A lipid compound containing a cholic acid derivative and use thereof

Abstract

The application relates to a lipid compound containing a bile acid derivative and an application thereof. The application provides a compound, a preparation method and a lipid nanoparticle and a pharmaceutical composition thereof, wherein the compound comprises at least one general structure of formula (I-1), or the compound is a pharmaceutically acceptable salt, a prodrug or an isomer thereof. The compound of the application improves the structural diversity, so that the structural diversity of the lipid molecule is increased. The compound takes an endogenous bile acid derivative as a mother nucleus, and introduces an ester bond as a degradable group, so that the biodegradability of the lipid molecule is improved, the in-vivo toxicity is reduced, and metabolism is accelerated. The compound has high transfection efficiency, good biocompatibility, simple synthesis steps and is suitable for bio-pharmaceutical industrialization.

Description

Technical Field

[0001] This invention generally relates to lipids with a bile acid derivative core, which can be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer-bound lipids, to form lipid nanoparticles for the delivery of therapeutic agents (e.g., nucleic acid molecules, including nucleic acid mimics such as locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholino)) in vitro and in vivo for therapeutic or preventative purposes, including vaccination. Background Technology

[0002] RNA therapy has a wide range of applications, including treating genetic diseases such as silencing disease-causing genes through RNA interference, modulating cardiovascular disease risk factors, inhibiting tumor progression, developing mRNA vaccines against infectious diseases, treating ophthalmic diseases such as macular degeneration, providing protein replacement therapy, and gene editing therapy combining CRISPR technology to treat genetic diseases. In addition, RNA technology is also used in early disease detection and personalized medicine diagnostic methods. These applications demonstrate the potential and diversity of RNA therapy in the medical field. RNA molecules mainly include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA).

[0003] mRNA therapy, a crucial component of RNA therapy, is a revolutionary medical technology. It involves introducing mRNA encoding specific proteins into the human body, utilizing the body's own cellular mechanisms to produce the desired proteins. These proteins can be used for vaccine development, treating tumors, infectious diseases, and autoimmune diseases. Compared to protein drugs, mRNA therapy offers advantages such as rapid, flexible, and cost-effective production. Furthermore, its non-mutagenic, easily degradable, and mass-producible nature demonstrates higher safety. mRNA vaccines, such as the COVID-19 mRNA vaccine, work by injecting fragments of viral mRNA into human cells to generate antigens, stimulating a specific immune response and forming immune memory.

[0004] The success of RNA therapy depends on an effective delivery system. Common delivery systems include lipid nanoparticles (LNPs) and polymer nanoparticles, among which LNP technology is currently leading in applications.

[0005] LNP is mainly composed of four components: ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol (PEG) modified lipids. It can effectively encapsulate and protect nucleic acid drugs such as mRNA, prevent them from being degraded during delivery, promote their delivery to specific cell types, help nucleic acids escape from the endostomy, and finally release them in the cytoplasm to perform their functions. Summary of the Invention

[0006] Ionizable lipids play a crucial role in LNPs, but their toxicity is an unavoidable issue in research and application. Therefore, designing carrier molecules with lower in vivo toxicity and better biocompatibility is currently key to LNP delivery. This patent uses endogenous bile acid derivatives as the ionizable lipid core and introduces ester bonds as degradable groups. While ensuring transfection efficiency, it improves the biodegradability of lipid molecules, accelerates metabolism, and further reduces the toxicity of ionizable lipids in vivo.

[0007] This invention provides a compound with a bile acid derivative as its core, comprising pharmaceutically acceptable salts, prodrugs, deuterated derivatives, or stereoisomers thereof, which can be used alone or in combination with other lipid components, such as neutral lipids, charged lipids, steroids (including, for example, all steroids), and / or their analogues and / or polymers, to form lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acid molecules, including nucleic acid mimics such as locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholino nucleic acids). In some cases, said lipid nanoparticles are used to deliver nucleic acids, such as antisense and / or messenger RNA. Methods for treating various diseases or disorders, such as those caused by infectious agents and / or protein deficiency, are also provided using these lipid nanoparticles. Specific embodiments are as follows: On one hand, the present invention provides a compound comprising at least one general structural formula (I-1) as follows: (I-1) Or the compound may be a pharmaceutically acceptable salt, prodrug, deuterated product, or isomer thereof; in, X1 is independently selected from any one of the following groups: , H; X2 is independently selected from any one of the following groups: , -CH2CH3, H; X3 is independently selected from any one of the following groups: , H; Y is independently selected from any one of the following groups: -O-, -N(R) a )-、-S-; and R a Independently -H or optionally substituted C1-C6 alkyl; Z is independently selected from any of the following groups: -C(=O)-, *-OC(=O)-**, *-N(R)- bC(=O)-** or does not exist; where *- indicates that it can be connected to equation (I-1). The site; -** indicates connection to the left side of equation (I-1) The site; and R b Independently -H or optionally substituted C1-C6 alkyl; m1, m2, m3 and m4 are each independently selected from integers between 1 and 18; L 1a L 1b and L 1c Each of the following structures is independently selected: -H, -S-SR1, -SR1, -C(=O)OR1, -OC(=O)R1, -OC(=O)OR1, -N(R2)R1, -C(=O)N(R2)R1, -OC(=O)N(R2)R1, -N(R2)C(=O)R1, -N(R2)C(=O)OR1, -N(R3)C(=O)N(R2)R1, -C(=O)NR1, -OR1; R1, R2, and R3 are each independently selected from the following structures: -H, alicyclic hydrocarbons, , , , , where n1, n2, x1, x2 and o are independently selected from integers from 0 to 12, and y is independently selected from 1, 2, 3 or 4; L2 is independently selected from the following structure: -R4G x R5G y -R4G y -R4G y R6, -G y -R4G x R5(G z G y 、; R4, R5, and R6 are each independently selected from the following structures: C1-C5 alkanes and C2-C5 alkenes; G x Independently selected from the following structures: -O-, -N(R) x )-, -S-, -SS-, -OC(=O)-, -C(=O)O-, -OP(=O)O-, -OS(=O)O-, -C(=O)N-, - N(C(=O) )-; R x Independently selected from the following structures: -H, -(CH2) m -OH, -(CH2) m -CN, -(CH2) m-H and alicyclic hydrocarbons, wherein m is independently selected from 0, 1, 2, 3, 4 or 5; G y It is independently selected from the following structure: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*、-(CH2) m -W1-(CH2) n -H, where m and n are independently selected from 0, 1, 2, 3, 4 or 5; W1 is independently selected from benzene rings or heterocycles; R* and R** are independently selected from the following structures: H, C1-C3 alkanes, -(CH2). m Q1, where m is independently selected from 1, 2, 3 or 4; Q1 is independently selected from the following structures: -OH, -CN; G z It is independently selected from the following structure: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*、-(CH2) m -W2-(CH2) n -H, where m and n are independently selected from 0, 1, 2, 3, 4 or 5; W2 is independently selected from benzene rings or heterocycles; R* and R** are independently selected from the following structures: H, C1-C3 alkanes, -(CH2). a Q2, where a is independently selected from 1, 2, 3, or 4; Q2 is independently selected from the following structures: -OH, -CN.

[0008] As a preferred embodiment of the compound described in this patent, R x It is an alicyclic hydrocarbon, R x A hydrocarbon having at least one or more of the following structural formulas (II-1), (II-2), (II-3), and (II-4), wherein structural formulas (II-1), (II-2), (II-3), and (II-4) are respectively: (Ⅱ-1) (Ⅱ-2) (Ⅱ-3) (Ⅱ-4) or R x It is a pharmaceutically acceptable salt, prodrug, or isomer of the structural formula (II-1), (II-2), (II-3), or (II-4); Among them, the “ in the structural formulas (Ⅱ-1), (Ⅱ-2), (Ⅱ-3) and (Ⅱ-4)” The key can be a single key or a double key.

[0009] As a preferred embodiment of the compound described in this patent, W1 and W2 are each independently a heterocycle. More preferably, W1 and W2 are each independently a nitrogen heterocycle, an oxygen heterocycle, or a sulfur heterocycle compound.

[0010] More preferably, W1 and W2 are independently acridine, acridine, tetrahydrofuran, furan, tetrahydropyrrole, pyrrole, thiophene, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyridine, piperidine, pyran, thiam, pyrazine, pyrimidine, pyrazine, piperazine, indole, quinoline, pteridine, or morpholine.

[0011] On the one hand, the present invention also provides a composition comprising excipients and a compound or an acceptable salt as described in the above technical solutions, wherein the excipients are selected from one or more of neutral phospholipids, steroids and polyethylene glycol.

[0012] Preferably, the composition can serve as a carrier for intracellular delivery of therapeutic drugs.

[0013] Preferably, the composition is nanoparticles.

[0014] As a preferred embodiment of the composition described in this patent, the steroid is a steroid of formula (Ⅲ-1): Ⅲ-1.

[0015] As a preferred embodiment of the composition described in this patent, the molar ratio of the compound to the steroid ranges from approximately (1:2) to (6:1).

[0016] Preferably, the molar ratio of the compound to the steroid is in the range of (1:0.5) to (1:1). More preferably, the molar ratio of the compound to the steroid is 1:0.77.

[0017] As a preferred embodiment of the composition described in this patent, the composition further includes polymer-bound lipids.

[0018] As a preferred embodiment of the composition described in this patent, the polymer-bound lipids include one or more polymer-bound lipids.

[0019] As a preferred embodiment of the composition described in this patent, the lipid bound to the polymer is one or more of polyinosinic acid, DSPE-PCB20, DMG-PEG2000, DMPE-PEG2000 and C14-PEG2000.

[0020] As a preferred embodiment of the composition described in this patent, the molar ratio of the compound to the lipid bound to the polymer is approximately (10:1) to (100:1).

[0021] Preferably, the molar ratio of the compound to the lipid bound to the polymer is in the range of (20:1) to (40:1). More preferably, the molar ratio of the compound to the lipid bound to the polymer is 20:1 or 33:1.

[0022] As a preferred embodiment of the composition described in this patent, the composition further includes structural lipids.

[0023] As a preferred embodiment of the composition described in this patent, the structural lipid comprises at least one of formulas (Ⅲ-2), (Ⅲ-3), and (Ⅲ-4): Ⅲ-2 Ⅲ-3 Ⅲ-4.

[0024] As a preferred embodiment of the composition described in this patent, the molar ratio of the compound to the structural lipid is approximately (2:1) to (10:1).

[0025] As a preferred embodiment of the composition described in this patent, the composition further includes a therapeutic agent or a preventative agent.

[0026] As a preferred embodiment of the composition described in this patent, the therapeutic or preventative agent comprises a nucleic acid drug or a nucleic acid-like drug.

[0027] As a preferred embodiment of the composition described in this patent, the nucleic acid drug includes a nucleic acid aptamer, circular RNA, ASO, siRNA, sgRNA, tRNA, or mRNA. Preferably, the mRNA includes at least one mRNA encoding an antigen or a fragment thereof or an epitope. More preferably, the antigen is a pathogenic antigen or a tumor-associated antigen. Preferably, the mRNA is a monocistronic mRNA or a polycistronic mRNA.

[0028] As a preferred embodiment of the composition described in this patent, the mRNA comprises one or more functional nucleotide analogs.

[0029] Preferably, the functional nucleotide analogues include one or more of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

[0030] As a preferred embodiment of the composition described in this patent, the nucleic acid-like drug includes locked nucleic acid (LNA), peptide nucleic acid (PNA), or morpholino.

[0031] In a preferred embodiment, the present invention provides a composition comprising a steroid, a structured lipid, a polymer-bound lipid, and the compounds described in the above-described technical solutions. The composition can serve as a carrier for intracellular delivery of therapeutic drugs. Preferably, the composition is in the form of nanoparticles.

[0032] The steroid, structured lipid and polymer-bound lipids are as described in the above technical solutions, and will not be repeated here.

[0033] Furthermore, this patent also provides a lipid nanoparticle comprising the compound or composition described in the above-described technical solution.

[0034] In another aspect, this patent also provides a pharmaceutical composition comprising the compound, composition, or lipid nanoparticles as described above, and pharmaceutically acceptable excipients or diluents.

[0035] Compared with the prior art, this patent has at least one or more of the following beneficial effects: The compounds of this invention improve structural diversity, thereby increasing the structural diversity of lipid molecules.

[0036] The compound uses an endogenous bile acid derivative as its core and introduces an ester bond as a degradable group, which improves the biodegradability of lipid molecules, reduces in vivo toxicity, and accelerates metabolism.

[0037] The compound exhibits high transfection efficiency, good biocompatibility, and simple synthesis steps, making it suitable for industrialization in the biopharmaceutical industry. Attached Figure Description

[0038] Figure 1 The 1H NMR spectrum of compound A3; Figure 2 The image shows the 1H NMR spectrum of compound A6. Figure 3 The image shows the 1H NMR spectrum of compound A7. Figure 4 The hydrogen nuclear magnetic resonance spectrum of compound A16 is shown below. Figure 5 The hydrogen nuclear magnetic resonance spectrum of compound A17; Figure 6 The 1H NMR spectrum of compound A18; Figure 7 The image shows the 1H NMR spectrum of compound A22. Figure 8 The 1H NMR spectrum of compound A23; Figure 9 The image shows the 1H NMR spectrum of compound A24. Figure 10 The 1H NMR spectrum of compound A25; Figure 11 The 1H NMR spectrum of compound A26; Figure 12 The 1H NMR spectrum of compound B1; Figure 13 The image shows the 1H NMR spectrum of compound B2. Figure 14 The 1H NMR spectrum of compound B3; Figure 15 The image shows the 1H NMR spectrum of compound C1. Figure 16 The 1H NMR spectrum of compound D1; Figures 17-19 This is a fluorescence imaging result of an LNP carrying Luciferase mRNA, 6 hours after intramuscular injection. Detailed Implementation

[0039] 1. General Technology The techniques and procedures described or cited herein include those that are generally well understood and / or commonly used by those skilled in the art using conventional methods, such as the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2003).

[0040] the term Unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following terminology will be applied, and where appropriate, terms used in the singular will also include the plural form, and vice versa. All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety. If any description of terminology set forth herein conflicts with any document incorporated herein by reference, the terminology set forth herein shall prevail.

[0041] Compound structure and type Table 1: Structural formulas of compounds 4 nanoparticle composition In one aspect, this document describes nanoparticle compositions comprising the lipid compounds described herein. In a particular embodiment, the nanoparticle composition comprises a compound according to formula (I-1) as described herein.

[0042] In some embodiments, the maximum size of the nanoparticle compositions provided herein is 1 μm or shorter (e.g., ≤1 μm, ≤900 nm, ≤800 nm, ≤700 nm, ≤600 nm, ≤500 nm, ≤400 nm, ≤300 nm, ≤200 nm, ≤175 nm, ≤150 nm, ≤125 nm, ≤100 nm, ≤75 nm, ≤50 nm or shorter) when measured, for example, by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. In one embodiment, the lipid nanoparticles provided herein have at least one size in the range of about 40 nm to about 200 nm. In one embodiment, said at least one size is in the range of about 40 nm to about 100 nm.

[0043] Nanoparticle compositions that can be used in conjunction with this disclosure include, for example, lipid nanoparticles (LNPs), lipoprotein nanoparticles, liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers may be functionalized and / or cross-linked with each other. The lipid bilayers may include one or more ligands, proteins, or channels.

[0044] The characteristics of nanoparticle compositions can depend on their components. For example, a nanoparticle composition containing cholesterol as a structural lipid may have different characteristics than a nanoparticle composition containing different structural lipids. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For example, a nanoparticle composition containing a higher molar fraction of phospholipids may have different characteristics than a nanoparticle composition containing a lower molar fraction of phospholipids. The characteristics may also vary depending on the preparation method and conditions of the nanoparticle composition.

[0045] Nanoparticle compositions can be characterized using a variety of methods. For example, microscopic examination (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0046] Size): The average size of the nanoparticle composition can range from tens of nanometers to hundreds of nanometers. For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm. In some embodiments, the average size of the nanoparticle composition may be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average size of the nanoparticle composition may be about 70 nm to about 100 nm. In some embodiments, the average size may be about 80 nm. In other embodiments, the average size may be about 100 nm.

[0047] The nanoparticle composition can be relatively homogeneous. A polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, such as its particle size distribution. A smaller polydispersity index (e.g., less than 0.3) generally indicates a narrower particle size distribution. The polydispersity index of the nanoparticle composition can be from about 0 to about 0.25, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition may be from about 0.10 to about 0.20.

[0048] Encapsulation efficiency: Encapsulation efficiency of the therapeutic and / or preventative agent describes the amount of the therapeutic and / or preventative agent encapsulated in or otherwise associated with the nanoparticle composition after preparation, relative to the initial amount provided. Encapsulation efficiency is desirablely high (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of the therapeutic and / or preventative agent in a solution containing the nanoparticle composition before and after the nanoparticle composition has been disrupted with one or more organic solvents or cleaning agents. Fluorescence can be used to measure the amount of free therapeutic and / or preventative agent (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or preventative agent may be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some implementations, the encapsulation efficiency may be at least 80%. In some implementations, the encapsulation efficiency may be at least 90%.

[0049] Apparent pKa: The zeta potential of a nanoparticle composition can be used to indicate the potential kinetics of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Nanoparticle compositions with relatively low positive or negative charges are generally desirable because substances with higher charges can interact undesirably with cells, tissues, and other components in the body. In some embodiments, the zeta potential of the nanoparticle composition may be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0050] In another embodiment, the self-replicating RNA may be formulated in liposomes. As a non-limiting example, the self-replicating RNA may be formulated in liposomes as described in International Publication No. WO20120067378, which is incorporated herein by reference in its entirety. In one aspect, the liposomes may contain lipids with pKa values ​​favorable for mRNA delivery. In another aspect, the liposomes may have a substantially neutral surface charge at physiological pH and thus be effective for immunization (see, for example, liposomes described in International Publication No. WO20120067378, which is incorporated herein by reference in its entirety).

[0051] In some embodiments, the nanoparticle composition comprises a lipid component, which includes at least one lipid, such as a compound according to one of formulas (I) to (III) (and their subformulas) as described herein. For example, in some embodiments, the nanoparticle composition may comprise a lipid component comprising a compound provided herein. The nanoparticle composition may also comprise one or more other lipid or non-lipid components as described below.

[0052] preparation According to this disclosure, the nanoparticle compositions described herein may comprise at least one lipid component and one or more additional components, such as therapeutic and / or preventative agents. The nanoparticle compositions may be designed for one or more specific applications or targets. The components of the nanoparticle compositions may be selected based on a specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, specific formulations of the nanoparticle compositions may be selected for a specific application or target based on, for example, the efficacy and toxicity of a specific combination of each component.

[0053] The lipid component of the nanoparticle composition may include, for example, lipids, phospholipids (e.g., unsaturated lipids, such as DOPE or DSPC, PEG lipids, and structural lipids) according to one of the formulas (I-1) described herein. Wherein, DOPE is dioleoylphosphatidylethanolamine, DSPC is distearylphosphatidylcholine, and PEG lipid is polyethylene glycol lipid. Each component of the lipid component may be provided in a specific fraction.

[0054] In one embodiment, this document provides a nanoparticle composition comprising a cationic or ionizable lipid compound, a therapeutic agent, and one or more excipients provided herein. In one embodiment, the cationic or ionizable lipid compound comprises a compound according to one of formulas (I-1) as described herein, and optionally one or more additional ionizable lipid compounds. In one embodiment, the one or more excipients are selected from neutral lipids, steroids, and polymer-bound lipids. In one embodiment, the therapeutic agent is encapsulated within or associated with lipid nanoparticles.

[0055] In one embodiment, this document provides a nanoparticle composition (i.e., lipid nanoparticles) comprising: i) 40 mol% to 50 mol% cationic lipids; ii) Neutral lipids; iii) Steroids; iv) Polymer-bound lipids; v) Therapeutic agents. Example

[0056] The embodiments in this section are provided by way of example only and are not intended to be limiting.

[0057] General methods General preparative HPLC (High Performance Liquid Chromatography) method: HPLC purification is performed on a SepaBean® machine U100-Test equipped with an evaporative light detector (ELSD) on a SepaFlash® rapid separation column, typically using dichloromethane as solvent A, methanol as solvent B, a methanol solution containing 7M ammonia as solvent C, petroleum ether as solvent D, and ethyl acetate as solvent E.

[0058] Typical LCMS (Liquid Chromatography-Mass Spectrometry) method: LCMS analysis is performed on a Thermo (LC-MS) system. Chromatography is performed on a SunFire C18, typically using water containing 0.1% trifluoroacetic acid (TFA) as solvent A and acetonitrile containing 0.1% formic acid as solvent B.

[0059] Example 1 Preparation of Class A compounds Step 1 Step 2 Step 3 Preparation of compound A1 Step 1: Preparation of intermediate A1-1 Preparation of compound A1-1-a Lithochoic acid (15.0 g, 39.83 mmol) and N,N-dimethylformamide (100 mL) were added to a round-bottom flask, followed by benzyl bromide (10.2 g, 51.75 mmol) and potassium carbonate (11.0 g, 79.67 mmol). The mixture was heated to 70 °C and stirred overnight under nitrogen protection. After the reaction was completed by TLC, an appropriate amount of water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. The crude product was then eluted by silica gel column chromatography with solvent A and solvent B mixed in a 20:1 mass ratio to give a white solid Al-1-a (15.7 g, 85% yield).

[0060] Preparation of compound A1-1-b A1-1-a (1.5 g, 3.21 mmol) was dissolved in acetonitrile (15 mL) and dichloromethane (15 mL) at room temperature. N,N-dimethyl-β-alanine (565 mg, 4.82 mmol), 1-methylimidazole (NMI) (1.0 g, 12.86 mmol), and tetramethylchlorourea hexafluorophosphate (TCFH) (1.8 g, 6.43 mmol) were added sequentially under nitrogen protection and stirred overnight at room temperature. After TLC detection, water was added, and the mixture was extracted three times with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with a 10:1 mass ratio of solvent A and solvent B to give a colorless oily product A1-1-b (1.7 g, 93% yield).

[0061] Preparation of compound A1-1 Intermediate A1-1-b (390 mg, 0.64 mmol) was dissolved in methanol (10 mL), and 10% Pd / C (117 mg, 30% wt) was added. The mixture was purged with hydrogen three times and stirred overnight at 35 °C. After the reaction was completed by TLC, the filtrate was collected, and the filter cake was washed twice with methanol. The filtrates were combined, and the solvent was concentrated to give a white solid A1-1 (295 mg, 89% yield).

[0062] Step 2: Preparation of intermediate A1-4 Preparation of compound A1-4-a Hexylamine (15.0 g, 148.2 mmol) was added to a flask containing ethyl formate (150 mL) at room temperature. The mixture was heated to 55 °C and refluxed overnight under nitrogen protection. After the reaction was completed by TLC monitoring, the product was concentrated to give a colorless oily substance A1-4-a (19.15 g, 99% yield).

[0063] Preparation of compound A1-4 Al-4-a (12.8 g, 98.8 mmol) was dissolved in dichloromethane (120 mL) at room temperature, and triethylamine (50.0 g, 494.2 mmol) was added. The mixture was cooled to 0 °C under nitrogen protection. Phosphorus oxychloride (18.2 g, 118.6 mmol) was dissolved in dichloromethane (60 mL) and slowly added dropwise to the mixture, stirring at 0 °C for 4 hours. After the reaction was detected by TLC, saturated sodium bicarbonate solution was added to quench the reaction. The mixture was extracted three times with petroleum ether, and the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with a mixture of solvents D and E in a 20:1 mass ratio to give a colorless oily product Al-4 (8.5 g, 77% yield).

[0064] Step 3: Preparation of compound A1 At room temperature, A1-1 (60 mg, 0.13 mmol) was dissolved in dichloromethane / methanol (8 mL, V) DCM V MeOH Dissolve compound A1-3 (19.7 mg, 0.13 mmol) in a 3:1 ratio, add compound A1-3 (19.7 mg, 0.13 mmol), and stir for 1 h. Then add compound A1-2 (23.5 mg, 0.13 mmol) and continue stirring for 0.5 h. Add compound A1-4 (15 mg, 0.13 mmol) and stir the mixture at room temperature for 16 h. TLC showed that the reaction was complete. The mixture was evaporated under reduced pressure and purified by preparative HPLC under a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6 to obtain a yellow oily substance A1 (41.1 mg, 36.1% yield). Its MS (ESI) was determined to be [M+H]. + = 910.9.

[0065] Preparation of compound A2 Step 1: Prepare intermediate A2-1 (same as intermediate A1-1) Step 2: Prepare intermediate A2-4 (same as intermediate A1-4) Step 3: Preparation of compound A2 The preparation procedure for this compound was the same as for A1, with intermediate A2-2 being n-butylamine and intermediate A2-3 being 1-tetradecanealdehyde. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A2 (22.8 mg, 21%). MS (ESI): [M+H] + = 854.9.

[0066] Preparation of compound A3 Step 1: Prepare intermediate A3-1 (same as intermediate A1-1) Step 2: Preparation of compound A3 The preparation procedure for this compound was the same as for A1, with intermediate A3-2 being n-hexylamine, intermediate A3-3 being 1-tetradecylaldehyde, and intermediate A3-4 being 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A3 (35.6 mg, 31%). MS (ESI): [M+2H] 2+ = 456.0.

[0067] Preparation of compound A4 Step 1: Prepare intermediate A4-1 (same as intermediate A1-1) Step 2: Preparation of compound A4 The preparation procedure for this compound was the same as for A1, with intermediate A4-2 being 1-decylamine, intermediate A4-3 being n-octanal, and intermediate A4-4 being 1-decylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A4 (57.1 mg, 50%). MS (ESI): [M+H] + = 910.9.

[0068] Preparation of compound A5 Step 1: Prepare intermediate A5-1 (same as intermediate A1-1) Step 2: Preparation of compound A5 The preparation procedure for this compound was the same as for A1, with intermediate A5-2 being 1-decylamine, intermediate A5-3 being 1-decanoal, and intermediate A5-4 being 1-decylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A5 (138.5 mg, 46.4%). MS (ESI): [M+H] + = 938.8.

[0069] Preparation of compound A6 Step 1: Prepare intermediate A6-1 (same as intermediate A1-1) Step 2: Preparation of intermediate A6-3 Preparation of compound A6-3-a 7-Tridecyl alcohol (2.0 g, 9.98 mmol) and adipic acid (3.0 g, 20.53 mmol) were dissolved in dichloromethane (40 mL). 4-Dimethylaminopyridine (DMAP) (1.2 g, 9.98 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (2.9 g, 14.97 mmol) were added sequentially under ice bath conditions. The reaction was carried out overnight at room temperature with stirring under nitrogen protection. After TLC detection, a suitable amount of water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with a mixture of solvent A and solvent B in a 20:1 mass ratio to give a colorless oil A6-3-a (2.5 g, 75%).

[0070] Preparation of compound A6-3 Intermediate A6-3-a (1.0 g, 3.04 mmol), pinacol borane (HBpin) (429 mg, 3.35 mmol), and 1-pyridine-4-piperidine (PPDP) (780 mg, 4.87 mmol) were dissolved in dichloromethane (40 mL). 1-pyridine-4-piperidine trifluoromethanesulfonate (Tf-PPDP) (2.3 g, 5.18 mmol) was added under ice bath conditions. The reaction was carried out under nitrogen protection and stirred at room temperature for 4 h. After TLC detection, a suitable amount of water was added, and the mixture was extracted three times with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with a 1:1 mass ratio of solvent A and solvent D to give a colorless oily product A6-3 (573 mg, 60%).

[0071] Step 3: Preparation of compound A6 The preparation procedure for this compound is the same as for A1, with intermediate A6-2 being 1-octylamine and intermediate A6-4 being 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A6 (93.2 mg, 25%). MS (ESI): [M+H] + = 1039.0.

[0072] Preparation of compound A7 Step 1: Prepare intermediate A7-1 (same as intermediate A1-1) Step 2: Preparation of intermediate A7-2 Preparation of compound A7-2-a N-bromohexyltriphenylphosphine (41.9 g, 97.81 mmol) was dissolved in tetrahydrofuran (280 mL), and KOtBu (11.5 g, 102.80 mmol) was added under ice bath. The reaction was carried out under nitrogen protection and stirred under ice bath for 2 h. Tetrahydro-2H-pyran-2-ol (5.0 g, 48.96 mmol) was dissolved in tetrahydrofuran (20 mL) and slowly added to the reaction system under ice bath. After the addition was complete, the reaction was carried out under nitrogen protection and stirred overnight at room temperature. TLC detected the complete reaction, and the filtrate was collected. The filter cake was washed twice with tetrahydrofuran, the filtrates were combined, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent A, to give a colorless oily substance A7-2-a (6.0 g, 72%).

[0073] Preparation of compound A7-2-b Compound A7-2-a (4.0 g, 23.49 mmol), phthalimide (5.2 g, 35.23 mmol), and triphenylphosphine (9.2 g, 35.23 mmol) were dissolved sequentially in diethyl ether (60 mL), and the reaction mixture was stirred for 0.5 h under nitrogen protection and an ice bath. Diisopropyl azodicarbonate (DIAD) (7.1 g, 35.23 mmol) was dissolved in diethyl ether (20 mL) and slowly added to the reaction mixture under an ice bath. After the addition was complete, the mixture was stirred overnight at room temperature under nitrogen protection. TLC detected the complete reaction, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with a 1:1 mass ratio of solvent A and solvent D to give a colorless oily compound A7-2-b (5.8 g, 83%).

[0074] Preparation of compound A7-2 Compound A7-2-b (5.8 g, 19.37 mmol) was dissolved in ethanol (60 mL) at room temperature, and 50% hydrazine hydrate (1.94 g, 38.74 mmol) was added. The mixture was heated to 80 °C and refluxed overnight under nitrogen protection. After the reaction was completed by TLC, the filtrate was collected and washed twice with dichloromethane. The filtrates were combined, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with a mixture of solvent A and solvent B in a 10:1 mass ratio to give a colorless oily compound A7-2 (2.7 g, 82%).

[0075] Step 3: Preparation of intermediate A7-3 Preparation of compound A7-3-a (same as A7-2-a) Preparation of compound A7-3 Intermediate A7-3-a (830 mg, 4.87 mmol) was dissolved in dichloromethane (20 mL), and Dess-Martin oxidant (4.10 mg, 9.75 mmol) was added under ice bath conditions. The reaction was carried out under nitrogen protection with stirring at room temperature for 3–4 h. After TLC detection, the mixture was diluted with dichloromethane, and the reaction was quenched with saturated sodium bicarbonate. The mixture was extracted twice with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent A:solvent D = 1:10, to give a colorless oily substance A7-3 (458 mg, 56%).

[0076] Step 4: Preparation of compound A7 The preparation procedure for this compound is the same as for A1, with intermediate A7-4 being a 1-decylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A7 (31.8 mg, 27%). MS (ESI): [M+H] + =962.9.

[0077] Preparation of compound A8 Step 1: Preparation of intermediate A8-1 Preparation of compound A8-1-a (same as A1-1-a) Preparation of compound A8-1-b The compound was prepared in the same manner as A1-1-b, by silica gel column chromatography, elution with solvent A: solvent B = 10:1, to obtain a colorless oily compound A8-1-b (738 mg, 74%).

[0078] Preparation of compound A8-1 The compound was prepared in the same manner as A1-1, by silica gel column chromatography, elution with solvent A: solvent B = 5:1, to give a white solid A8-1 (350 mg, 56%).

[0079] Step 2: Preparation of compound A8 The preparation procedure for this compound was the same as for A1, with intermediate A8-2 being 1-hexylamine, intermediate A8-3 being 1-hexadecaldehyde, and intermediate A8-4 being 1-hexylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A8 (26.8 mg, 27%). MS (ESI): [M+H] + = 924.9.

[0080] Preparation of compound A9 Step 1: Prepare intermediate A9-1 (same as intermediate A8-1) Step 2: Preparation of compound A9 The preparation procedure for this compound was the same as for A1, with intermediate A9-2 being 1-butylamine, intermediate A9-3 being 1-hexadecaldehyde, and intermediate A9-4 being 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A9 (32.0 mg, 32%). MS (ESI): [M+H] + = 924.9.

[0081] Preparation of compound A10 Step 1: Prepare intermediate A10-1 (same as intermediate A8-1) Step 2: Preparation of compound A10 The preparation procedure for this compound was the same as for A1, with intermediate A10-2 being 1-tetradecylamine, intermediate A10-3 being 1-hexylaldehyde, and intermediate A10-4 being 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A10 (46.4 mg, 47%). MS (ESI): [M+2H] 2+ = 463.1.

[0082] Preparation of compound A11 Step 1: Prepare intermediate A11-1 (same as intermediate A8-1) Step 2: Preparation of compound A11 The preparation procedure for this compound was the same as for A1, with intermediate A11-2 being 1-hexylamine, intermediate A11-3 being 1-hexadecaldehyde, and intermediate A11-4 being 1-decylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A11 (40.0 mg, 41%). MS (ESI): [M+H] + = 981.0.

[0083] Preparation of compound A12 Step 1: Prepare intermediate A12-1 (same as intermediate A8-1) Step 2: Preparation of compound A12 The preparation procedure for this compound was the same as for A1, with intermediate A12-2 being 1-hexadecanine, intermediate A12-3 being 1-hexylaldehyde, and intermediate A12-4 being 1-dodecylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A12 (50.6 mg, 49%). MS (ESI): [M+2H] 2+ = 505.2.

[0084] Preparation of compound A13 Step 1: Prepare intermediate A13-1 (same as intermediate A8-1) Step 2: Preparation of compound A13 The preparation procedure for this compound was the same as for A1, with intermediate A13-2 being 1-octylamine, intermediate A13-3 being 1-decylaldehyde, and intermediate A13-4 being 1-tetradecylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A13 (54.2 mg, 51%). MS (ESI): [M+2H] 2+ = 491.2.

[0085] Preparation of compound A14 Step 1: Prepare intermediate A14-1 (same as intermediate A8-1) Step 2: Preparation of intermediate A14-4 Preparation of compound A14-4-a At room temperature, 4-aminobutyric acid (10.0 g, 97.0 mmol) was dissolved in N,N-dimethylformamide (35 mL), and formic acid (8.9 g, 193.9 mmol) was added. Under nitrogen protection, the mixture was stirred at 110 °C for about 4 hours. After the reaction was completed by TLC, the mixture was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated, and ethyl acetate was added to precipitate a second time, followed by filtration. The two filter cakes were combined and dried under vacuum to give a white solid A14-4-a (12.6 g, 99.2%).

[0086] Preparation of compound A14-4-b At 0 °C, A14-4-a (1.5 g, 11.4 mmol), n-butanol (0.93 g, 12.6 mmol), and 4-dimethylaminopyridine (DMAP) (0.28 g, 2.3 mmol) were dissolved in dichloromethane (30 mL). 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (2.4 g, 12.6 mmol) was added to the reaction mixture and stirred overnight at room temperature. After TLC detection, saturated sodium bicarbonate solution was added, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent D:solvent E = 4:1, to give a colorless oily substance A14-4-b (1.1 g, 54%).

[0087] Preparation of compound A14-4 The compound was prepared in the same manner as A1-1-b by silica gel column chromatography, elution with solvent D: solvent E = 20:1, yielding a colorless oily compound A14-4 (125 mg, 55%).

[0088] Step 3: Preparation of compound A14 The preparation procedure for this compound was the same as for A1, with intermediate A14-2 being 1-butylamine and intermediate A14-3 being 1-octadecaldehyde. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A14 (50.0 mg, 24%). MS (ESI): [M+2H] 2+ = 492.1.

[0089] Preparation of compound A15 Step 1: Prepare intermediate A15-1 (same as intermediate A8-1) Step 2: Prepare intermediate A15-3 (same as intermediate A6-3) Step 3: Preparation of compound A15 The preparation procedure for this compound is the same as for A1, with intermediate A15-2 being 1-octylamine and intermediate A15-4 being 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A15 (121.0 mg, 37%). MS (ESI): [M+2H] 2+ = 527.0.

[0090] Preparation of compound A16 Step 1: Prepare intermediate A16-1 (same as intermediate A8-1) Step 2: Prepare intermediate A16-2 (same as intermediate A7-2) Step 3: Prepare intermediate A16-3 (same as intermediate A7-3) Step 4: Preparation of compound A16 The preparation procedure for this compound is the same as for A1, with intermediate A16-4 being a 1-hexylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A16 (82.0 mg, 29%). MS (ESI): [M+H] + = 920.9.

[0091] Preparation of compound A17 Step 1: Prepare intermediate A17-1 (same as intermediate A8-1) Step 2: Prepare intermediate A17-2 (same as intermediate A7-2) Step 3: Prepare intermediate A17-3 (same as intermediate A7-3) Step 4: Preparation of compound A17 The preparation procedure for this compound is the same as for A1, with intermediate A17-4 being a 1-decylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A17 (81.0 mg, 27%). MS (ESI): [M+H] + = 976.9.

[0092] Preparation of compound A18 Step 1: Prepare intermediate A18-1 (same as intermediate A8-1) Step 2: Prepare intermediate A18-2 (same as intermediate A7-2) Step 3: Preparation of intermediate A18-3 The compound was prepared in the same manner as A7-3, by silica gel column chromatography, elution with solvent D: solvent E = 20:1, to give a colorless oily compound A18-3 (992 mg, 62%).

[0093] Step 4: Preparation of compound A18 The preparation procedure for this compound is the same as for A1, with intermediate A18-4 being a 1-hexylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A18 (138.0 mg, 46%). MS (ESI): [M+2H] 2+ = 509.2.

[0094] Preparation of compound A19 Step 1: Preparation of intermediate A19-1 Preparation of compound A19-1-a (same as A1-1-a) Preparation of compound A19-1-b The compound was prepared in the same manner as A1-1-b by silica gel column chromatography with solvent A: solvent B = 10:1 elution, yielding a colorless oily compound A19-1-b (2.0 g, 87%).

[0095] Preparation of compound A19-1 The compound was prepared in the same manner as A1-1, by silica gel column chromatography, elution with solvent A: solvent B = 5:1, to give a white solid A19-1 (1.06 g, 62%).

[0096] Step 2: Preparation of compound A19 The preparation procedure for this compound was the same as for A1, with intermediate A19-2 being 1-dodecylamine, intermediate A19-3 being 1-dodecylaldehyde, and intermediate A19-4 being 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A19 (69.4 mg, 24%). MS (ESI): [M+H] + = 995.0.

[0097] Preparation of compound A20 Step 1: Preparation of intermediate A20-1 Preparation of compound A20-1-a (same as A1-1-a) Preparation of compound A20-1-b A20-1-a (2.00 g, 4.28 mmol) was dissolved in acetonitrile (20 mL) and dichloromethane (20 mL) at room temperature. Then, 6-bromohexanoic acid (1.00 g, 5.14 mmol), 1-methylimidazole (NMI) (1.05 g, 12.86 mmol), and tetramethylchlorourea hexafluorophosphate (TCFH) (1.80 g, 6.43 mmol) were added sequentially. The reaction was carried out overnight at room temperature under nitrogen protection with stirring. After the reaction was complete as detected by TLC, a suitable amount of water was added, and the mixture was extracted three times with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. The crude product was then subjected to silica gel column chromatography with solvent A:solvent D = 1:1 elution to give a pale yellow oil, A20-1-b (2.5 g, 76%).

[0098] Preparation of compound A20-1-c Intermediate A20-1-b (2.50 mg, 3.88 mmol) was dissolved in N,N-dimethylformamide (25 mL) at room temperature, followed by the addition of dimethylamine (1.75 g, 38.8 mmol) and DIPEA (1.00 g, 7.77 mmol). The reaction was carried out overnight at room temperature under nitrogen protection with stirring. After TLC detection, water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent A:solvent B = 10:1, to give a pale yellow oil, A20-1-c (2.2 g, 93%).

[0099] Preparation of intermediate A20-1 The compound was prepared in the same manner as A1-1 by silica gel column chromatography, eluting with solvent A: solvent B = 10:1 to give a colorless oily compound A20-1 (1.6 g, 88%).

[0100] Step 2: Preparation of compound A20 The preparation procedure for this compound was the same as for A1. Intermediate A20-2 was 1-tetradecylamine, intermediate A20-3 was 1-octylaldehyde, and intermediate A20-4 was 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A20 (175.4 mg, 62%). MS (ESI): [M+H] + = 981.0.

[0101] Preparation of compound A21 Step 1: Preparation of intermediate A21-1 The compound was prepared in the same manner as A20-1, by silica gel column chromatography, elution with solvent A: solvent B = 10:1, to give a pale yellow solid A21-1 (725 mg, 65%).

[0102] Step 2: Preparation of compound A21 The preparation procedure for this compound was the same as for A1. Intermediate A21-2 was 1-octylamine, intermediate A21-3 was 1-octylaldehyde, and intermediate A21-4 was 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A21 (168.9 mg, 67%). MS (ESI): [M+H] + = 924.9.

[0103] Preparation of compound A22 Step 1: Preparation of intermediate A22-1 The compound was prepared in the same manner as A20-1, by silica gel column chromatography, elution with solvent A: solvent B = 10:1, to give a white solid A22-1 (295 mg, 89%).

[0104] Step 2: Preparation of compound A22 The preparation procedure for this compound is the same as for A1. Intermediate A22-2 is 1-tetradecylamine, intermediate A22-3 is 1-butylaldehyde, and intermediate A22-4 is 1-hexylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A22 (97.0 mg, 56%). MS (ESI): [M+H] + = 894.9.

[0105] Preparation of compound A23 Step 1: Preparation of intermediate A23-1 The compound was prepared in the same manner as A20-1, by silica gel column chromatography, elution with solvent A: solvent B = 10:1, to give a white solid A23-1 (310 mg, 83%).

[0106] Step 2: Preparation of compound A23 The preparation procedure for this compound was the same as for A1. Intermediate A23-2 was 1-octylamine, intermediate A23-3 was 1-octylaldehyde, and intermediate A23-4 was 1-hexylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A23 (97.0 mg, 58%). MS (ESI): [M+H] += 870.8.

[0107] Preparation of compound A24 Step 1: Preparation of intermediate A24-1 Preparation of compound A24-1-a (same as A1-1-a) Preparation of compound A24-1-b A24-1-a (800 mg, 1.71 mmol) was dissolved in tetrahydrofuran (20 mL) at room temperature, followed by the addition of p-nitrobenzene chloroformate (518 mg, 2.57 mmol) and pyridine (407 mg, 5.14 mmol). The mixture was heated to 45 °C and stirred under nitrogen protection for 6 h. After the reaction was completed by TLC, the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent A:solvent D = 1:1, to give a white solid A24-1-b (805 mg, 74%).

[0108] Preparation of compound A24-1-c A24-1-b (800 mg, 1.27 mmol) was dissolved in dichloromethane (20 mL) at room temperature, followed by the sequential addition of N,N-dimethyl-1,3-propanediamine (142 mg, 1.39 mmol) and N,N-diisopropylethylamine (327 mg, 2.53 mmol). The reaction was carried out overnight at room temperature under nitrogen protection with stirring. After the reaction was complete as detected by TLC, the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent A:solvent B = 10:1, to give a white solid A24-1-c (650 mg, 86%).

[0109] Preparation of compound A24-1 The preparation method of this compound is the same as that of A1-1. The solvent is concentrated to obtain a crude product, and the crude product is slurried with dichloromethane to obtain a white solid A24-1 (374 mg, 75%).

[0110] Step 2: Preparation of compound A24 The preparation procedure for this compound was the same as for A1. Intermediate A24-2 was 1-butylamine, intermediate A24-3 was 1-hexadecaldehyde, and intermediate A24-4 ​​was 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A24 (180.0 mg, 64%). MS (ESI): [M+H] + = 939.9.

[0111] Preparation of compound A25 Step 1: Prepare intermediate A25-1 (same as A24-1) Step 2: Prepare intermediate A25-2 (same as intermediate A7-2) Step 3: Prepare intermediate A25-3 (same as intermediate A7-3) Step 4: Preparation of compound A25 The preparation procedure for this compound is the same as for A1, with intermediate A24-4 ​​being a 1-decylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A25 (121.7 mg, 43%). MS (ESI): [M+H] + = 992.0.

[0112] Preparation of compound A26 Step 1: Preparation of intermediate A26-1 Preparation of compound A26-1-a (same as A1-1-a) Preparation of compound A26-1-b (same as A24-1-b) Preparation of compound A26-1-c A26-1-b (1 g, 1.58 mmol) was dissolved in pyridine (10 mL) at room temperature, and the intermediate N,N-dimethylpropanolamine (326 mg, 3.16 mmol) was added. The reaction was carried out overnight at room temperature under nitrogen protection with stirring. TLC analysis was performed, and after the reaction was complete, the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent A:solvent B = 20:1, to give a pale yellow oil, A26-1-c (750 mg, 80%).

[0113] Step 2: Preparation of compound A26 The preparation procedure for this compound is the same as for A1. Intermediate A26-2 is 1-tetradecylamine, intermediate A26-3 is 1-octylaldehyde, and intermediate A26-4 is 1-hexylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound A26 (92.0 mg, 50%). MS (ESI): [M+H] + = 940.9.

[0114] 6.2 Example 2 Preparation of Class B compounds Step 1 Step 2 Preparation of compound B1 Step 1: Preparation of intermediate B1-1 Preparation of compound B1-1-a The compound was prepared in the same manner as A1-1-a by silica gel column chromatography, elution with solvent A: solvent B = 10:1, yielding a pale yellow oily compound B1-1-a (19 g, 77%).

[0115] Preparation of compound B1-1-b The compound was prepared in the same manner as A20-1-b by silica gel column chromatography, eluting with solvent D: solvent E = 3:1, to obtain a colorless oily compound B1-1-b (3.0 g, 38%).

[0116] Preparation of compound B1-1-c The compound was prepared in the same manner as A20-1-c by silica gel column chromatography with solvent A: solvent B = 10:1 elution, yielding a white solid B1-1-c (2.4 g, 86%).

[0117] Preparation of compound B1-1 The compound was prepared in the same manner as A1-1, and the solvent was concentrated to give a white solid B1-1 (1.8 g, 90%).

[0118] Step 2: Preparation of compound B1 The preparation procedure for this compound was the same as for A1, with intermediate B1-2 being 1-octylamine, intermediate B1-3 being 1-hexadecaldehyde, and intermediate B1-4 being 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound B1 (115.0 mg, 41%). MS (ESI): [M+H] + = 996.9.

[0119] Preparation of compound B2 Step 1: Prepare intermediate B2-1 (same as B1-1) Step 2: Preparation of compound B2 The preparation procedure for this compound is the same as for A1. Intermediate B2-2 is 1-tetradecylamine, intermediate B2-3 is 1-octylaldehyde, and intermediate B2-4 is 1-octylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound B2 (70.0 mg, 24%). MS (ESI): [M+H] + = 968.9.

[0120] Preparation of compound B3 Step 1: Prepare intermediate B3-1 (same as B1-1) Step 2: Preparation of intermediate B3-3 Preparation of compound B3-3-a 2-Hexyldecanoic acid (2.0 g, 7.80 mmol) and 1,6-hexanediol (2.8 g, 23.40 mmol) were dissolved in dichloromethane (30 mL). Under ice bath conditions, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (1.9 g, 10.14 mmol), 4-dimethylaminopyridine (DMAP) (191 mg, 1.56 mmol), and N,N-diisopropylethylamine (1.5 g, 11.70 mmol) were added sequentially. The reaction was carried out overnight at room temperature under nitrogen protection with stirring. TLC analysis was performed. After the reaction was complete, a suitable amount of water was added, and the mixture was extracted three times with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent A, to give a pale yellow oil B3-3-a (1.87 g, 67%).

[0121] Preparation of compound B3-3 B3-3-a (900 mg, 2.52 mmol) was dissolved in dichloromethane (20 mL), and pyridine chlorochromate (PCC) (626 mg, 2.90 mmol) was added under ice bath conditions. The reaction was carried out under nitrogen protection with stirring at room temperature for 2 h. TLC was performed. After the reaction was complete, the filtrate was collected, and the filter cake was washed twice with dichloromethane. The filtrates were combined, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent A:solvent D = 1:1, to give a colorless oily substance B3-3 (270 mg, 30%).

[0122] Step 3: Preparation of compound B3 The preparation procedure for this compound is the same as for A1, with intermediate B3-2 being 1-butylamine and intermediate B3-4 being 1-hexylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound B3 (144.0 mg, 51%). MS (ESI): [M+H] + = 1027.0.

[0123] 6.3 Example 3 Preparation of Class C compounds Step 1 Step 2 Preparation of compound C1 Step 1: Preparation of intermediate C1-1 Preparation of compound C1-1-a The compound was prepared in the same manner as B1-1-a by silica gel column chromatography with solvent A: solvent B = 10:1 elution, yielding a white solid C1-1-a (5.2 g, 85%).

[0124] Preparation of compound C1-1-b The compound was prepared in the same manner as B1-1-b by silica gel column chromatography, eluting with solvent D: solvent E = 3:1, to give a white solid C1-1-b (1.23 g, 63%).

[0125] Preparation of compound C1-1-c The compound was prepared in the same manner as B1-1-c by silica gel column chromatography, eluting with solvent A: solvent B = 10:1, to give a white solid C1-1-c (1.0 g, 89%).

[0126] Preparation of compound C1-1 The compound was prepared in the same manner as B1-1, and the solvent was concentrated to give a white solid (827 mg, 97%).

[0127] Step 2: Prepare compound C1-2 (same as compound A7-2) Step 3: Prepare compound C1-3 (same as compound A7-3) Step 4: Preparation of compound C1 The preparation procedure for this compound is the same as for A1, with intermediate C1-4 being 1-decylisocyanate. Purification was performed using a gradient program of solvent A:solvent B:solvent C = 100:0:0 to 70:24:6, yielding a yellow oily compound C1 (66.0 mg, 23%). MS (ESI): [M+H] + =1008.9.

[0128] 6.4 Example 4 Preparation of Class D compounds Step 1 Step 2 Step 3 Preparation of compound D1 Step 1: Prepare intermediate D1-1 (same as A8-1) Step 2: Preparation of intermediate D1-2 Preparation of compound D1-2-a Undecanoic acid (10.0 g, 53.68 mmol) and N-bromosuccinimide (NBS) (14.3 g, 80.52 mmol) were added to a round-bottom flask, followed by trifluoroacetic acid (TFA) (30 mL) and concentrated sulfuric acid (1.5 mL). The mixture was heated to 85 °C and stirred overnight under nitrogen protection. After the reaction was completed by TLC, a suitable amount of water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. The crude product was then subjected to silica gel column chromatography with solvent A:solvent D = 1:4 elution to give a colorless oil D1-2-a (7.0 g, 49%).

[0129] Preparation of compound D1-2-b Compound D1-2-a (3.0 g, 11.31 mmol) was dissolved in acetonitrile (40 mL) and dichloromethane (20 mL) at room temperature. Tetradecyl alcohol (3.6 g, 16.97 mmol), 1-methylimidazole (NMI) (2.3 g, 28.28 mmol), and tetramethylchlorourea hexafluorophosphate (TCFH) (4.1 g, 14.71 mmol) were added sequentially. The reaction was carried out overnight at room temperature under nitrogen protection with stirring. After the reaction was complete as detected by TLC, water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to give the crude product. Silica gel column chromatography was performed, eluting with solvent D:solvent E = 9:1, to give a colorless oil D1-2-b (3.7 g, 71%).

[0130] Preparation of compound D1-2 Compound D1-2-b (3.7 g, 8.02 mmol) was dissolved in N,N-dimethylformamide (40 mL) at room temperature, followed by the addition of n-octylamine (1.6 g, 12.02 mmol) and potassium carbonate (3.0 g, 24.05 mmol). The mixture was heated to 80 °C and stirred overnight under nitrogen protection. After the reaction was completed by TLC, water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography with DCM elution yielded a colorless oil D1-2 (2.6 g, 64%).

[0131] Step 3: Preparation of compound D1 Intermediate D1-1 (100 mg, 0.20 mmol) was dissolved in N,N-dimethylformamide (2 mL) at room temperature. Intermediate D1-2 (125 mg, 0.25 mmol), HATU (101 mg, 0.27 mmol), and DIPEA (66 mg, 0.51 mmol) were added sequentially. The mixture was heated to 35 °C and stirred overnight under nitrogen protection. After the reaction was complete as detected by TLC, a suitable amount of water was added, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain the crude product. Silica gel column chromatography was performed, eluting with solvent A:solvent B = 10:1, to give a yellowish-brown oily substance D1 (117 mg, 59%), MS (ESI): [M+H]. + = 981.9.

[0132] Preparation of compound D2 Step 1: Prepare intermediate D2-1 (same as A1-1) Step 2: Prepare intermediate D2-2 (same as D1-2) Step 3: Preparation of compound D2 The preparation method for this compound was the same as for D1, using silica gel column chromatography with solvent A:solvent B = 10:1 elution to give a yellowish-brown oily compound D2 (164 mg, 81%), MS (ESI): [M+H]. + = 967.9.

[0133] Preparation of compound D3 Step 1: Prepare intermediate D3-1 (same as A1-1) Step 2: Preparation of intermediate D3-2 The compound was prepared in the same manner as D1-2, by silica gel column chromatography and DCM elution, yielding a colorless oily substance D3-2 (400 mg, 38%).

[0134] Step 3: Preparation of compound D3 The preparation method for this compound was the same as for D1, using silica gel column chromatography with solvent A:solvent B = 10:1 elution to give a pale yellow oil D3 (230 mg, 61%), MS (ESI): [M+H]. + = 911.9.

[0135] 6.5 Example 5 Preparation of Class E compounds Step 1 Step 2 Step 3 Preparation of compound E1 Step 1: Prepare intermediate E1-1 (same as B1-1) Step 2: Prepare intermediate E1-2 (same as D1-2) Step 3: Preparation of compound E1 The compound was prepared using the same method as D1-2, by silica gel column chromatography with solvent A:solvent B = 10:1 elution, yielding a yellowish-brown oily compound E1 (87 mg, 44%). MS (ESI): [M+H] + = 997.9.

[0136] 6.6 Example 6: Preparation and Characterization of Lipid Nanoparticles In brief, the cationic lipids (i.e., the prepared compounds), DSPC, cholesterol, and PEG-lipids provided herein were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5, and the mRNA was diluted in 10 mM to 50 mM citrate buffer at pH 4. LNPs were prepared by mixing the lipid-ethanol solution and the mRNA-water solution at a volume ratio of 1:3 using a microfluidic device at a total flow rate ranging from 6 to 30 mL / min, resulting in a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Ethanol was removed by dialysis and replaced with PBS (i.e., phosphate-buffered saline). Finally, the lipid nanoparticles were filtered through a 0.22 μm sterile filter.

[0137] The size of lipid nanoparticles was determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 90° scattering detection mode. 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 apparent pKa of the LNP formulations was correlated with the in vivo delivery efficiency of LNPs for nucleic acids. The apparent pKa ranged from approximately 5 to 7. The apparent pKa of each formulation was determined using fluorescence-based analysis of 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS). As described above, LNP formulations containing cationic lipids / DSPC / cholesterol / DMGPEG (50 / 10 / 38.5 / 1.5 mol%) in PBS were prepared. A 300 μM stock solution of TNS in distilled water was prepared. The LNP formulation was diluted to 0.1 mg / mL total lipids in 3 mL of a buffer solution containing 50 mM sodium citrate, 50 mM sodium phosphate, 50 mM sodium borate, and 30 mM sodium chloride (pH 3 to 9). Aliquots of the sample were added to obtain a final concentration of 0.1 mg / mL, and after vortex mixing, fluorescence intensity was measured at room temperature using an excitation wavelength of 325 nm and an emission wavelength of 435 nm on a Molecular Devices Spectramax iD3 spectrometer. S-curve best-fit analysis was applied to the fluorescence data, and the pKa value was measured as the pH at which the half-maximum fluorescence intensity was produced.

[0138] 6.7 Example 7: Animal Research The efficacy of the nanoparticle composition was evaluated using bioluminescence studies. Mice (n=3) were administered a 0.25 mg / kg (mpk) dose of the formulation via intramuscular injection, and bioluminescence was measured at 6 hours. The MC3 formulation was evaluated, and in some cases, controls (e.g., PBS controls) were evaluated for comparison.

[0139] The characteristics of the test lipid nanoparticles measured by the self-test group, including the F-luc luminescence intensity, are listed in Table 2 below.

[0140] Table 2: Properties of nanoparticles formed from compounds Note: A, B, C, and D represent the normalized results after comparing the luminous flux of the compound and MC3, with the following ratio ranges: A: ≥1 B: ≥0.1 and <1 C: <0.1.

[0141] 6.8 Example 8: Intramuscular Injection In vivo imaging detection A formulation containing compound (I-1) was prepared and administered intramuscularly at a dose of 0.25 mpk. Female SPF-grade BALB / c mice (n=3) aged 6-8 weeks were selected as test animals and housed in an SPF-grade animal facility at a temperature of 20-26℃ and humidity of 40-70%, with adequate feed and water. The control group was compared with MC3-LNP in vivo. Six hours after administration, 200 μL of D-fluorescein potassium salt at a concentration of 15 mg / mL was injected intraperitoneally, and in vivo imaging was performed 5 minutes after injection. Results are as follows... Figure 17-19 As shown, A3, A5, and A17 have a stronger total fluorescence than MC3, meaning they have a stronger ability to express mRNA in vivo than MC3.

[0142] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications and variations to the above embodiments within the scope of the present invention.

Claims

1. A lipid compound containing a bile acid derivative, characterized in that, The compound comprises at least one general structural formula as shown in formula (I-1): (I-1) Or the compound may be a pharmaceutically acceptable salt, prodrug, deuterated product, or isomer thereof; in, X1 is independently selected from any one of the following groups: , H; X2 is independently selected from any one of the following groups: , -CH2CH3, H; X3 is independently selected from any one of the following groups: , H; Y is independently selected from any one of the following groups: -O-, -N(R) a )-、-S-; and R a Independently -H or optionally substituted C1-C6 alkyl; Z is independently selected from any of the following groups: -C(=O)-, *-OC(=O)-**, *-N(R)- b C(=O)-** or does not exist; where *- indicates that it can be connected to equation (I-1). The site; -** indicates connection to the left side of equation (I-1) The site; and R b Independently -H or optionally substituted C1-C6 alkyl; m1, m2, m3 and m4 are each independently selected from integers between 1 and 18; L 1a L 1b and L 1c Each of the following structures is independently selected: -H, -S-SR1, -SR1, -C(=O)OR1, -OC(=O)R1, -OC(=O)OR1, -N(R2)R1, -C(=O)N(R2)R1, -OC(=O)N(R2)R1, -N(R2)C(=O)R1, -N(R2)C(=O)OR1, -N(R3)C(=O)N(R2)R1, -C(=O)NR1, -OR1; R1, R2, and R3 are each independently selected from the following structures: -H, alicyclic hydrocarbons, , , , , where n1, n2, x1, x2 and o are independently selected from integers from 0 to 12, and y is independently selected from 1, 2, 3 or 4; L2 is independently selected from the following structure: -R4G x R5G y -R4G y -R4G y R6, -G y -R4G x R5(G z G y 、; R4, R5, and R6 are each independently selected from the following structures: C1-C5 alkanes and C2-C5 alkenes; G x Independently selected from the following structures: -O-, -N(R) x )-, -S-, -SS-, -OC(=O)-, -C(=O)O-, -OP(=O)O-, -OS(=O)O-, -C(=O)N-, - N(C(=O) )-; R x Independently selected from the following structures: -H, -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -H and alicyclic hydrocarbons, wherein m is independently selected from 0, 1, 2, 3, 4 or 5; G y It is independently selected from the following structure: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*、-(CH2) m -W1-(CH2) n -H, where m and n are independently selected from 0, 1, 2, 3, 4 or 5; W1 is independently selected from benzene rings or heterocycles; R* and R** are independently selected from the following structures: H, C1-C3 alkanes, -(CH2). m Q1, where m is independently selected from 1, 2, 3 or 4; Q1 is independently selected from the following structures: -OH, -CN; G z It is independently selected from the following structure: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*、-(CH2) m -W2-(CH2) n -H, where m and n are independently selected from 0, 1, 2, 3, 4 or 5; W2 is independently selected from benzene rings or heterocycles; R* and R** are independently selected from the following structures: H, C1-C3 alkanes, -(CH2). a Q2, where a is independently selected from 1, 2, 3, or 4; Q2 is independently selected from the following structures: -OH, -CN.

2. The compound according to claim 1, characterized in that, R x It is an alicyclic hydrocarbon, R x A hydrocarbon having at least one or more of the following structural formulas (II-1), (II-2), (II-3), and (II-4), wherein structural formulas (II-1), (II-2), (II-3), and (II-4) are respectively: (Ⅱ-1) (Ⅱ-2) (Ⅱ-3) (Ⅱ-4) Or R x It is a pharmaceutically acceptable salt, prodrug, or isomer of the structural formula (II-1), (II-2), (II-3), or (II-4); Among them, the structural formulas (Ⅱ-1), (Ⅱ-2), (Ⅱ-3) and (Ⅱ-4) contain " The key can be a single key or a double key.

3. The compound according to claim 1, characterized in that, W1 and W2 are each independently heterocyclic compounds, and W1 and W2 are each independently nitrogen heterocyclic, oxygen heterocyclic, or sulfur heterocyclic compounds.

4. A composition, characterized in that, The composition comprises an excipient and a compound or an acceptable salt as described in any one of claims 1-3, wherein the excipient is selected from one or more neutral phospholipids, steroids and polyethylene glycol.

5. The composition according to claim 4, characterized in that, The steroid has the structure of formula (Ⅲ-1): Ⅲ-1。 6. The composition according to claim 4 or 5, characterized in that, The molar ratio of the compound to the steroid is in the range of (1:2) to (6:1).

7. The composition according to claim 4, characterized in that, The composition also includes polymer-bound lipids, wherein the polymer-bound lipids include one or more types.

8. The composition according to claim 7, characterized in that, The molar ratio of the compound to the lipid bound to the polymer ranges from (10:1) to (100:1); and / or The lipids bound to the polymer are one or more of polyinosinic acid, DSPE-PCB20, DMG-PEG2000, DMPE-PEG2000 and C14-PEG2000.

9. The composition according to claim 4, characterized in that, The composition further includes structural lipids, said structural lipids comprising at least one of formulas (III-2), (III-3), and (III-4): Ⅲ-2 Ⅲ-3 Ⅲ-4。 10. The composition according to claim 9, characterized in that, The molar ratio of the compound to the structural lipid is in the range of (2:1) to (10:1).

11. The composition according to claim 4, characterized in that, The composition may also include a therapeutic or preventative agent; The therapeutic or preventive agent is a nucleic acid drug or a nucleic acid-like drug.

12. The composition according to claim 11, characterized in that, The nucleic acid drugs include nucleic acid aptamers, circular RNA, ASO, siRNA, sgRNA, tRNA, or mRNA; The mRNA includes at least one mRNA encoding an antigen or a fragment thereof or an epitope, wherein the antigen is a pathogenic antigen or a tumor-associated antigen; and / or The mRNA is either a monocistronic mRNA or a polycistronic mRNA.

13. The composition according to claim 11, wherein the nucleic acid-like drug comprises locked nucleic acid (LNA), peptide nucleic acid (PNA), or morpholino.

14. The composition according to claim 12, characterized in that, The mRNA includes one or more functional nucleotide analogs; The functional nucleotide analogue is selected from one or more of the following: pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

15. A lipid nanoparticle, characterized in that, It comprises the compound as described in any one of claims 1-3 or the composition as described in any one of claims 4-14.

16. A pharmaceutical composition, characterized in that, It comprises a compound as described in any one of claims 1-3, a composition as described in any one of claims 4-14, or lipid nanoparticles as described in claim 15, and a pharmaceutically acceptable excipient or diluent.

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