Ionizable lipids, stereoisomers or pharmaceutically acceptable salts thereof, and uses thereof

Abstract

The application provides an ionizable lipid, a stereoisomer or a pharmaceutically acceptable salt thereof and application thereof and application thereof in preparation of a lipid nanoparticle, an mRNA drug, and has the structure shown in the following formula: wherein X is a 4-10 membered heterocycle containing one or more heteroatoms of nitrogen, sulfur and oxygen; Y is selected from -C- or N; G1, G2, G3, G4 and G5 are each independently a single bond, -O(C=O)- or the like; L1, L2, L3, L4, L5, L6 and L7 are each independently a divalent linking group or null, and the divalent group has the structure of - (L a -L b -L c ) -, wherein L a and L c are the same or different and are each independently a substituted or unsubstituted C1-C 12 alkylene or the like; R1, R2 and R3 are each independently H, a 4-10 membered heterocycle containing one or more heteroatoms of nitrogen, sulfur and oxygen, a substituted or unsubstituted C1-C 24 alkylene containing or not containing one or more heteroatoms of nitrogen, sulfur and oxygen, C2-C 24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene, wherein the substituent is a hydrocarbon group, a carboxyl group, an acyl group or an alkoxy group. The technical scheme of the application has the characteristics of high delivery efficiency, high transfection efficiency, low toxicity, safety and effectiveness.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to an ionizable lipid, its stereoisomers or pharmaceutically acceptable salts and their applications. Background Technology

[0002] Messenger RNA (mRNA) technology utilizes the organism's own cellular mechanisms to produce specific proteins, thereby achieving the purpose of treating or preventing diseases. However, the inherent negative charge and large molecular weight of mRNA limit its internalization efficiency and lead to rapid clearance. Endosomal / lysosomal barriers result in inefficient mRNA transfection, and in vivo / in vitro RNases cause rapid degradation. These challenges limit the application potential of mRNA technology. To address these challenges, currently marketed nucleic acid drugs all use lipid nanoparticles (LNPs) as delivery carriers to encapsulate and deliver RNA to designated sites in the body. Examples include Onpattro, a siRNA liposome product for treating familial amyloid polyneuropathy, which uses the ionizable lipid Dlin-MC3-DMA as a delivery carrier; and two vaccines for treating SARS-CoV-2, BNT162b2 and mRNA-1273, which use the ionizable lipids SM102 and ALC-0315, respectively, as delivery carriers. Ionizable lipid nanodelivery systems have achieved great success in the field of therapeutic nucleic acid drug delivery, especially mRNA technology, which is regarded as a disruptive drug development platform. This has enabled researchers to expand the research field of mRNA technology from infectious disease vaccines to areas such as tumor immunotherapy, protein replacement therapy, gene editing, and autoimmune diseases. Its broad application prospects are leading a revolution in the biomedical field.

[0003] LNPs are typically prepared from ionizable lipids, accessory lipids, cholesterol, and polyethylene glycol lipids in a specific ratio. Ionizable lipids play a crucial role, usually consisting of a hydrophilic amino head, a number and length of hydrophobic alkane tails, and a degradable linker. The amino head is often a tertiary amine. Due to their ionizable nature, ionizable lipids with a suitable pKa are electrically neutral under physiological conditions. This not only protects mRNA from degradation by RNases in the environment but also delivers mRNA to target cells or organs. Once endocytosed, the endosomal pH decreases, causing the ionizable lipids to become positively charged. This leads to electrostatic interactions with the negatively charged membrane lipids in the endosomal region, resulting in changes in osmotic pressure. Furthermore, the positively charged membrane lipids and ionizable lipids form neutral electron pairs, which, in conjunction with the hydrophobic lipid tails, fuse to cause membrane flipping. In summary, LNPs release mRNA into the cytoplasm through the proton sponge effect and membrane fusion mechanisms, thereby expressing specific encoded proteins to exert therapeutic effects.

[0004] Beyond the field of infectious diseases, tumor immunotherapy is another important area of ​​application for mRNA technology. However, the highly immunosuppressive microenvironment of tumors and the lack of infiltrating immune cells limit the immune recognition process. Research on infectious disease vaccines has largely focused on the design of low-immunogenic, ionizable lipid delivery vectors. The innate immune system plays a significant role in specifically recognizing, destroying, and remembering tumor cells without causing side effects on normal tissues. Tumor recognition and clearance are typically driven by antigen-presenting cells. Immune adjuvants are danger signaling molecules that promote the generation of antigen signals and co-stimulatory signals by targeting Toll-like receptors and other pattern recognition receptors, thereby enhancing adaptive immune responses and leading to the maturation and activation of antigen-presenting cells.

[0005] In summary, clinically approved ionizable lipids are mainly low in immunogenicity, while malignant tumors often require full activation of the immune system. Therefore, developing ionizable lipid compounds with high lysosomal escape efficiency, adjuvant activity, and high safety remains an important research direction in this field. Summary of the Invention

[0006] To address the problems in the prior art, the present invention aims to provide an ionizable lipid with high lysosomal escape efficiency and adjuvant activity, its stereoisomers or pharmaceutically acceptable salts, and their applications.

[0007] On one hand, the present invention provides an ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt thereof, having the structure shown in formula (I): (I) Wherein, X is a 4- to 10-membered heterocycle containing one or more heteroatoms of nitrogen, sulfur, and oxygen, or a branched C2-C ring containing or not containing one or more heteroatoms of nitrogen, sulfur, and oxygen. 24 Alkyl, branched C2-C 24 alkenyl, C2-C 24 The substituents of the divalent linker are alkyl, C3-C8 cycloalkyl, or C3-C8 cycloalkylene, wherein the substituents of the divalent linker are hydrocarbon, carboxyl, acyl, or alkoxy groups; Y is selected from -C- or N; G1, G2, G3, G4, and G5 are each independently a single bond, -O(C=O)-, -(C=O)O-, -(C=O)-, -O-, and -S(O). P -、-SS-、-Se-Se-、-(C=O)S-、-S(C=O)-、-NRa(C=O)-、-(C=O)NRa-、-NRa(C=O)NRa-、-O(C=O)NRa- or -NRa(C=O)O-; where p = 0, 1 or 2; Ra is H or Cl-C 12 hydrocarbon group; L1, L2, L3, L4, L5, L6, and L7 are each independently a divalent linker or none; the divalent linker has -(L a -L b -L c The structure of )-, where L a and L c Whether they are the same or different, each is independently substituted or unsubstituted C1-C. 12 Alkylene, C2-C 12 alkenyl, C3-C8 cycloalkylene, or C3-C8 cycloalkylene, wherein the substituent is a hydrocarbon, carboxyl, acyl, or alkoxy group; L b Selected from single bonds, -O(C=O)-, -(C=O)O-, -(C=O)-, -O-, -S(O) P -、-(C=O)S-、-S(C=O)-、-NRa(C=O)-、-(C=O)NRa-、-NRa(C=O)NRa-、-O(C=O)NRa- or -NRa(C=O)O-; where p = 0, 1 or 2; Ra is H or Cl-C 12 hydrocarbon group; R1, R2, and R3 are each independently H, a 4- to 10-membered heterocycle containing one or more heteroatoms of nitrogen, sulfur, or oxygen, or a substituted or unsubstituted C1-C ring with or without one or more heteroatoms of nitrogen, sulfur, or oxygen. 24 Alkylene, C2-C 24 The substituent is an alkenyl group, a C3-C8 cycloalkyl group, or a C3-C8 cycloalkenyl group, wherein the substituent is a hydrocarbon group, a carboxyl group, an acyl group, or an alkoxy group.

[0008] Preferably, the structure of formula (I) is as follows: or , where m and n are each independent positive integers from 1 to 20.

[0009] Preferably, the structure of formula (I) is as follows: or , where m and n are each independent positive integers from 1 to 20.

[0010] Preferably, the structure of formula (I) is as follows: , or , where m and n are each independent positive integers from 1 to 20.

[0011] Preferably, the structure of formula (I) is as follows: , , , , , , , , or .

[0012] On the other hand, the present invention provides the use of the above-mentioned ionizable lipids, their stereoisomers or pharmaceutically acceptable salts in the preparation of lipid nanoparticles: the lipid nanoparticles comprising the ionizable lipids, their stereoisomers or pharmaceutically acceptable salts as described in any one of claims 1-5.

[0013] Preferably, the lipid nanoparticles also contain bioactive substances.

[0014] Preferably, the bioactive substance is mRNA.

[0015] Preferably, the lipid nanoparticles further comprise polyethylene glycol lipids, steroidal lipids, and neutral lipids.

[0016] On the other hand, the present invention provides the use of the above-mentioned ionizable lipids, their stereoisomers, or pharmaceutically acceptable salts in the preparation of mRNA drugs.

[0017] The technical solution of this invention has the characteristics of high delivery efficiency, high transfection efficiency, low toxicity, safety and effectiveness. Attached Figure Description

[0018] Other features, objects, and advantages of the invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings.

[0019] Figure 1 This is a schematic diagram of the lipid molecule synthesis library used in Example 14 of the present invention; Figure 2 This is a fluorescence intensity diagram of LNP constructed from the four histamine heads A1, A2, A3, and A4 in Example 14 of the present invention; Figure 3 This is a fluorescence intensity diagram of LNPs constructed from ionizable lipid tails T1, T2, T3, T4, T5, T6, T7, T8 and T9 in Example 14 of the present invention. Figure 4 This is a fluorescence intensity diagram of LNPs constructed from ionizable lipids A1L1T6, A1L2T6, A1L3T6, A1L4T6, A1L5T6 and A1L6T6 in Example 14 of the present invention; Figure 5This is the fluorescence intensity map of the LNPs constructed from A1L6T6, A1L6T6-V1, A1L6T6-V2, A1L6T6-V3, A1L6T6-V4, A1L6T6-V5, A1L6T6-V6, A1L6T6-V7, and A1L6T6-V8 in Example 15 of this invention; Figure 6 This is the fluorescence intensity diagram of the LNP constructed using A1L6T6-V2, N2L6T6-V2, and SM102 in Example 14 of this invention; Figure 7 The high-resolution mass spectrum of the ionizable lipid compound A1L6T6-V1 in Example 3 of the present invention is shown. Figure 8 The high-resolution mass spectrum of the ionizable lipid compound A1L6T6-V2 in Example 4 of the present invention is shown. Figure 9 The high-resolution mass spectrum of the ionizable lipid compound A1L6T6-V3 in Example 5 of the present invention is shown. Figure 10 The high-resolution mass spectrum of the ionizable lipid compound A1L6T6-V4 in Example 6 of the present invention is shown. Figure 11 The high-resolution mass spectrum of the ionizable lipid compound A1L6T6-V5 in Example 7 of the present invention is shown. Figure 12 The high-resolution mass spectrum of the ionizable lipid compound A1L6T6-V6 in Example 8 of the present invention is shown. Figure 13 The high-resolution mass spectrum of the ionizable lipid compound A1L6T6-V7 in Example 9 of the present invention is shown. Figure 14 The high-resolution mass spectrum of the ionizable lipid compound A1L6T6-V8 of Example 10 of the present invention is shown. Figure 15 This shows a high-resolution mass spectrum of the ionizable lipid compound N2L6T6-V2 from Example 11 of the present invention. Detailed Implementation

[0020] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art. The same reference numerals in the drawings denote the same or similar structures, and therefore repeated descriptions of them will be omitted.

[0021] The embodiments of this invention innovatively employ a modular design strategy, using structurally diverse polyamines as the core framework. Through systematic and extensive permutations and combinations with various hydrophobic tail chains, linkers, and ionizable amine heads, a novel library of ionizable lipids with high chemical diversity is constructed. This invention, through this efficient combinatorial chemistry strategy, successfully develops a new generation of ionizable lipids that combine excellent nucleic acid delivery efficiency with rational immune activation functions, providing a powerful and customizable tool platform for the development of vaccines, tumor immunotherapy drugs, and gene therapy products.

[0022] As used herein, unless otherwise stated, "lipid" refers to a group of organic compounds, including but not limited to fatty acid esters, typically characterized by poor water solubility but soluble in nonpolar organic solvents. It encompasses biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Furthermore, lipids also include lipidoids, i.e., amphiphilic compounds with lipid-like physical properties.

[0023] The terms “ionizable lipid” and “ionizable lipid molecule” and “ionizable lipid compound” are used interchangeably and all refer to lipid compounds having the structure of Formula I, or their pharmaceutically acceptable salts, tautomers or stereoisomers.

[0024] The term "lipid nanoparticle" or "LNP" herein refers to nanoscale particles composed of one or more lipid components, used for encapsulating and delivering bioactive components (e.g., nucleic acids), with a hydrated particle size or electron microscopic observation size between 1 nm and 1000 nm. In some embodiments, the LNP comprises a bioactive component partially or completely encapsulated within a lipid shell. For example, in some embodiments, the bioactive component is an mRNA molecule encoding a protein, which binds to an ionizable lipid molecule via electrostatic interactions and self-assembles into a nanoparticle in the presence or absence of other lipid molecules, during which the mRNA molecule is encapsulated in a non-free state within the lipid nanoparticle. Other lipid components that can co-assemble with the ionizable lipids described herein to form an LNP include, but are not limited to, neutral lipids, charged lipids (such as DOPA, DOTAP), sterols, polymer-bound lipids, and zwitterionic lipids.

[0025] The term "ionizable lipid" refers to a lipid with a designed pKa that protonates and becomes positively charged in response to the pH of its environment, but deprotonates and remains uncharged at certain pH values. The protonation and deprotonation of an ionizable lipid molecule depend on its pKa; when the ambient pH is below the pKa, the ionizable lipid protonates and becomes positively charged, while conversely, when the ambient pH is above the pKa, the ionizable lipid deprotonates and becomes uncharged. In some embodiments, the ionizable lipid remains positively charged under neutral conditions. In some embodiments, the positive charge of the ionizable lipid originates from the presence of secondary and tertiary amines. In some embodiments, the pKa of the ionizable lipid is between 6.5 and 9.

[0026] The term "polymer-bound lipid" refers to a molecule that simultaneously possesses a lipid moiety and a polymer moiety, wherein the polymer moiety is covalently linked to the lipid moiety. Such lipids include polyethylene glycol-modified lipids, such as DMG-PEG2000.

[0027] The term "neutral lipids" refers to lipid molecules that exist in a neutral form within a specific pH range, including uncharged molecules or neutral zwitterionic forms. In some embodiments, the selected pH value or range may correspond to the pH conditions of the target application environment, such as physiological pH conditions. Neutral lipids described herein include, but are not limited to, distearylphosphatidylcholine (DSPC), dipalmitoyllecithin (DPPC), dimyristoyllecithin (DMPC), dioleoyllecithin (DOPC), 1-palmitoyl-2-oleoyllecithin (POPC), disorhoylphosphatidylcholine (DEPC), dilauroyllecithin (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), distearylphosphatidic acid (DSPA), dipalmitoylphosphatidic acid (DPPA), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylserine (DPPS), and dioleoylphosphatidylserine (DOPS). The neutral lipids described herein may be of synthetic origin or derived from natural sources or compounds (including isolated or modified natural components). In some embodiments, the neutral lipid is DSPC.

[0028] The term "steroidal lipid" refers to lipid compounds whose core structural feature is a cyclopentane-polyhydrophenanthrene core. This definition encompasses natural and synthetic molecules with this basic framework, including but not limited to cholesterol, β-sitosterol, stigmasterol, fucosterol, campesterol, brassosterol, ergosterol, 9,11-dehydroergosterol, cholesterol, dehydrocholesterol, bile acids, and deoxycholic acid. In some embodiments, the steroidal lipid is cholesterol.

[0029] As used herein and unless otherwise stated, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group consisting only of carbon and hydrogen atoms. This includes, but is not limited to, methyl, ethyl, propyl (n-propyl, isopropyl), butyl (n-butyl, isobutyl, sec-butyl, tert-butyl), and straight-chain or branched forms with five to twenty-four carbon atoms (C5-C24), such as pentyl (n-pentyl, isopentyl, sec-pentyl, neopentyl), hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. The alkyl group may be cyclic or acyclic, branched or straight-chain, and may optionally be modified by one or more substituents, including alkyl, cycloalkyl, alkoxy, amino, ether, halogen, hydroxyl, nitro, silyl, sulfoxide, sulfonate, carboxylic acid ester, or thiol, etc.

[0030] As used herein and unless otherwise stated, the term "alkenyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, including its unsubstituted form and derivatives with substituents. The term "alkenyl" also includes groups having "cis" and "trans" configurations, or "E" and "Z" configurations. In substituted alkenyl groups, the substituent may be attached to any carbon atom (whether or not it forms a double bond), as long as it does not compromise the chemical stability of the structure. The permitted types of substituents are the same as common substituents for alkyl groups, such as alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups. Common examples of alkenyl groups include vinyl, allyl, cyclopentenyl, and 5-hexenyl.

[0031] As used herein and unless otherwise stated, the term "alkylene" refers to a divalent saturated hydrocarbon group formed by removing two hydrogen atoms from a saturated alkane molecule. Its structure can be classified as straight-chain (e.g., -CH2CH2-, -CH2CH2CH2-) or branched-chain (e.g., -CH(CH3)-, -CH2CH(CH3)-). This group can exist as an unsubstituted structure or can be attached to various substituents such as alkyl, alkoxy, halogen, hydroxyl, and amino groups.

[0032] As used herein and unless otherwise stated, the term "alkenyl" refers to a divalent group formed by further abstracting a hydrogen atom from an alkenyl molecule, corresponding to a straight-chain or branched acyclic unsaturated hydrocarbon group containing at least one carbon-carbon double bond. The term covers groups with a specific range of carbon numbers, such as alkenyl groups having 2 to 6 carbon atoms.

[0033] As used herein and unless otherwise stated, the term "heterocyclic" refers to a non-aromatic cyclic structure that must contain at least one nitrogen atom as a heteroatom and may optionally contain one to three additional heteroatoms, each independently selected from nitrogen and sulfur. Such heterocyclic structures encompass monocyclic and bicyclic systems (including bridged and fused ring structures). Representative examples include, but are not limited to: aziridinyl, diaziridinyl, thiaaziridinyl, aziridinyl butyl, diaziridinyl, triaziridinyl, thiadiaziridinyl, thiadiaziridinyl, pyrrolidinyl, pyrazolyl, imidazolinyl, isothiazolyl, thiazolinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, aziridinyl heptyl, and aziridinyl octyl. The heterocyclic structures contain a total of one to four identical or different heteroatoms selected from nitrogen and sulfur. In some embodiments, the heterocycle may contain one to three nitrogen atoms; in other embodiments, one or two nitrogen atoms; and in still other embodiments, only one nitrogen atom. Furthermore, the term "heterocyclic" should be understood to encompass all structurally feasible isomers. Where structural conditions permit, heterocyclic groups can be linked to the rest of the molecule via carbon atoms (carbon linkages) or nitrogen atoms (nitrogen linkages) on the ring.

[0034] As used herein, unless otherwise stated, "amine" or "amino" may be used interchangeably to refer to a functional group containing a basic nitrogen atom with a lone pair.

[0035] As used herein, the term "pharmaceutically acceptable salt" refers to an organic or inorganic salt formed from an ionizable lipid of the present invention and a pharmaceutically acceptable acid or base. Such salts include, but are not limited to: acid addition salts derived from inorganic or organic acids, wherein examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; examples of organic acids include acetic acid, citric acid, tartaric acid, maleic acid, succinic acid, fumaric acid, lactic acid, gluconic acid, ascorbic acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, etc.; and base addition salts derived from inorganic or organic bases, wherein examples of inorganic bases include salts of sodium, potassium, calcium, magnesium, and ammonium; and examples of organic bases include isopropylamine, triethylamine, ethanolamine, triethanolamine, choline, glucosamine, arginine, lysine, etc. The salt may contain one or more charged centers, and correspondingly be accompanied by one or more pharmaceutically acceptable counterions. The counterions may be any inorganic or organic fraction that is stable under physiological conditions and suitable for pharmaceutical use.

[0036] The compounds described herein may contain one or more chiral centers, and therefore may exist as enantiomers, diastereomers, and other stereoisomers. Each stereoisomer may be classified according to its absolute configuration as (R)- or (S)-, and for amino acid compounds as (D)- or (L)-. Unless otherwise expressly stated, this document is intended to cover all such stereoisomers, including racemic mixtures and optically pure single isomers. Optically active (+)- and (-)-, (R)- and (S)-, or (D)- and (L)- isomers can be obtained by chiral synthesis or chiral reagents, or separated by conventional resolution methods such as chromatography or fractional crystallization. If the compounds described herein contain olefinic double bonds or other geometrically asymmetric centers, unless otherwise specified, this should be understood to include both E- and Z-type geometric isomers. Furthermore, this document also covers all possible tautomers.

[0037] Embodiments of the present invention provide an ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt thereof, having the structure shown in formula (I): (I) Wherein, X is a 4- to 10-membered heterocycle containing one or more heteroatoms of nitrogen, sulfur, and oxygen, or a branched C2-C ring containing or not containing one or more heteroatoms of nitrogen, sulfur, and oxygen. 24 Alkyl, branched C2-C 24 alkenyl, C2-C 24 The substituents are alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkyl, wherein the substituents are hydrocarbon, carboxyl, acyl or alkoxy; Y is selected from -C- or N; G1, G2, G3, G4, and G5 are each independently a single bond, -O(C=O)-, -(C=O)O-, -(C=O)-, -O-, and -S(O). P -、-SS-、-Se-Se-、-(C=O)S-、-S(C=O)-、-NRa(C=O)-、-(C=O)NRa-、-NRa(C=O)NRa-、-O(C=O)NRa- or -NRa(C=O)O-; where p = 0, 1 or 2; Ra is H or Cl-C 12 hydrocarbon group; L1, L2, L3, L4, L5, L6, and L7 are each independently a divalent linker or none; the divalent linker has -(L a -L b -L c The structure of )-, where L a and L cWhether they are the same or different, each is independently substituted or unsubstituted C1-C. 12 Alkylene, C2-C 12 alkenyl, C3-C8 cycloalkylene, or C3-C8 cycloalkylene, wherein the substituent is a hydrocarbon, carboxyl, acyl, or alkoxy group; L b Selected from single bonds, -O(C=O)-, -(C=O)O-, -(C=O)-, -O-, -S(O) P -、-(C=O)S-、-S(C=O)-、-NRa(C=O)-、-(C=O)NRa-、-NRa(C=O)NRa-、-O(C=O)NRa- or -NRa(C=O)O-; where p = 0, 1 or 2; Ra is H or Cl-C 12 hydrocarbon group; R1, R2, and R3 are each independently H, a 4- to 10-membered heterocycle containing one or more heteroatoms of nitrogen, sulfur, or oxygen, or a substituted or unsubstituted C1-C ring with or without one or more heteroatoms of nitrogen, sulfur, or oxygen. 24 Alkylene, C2-C 24 The substituent is an alkenyl group, a C3-C8 cycloalkyl group, or a C3-C8 cycloalkenyl group, wherein the substituent is a hydrocarbon group, a carboxyl group, an acyl group, or an alkoxy group.

[0038] Preferably, the structure of formula (I) is as follows: , , , , , or , where m and n are each independent positive integers from 1 to 20.

[0039] Furthermore, in embodiments of the present invention, R1, R2, and R3 in the following formula (I) are each independently one of the following structures:

[0040] Furthermore, in the embodiments of the present invention, the compounds of formula (I) are those listed in Table 1: Table 1: Structure table of compounds of formula (I)

[0041] The embodiments of the present invention provide the application of the above-described ionizable lipids, their stereoisomers, or pharmaceutically acceptable salts in the preparation of lipid nanoparticles and mRNA drugs.

[0042] The construction of nanodelivery systems includes lipid nanoparticles, liposomes, micelles, lipid polymer hybrid nanoparticles, etc.

[0043] The ionizable lipid compounds of this invention are combined with one or more of the following: auxiliary lipids, steroidal lipids, and polyethylene glycol lipids, to prepare lipid nanoparticles.

[0044] The auxiliary lipid is preferably a non-cationic lipid, and the lipid is selected from one or more of the following: distearylphosphatidylcholine (DSPC), dipalmitoyllecithin (DPPC), dimyristoyllecithin (DMPC), dioleoyllecithin (DOPC), 1-palmitoyl-2-oleoyllecithin (POPC), disorhoylphosphatidylcholine (DEPC), dilauroyllecithin (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), distearylphosphatidic acid (DSPA), dipalmitoylphosphatidic acid (DPPA), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylserine (DPPS), and dioleoylphosphatidylserine (DOPS).

[0045] The steroidal lipid is preferably cholesterol, selected from one or more of β-sitosterol, stigmasterol, fucosterol, campesterol, brassosterol, ergosterol, 9,11-dehydroergosterol, cholesterol, dehydrocholesterol, cholic acid, and deoxycholic acid.

[0046] The polyethylene glycol lipid is selected from one or more of the following: distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol (DPPE-mPEG), 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (DMG-PEG), and 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0315).

[0047] Preferably, in the lipid nanoparticles of this embodiment, the polyethylene glycol lipid is DMG-PEG2000, the steroidal lipid is cholesterol, and the neutral lipid is DSPC.

[0048] Preferably, the lipid nanoparticles of the present invention comprise ionizable lipids, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of ionizable lipids, DSPC, cholesterol, and DMG-PEG2000 is (40-60):(5-20):(30-50):(0.5-5), preferably (45-55):(8-12):(35-50):(1-3); in one embodiment of the present invention, the molar ratio is 40:10:48.5:1.5.

[0049] The ionizable lipid nanoparticles of the present invention can be prepared using conventional lipid nanoparticle preparation methods in the art, such as microfluidic mixing, ethanol injection, thin film hydration, high-pressure homogenization, and emulsification precipitation.

[0050] Furthermore, the lipid nanoparticles formed by combining ionizable lipid compounds with auxiliary lipids, sterols, and polyethylene glycol lipids in the embodiments of the present invention can encapsulate bioactive components.

[0051] Furthermore, the bioactive component is preferably one or more of nucleic acids, peptides, proteins, antibodies, antibiotics, antitumor agents, immunomodulators, anti-inflammatory agents, or small molecules.

[0052] Furthermore, the nucleic acid is preferably one or more of DNA, plasmid (pDNA), RNA, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), self-replicating RNA (saRNA), or circular RNA (circRNA).

[0053] Furthermore, when the bioactive substance is nucleic acid, in the ionizable lipid nanoparticles, the molar ratio (i.e., NP ratio) of the ionizable nitrogen atoms in the ionizable lipid molecules to the phosphate groups in the nucleic acid molecules is (1-60):1, preferably (3-30):1.

[0054] Furthermore, the hydrated particle size of the ionizable lipid nanoparticles is 30-300 nm, preferably 75-150 nm.

[0055] Furthermore, ionizable lipid nanomedicines loaded with bioactive components are used to prepare drugs for the prevention and / or treatment of cancer, inflammation, fibrotic diseases, autoimmune diseases, and rare diseases.

[0056] Furthermore, the ionizable lipids provided in this embodiment of the invention are used to deliver siRNA to specific cells or organs.

[0057] Furthermore, the ionizable lipids provided in this embodiment of the invention are used to deliver mRNA to specific cells or organs.

[0058] Furthermore, the lipid nanoparticles of this invention can deliver bioactive substances via oral, inhalation, transdermal, or injection routes.

[0059] Furthermore, the injection methods include intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, and intrathecal injection.

[0060] In another aspect, embodiments of the present invention provide a pharmaceutical composition comprising the lipid composition or lipid nanoparticles of the present invention, and pharmaceutically acceptable excipients.

[0061] Furthermore, pharmaceutically acceptable excipients include carriers, adjuvants, and diluents.

[0062] The present invention will be illustrated below with specific examples of the compounds of formula (I) in Table 2: Table 2: Structure Table of Specific Embodiments

[0063] Example 1

[0064] The synthetic steps of compound A1L1T1 are as follows:

[0065] Intermediate A1L1:

[0066] A 15 mL aqueous solution of 1-(2-chloroethyl)piperidine hydrochloride (5.0 g, 27 mmol) was added to 15 mL of ethylenediamine (5 equivalents) aqueous solution. The reaction mixture was stirred and reacted at 80 °C for 24 hours. After the reaction was complete, sodium hydroxide was added until the solution was saturated. The aqueous phase was extracted three times with dichloromethane to obtain the organic phase, which was then washed three times with saturated brine. The organic layer was separated, dried on anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. Subsequently, it was purified by silica gel column chromatography (100% dichloromethane → 100% 75:22:3 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was the intermediate A1L1.

[0067] Compound A1L1T1: 3,3'-((2-((3-(dodecyloxy)-3-oxopropyl)(2-piperidin-1-ylethyl)amino)ethyl)azadiyl)dipropionate didodecyl ester

[0068] Intermediate A1L1 (1 g, 5.8 mmol) was added to dodecyl acrylate (3.5 equivalents), and the reaction mixture was stirred and reacted at 80 °C for 48 hours. The mixture was purified by direct silica gel column chromatography (100% dichloromethane → 100% 89.4:10.0:0.6 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was compound A1L1T1.

[0069] Example 2

[0070] The synthetic steps of compound A1L6T6 are as follows:

[0071] Intermediate A1L6: 2-((2-aminoethyl)dithioetheryl)-N-(2-(piperidin-1-yl)ethyl)ethane-1-amine

[0072] A 15 mL aqueous solution of 1-(2-chloroethyl)piperidine hydrochloride (5.0 g, 27 mmol) was added to 15 mL of an aqueous solution of 2,2'-dithiodimethyldiethylamine (5 equivalents). The reaction mixture was stirred and reacted at 80 °C for 24 hours. After the reaction was complete, sodium hydroxide was added until the solution was saturated. The aqueous phase was extracted three times with dichloromethane to obtain the organic phase, which was then washed three times with saturated brine. The organic layer was separated, dried on anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. Subsequently, it was purified by silica gel column chromatography (100% dichloromethane → 100% 83.5:15.0:1.5 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which is intermediate A1L6.

[0073] Intermediate T6: 2-Hexyldecyl acrylate

[0074] 12 g (50 mmol) of 2-hexyl-1-decyl alcohol was dissolved in 20 mL of dichloromethane and cooled to 0 °C. Triethylamine (1.3 equivalents) was dissolved in 15 mL of dichloromethane and added dropwise through a separating funnel while stirring vigorously for at least 1 hour. Acryloyl chloride (1.5 equivalents) was dissolved in 15 mL of dichloromethane and added dropwise through a separating funnel while stirring vigorously for at least 1 hour. The reaction mixture was then allowed to react at room temperature for 24 hours. After the reaction was complete, the reaction mixture was filtered, washed 4-5 times with saturated brine, and the organic layer was collected, dried over anhydrous potassium carbonate, and concentrated by rotary evaporation under vacuum. The solution was then purified by silica gel column chromatography (100% dichloromethane) to obtain a colorless oily liquid, which was intermediate T6.

[0075] Compound A1L6T6: bis(2-hexyldecyl)3,3'-((2-((2-3-((2-hexyldecyl)oxy)-3-oxopropylamino)ethyl)dithioalkyl)ethyl)azadiyl)dipropionate

[0076] Intermediate A1L6 (1 g, 3.8 mmol) was added to 2-hexyldecyl acrylate (3.5 equivalents), and the reaction mixture was stirred and reacted at 80 °C for 48 hours. The mixture was purified by direct silica gel column chromatography (100% dichloromethane → 100% dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was A1L6T6.

[0077] Example 3

[0078] The synthetic steps of compound A1L6T6-V1 are as follows:

[0079] Intermediate A1L6 (1 g, 3.8 mmol) was added to 2-hexyldecyl acrylate (0.9 equivalents), and the reaction mixture was stirred and reacted at room temperature for 48 hours. After the reaction was completed, the mixture was purified by direct silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was A1L6T6-V1.

[0080] like Figure 7 The high-resolution mass spectra of the ionizable lipid compound A1L6T6-V1 show that the molecular weight of A1L6T6-V1 is [M+H]. + The molecular weight was 560.43 g / mol, consistent with the theoretical molecular weight of 559.42 g / mol.

[0081] Example 4

[0082] Synthetic steps of compound A1L6T6-V2

[0083] Intermediate 2T6-L6-Boc:

[0084] 1 g (1.2 mmol) of tert-butyl (2-((2-aminoethyl)dithioalkyl)ethyl)carbamate was added to 2-hexyldecyl acrylate (2.5 equivalents), and the reaction was carried out at 80 °C for 48 hours. The mixture was then purified by silica gel column chromatography (100% dichloromethane → 100% dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was 2T6-L6-Boc.

[0085] Intermediate 2T6-L6: bis(2-hexyldecyl)3,3'-((2-((2-aminoethyl)dithio)ethyl)azadipropionate

[0086] 2T6-L6-Boc (1 g, 1.2 mmol) was dissolved in 20 mL of dichloromethane and cooled to 0 °C. Trifluoroacetic acid (15 equivalents) was added dropwise while stirring for 1 hour, followed by stirring overnight at room temperature. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was slowly added at 0 °C to quench the reaction. The organic layer was separated, washed three times with saturated brine, dried over anhydrous potassium carbonate, filtered, and rotary evaporated. No further purification was required to obtain 2T6-L6.

[0087] Compound A1L6T6-V2: bis(2-hexyldecyl)3,3'-((2-((2-((2-(piperidin-1-yl)ethyl)amino)ethyl)dithioalkyl)ethyl)azadipropionate

[0088] 2T6-L6 (1 g, 1.3 mmol) was dissolved in 10 mL of anhydrous ethanol, followed by the addition of N,N-diisopropylethylamine (DIPEA, 1.2 equivalents) and 1-(2-chloroethyl)piperidine (0.9 equivalents), and the reaction was carried out at 50 °C for 48 hours. After the reaction was completed, the reaction mixture was filtered, and the crude oil obtained by vacuum rotary evaporation was dissolved in dichloromethane, washed three times with saturated brine, and the organic layer was collected, dried over anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. The solution was then purified by silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was compound A1L6T6-V2.

[0089] like Figure 8 High-resolution mass spectrometry of the ionizable lipid compound A1L6T6-V2 revealed that its molecular weight [M+H] was [M+H]. + The molecular weight was 856.70 g / mol, consistent with the theoretical molecular weight of 855.69 g / mol.

[0090] Example 5

[0091] The synthetic steps of compound A1L6T6-V3 are as follows:

[0092] Intermediate T6-L6-T6: bis(2-hexadecyl)3,3'-((dithioalkylbis(ethane-2,1-diyl))bis(azoalkyl))dipropionate.

[0093]

[0094] 2,2'-Dithiodimethyldiethylamine (1 g, 6.5 mmol) was added to 2-hexyldecyl acrylate (2 equivalents), and the reaction mixture was stirred and reacted at room temperature for 48 hours. After the reaction was completed, the mixture was purified by direct silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which is the intermediate T6-L6-T6.

[0095] Compound A1L6T6-V3: 2-hexyldecyl 3-({2-[2-({3-[(2-hexyldecyl)oxy]-3-oxopropyl}[2-(piperidin-1-yl)ethyl]amino)ethyl]dithioalkyl}ethylamino)propionate.

[0096] T6-L6-T6 (1 g, 1.3 mmol) was dissolved in 10 mL of anhydrous ethanol, followed by the addition of N,N-diisopropylethylamine (DIPEA, 1.2 equivalents) and 1-(2-chloroethyl)piperidine (0.9 equivalents), and the reaction was carried out at 50 °C for 48 hours. After the reaction was completed, the reaction mixture was filtered, and the crude oil obtained by vacuum rotary evaporation was dissolved in dichloromethane, washed three times with saturated brine, and the organic layer was collected, dried over anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. The solution was then purified by silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was compound A1L6T6-V3.

[0097] like Figure 9 High-resolution mass spectrometry of the ionizable lipid compound A1L6T6-V3 revealed that its molecular weight [M+H] was [M+H]. +The molecular weight was 856.70 g / mol, consistent with the theoretical molecular weight of 855.69 g / mol.

[0098] Example 6

[0099] The synthetic steps of compound A1L6T6-V4 are as follows:

[0100] Intermediate 2A1-L6-Boc: tert-butyl(2-((2-(bis(2-piperidin-1-ylethyl)amino)ethyl)dithioalkyl)ethyl)carbamate

[0101] 1 g of tert-butyl (2-((2-aminoethyl)dithioalkyl)ethyl)carbamate (1.2 mmol) and 1-(2-chloroethyl)piperidine (1.8 equivalents) were dissolved in 10 mL of anhydrous ethanol, followed by the addition of N,N-diisopropylethylamine (DIPEA, 1.8 equivalents), and the reaction was carried out at 80 °C for 48 hours. After the reaction was completed, the reaction mixture was filtered, and the crude oil obtained by vacuum rotary evaporation was dissolved in dichloromethane, washed three times with saturated brine, and the organic layer was collected, dried over anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. Subsequently, the mixture was purified by silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was the intermediate 2A1-L6-Boc.

[0102] Intermediate 2A1-L6-NH2: 2-[(2-aminoethyl)dithioalkyl]-N,N-bis[2-(piperidin-1-yl)ethyl]ethylamine

[0103] 2A1-L6-Boc (1 g, 2.1 mmol) was dissolved in 20 mL of dichloromethane and cooled to 0 °C. Trifluoroacetic acid (15 equivalents) was added dropwise while stirring for 1 hour, followed by stirring overnight at room temperature. After the reaction was complete, the reaction was quenched by slowly adding saturated sodium bicarbonate aqueous solution at 0 °C. The organic layer was separated, washed three times with saturated brine, dried over anhydrous potassium carbonate, filtered, and rotary evaporated. No further purification was required to obtain intermediate 2A1-L6.

[0104] Compound A1L6T6-V4: 3-((2-((2-(bis(2-piperidin-1-ylethyl)amino)ethyl)dithioalkyl)ethyl)amino)hexyldecyl propionate.

[0105] 2A1-L6 (1 g, 2.6 mmol) was added to 2-hexyldecyl acrylate (0.9 equivalents), and the reaction mixture was stirred and reacted at room temperature for 48 hours. After the reaction was completed, the mixture was purified by direct silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was compound A1L6T6-V4.

[0106] like Figure 10 High-resolution mass spectrometry of the ionizable lipid compound A1L6T6-V4 revealed that its molecular weight [M+H] was [M+H]. + The molecular weight was 671.53 g / mol, consistent with the theoretical molecular weight of 670.53 g / mol.

[0107] Example 7

[0108] The synthetic steps of compound A1L6T6-V5 are as follows:

[0109] Compound A1L6T6-V5: bis(2-hexyldecyl)3,3'-((2-((2-(bis(2-(piperidin-1-yl)ethyl)amino)ethyl)dithioalkyl)ethyl)azadiyl)dipropionate

[0110] 2T6-L6 (1 g, 1.3 mmol) was dissolved in 10 mL of anhydrous ethanol, followed by the addition of N,N-diisopropylethylamine (DIPEA, 2.5 equivalents) and 1-(2-chloroethyl)piperidine (2.5 equivalents), and the reaction was carried out at 80 °C for 48 hours. After the reaction was completed, the reaction mixture was filtered, and the crude oil obtained by vacuum rotary evaporation was dissolved in dichloromethane, washed three times with saturated brine, and the organic layer was collected, dried over anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. The solution was then purified by silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was compound A1L6T6-V5.

[0111] like Figure 11 High-resolution mass spectrometry of the ionizable lipid compound A1L6T6-V5 revealed that its molecular weight [M+H] was [M+H]. + The molecular weight is 967.80 g / mol, which is consistent with the theoretical molecular weight of 966.80 g / mol.

[0112] Example 8

[0113] The synthetic steps of compound A1L6T6-V6 are as follows:

[0114] Intermediate A1-L6-A1: 2,2'-dithiobis(N-(2-(piperidin-1-yl)ethyl)ethylamine)

[0115] 2,2'-Dithiodimethyldiethylamine (1 g, 6.5 mmol) and 1-(2-chloroethyl)piperidine (1.8 equivalents) were dissolved in 10 mL of anhydrous ethanol, followed by the addition of N,N-diisopropylethylamine (DIPEA, 1.8 equivalents), and the reaction was carried out at 80 °C for 48 hours. After the reaction was completed, the reaction mixture was filtered, and the crude oil obtained by vacuum rotary evaporation was dissolved in dichloromethane, washed three times with saturated brine, and the organic layer was collected, dried over anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. The solution was then purified by silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was the intermediate A1-L6-A1.

[0116] Compound A1L6T6-V7: 2-hexyldecyl 3-((2-piperidin-1-ylethyl)(2-((2-[(2-piperidin-1-ylethyl)amino]ethyl)dithioalkyl)ethyl)amino)propionate.

[0117] A1-L6-A1 (1 g, 6.5 mmol) was added to 2-hexyldecyl acrylate (0.9 equivalents), and the reaction mixture was stirred and reacted at 80 °C for 48 hours. After the reaction was completed, the mixture was purified by direct silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was the intermediate A1L6T6-V6.

[0118] like Figure 12 High-resolution mass spectrometry of the ionizable lipid compound A1L6T6-V6 revealed that its molecular weight [M+H] was [M+H]. + The molecular weight was 671.53 g / mol, consistent with the theoretical molecular weight of 670.53 g / mol.

[0119] Example 9

[0120] The synthetic steps of compound A1L6T6-V7 are as follows:

[0121] Compound A1L6T6-V7: bis(2-hexyldecyl)3,3'-[(dithionidylbis(ethane-2,1-diyl))bis((2-piperidin-1-ylethyl)azadiyl)]dipropionate

[0122] T6-L6-T6 (1 g, 1.3 mmol) was dissolved in 10 mL of anhydrous ethanol, followed by the addition of N,N-diisopropylethylamine (DIPEA, 2.5 equivalents) and 1-(2-chloroethyl)piperidine (2.5 equivalents), and the reaction was carried out at 80 °C for 48 hours. After the reaction was completed, the reaction mixture was filtered, and the crude oil obtained by vacuum rotary evaporation was dissolved in dichloromethane, washed three times with saturated brine, and the organic layer was collected, dried on anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. The solution was then purified by silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was compound A1L6T6-V7.

[0123] like Figure 13 High-resolution mass spectrometry of the ionizable lipid compound A1L6T6-V7 revealed that its molecular weight [M+H] was [M+H]. + The molecular weight is 967.80 g / mol, which is consistent with the theoretical molecular weight of 966.80 g / mol.

[0124] Example 10

[0125] The synthetic steps of compound A1L6T6-V8 are as follows:

[0126] Intermediate T6-L6: 2-hexyldecyl 3-((2-((2-aminoethyl)dithio)ethyl)amino)propionic acid

[0127] 2,2'-Dithiodimethyldiethylamine (1 g, 6.5 mmol) was added to 2-hexyldecyl acrylate (1.1 equivalents), and the reaction mixture was stirred and reacted at room temperature for 48 hours. After the reaction was completed, the mixture was purified by direct silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was intermediate T6-L6.

[0128] Compound A1L6T6-V8: 3-((2-((2-(bis(2-(piperidin-1-yl)ethyl)amino)ethyl)dithioalkyl)ethyl)(2-(piperidin-1-yl)ethyl)amino)propionate 2-hexyldecyl ester.

[0129] T6-L6 (1 g, 2.2 mmol) and 1-(2-chloroethyl)piperidine (4 equivalents) were dissolved in 10 mL of anhydrous ethanol, followed by the addition of N,N-diisopropylethylamine (DIPEA, 4 equivalents), and the reaction was carried out at 80 °C for 48 hours. After the reaction was completed, the reaction mixture was filtered, and the crude oil obtained by vacuum rotary evaporation was dissolved in dichloromethane, washed three times with saturated brine, and the organic layer was collected, dried over anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. The solution was then purified by silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was compound A1L6T6-V8.

[0130] like Figure 14 High-resolution mass spectrometry of the ionizable lipid compound A1L6T6-V8 revealed that its molecular weight [M+H] was [M+H]. + The molecular weight was 782.64 g / mol, consistent with the theoretical molecular weight of 781.63 g / mol.

[0131] Example 11

[0132] The synthetic steps of compound N2L6T6-V2 are as follows:

[0133] Compound N2L6T6-V2: 3-ethyl-13-[3-((2-hexyldecyl)oxy)-3-oxopropyl]-9,10-dithio-3,6,13-triazahexadecane-16-acid 2-hexyldecyl ester.

[0134] 2T6-L6 (1 g, 1.3 mmol) was dissolved in 10 mL of anhydrous ethanol, followed by the addition of sodium hydroxide (1.0 equivalent), N,N-diisopropylethylamine (DIPEA, 1.1 equivalent), and 1-(2-chloroethyl)piperidine (1.0 equivalent). The reaction mixture was reacted at 50 °C for 48 hours. After the reaction was complete, the reaction mixture was filtered, and the crude oil obtained by vacuum rotary evaporation was dissolved in dichloromethane, washed three times with saturated brine, and the organic layer was collected, dried over anhydrous potassium carbonate, and concentrated by vacuum rotary evaporation. The solution was then purified by silica gel column chromatography (100% dichloromethane → 100% 89:10:1 dichloromethane / methanol / ammonium hydroxide) to obtain a yellow oily liquid, which was compound N2L6T6-V2.

[0135] like Figure 15 High-resolution mass spectrometry of the ionizable lipid compound N2L6T6-V2 revealed that its molecular weight [M+H] was [M+H]. + The molecular weight was 844.70 g / mol, consistent with the theoretical molecular weight of 843.69 g / mol.

[0136] Example 12

[0137] Example 1: Preparation of Lipid Nanoparticles Loaded with Nucleic Acids (mRNA-LNP) The ionizable lipids, 1,2-distearate-sn-glycerol-3-phosphorylcholine (DSPC) cholesterol, and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) from Examples 1-11 were dissolved in anhydrous ethanol. The four components were mixed in different proportions to form an organic phase. Messenger RNA (mRNA) was dissolved in sodium acetate buffer (pH=4.0, 50 mM) as an aqueous phase. An appropriate amount of organic and aqueous phases were drawn into a syringe and mixed through a microfluidic chip. The volume ratio of the aqueous phase to the ethanol phase was 3:1, the total flow rate was 12 mL / min, and the nitrogen-phosphorus ratio was 18. Three volumes of phosphate buffer (pH=7.4) were added. After standing for 15 min, the prepared mRNA-LNP was concentrated by ultrafiltration until neutral using an ultrafiltration tube. After filtration through a 0.22 μm filter membrane, subsequent characterization was performed.

[0138] Example 2: Preparation of nucleic acid-loaded lipid nanoparticles (mRNA-LNP): The ionizable lipids, 1,2-distearate-sn-glycerol-3-phosphorylcholine (DSPC) cholesterol, and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) from Examples 1-11 were dissolved in anhydrous ethanol. The four components were mixed in different proportions to form an organic phase. Messenger RNA (mRNA) was dissolved in sodium acetate buffer (pH=4.0, 20 mM) as the aqueous phase. The volume ratio of the aqueous phase to the ethanol phase was 3:1, and the nitrogen-phosphorus ratio was 18. An appropriate amount of organic phase was pipetted into an EP tube, and three times the volume of the aqueous phase solution was added to the organic phase. The mixture was quickly mixed by pipetting, and then three times the volume of phosphate buffer (pH=7.4) was added. After standing for 15 min, the prepared mRNA-LNP was concentrated by ultrafiltration until neutral. After filtration through a 0.22 μm filter membrane, it was used for subsequent characterization.

[0139] Example 13

[0140] Table 3 below shows the pKa measurements of the ionizable lipids prepared according to Preparation Example 1 or Preparation Example 2 in Example 12: The apparent pKa of lipid nanoparticles was determined according to the method described by Staci et al. (Mol. Ther., 2018, 26,(6), 1509-1519).

[0141] Lipid nanoparticles were prepared at a concentration of 40 μg / mL mRNA. A 300 μM stock solution was prepared by dissolving 6-(p-toluidine)-2-naphthalenesulfonic acid (TNS) in dimethyl sulfoxide. Detection buffers were prepared using 10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodium citrate, and 150 mM sodium chloride. A series of buffer solutions with pH values ​​between 3 and 11 were prepared using 1 M sodium hydroxide and 1 M hydrochloric acid. In a black transparent 96-well plate, 90 μL of buffer, 3.26 μL of lipid nanoparticle sample, and 2 μL of 300 μM TNS reagent solution were thoroughly mixed per well. The plates were read using a microplate reader (Ex: 321 / Em: 447). An S-shaped curve of fluorescence versus buffer pH was constructed based on the obtained fluorescence values. The logarithm of the inflection point of the curve is the apparent pKa according to the LNP formula. The results are shown in Table 3 below.

[0142] Table 3: pKa values

[0143] Example 14

[0144] In vivo expression detection of mRNA-LNP prepared from different ionizable lipids: To evaluate the expression efficiency of lipid nanoparticles constructed from ionizable lipids, mRNA encoding luciferase was encapsulated in LNPs containing ionizable lipids, DSPC, cholesterol, and DMG-PEG2000. This LNP was injected subcutaneously into female C57BL / 6 mice (7 weeks old) via the dorsal side. Hair was removed from the back of the mice before imaging. Six hours after injection, 100 μL of a 30 mg / mL D-luciferin potassium solution was injected intraperitoneally. Ten minutes after substrate injection, the C57BL / 6 mice were anesthetized with isoflurane using a gas anesthesia machine. In vivo IVIS imaging was then performed, and the bioluminescence intensity at the dorsal injection site was measured to evaluate the expression efficiency of the lipid nanoparticles.

[0145] like Figure 1 As shown in Example 1, with the linker L1 unchanged, and the amino head A1, A2, A3, A4 and lipid tail T1, T2, T3, T4, T5, T6, T7, T8, T9 combined, a total of 36 ionizable lipids were prepared.

[0146] Lipids were grouped according to the same amino head, resulting in four groups (A1, A2, A3, and A4), with nine ionizable lipids in each group. Lipids with the same amino head were mixed together, and mRNA-LNPs were prepared. Drug administration and imaging were then performed at a dose of 0.25 mg / kg. Figure 2As shown, each group had 3 parallel samples, and the group with the A1 amino head had the highest transfection efficiency.

[0147] Nine groups were formed based on the same lipid tail (T1, T2, T3, T4, T5, T6, T7, T8, and T9), with four ionizable lipids in each group. Lipids with the same lipid tail were mixed together, and mRNA-LNP was prepared. Drug administration and imaging were then performed at a dose of 0.25 mg / kg. Figure 3 As shown, each group had 3 parallel samples, and the group with lipid tail T6 characteristics had the highest transfection efficiency.

[0148] Following Example 1 or Example 2, with the amino head A1 and lipid tail T6 remaining unchanged, and... Figure 1 The six linkers L1, L2, L3, L4, L5, and L6 were combined to prepare six ionizable lipids (A1L1T6, A1L2T6, A1L3T6, A1L4T6, A1L5T6, and A1L6T6), which were then encapsulated to construct LNPs encoding Luc mRNA. The dosage was 0.25 mg / kg. Figure 4 As shown, the ionizable lipid A1L6T6 exhibits the highest transfection efficiency.

[0149] The ionizable lipids A1L6T6-V2 prepared according to Example 4, N2L6T6-V2 prepared according to Example 11, and the commercially available ionizable lipid SM102 were used to prepare mRNA-LNP, followed by drug administration and imaging. The dosage was 0.125 mg / kg. Figure 6 As shown, ionizable lipids A1L6T6-V2 and N2L6T6-V2 exhibited high transfection efficiencies similar to SM102.

[0150] The structure of SM102 is as follows:

[0151] Example 15

[0152] In vitro expression detection of mRNA-LNP prepared from different ionizable lipids: Nanoparticle preparation: LNPs loaded with green fluorescent protein mRNA were prepared according to Preparation Example 2 in Example 12.

[0153] Cell plating: DC2.4 cells were digested to obtain a single-cell suspension, and the cell density was adjusted to 1 x 10⁻⁶. 5 / wells were seeded into 24-well cell culture plates and incubated overnight at 37 ℃ in a 5% CO2 cell culture incubator.

[0154] Experimental groups: Candidate ionizable lipids A1L6T6, A1L6T6-V1, A1L6T6-V2, A1L6T6-V3, A1L6T6-V4, A1L6T6-V5, A1L6T6-V6, A1L6T6-V7 and A1L6T6-V8 were selected as experimental groups, and the saline group served as the negative control group.

[0155] Cell transfection: Add mRNA-LNP to a 24-well plate at a concentration of 1 μg / mL. Incubate the cell culture plate at 37 °C in a 5% CO2 cell culture incubator for 24 hours.

[0156] Flow cytometry: After digestion of DC2.4 cells, single-cell suspensions were obtained, and live / dead dye (Zombie R718™ Fixable Viability Kit, 1:1000) was added. The cells were incubated at room temperature for 15 minutes, followed by flow cytometry detection of FITC channel positive signals. Figure 5 As shown, flow cytometry results of DC2.4 cell transfection showed that A1L6T6-V2 had the highest efficiency and average fluorescence intensity.

[0157] In summary, the ionizable lipids provided by this invention, especially A1L6T6-V2 and N2L6T6-V2, have the characteristics of high delivery efficiency, high transfection efficiency, low toxicity, safety and effectiveness.

[0158] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. An ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt, characterized in that, It has the structure shown in equation (I): （I） Wherein, X is a 4- to 10-membered heterocycle containing one or more heteroatoms of nitrogen, sulfur, and oxygen, or a branched C2-C ring containing or not containing one or more heteroatoms of nitrogen, sulfur, and oxygen. 24 Alkyl, branched C2-C 24 alkenyl, C2-C 24 The substituents are alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkyl, wherein the substituents are hydrocarbon, carboxyl, acyl or alkoxy; Y is selected from -C- or N; G1, G2, G3, G4, and G5 are each independently a single bond, -O(C=O)-, -(C=O)O-, -(C=O)-, -O-, and -S(O). P -、-SS-、-Se-Se-、-(C=O)S-、-S(C=O)-、-NRa(C=O)-、-(C=O)NRa-、-NRa(C=O)NRa-、-O(C=O)NRa- or -NRa(C=O)O-; where p = 0, 1 or 2; Ra is H or Cl-C 12 hydrocarbon group; L1, L2, L3, L4, L5, L6, and L7 are each independently a divalent linker or none; the divalent linker has -(L a -L b -L c The structure of )-, where L a and L c Whether they are the same or different, each is independently substituted or unsubstituted C1-C. 12 Alkylene, C2-C 12 alkenyl, C3-C8 cycloalkylene, or C3-C8 cycloalkylene, wherein the substituent is a hydrocarbon group, carboxyl group, acyl group, or alkoxy group; L b Selected from single bonds, -O(C=O)-, -(C=O)O-, -(C=O)-, -O-, -S(O) P -、-(C=O)S-、-S(C=O)-、-NRa(C=O)-、-(C=O)NRa-、-NRa(C=O)NRa-、-O(C=O)NRa- or -NRa(C=O)O-; where p = 0, 1 or 2; Ra is H or Cl-C 12 hydrocarbon group; R1, R2, and R3 are each independently H, a 4- to 10-membered heterocycle containing one or more heteroatoms from nitrogen, sulfur, and oxygen, and are either substituted or unsubstituted C1-C rings with or without one or more heteroatoms from nitrogen, sulfur, and oxygen. 24 Alkylene, C2-C 24 The substituent is an alkenyl group, a C3-C8 cycloalkyl group, or a C3-C8 cycloalkenyl group, wherein the substituent is a hydrocarbon group, a carboxyl group, an acyl group, or an alkoxy group.

2. The ionizable lipid, its stereoisomer, or pharmaceutically acceptable salt according to claim 1, characterized in that: The structure of equation (I) is as follows: or , where m and n are each independent positive integers from 1 to 20.

3. The ionizable lipid, its stereoisomer, or pharmaceutically acceptable salt according to claim 1, characterized in that: The structure of equation (I) is as follows: or , where m and n are each independent positive integers from 1 to 20.

4. The ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt according to claim 1, characterized in that: The structure of equation (I) is as follows: , or , where m and n are each independent positive integers from 1 to 20.

5. The ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt according to claim 1, characterized in that: The structure of equation (I) is as follows: , , , , , , , , or .

6. The use of the ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt according to any one of claims 1-5 in the preparation of lipid nanoparticles, characterized in that: The lipid nanoparticles comprise the ionizable lipids, stereoisomers thereof, or pharmaceutically acceptable salts as described in any one of claims 1-5.

7. The use of the ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt according to claim 6 in the preparation of lipid nanoparticles, characterized in that: The lipid nanoparticles also contain bioactive substances.

8. The use of the ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt according to claim 7 in the preparation of lipid nanoparticles, characterized in that: The bioactive substance is mRNA.

9. The use of the ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt according to claim 6 in the preparation of lipid nanoparticles, characterized in that: The lipid nanoparticles also contain polyethylene glycol lipids, steroidal lipids, and neutral lipids.

10. The use of the ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt according to any one of claims 1-5 in the preparation of an mRNA drug.

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