz,1H),3.75(m,4H),3.57(t,J=6.4Hz,2H),3.37(s,2H),2.68(q,J=6.9Hz,6H),2.61(t, J=6.1Hz,2H),1.95(dq,J=13.8,7.1Hz,8H),1.45(q,J=6.8Hz,4H),1.30–1.09(m,36H),0.80(t,J=6.9Hz,6H). 13C NMR (151MHz, CDCl3) δ175.04,173.44,151.20,151.11,149.88,149.49,148.16,136.13,1 35.63,130.22,130.11,127.99,127.92,127.63,126.13,125.98,125.73,122.40,121.70, 117.01,116.66,98.77,98.02,75.64,47.38,46.10,43.44,41.61,33.92,31.53,29.70,29 .67,29.62,29.57,29.53,29.35,29.32,27.89,27.23,27.20,25.63,25.34,22.58,14.09. ESI-HRMS:C 63 H 92 Cl2N5O3 + m / z[M+H] + The calculated value is 1036.6572, and the measured value is 1036.6534.

[0148] Example 14 Synthetic route and characterization of cationic lipid 6-0

[0149] At room temperature, compounds A6 (93.4 mg, 310.8 μmol), Al2 (256 mg, 373 μmol), TCFH (165 mg, 600 μmol), and NMI (83 mg, 900 μmol) were added to a 50 mL reaction flask, dissolved in DMF (5 mL), and stirred for 16 h. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and then deionized water (20 mL) and dichloromethane (20 mL × 2) were added to the concentrated system for extraction. The organic layer was separated, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the crude product obtained by concentrated organic layer under reduced pressure was purified by silica gel column chromatography (eluent: MeOH:DCM = 1:10) to give a light yellow oily compound 6-0 (78 mg, 42.3%). 1H NMR(600MHz,Chloroform-d)δ8.31(d,J=6.4Hz,1H),8.07(s,1H),7.91(d,J=8.4Hz,1H),7.85(d,J=8.5Hz,1H),7.73(t,J=7 .7Hz,1H),7.58(t,J=7.7Hz,1H),6.53(d,J=6.4Hz,1H),5.44–5.29(m,8H),4.33(qd,J=11.6,5.6Hz,2H),3.91–3.86(m,2H) ,3.62(q,J=4.0Hz,2H),3.27–3.25(m,1H),3.18(s,3H),3.07–3.00(m,1H),2.91(dd,J=13.8,8.2Hz,1H),2.76(q,J=6.8Hz, 6H), 2.73–2.68 (m, 6H), 2.04 (p, J = 7.1Hz, 8H), 1.66 (qq, J = 11.7, 5.8, 5.2Hz, 4H), 1.46–1.19 (m, 36H), 0.89 (t, J = 6.9Hz, 6H). 13 C NMR (151MHz, CDCl3) δ174.89,172.43,154.04,145.07,132.17,130.24,130.07,128.06 ,127.91,126.62,123.51,121.58,117.48,97.87,77.24,77.03,76.82,64.36,49.06,47 .51,44.02,39.97,39.87,38.84,36.47,31.54,31.42,29.71,29.66,29.44,29.36,29.2 6,29.24,29.22,29.20,29.18,29.14,28.52,28.49,28.31,27.22,25.65,22.59,14.10. ESI-HRMS:C 55 H 90 N3O3S4 + m / z[M+H] + The calculated value is 968.5860, and the measured value is 968.5876.

[0150] Example 15: Synthetic route and characterization of cationic lipid 7-0

[0151] The synthetic route of compound 7-0 is similar to that of compound 6-0, yielding a white oily compound 7-0 (63 mg, 29.3%). 1H NMR(600MHz,Chloroform-d)δ8.46(d,J=5.6Hz,1H),7.98(d,J=2.2Hz,1H),7.74(d,J=9.0Hz,1H),7.42(dd,J=8.9,2.1Hz, 1H),7.07(s,1H),6.32(d,J=5.6Hz,1H),5.43–5.29(m,8H),4.34(qd,J=11.6,5.7Hz,2H),3.87–3.83(m,2H),3.47(q,J=4.3 Hz,2H),3.27(dq,J=8.0,5.8Hz,1H),3.14(s,3H),3.03(dd,J=13.8,6.2Hz,1H),2.92(dd,J=13.8,8.0Hz,1H),2.79–2.74(m ,6H),2.70(tt,J=7.1,3.5Hz,6H),2.04(p,J=7.1Hz,8H),1.67(q,J=7.5Hz,4H),1.41–1.21(m,36H),0.89(t,J=6.9Hz,6H). 13 C NMR (151MHz, CDCl3) δ174.36,172.43,130.23,130.08,128.04,127.92,125.96,12 2.35,116.85,97.91,77.25,77.04,76.83,64.43,49.05,47.60,43.33,40.03,39.8 8,38.85,36.32,31.54,29.71,29.66,29.44,29.43,29.36,29.33,29.26,29.24,29 .22,29.20,29.18,28.52,28.49,28.32,27.23,27.21,25.65,22.59,14.13,14.10. ESI-HRMS:C 55 H 89 ClN3O3S4 + m / z[M+H] + The calculated value is 1002.5470, and the measured value is 1002.5463.

[0152] Example 16: Synthetic route and characterization of cationic lipid 8-0

[0153] The synthetic route of compound 8-0 is similar to that of compound 6-0, yielding a white oily compound 8-0 (87 mg, 46.6%). 1H NMR(600MHz,Chloroform-d)δ8.48(d,J=5.3Hz,1H),8.36(d,J=5.4Hz,1H),7.91(dd,J=13.5,8.4Hz,2H),7.80(t,J=8.9Hz,2H),7.56(dt,J=12.0,7 .6Hz,2H),7.36(t,J=7.6Hz,1H),7.20(t,J=7.6Hz,1H),6.84(q,J=7.9,4 .6Hz,1H),6.35(d,J=5.4Hz,1H),6.26–6.06(m,2H),5.33(dtd,J=22.8,10 .7,5.1Hz,8H),4.84(p,J=6.3Hz,1H),3.78(t,J=5.5Hz,2H),3.76–3.72( m,2H),3.61(d,J=6.3Hz,2H),3.40(q,J=4.8Hz,2H),2.74(t,J=6.8Hz,4H) ,2.71(t,J=6.2Hz,2H),2.61(dd,J=7.4,4.9Hz,2H),2.01(dq,J=13.5,7.0 Hz, 8H), 1.50 (q, J = 7.0Hz, 4H), 1.34–1.22 (m, 36H), 0.87 (t, J = 6.9Hz, 6H). 13 C NMR (151MHz, CDCl3) δ174.46,173.13,150.52,150.50,149.90,149.56,148.10,147.25,130. 20,130.10,129.49,129.45,129.30,128.46,127.98,127.92,124.97,120.53,120.04,118.81 ,118.60,98.36,97.78,75.33,47.35,46.08,43.00,41.66,33.96,31.52,29.76,29.70,29.66 ,29.59,29.54,29.52,29.43,29.35,29.30,27.86,27.23,27.20,25.63,25.31,22.57,14.08. ESI-HRMS:C 63 H 94 N5O3 + m / z[M+H] + The calculated value is 968.7351, and the measured value is 968.7362.

[0154] Example 17 Synthetic route and characterization of cationic lipid 9-0

[0155] The synthetic route of compound 9-0 is similar to that of compound 6-0, yielding a pale yellow oily compound 9-0 (78 mg, 42.3%). 1 H NMR(600MHz,Chloroform-d)δ8.51(d,J=5.4Hz,1H),7.98(d,J=8.4Hz,1H),7.81(d,J=8.4Hz,1H),7.63(t, J=7.7Hz,1H),7.45(t,J=7.6Hz,1H),6.80(s,1H),6.33(d,J=5.4Hz,1H),5.35(dq,J=22.2,8.4,7.8Hz,8H), 4.86(p,J=6.2Hz,1H),3.84(t,J=5.2Hz,2H),3.47(d,J=6.0Hz,2H),3.13(s,3H),2.76(t,J=6.9Hz,4H),2.7 3–2.66(m,4H),2.06–2.02(m,8H),1.52(dt,J=11.1,6.1Hz,4H),1.37–1.20(m,36H),0.89(d,J=6.6Hz,6H). 13 C NMR (151MHz, CDCl3) δ174.24,172.75,130.21,130.14,127.98,127.93,125.01 ,120.49,97.62,77.25,77.04,76.82,74.93,65.59,58.98,50.77,47.51,43.0 1,36.28,34.04,31.53,29.70,29.67,29.60,29.57,29.54,29.51,29.35,29.3 1,28.44,27.24,27.20,25.63,25.31,22.58,19.19,14.12,14.08,13.73,8.37. ESI-HRMS:C 53 H 86 N3O3 + m / z[M+H] + The calculated value is 812.6664, and the measured value is 812.6653.

[0156] Example 18: Synthetic route and characterization of cationic lipid 10-0

[0157] The synthetic route of compound 10-0 is similar to that of compound 6-0, yielding a white oily compound 10-0 (70 mg, 36.3%). 1H NMR(600MHz,Chloroform-d)δ8.47(d,J=5.3Hz,1H),8.29(d,J=5.4Hz,1H),7. 88(d,J=4.9Hz,2H),7.74(d,J=8.9Hz,1H),7.68(d,J=9.0Hz,1H),7.33(d,J=8. 7Hz,1H),7.12(d,J=8.7Hz,1H),6.88(s,2H),6.35(d,J=5.4Hz,1H),6.07(d,J =5.6Hz,1H),5.41–5.28(m,8H),4.34(qd,J=11.6,5.6Hz,2H),3.83(t,J=5.3Hz ,2H),3.79(t,J=6.2Hz,2H),3.63(t,J=6.0Hz,2H),3.38(d,J=6.3Hz,2H),3.2 7(p,J=5.9Hz,1H),3.04(dd,J=13.7,6.1Hz,1H),2.92(dd,J=13.8,8.2Hz,1H), 2.77(t,J=7.0Hz,6H),2.70(q,J=6.8Hz,4H),2.66(t,J=6.1Hz,2H),2.04(q,J =7.1Hz, 8H), 1.66 (h, J = 7.8Hz, 4H), 1.42–1.20 (m, 32H), 0.88 (d, J = 7.1Hz, 6H). 13 C NMR (151MHz, CDCl3) δ174.57,173.00,151.49,150.90,150.34,149.55,148.71,147.98,135. 39,135.35,130.23,130.07,128.04,127.91,125.68,125.63,122.17,121.77,117.16,116.95 ,98.68,97.98,64.54,48.96,47.32,46.10,43.42,41.66,39.88,38.83,31.53,29.66,29.44 ,29.43,29.35,29.26,29.23,29.22,29.20,28.53,28.50,27.22,27.21,25.64,22.58,14.10. ESI-HRMS:C 65 H 96 Cl2N5O3S4 + m / z[M+H] + The calculated value is 1192.5768, and the measured value is 1192.5734.

[0158] Example 19: Synthetic route and characterization of cationic lipid 11-0

[0159] The synthetic route of compound 11-0 is similar to that of compound 6-0, yielding a white oily compound 11-0 (65 mg, 54.5%). 1 H NMR(600MHz,Chloroform-d)δ8.48(d,J=5.3Hz,1H),8.36(d,J=5.4Hz,1H),7.91(dd ,J=13.5,8.4Hz,2H),7.80(t,J=8.9Hz,2H),7.56(dt,J=12.0,7.6Hz,2H),7.36(t,J =7.6Hz,1H),7.20(t,J=7.6Hz,1H),6.84(q,J=7.9,4.6Hz,1H),6.35(d,J=5.4Hz,1H ),6.26–6.06(m,2H),5.33(dtd,J=22.8,10.7,5.1Hz,8H),4.34(qd,J=11.6,5.6Hz, 2H),3.83(t,J=5.3Hz,2H),3.79(t,J=6.2Hz,2H),3.63(t,J=6.0Hz,2H),3.38(d,J= 6.3Hz,2H),3.27(p,J=5.9Hz,1H),3.04(dd,J=13.7,6.1Hz,1H),2.92(dd,J=13.8,8 .2Hz,1H),2.77(t,J=7.0Hz,6H),2.70(q,J=6.8Hz,4H),2.66(t,J=6.1Hz,2H),2.04 (q, J=7.1Hz, 8H), 1.66 (h, J=7.8Hz, 4H), 1.42–1.20 (m, 32H), 0.88 (d, J=7.1Hz, 6H). 13C NMR (151MHz, CDCl3) δ172.16,171.18,149.42,149.26,147.09,130.58,130.30,130.23,129. 35,128.93,128.68,128.59,128.50,128.18,126.22,123.46,119.92,101.67,68.63,48.32, 42.92,42.42,40.45,38.45,38.22,32.04,30.49,30.36,30.31,29.88,29.30,29.17,28.94, 28.90,28.89,28.87,28.20,27.80,27.38,27.32,26.81,26.59,26.20,22.74,22.66,13.68. ESI-HRMS:C 65 H 98 N5O3S4 + m / z[M+H] + The calculated value is 1124.6547, and the measured value is 1124.6585.

[0160] Example 20 Preparation of lipid nanoparticles

[0161] The neutral lipid in the lipid nanoparticles described in this embodiment is distearate phosphatidylcholine (DSPC), and the PEG lipid is DMG-PEG 2000.

[0162] Cationic lipids, DSPC, cholesterol (Chol), and DMG-PEG 2000 were dissolved in anhydrous ethanol to prepare a mixed solution with a molar ratio of 50:10:38.5:1.5. mRNA was dissolved in a citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with a concentration of 1 mg / mL. Lipid nanoparticles were prepared by mixing cationic lipids and mRNA at a mass ratio of 20:1 using microfluidic technology (600 μL organic phase, 1800 μL aqueous phase, organic phase:aqueous phase flow rate ratio of 1:3). The mixture was then ultrafiltered using ultrafiltration centrifuge tubes with the medium replaced with 1×PBS, and filtered through a 0.22 μm filter membrane for sterilization to obtain a lipid nanoparticle suspension.

[0163] The cationic lipid and mRNA test samples are shown in Table 1:

[0164] Table 1. Raw Material List for Lipid Nanoparticles

[0165] The mRNA encapsulation efficiency of lipid nanoparticles prepared using cationic lipids 1-0 to 5-0 was detected, and the results are shown in Figure 1. The lipid nanoparticles prepared using cationic lipids 1-0 to 5-0 exhibit excellent encapsulation properties.

[0166] Example 21: Stability Study of Lipid Nanoparticles

[0167] The lipid nanoparticle suspension (formulation 11) prepared using compound 3-0 with OVA mRNA as the reporter gene in Example 20 was placed at 4°C, and the particle size, distribution and encapsulation efficiency were detected on day 1, day 3, day 5 and day 7.

[0168] The results are shown in Figure 2. After being placed at 4°C for 7 days, the lipid nanoparticles showed no significant changes in particle size and PDI, and the encapsulation efficiency remained basically unchanged over the 7 days (Day 1: 95.10%; Day 3: 96.07%; Day 7: 96.03%), indicating that the formulation can protect mRNA from degradation to a certain extent and has good stability.

[0169] Example 22 In vitro safety study of lipid nanoparticles

[0170] Cell seeding: DC2.4 cells were routinely cultured in RPMI-1640 medium, digested with trypsin, and counted. Cells were then further cultured in RPMI-1640 medium at a density of 1.5 × 10⁶ cells per well. 4 The cells were seeded at a density of 2 × 10⁶ cells / well in 96-well plates and cultured overnight in a cell culture incubator; 293T cells were cultured in DMEM medium and seeded again at a density of 2 × 10⁶ cells / well in the same manner. 4 The cells were inoculated at a density in 96-well plates and cultured overnight.

[0171] Drug solution preparation: The mRNA content in the lipid nanoparticles used was determined using Ribogreen reagent. RPMI-1640 medium containing 0, 0.05 μg / mL, and 0.10 μg / mL mRNA was prepared using formulation 9 with EGFP mRNA as a reporter gene prepared in Example 20 and lipid nanoparticles prepared with EGFP mRNA and DLin-MC3-MDA according to the method in Example 20. The specific steps are as follows: The initial LNP-mRNA concentration was 0.05 mg / mL. Taking a dosage concentration of 0.5 μg / mL as an example, 10 μL of the original solution was added to 990 μL of RPMI-1640 medium and mixed thoroughly. The drug solutions of 0.25 μg / mL, 0.1 μg / mL, and 0.05 μg / mL were calculated and diluted sequentially with a 0.5 μg / mL drug solution according to the final concentration; the DMEM culture medium containing mRNA concentrations of 0, 0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, and 0.5 μg / mL were prepared in the same manner.

[0172] Drug administration: RPMI-1640 medium containing mRNA concentrations of 0, 0.05 μg / mL, and 0.10 μg / mL was prepared for the DC2.4 group; DMEM medium containing mRNA concentrations of 0, 0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, and 0.5 μg / mL was prepared for the 293T group. The original medium was discarded, and after washing three times with PBS, the medium was replaced with the prepared mRNA-containing medium and incubated for 24 hours.

[0173] Measurement: Discard the original culture medium, wash 3 times with PBS, replace with fresh culture medium, add 10 μL of CCK-8 to each well, and continue to incubate in an incubator for 1.5 h. Measure the absorbance at 450 nm.

[0174] The test results are shown in Figures 3 and 4. It can be seen that when mRNA ≤ 0.1 μg / mL, the vector did not show obvious toxicity in either the DC2.4 or 293T cell lines, and has good biocompatibility.

[0175] Example 23: In vivo expression investigation of lipid nanoparticles

[0176] Female BALB / c mice aged 6-8 weeks were randomly divided into 6 groups, with 3 mice in each group.

[0177] Groups and administration regimens: The mRNA content in the lipid nanoparticles used was determined using Ribogreen reagent. The initial mRNA concentration in the LNP-mRNA suspension was 0.5 mg / mL. Taking a mouse weighing 20 g as an example, 20 μL of the original solution was added to 180 μL of PBS solution and mixed before being administered via tail vein. The dosage was 0.5 mg / kg (different groups were administered according to the same Luc mRNA content).

[0178] Control group: Lipid nanoparticles prepared using Luc mRNA and DLin-MC3-DMA compound (solvent: PBS, code name: DLin-MC3-DMA) obtained according to the method in Example 20 were injected intravenously in a volume of 0.2 mL.

[0179] Experimental group: The lipid nanoparticles prepared in Example 20 using Luc mRNA and cationic lipids 1-0 to 5-0 (solvent is PBS, code 1-0 to 5-0) were injected intravenously in a volume of 0.2 mL.

[0180] At predetermined time points (2, 4, and 6 hours post-injection), mice were intraperitoneally injected with D-Luciferin (150 mg / kg). In vivo imaging was performed using a small animal in vivo imaging system to observe the distribution of the formulation within the animals. After imaging, the mice were euthanized, and the heart, liver, spleen, lungs, kidneys, and lymph nodes were collected for ex vivo tissue imaging. The results are shown in Figures 5-6. In the control group, LNP-mRNA was mainly distributed in the liver; compared to the control group, the spleen of the lipid nanoparticle-treated group (compound 3-0) showed a significant bioluminescent signal.

[0181] Further quantitative analysis of bioluminescent signals in isolated tissues was performed using the IVIS Spectrum imaging system to assess mRNA expression levels in different organs. In the control group, the bioluminescent signal in the liver was significantly higher than that in the spleen, with a liver-to-spleen protein expression ratio of 11.88:1. In contrast, the ratio for lipid nanoparticles prepared with compound 1-0 was 5.32:1, and for those prepared with compound 4-0, it was 1:3.29. The delivery effects of lipid nanoparticles prepared with compounds 2-0, 3-0, and 5-0 compared to the control group are shown in Figure 7. In comparison, mRNA delivered via lipid nanoparticles prepared with different compounds all exhibited a certain degree of spleen-targeting in vivo. In particular, the transfection efficiency of the lipid nanoparticle delivery system prepared with compound 3-0 in the spleen was significantly higher than that in the liver, demonstrating excellent spleen-targeting properties. Highly efficient mRNA expression in the spleen holds promise for activating systemic immune responses, thereby contributing to the immunotherapy of related diseases.

[0182] Example 24 In vivo safety study of lipid nanoparticles

[0183] A B16F10 subcutaneous xenograft model was constructed and randomly divided into 3 groups of 6 animals each.

[0184] Groups and administration regimens: The mRNA content in the lipid nanoparticles used was determined using Ribogreen reagent. The initial mRNA concentration in the LNP-mRNA suspension was 0.5 mg / mL. Taking a mouse weighing 20 g as an example, 20 μL of the original solution was added to 180 μL of PBS solution and administered via tail vein. The dosage was 0.5 mg / kg (different groups were administered according to the same OVA mRNA content).

[0185] Control group: Phosphate-buffered saline (PBS), intravenously injected every three days, 0.2 mL in volume, for four consecutive administrations.

[0186] Control group: Lipid nanoparticles prepared using OVA mRNA and DLin-MC3-DMA compound (solvent: PBS, code name: DLin-MC3-DMA) obtained according to the method in Example 20 were administered intravenously once every three days, with a volume of 0.2 mL, for four consecutive administrations.

[0187] Experimental group: The lipid nanoparticles prepared in Example 20 using OVA mRNA and cationic lipid 3-0 (solvent is PBS, code 3-0) were injected intravenously once every three days, with a volume of 0.2 mL, for four consecutive administrations.

[0188] During the experiment, the animals' weight changes were recorded every two days; after the treatment, blood was collected for blood biochemistry tests to assess the safety of the preparation.

[0189] The animals did not experience a significant decrease in body weight during treatment (Figure 8); all blood biochemical indicators were within the normal range and showed no significant difference compared to the control group (Figure 9). These results indicate that the preparation did not adversely affect the overall health of the animals during treatment and has good biocompatibility and safety.

[0190] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and specifications of the technical solutions of the embodiments of the present invention.

Claims

1. A cationic lipid compound comprising a quinoline, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, The structure is shown in formula I: wherein R b selected from H, C1-C40 alkyl, -C(O)-C1-C40 alkyl; R a1 R a2 Independently, they are H, F, Cl, Br, and I; R c1 R c2 Independently H, C1-C8 alkyl; A is selected from -CH2-, -C(O)-, C1-C10 alkylene-NHC(O)-; R d For R 1 Selected from C1-C40 alkyl, C2-C40 alkenyl, or C2-C40 alkynyl; R 2 selected from C1-C40alkyl, C2-C40alkenyl or C2-C40alkynyl; m 1 m 2 n can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 independently.

2. The cationic lipid compound containing quinoline according to claim 1, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, The structure is shown in formula II: wherein R b selected from H, C1-C10 alkyl, -C(O)-C1-C10 alkyl; R a1 , R a2 independently H, F, Cl, Br, I; R c1 R c2 Independently, it is H or a C1-C5 alkyl group; A is selected from -CH2-, -C(O)-, C1-C6 alkylene-NHC(O)-; R d For R 1 selected from C1-C30alkyl, C2-C30alkenyl or C2-C30alkynyl; R 2 selected from C1-C30alkyl, C2-C30alkenyl or C2-C30alkynyl; m 1 , m 2 , n are independently 1, 2, 3, 4, 5, 6, 7, or 8.

3. A cationic lipid compound containing quinoline according to claim 2, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that: wherein R b selected from H, C1-C5 alkyl, -C(O)-C1-C5 alkyl; R a1 , R a2 independently H, F, Cl, Br, I; R c1 , R c2 independently H, C1-C3alkyl; A is selected from -CH2-, -C(O)-, -(CH2) q NHC(O)-; R d for R 1 selected from C6-C24alkyl, C6-C24alkenyl or C6-C24alkynyl; R 2 selected from C6-C24alkyl, C4-C24alkenyl or C6-C24alkynyl; m 1 , m 2 , n, q are independently 1, 2, 3, 4, 5 or 6.

4. The cationic lipid compound containing quinoline according to claim 1, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, The quinoline-containing cationic lipid compound has the following structure:

5. A lipid nano-composition characterized in that, It comprises a lipid component, said lipid component comprising any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

6. The lipid nano-composition according to claim 5, characterized in that, The lipid component further comprises neutral phospholipids, cholesterol, and PEG lipids; Preferably, the neutral phospholipid is selected from one or more of the following compounds: distearylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, phosphatidic acid, and sphingomyelin; The PEG lipid is selected from one or more of the following compounds: PEG-modified distearylphosphatidylethanolamine, PEG-modified dimyristoylglycerol, PEG-modified dipalmitoylphosphatidylethanolamine, PEG-modified dimyristoylphosphatidylethanolamine, PEG-modified dilauroylphosphatidylethanolamine, PEG-modified ceramide, and mPEG-modified bis(tetradecyl)acetamide; more preferably, the molecular weight of the PEG or mPEG is 1000-5000 Da, and most preferably, DMG-PEG 2000.

7. The lipid nanocomposition according to any one of claims 5-6, characterized in that, It further includes therapeutic and / or preventive agents, wherein the therapeutic and / or preventive agents are nucleic acids.

8. The lipid nano-composition according to claim 7, characterized in that, The nucleic acid is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA, snRNA, tRNA, rRNA, circRNA, saRNA, antisense oligonucleotide, lncRNA, ssDNA, mRNA, nucleic acid aptamers or ribozymes, with mRNA being preferred.

9. A pharmaceutical composition comprising the lipid nanocomposition of any one of claims 5-8 and a pharmaceutically acceptable carrier.

10. Use of the compound according to any one of claims 1-4 in the preparation of a medicament for targeting the liver or spleen to treat infectious diseases, genetic defects, autoimmune diseases or tumors.

Images