Aminolipid compounds, their production method and use

Abstract

The present invention relates to an amino lipid compound having the following structural formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof, and its use as a component of a lipid nanoparticle formulation for delivering a therapeutic agent. The present invention also relates to compositions, particularly lipid nanoparticles, comprising said amino lipid compound, and uses thereof. TIFF2025525597000117.tif39170

Description

[Technical Field]

[0001] The present disclosure is in the field of pharmaceuticals, and specifically relates to amino lipid compounds, their preparation methods and uses. [Background technology]

[0002] Gene therapy is the artificial delivery of genes with specific genetic information to target cells, and the expressed target proteins can regulate, treat, and even cure diseases caused by congenital or acquired genetic defects. Both nucleic acids and cell membranes are negatively charged. Therefore, naked nucleic acids are difficult to directly introduce into cells and are easily degraded by nucleases in the cytoplasm, making it impossible to achieve the effects of gene transfer and gene therapy. Therefore, it is necessary to use external forces or vectors to carry out gene delivery.

[0003] Gene vectors are generally divided into viral vectors and non-viral vectors. Viral vectors have high transfection efficiency in vivo and in vitro, but also have many defects such as high toxicity, strong immune response, small gene capacity, poor targeting, and complicated manufacturing methods. Non-viral vectors have attracted increasing attention due to their advantages such as easy manufacturing, transportation, storage, safety, efficacy, and non-immunogenicity.

[0004] Lipid nanoparticles (LNPs) are currently the mainstream non-viral vectors commonly used in vaccines due to their ease of absorption by antigen-presenting cells. While numerous compounds have been reported for use in lipid nanoparticles, their use in vaccine delivery has been found to result in low delivery efficiency and poor pharmaceutical stability. Furthermore, they have issues such as long half-lives, high toxicity, and poor safety, making them unsuitable for clinical application and unable to meet the demands of modern vaccine formulations.

[0005] When commercially available cationic liposomal compounds like MC3 were actually used for vaccine delivery, protein expression levels were insufficient, resulting in low levels of antibody production in vivo. Furthermore, in a Phase 2 clinical study of patisiran reported in the paper "Pharmacokinetics of Patisiran, the First Approved RNA Interference Therapy in Patients With Hereditary Transthyretin-Mediated Amyloidosis" by Xiaopin GZhang et al., The Journal of Clinical Pharmacology, 2020, 60(5), 573-585, cationic liposomal MC3 had a long clearance phase half-life of 14.6 to 28.7 days. [ka]

[0006] Therefore, there is a strong demand for the development of substances useful for delivering therapeutic agents into cells that have strong delivery capabilities, excellent stability, an appropriate clearance phase half-life, and / or high safety (i.e., low incidence of side effects). Summary of the Invention

[0007] As a result of extensive research, the present inventors have surprisingly found that amino lipid compounds having a cycloalkyl structural unit and another substituent at the meta position relative to the amine substituent of the cycloalkyl group have excellent delivery ability, stability, and safety, are useful for delivering physiologically active agents (e.g., nucleic acids) into cells, and have the function of increasing protein expression levels.

[0008] Thus, the present disclosure provides amino lipid compounds having a cycloalkyl structural unit and further having another substituent at the meta position relative to the amine substituent of the cycloalkyl.The amino lipid compounds of the present disclosure are stable in the circulation in the body, are rapidly degraded in endosomes / lysosomes, and have significantly enhanced delivery efficiency.

[0009] In the present disclosure, amino lipid compounds having a cycloalkyl structural unit and another substituent at the meta position relative to the amine substituent of the cycloalkyl have good biological activity, high protein expression levels, significant immune activity, and good stability when used as vaccine vectors, allowing them to be stored, transported, and used at room temperature, with a low incidence of adverse reactions.

[0010] The present disclosure also provides a method for preparing the amino lipid compound or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the raw materials are readily available, the reaction conditions are mild, the reaction selectivity is good, the yield is high, the equipment requirements are low, and the operation is simple.

[0011] The present disclosure also provides compositions (e.g., lipid nanoparticles) comprising the amino lipid compounds or pharmaceutically acceptable salts or stereoisomers thereof.

[0012] In one aspect, the present disclosure provides a compound represented by the following structural formula I, or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [In formula: L 1 and L 2 are the same or different, and each independently represents C1-C 12 Alkylene, C2-C 12 Alkenylene or C 2- C 12 alkynylene; preferably, L 1 and L 2 are the same or different, and each independently represents C3-C 10 Alkylene, C3-C 10 Alkenylene or C 3- C 10 alkynylene; more preferably, L 1 and L 2 are the same or different, and each independently represents C3-C 10 alkylene; most preferably, L1 and L 2 are the same or different and each independently selected from C5-C8 alkylene; G 1 or G 2 are the same or different and are each independently selected from —O—(C═O)—, —(C═O)—O—, —C(═O)—, —O—, —C(═O)—S—, and —SC(═O)—; preferably, G 1 or G 2 are the same or different and are each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, and -O-; most preferably, G 1 or G 2 are the same or different and are each independently selected from -O-(C=O)-, -(C=O)-O-; R 1 and R 2 are the same or different and each independently represent a C5-C bonded via any carbon atom. 27 Alkyl, C5-C containing one or more double bonds 27 alkenyl; preferably, R 1 and R 2 are the same or different, and each independently represents a C8-C bonded via any carbon atom. 20 Alkyl, C8-C containing one or more double bonds 20 alkenyl; more preferably, R 1 and R 2 are the same or different, and each independently represents a C9-C bonded via any carbon atom. 17 Alkyl, C9-C containing one or two double bonds 18 alkenyl; most preferably, R 1 and R 2 are the same or different and are each independently [ka] Selected from; R 3is selected from halogen, hydroxyl, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, and C1-C6 alkylcarbonylamino; preferably, R 3 is selected from halogen, hydroxyl, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; more preferably, R 3 is selected from halogen, hydroxyl, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 alkylcarbonylamino; most preferably, R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido; n is selected from 1, 2, and 3.

[0013] The present disclosure further provides a compound represented by the above structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 and L 2 are the same or different, and each independently represents C3-C 10 Alkylene, C3-C 10 Alkenylene or C 3- C 10 alkynylene; G 1 or G 2 are the same or different and are each independently selected from —O—(C═O)—, —(C═O)—O—, —C(═O)—, and —O—; R 1 and R 2 are the same or different, and each independently represents a C8-C bonded via any carbon atom. 20 Alkyl, C8-C containing one or more double bonds 20 alkenyl; R 3is selected from halogen, hydroxyl, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, and C1-C6 alkylcarbonylamino; n is selected from 1, 2, and 3.

[0014] The present disclosure further provides a compound represented by the above structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 and L 2 are the same or different, and each independently represents C3-C 10 alkylene; G 1 or G 2 are the same or different and are each independently selected from —O—(C═O)— and —(C═O)—O—; R 1 and R 2 are the same or different, and each independently represents a C9-C bonded via any carbon atom. 17 Alkyl, C9-C containing one or two double bonds 18 alkenyl; R 3 is selected from halogen, hydroxyl, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl, and C1-C4 alkylcarbonylamino; n is selected from 1, 2, and 3.

[0015] Furthermore, the present disclosure provides a compound represented by the above structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 and L 2 are the same or different and each independently selected from C5-C8 alkylene; G 1 or G 2 are the same or different and are each independently selected from —O—(C═O)— and —(C═O)—O—; R 1 and R 2are the same or different and are each independently [ka] Selected from; R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido; n is selected from 1, 2, and 3.

[0016] The present disclosure further provides a compound represented by the above structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is selected from the following: [Table 1] [Table 2] [Table 3]

[0017] In yet another aspect, the disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: [ka] .

[0018] In yet another aspect, the disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: [ka] .

[0019] Additionally, the present disclosure provides a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof: [ka] .

[0020] In yet another aspect, the disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: [ka] .

[0021] Additionally, the present disclosure provides a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof: [ka] .

[0022] In yet another aspect, the disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: [ka] .

[0023] Additionally, the present disclosure provides a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof: [ka] .

[0024] In yet another aspect, the disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: [ka] .

[0025] Additionally, the present disclosure provides a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof: [ka] .

[0026] In one aspect, the present disclosure provides lipid nanoparticles (LNPs) containing a compound of structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, according to any of the above embodiments.

[0027] In some embodiments, lipid nanoparticles refer to nanometer-scale (eg, 1 nm to 1000 nm) particles comprising one or more lipids.

[0028] In some embodiments, the lipid nanoparticles are 20 nm to 800 nm, 20 nm to 500 nm, 20 nm to 400 nm, 20 nm to 300 nm, 20 nm to 200 nm, 20 nm to 100 nm, 30 nm to 700 nm, 30 nm to 500 nm, 30 nm to 300 nm, 30 nm to 200 nm, 30 nm to 100 nm, 40 nm to 800 nm, 40 nm to 600 nm, 40 nm to 500 nm, 40 nm to 300 nm, 40 nm to 200 nm, 40 nm to The average diameter is 100 nm, 50 nm to 800 nm, 50 nm to 600 nm, 50 nm to 500 nm, 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 200 nm, 50 nm to 100 nm, 60 nm to 800 nm, 60 nm to 600 nm, 60 nm to 500 nm, 60 nm to 400 nm, 60 nm to 300 nm, 60 nm to 200 nm, or 60 nm to 100 nm. In some particular embodiments, the lipid nanoparticles have an average diameter of 26 nm, 31 nm, 36 nm, 41 nm, 46 nm, 51 nm, 56 nm, 61 nm, 66 nm, 71 nm, 76 nm, 81 nm, 86 nm, 91 nm, 96 nm, 101 nm, 106 nm, 111 nm, 116 nm, 121 nm, 126 nm, 131 nm, 136 nm, 141 nm, 146 nm, 151 nm, 156 nm, 161 nm, 166 nm, 171 nm, 176 nm, 181 nm, 186 nm, 191 nm, 196 nm, 201 nm, 206 nm, 211 nm, 216 nm, 221 nm, 226 nm, 231 nm, 236 nm, 241 nm, 246 nm, or 249 nm. Here, the mean diameter of lipid nanoparticles can be expressed as the z-average value determined by dynamic light scattering.

[0029] In some embodiments, the lipid nanoparticles further comprise one or more of a helper lipid, a structural lipid, and a polymer-lipid (eg, a polyethylene glycol-lipid).

[0030] In some embodiments, the lipid nanoparticles comprise (1) a helper lipid, (2) a structural lipid, (3) a polymer-lipid, and (4) a compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0031] In some embodiments, the helper lipid of the lipid nanoparticle comprises a phospholipid species. Phospholipids are typically semi-synthetic and may be naturally occurring or chemically modified. In some optional specific examples, the helper lipid of the lipid nanoparticle is a phospholipid. In some embodiments, the phospholipid species of the lipid nanoparticle comprises one or more of DSPC (distearoylphosphatidylcholine), DOPE (dioleylphosphatidylethanolamine), DOPC (dioleyllecithin), DOPS (dioleylphosphatidylserine), DSPG (1,2-octacosanoyl-sn-glycerol-3-phospho-(1'-rac-glycerol)), DPPG (dipalmitoylphosphatidylglycerol), DPPC (dipalmitoylphosphatidylcholine), DGTS (1,2-dipalmitoyl-sn-glycerol-3-O-4'-(N,N,N-trimethyl)homoserine), and lysolecithin. In some embodiments, the helper lipid of the lipid nanoparticle is one or more selected from DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid of the lipid nanoparticle is DSPC and / or DOPE.

[0032] In some embodiments, the structural lipid of the lipid nanoparticles comprises a sterol. In one optional specific example, the structural lipid of the lipid nanoparticles is a sterol. In some embodiments, the sterol of the lipid nanoparticles comprises one or more of 20α-hydroxycholesterol, cholesterol, cholesterol esters, steroid hormones, steroid vitamins, bile acids, cholesterol, ergosterol, β-sitosterol, and oxidized cholesterol derivatives. In some embodiments, the structural lipid of the lipid nanoparticles comprises at least one of cholesterol, cholesterol esters, steroid hormones, steroid vitamins, and bile acids. In some embodiments, the structural lipid of the lipid nanoparticles is cholesterol. In one optional specific example, the structural lipid of the lipid nanoparticles is highly purified cholesterol, particularly injectable levels of highly purified cholesterol, such as CHO-HP (produced by AVT). In other embodiments, the structural lipid is 20α-hydroxycholesterol.

[0033] In some embodiments, the polymer-lipid of a lipid nanoparticle refers to a conjugate comprising a polymer and a lipid bound to the polymer. The polymer-lipid (e.g., polyethylene glycol-lipid) in the lipid nanoparticle can improve the stability of the lipid nanoparticle in the body.

[0034] In some embodiments, the lipid of the polymer-lipid used to form the lipid nanoparticles comprises one or more of myristoyl diglyceride (1,2-dimyristoyl-sn-glycerol, DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipid, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG), and 1,2-dipalmitoyl-rac-glycerol (DPG).

[0035] In some embodiments, the polymer in the polymer-lipid used to form the lipid nanoparticles comprises one or both of a hydrophilic polymer and an amphoteric polymer.

[0036] In some embodiments, the polymer in the polymer-lipid used to form the lipid nanoparticles is a hydrophilic polymer, hi other embodiments, the polymer in the polymer-lipid used to form the lipid nanoparticles is an amphoteric polymer.

[0037] In some embodiments, the hydrophilic polymer is polyethylene glycol (PEG), polyoxazolines (POX), polyglycerols (PGs), poly(hydroxypropyl methacrylate) (PHPMA), poly-2-hydroxyethyl methacrylate (PHEMA), poly-N-(2-hydroxypropyl) methacrylamide (poly(N-(2-hydroxypropyl)methacrylamide)), or the like. The hydrophilic polymer may comprise one or more of the following: poly(hydroxypropyl)methacrylamide (HPMA), poly(vinylpyrrolidone) (PVP), poly-N,N-dimethylacrylamide (PDMA), poly(N-acryloyLmorpholine) (PAcM), polyaminoacids, glycosaminoglycans (GAGs), heparin, hyaluronic acid (HA), polysialic acid (PSA), elastin-like polypeptides (ELPs), serum albumin, and CD47. In one optional specific example, the hydrophilic polymer comprises polyethylene glycol.

[0038] Thus, polymer-lipids include one or more of polyethylene glycol-lipid (PEG-lipid), polyoxazoline-lipid, polyglycerol-lipid, polyhydroxypropyl methacrylate-lipid, poly-2-hydroxyethyl methacrylate-lipid, poly-N-(2-hydroxypropyl)methacrylamide-lipid, polyvinylpyrrolidone-lipid, poly-N,N-dimethylacrylamide-lipid, poly-N-acryloylmorpholine-lipid, glycosaminoglycan-lipid, heparin-lipid, hyaluronic acid-lipid, polysialic acid-lipid, elastin-like-lipid, serum albumin-lipid, and CD47-lipid. Note that "PEG-lipid" refers to a conjugate of polyethylene glycol and a lipid, "polyoxazoline-lipid" refers to a conjugate formed by combining polyoxazoline and a lipid, and "polyglycerin-lipid" refers to a conjugate formed by combining polyglycerin and a lipid. The same applies to other polymer-lipids.

[0039] In some embodiments, the polymer-lipid comprises a PEG-lipid. In one optional specific example, the polymer-lipid is a PEG-lipid. In some embodiments, the PEG-lipid comprises one or more of PEG-myristoyl diglyceride (PEG-DMG), PEG-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-diacylglycerol (PEG-DAG), PEG-dialkyloxypropyl (PEG-DAA), PEG-phospholipid, PEG-ceramide (PEG-Cer), PEG-1,2-distearoyl-rac-glycerol (PEG-DSG), and PEG-1,2-dipalmitoyl-rac-glycerol (PEG-DPG). The PEG-lipid is preferably one or more of PEG-DMG, PEG-DSG, and PEG-DPG. PEG-DMG is a polyethylene glycol derivative of 1,2-glyceryl dimyristate. In some embodiments, the average molecular weight of the PEG in the PEG-lipid is about 2000 to 5000. In one optional specific example, the average molecular weight of the PEG in the PEG-lipid is about 2000. In some embodiments, the PEG-lipid is PEG2000-DMG.

[0040] In certain embodiments, the amphoteric polymer comprises one or more of poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-based polymers, and phosphorylcholine polymers. In some embodiments, the amphoteric polymer comprises one or more of poly(carboxybetaine acrylamide) (pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyLphosphorylcholine), poly(vinyl-pyridinio propanesulfonate), polyvinylimidazolium betaine (poly(carboxybetaine) based on vinylimidazole), polyvinylimidazolium sulfobetaine (poly(sulfobetaine) based on vinylimidazole), and polyvinylpyridyl sulfobetaine (poly(sulfobetaine) based on vinylpyridine).

[0041] Correspondingly, the polymer-lipid comprises one or more of polyhydroxybetaine-lipid, polysulfobetaine-lipid, phosphobetaine-polymer-lipid, and phosphocholine polymer-lipid. In some embodiments, the polymer-lipid comprises one or more of poly(carboxybetaine acrylamide)-lipid, poly(carboxybetaine methacrylate)-lipid, poly(sulfobetaine methacrylate)-lipid, poly(methacryloyloxyethyl phosphorylcholine)-lipid, poly(vinylpyridinylpropanesulfonate)-lipid, polyvinylimidazolinylbetaine-lipid, polyvinylimidazolinylsulfobetaine-lipid, and polyvinylpyridinylsulfobetaine-lipid.

[0042] Additionally, in some embodiments, polymers applied to nanoparticles as described in Hoan Thi, Thai Thanh et al., "The Importance of Poly(ethylene glycol) Alternatives for Overcomin PEG Immunogenicity in Drug Delivery and Bioconjugation," Polymers, vol. 12, 2298, are also incorporated herein.

[0043] In some embodiments, the lipid nanoparticles comprise (1) a helper lipid, (2) a structural lipid, (3) a PEG-lipid, and (4) a compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, and the lipid nanoparticles comprise about 25% to 75%, for example about, based on the total amount of the compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, the helper lipid, the structural lipid, and the PEG-lipid. The compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof is contained in an amount (mol %) of 25% to 28%, 28% to 32%, 32% to 35%, 35% to 40%, 40% to 42%, 42% to 45%, 45% to 48%, 48% to 55%, 55% to 65%, 65% to 75%, 45% to 46.3%, 46.3% to 48%, 48% to 49.5%, 49.5% to 50%, 50% to 55%, or 60% to 65%.

[0044] In some embodiments, the lipid nanoparticles comprise (1) a helper lipid, (2) a structural lipid, (3) a PEG lipid, and (4) a compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, and the lipid nanoparticles contain about 5% or more of the compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, the helper lipid, the structural lipid, and the PEG lipid. The helper lipid is contained in an amount (mol %) of about 5% to 45%, for example, about 5% to 10%, 10% to 16%, 16% to 25%, 25% to 33.5%, 33.5% to 37%, 37% to 40%, 40% to 42%, 42% to 45%, 5% to 9%, 9% to 9.4%, 9.4% to 10%, 10% to 10.5%, 10.5% to 11%, 11% to 15%, 15% to 16%, 16% to 18%, 18% to 20%, or 20% to 25%.

[0045] In some embodiments, the lipid nanoparticles comprise (1) a helper lipid, (2) a structural lipid, (3) a PEG-lipid, and (4) a compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, and the lipid nanoparticles have a total content of about 0% to 55%, for example, about 0% to 10%, 10% to 15.5%, 15.5% to 22.5%, 22.5% to 35%, 35% to 36.5%, 36.5% to 39.5%, based on the total amount of the compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, the helper lipid, the structural lipid, and the PEG-lipid. %, 39.5% to 40.5%, 40.5% to 41.5%, 41.5% to 45%, 45% to 46.5%, 46.5% to 50%, 15.5% to 18.5%, 18.5% to 22.5%, 22.5% to 23.5%, 23.5% to 28.5%, 28.5% to 33.5%, 33.5% to 35%, 36.5% to 38%, 38% to 38.5%, 38.5% to 39%, 39% to 39.5%, 41.5% to 42.5%, 42.5% to 42.7%, 42.7% to 43%, 43% to 43.5%, 43.5% to 45%, or 46.5% to 48.5% (mol %).

[0046] In some embodiments, the lipid nanoparticles comprise (1) a helper lipid, (2) a structural lipid, (3) a PEG-lipid, and (4) a compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, and the lipid nanoparticles comprise the PEG-lipid in an amount (mol %) of about 0.5% to 5%, e.g., about 0.5% to 1%, 1% to 1.5%, 1.5% to 2%, 2% to 2.5%, 2.5% to 3%, 3% to 3.5%, 3.5% to 4%, 4% to 4.5%, 4.5% to 5%, 1.5% to 1.6%, or 1.6% to 2%, based on the total amount of the compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, the helper lipid, the structural lipid, and the PEG-lipid.

[0047] In one embodiment of the invention, the lipid nanoparticles comprise (1) a helper lipid, (2) a structural lipid, (3) a PEG-lipid, and (4) a compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the molar ratio of the compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof:helper lipid:structural lipid:PEG-lipid is about (25-75):(5-45):(0-55):(25-65):(5-42):(10-55):(0.5-4), (28-60):(5-42):(15.5-53.5):(0.5-3.5), or (35-60):(5-40):(18.5-53.5):(1.5-3). For example, 45:11:41.5:2.5, 42:10.5:45:2.5, 42:16:39.5:2.5, 40:16:41.5:2.5, 40:18:39.5:2.5, 35:16:46.5:2.5, 35:25:36.5:3.5, 28:33.5:35:3.5, 32:37:40.5: 0.5, 35:40:22.5:2.5, 40:42:15.5:2.5, 45:10:42.5:2.5, 40:20:38.5:1.5, 45:15:38.5:1.5, 55:5:38.5:1.5, 60:5:33.5:1.5, 45:20:33.5:1.5, 50:20:28.5:1 .5, 55:20:23.5:1.5, 60:20:18.5:1.5, 40:15:43.5:1.5, 50:15:33.5:1.5, 55:15:28.5:1.5, 60:15:23.5:1.5, 40:10:48.5:1.5, 45:10:43.5:1.5, 55:10:33.5: 1.5, 40:5:53.5:1.5, 45:5:48.5:1.5, 50:5:43.5:1.5, 48:10:40.5:1.5, 50:10:38.5:1.5, 50:9:38:3, 49.5:10:39:1.5, 46.3:9.4:42.7:1.6 or 45:9:43:3.

[0048] In some embodiments, the lipid nanoparticles comprise DOPE, cholesterol (e.g., CHO-HP), PEG-lipid, and a compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the molar ratio of the compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof:DOPE:cholesterol:PEG-lipid is about (28-60):(5-42):(15.5-53.5):(0.5-3.5).

[0049] In some embodiments, the lipid nanoparticles comprise DOPE, cholesterol (e.g., CHO-HP), PEG-lipid, and a compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the molar ratio of compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof:helper lipid:structural lipid:PEG-lipid is about 45:11:41.5:2.5, 42:10.5:45:2.5, 42:16:39.5:2.5, 40:16:41.5:2.5, 40:18:39.5:2.5, 35:16:46.5:2.5, 35:25:36.5:3.5, 28:33.5:35:3.5, 32:37:40.5:0.5, 35:40:22 .5:2.5, 40:42:15.5:2.5, 45:10:42.5:2.5, 40:20:38.5:1.5, 45:15:38.5:1.5, 55:5:38.5:1.5, 60:5:33.5:1.5, 45:20:33.5:1.5, 50:20:28.5:1.5, 55:20:23.5:1.5, 60:20:18.5:1.5 , 40:15:43.5:1.5, 50:15:33.5:1.5, 55:15:28.5:1.5, 60:15:23.5:1.5, 40:10:48.5:1.5, 45:10:43.5:1.5, 55:10:33.5:1.5, 40:5:53.5:1.5, 45:5:48.5:1.5, or 50:5:43.5:1.5.

[0050] In some embodiments, the lipid nanoparticles comprise DSPC, cholesterol (e.g., CHO-HP), PEG-lipid, and a compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the molar ratio of the compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof:DSPC:cholesterol:PEG-lipid is about (35-60):(5-40):(18.5-53.5):(1.5-3).

[0051] In some embodiments, the lipid nanoparticles comprise DSPC, cholesterol (e.g., CHO-HP), PEG-lipid, and a compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the molar ratio of compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof:helper lipid:structural lipid:PEG-lipid is about 48:10:40.5:1.5, 50:10:38.5:1.5, 50:9:38:3, 49.5:10:39:1.5, 46.3:9.4:42.7:1.6, 45:9:43:3, 45:11:41.5:2.5, 42:10.5:45:2.5, 42:16:39.5:2.5, 40:16:41.5: 2.5, 40:18:39.5:2.5, 35:40:22.5:2.5, 40:20:38.5:1.5, 45:15:38.5:1.5, 55:5:38.5:1.5, 60:5:33.5:1.5, 45:20:33.5:1.5, 50:20:28.5:1.5, 55:20:23.5:1.5, 60:20:18.5:1.5, 40:15:43.5:1.5, 50:15:33.5:1.5, 55:15:28.5:1.5, 60:15:23.5:1.5, 40:10:48.5:1.5, 45:10:43.5:1.5, 55:10:33.5:1.5, 40:5:53.5:1.5, 45:5:48.5:1.5, or 50:5:43.5:1.5.

[0052] In some embodiments, the lipid nanoparticles comprise (1) a helper lipid, (2) a structural lipid, (3) a PEG-lipid, (4) a compound of formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, and (5) an active ingredient (e.g., RNA or DNA), or a pharmaceutically acceptable carrier, diluent, or excipient (e.g., a buffer).

[0053] In some embodiments, the lipid nanoparticles comprise (1) a helper lipid, (2) a structural lipid, (3) a PEG-lipid, (4) a compound of formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, (5) an active ingredient, and (6) a pharmaceutically acceptable carrier, diluent, or excipient.

[0054] In another aspect, the present disclosure provides a pharmaceutical composition containing the lipid nanoparticles of the present disclosure, or a compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof. Because the compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, contains a long nonpolar residue, all of the resulting compounds have hydrophobic characteristics, and at the same time, hydrophilic characteristics due to the amino group. This amphiphilic characteristic can be used to form lipid nanoparticles, such as lipid bilayers, micelles, liposomes, etc.

[0055] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient, and a compound of structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, and one of the lipid nanoparticles of the present disclosure.

[0056] In some embodiments, the pharmaceutical composition is a lipid nanoparticle.

[0057] Lipid nanoparticles containing the compound represented by structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof have excellent properties for encapsulating bioactive ingredients, and the lipid nanoparticles of the present disclosure containing bioactive ingredients can deliver any one or more of a variety of therapeutic agents to cells, tissues, or organs. The present disclosure provides the use of lipid nanoparticles of the present disclosure for delivering bioactive ingredients (e.g., DNA, RNA, etc.) to cells, tissues, or organs, and further provides the use of lipid nanoparticles or pharmaceutical compositions containing bioactive ingredients (e.g., DNA, RNA, etc.) for producing polypeptides and / or proteins of interest, producing pharmaceuticals, producing pharmaceuticals for transferring nucleic acids, and preventing and / or treating diseases.

[0058] The present disclosure provides use of a compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, or lipid nanoparticles for delivering a biologically active ingredient (e.g., DNA, RNA, etc.) to a cell, tissue, or organ.

[0059] In some embodiments, the use includes contacting a cell, tissue, or organ with lipid nanoparticles containing a bioactive component (e.g., a nucleic acid (e.g., mRNA) encoding a polypeptide and / or protein of interest).

[0060] In some embodiments, the tissue or organ is selected from spleen, liver, kidney, lung, femur, ocular tissue, vascular endothelium of blood vessels, lymphatic vessels, and tumor tissue.

[0061] In some embodiments, the cell is a mammalian cell. As used herein, a mammalian cell may be any mammalian cell. In some embodiments, a mammal includes one or more of a human, a mouse, a rat, a pig, a cat, a dog, a horse, a goat, a cow, and a monkey. In some embodiments, the mammal is a human.

[0062] In some embodiments, the mammalian cell is a cell within a mammal.

[0063] The present disclosure provides methods for producing a polypeptide and / or protein of interest, comprising contacting a cell, tissue, or organ with a lipid nanoparticle or pharmaceutical composition of the present disclosure comprising a bioactive component (e.g., a nucleic acid (e.g., mRNA) encoding the polypeptide and / or protein of interest).

[0064] In some embodiments, after contacting the cells with the lipid nanoparticles or pharmaceutical composition, the nucleic acid is taken up by the cells, and the taken up nucleic acid is utilized by the cells to produce the desired polypeptide and / or protein.

[0065] In some embodiments, the cells, tissues, or organs are the same as those used in the delivery of bioactive components (e.g., DNA, RNA, etc.) to cells, tissues, or organs using a compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, or lipid nanoparticles, and are not described again herein.

[0066] In some embodiments, the methods for producing a polypeptide and / or protein of interest are performed in vivo or partially in vivo.

[0067] In yet another aspect, the present disclosure further provides the use of a compound represented by structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, lipid nanoparticle, or pharmaceutical composition of the present disclosure in the manufacture of a medicament.

[0068] In some embodiments, the medicament is indicated for the treatment and / or prevention of a disease.

[0069] In some embodiments, the disease is selected from a rare disease, an infectious disease, a cancer, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

[0070] In some embodiments, the cancer is one or more selected from lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, and prostate cancer. In some embodiments, the genetic disease is one or more selected from hemophilia, thalassemia, and Gaucher disease.

[0071] In some embodiments, the rare disease includes Brittle Bone Disease, Wilson Disease, Spinal Muscular Atrophy (SMA), Huntingdon's Disease, Rett Syndrome, Amyotrophic Lateral Sclerosis (ALS), Duchenne Type Muscular Dystrophy, Friedrichs Ataxia, methylmalonic acidemia (MMA), Cystic Fibrosis (CF), Glycogen Storage Disease 1a (GSD1a), Glycogen Storage Disease III (GSD1b), and the like. This includes one or more of the following: Crigler-Najjar syndrome, ornithine carbatransmylase deficiency (OTCD), propionic acidemia (PA), phenylpropionuria (PKU), hemophilia A, hemophilia B, β-thalassemia, Lafora disease, Dravet syndrome (DS), Alexander disease, Leber's congenital amaurosis (LCA), myelodysplastic syndrome (MDS), and homocystinuria due to CBS deficiency. Also incorporated herein are the rare diseases listed at www.orpha.net / consor / cgi-bin / Disease_Search_List.php and Rarediseases.info.nih.gov / diseases.

[0072] In some embodiments, the medicament is used in, for example, gene therapy, protein replacement therapy, antisense therapy, or interfering RNA therapy, or genetic vaccination.

[0073] In some embodiments, the pharmaceutical is a nucleic acid pharmaceutical. In some embodiments, the nucleic acid in the nucleic acid pharmaceutical comprises at least one of RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA), and cas9 mRNA.

[0074] In some embodiments, the medicament is a vaccine. In some embodiments, the medicament is used to produce an antigen or a portion thereof of a pathogen.

[0075] In some embodiments, the pharmaceutical agent is a genetic vaccine. In some embodiments, the pharmaceutical agent is a nucleic acid vaccine. In some embodiments, the pharmaceutical agent is an mRNA vaccine.

[0076] In some embodiments, the genetic vaccination is used to treat and / or prevent one or more of cancer, allergy, toxicity, and pathogen infection.

[0077] In some embodiments, the pathogen is one or more selected from a virus, a bacterium, or a fungus.

[0078] In some embodiments, the pharmaceutical agent is a gene therapy agent. In some embodiments, the pharmaceutical agent is used for the production of a protein associated with a genetic disease.

[0079] In some embodiments, the medicament is used for the production of an antibody, such as an scFV or a nanobody.

[0080] In yet another aspect, the present disclosure also provides use of a compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, lipid nanoparticle, or pharmaceutical composition in the manufacture of a medicament for nucleic acid transfer.

[0081] In some embodiments, the nucleic acid in the pharmaceutical agent for nucleic acid transfer is selected from RNA, antisense oligonucleotides, and DNA.

[0082] In some embodiments, the RNA is selected from messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA), and cas9 mRNA, or a mixture thereof.

[0083] In some embodiments, the DNA is a plasmid.

[0084] Furthermore, the present disclosure provides a pharmaceutical product comprising a compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, or lipid nanoparticles.

[0085] Furthermore, the present disclosure also provides a method for preventing and / or treating a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle, pharmaceutical composition, or medicament comprising a compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, and a biologically active ingredient (e.g., a nucleic acid (e.g., mRNA) encoding a polypeptide and / or protein of interest).

[0086] In some embodiments, the diseases and disorders refer to a description of the use of a compound of Formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, lipid nanoparticle, or pharmaceutical composition disclosed herein in the manufacture of a medicament.

[0087] In yet another aspect, the present disclosure provides the use of pharmaceutical compositions, particularly lipid nanoparticles, in the manufacture of medicaments for, for example, gene therapy, gene vaccination, antisense therapy, or treatment with interfering RNA. Preferably, gene therapy is useful for the treatment of cancer and genetic diseases. The cancer is preferably one or more selected from lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain tumor, lymphoma, blood cancer, or prostate cancer; the genetic disease is preferably one or more selected from hemophilia, thalassemia, and Gaucher disease. The genetic vaccine is preferably used for the treatment of cancer, allergy, toxicity, and pathogen infection. The pathogen is preferably one or more selected from viruses, bacteria, and fungi.

[0088] In yet another aspect, the present disclosure relates to the use of the compound or its pharmaceutically acceptable salt or stereoisomer, and lipid nanoparticles containing the compound or its pharmaceutically acceptable salt or stereoisomer, in the manufacture of a pharmaceutical for nucleic acid transfer.Preferably, the nucleic acid is selected from RNA, DNA, and antisense oligonucleotide;Preferably, the RNA is selected from messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), and small nuclear RNA (snRNA);Preferably, the DNA is a plasmid.

[0089] In yet another aspect, the present disclosure further provides a method for preparing a compound of formula (I) according to the following general scheme: General Scheme 1: [ka] however, n, L1, L2, G1, G2, R1, R2, R3 have the same meanings as defined above in this disclosure for compounds of formula (I); X is selected from halogens; preferably, X is selected from bromine.

[0090] Specifically, the compound of formula (I) of the present disclosure can be obtained by a substitution reaction of intermediate compound (II) with intermediate compound (III) in an organic solvent in the presence or absence of an acid binder and an iodide at room temperature.

[0091] Here, the organic solvent may be selected from nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE); the acid binder may be selected from organic bases and inorganic bases, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA; and the iodide may be potassium iodide.

[0092] General Scheme 2 [ka] however, n, L1, L2, G1, G2, R1, R2, R3 have the same meanings as defined above in this disclosure for compounds of formula (I); Y is (=O).

[0093] Specifically, the compound of formula (I) of the present disclosure can be obtained by reducing intermediate compound (II) with intermediate compound (V) in the presence of a reducing agent in an organic solvent at room temperature.

[0094] Here, the organic solvent may be selected from nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE); and the reducing agent may be selected from sodium triacetoxyborohydride.

[0095] Furthermore, in the compound of formula (I) of the present disclosure, when the group L1 and the group L2 are the same, R1 and R2 are the same, and G1 and G2 are the same, the compound of formula (I) of the present disclosure can be prepared by the following general scheme 3: General Scheme 3 [ka] however, n, L1, L2, G1, G2, R1, R2, R3 have the same meanings as defined earlier in this disclosure for compounds of formula (I); and L = L1 = L2, G = G1 = G2, and R = R1 = R2.

[0096] Specifically, the compound of formula (I) of the present disclosure can be obtained by reducing intermediate compound (IV) with intermediate compound (VI) in the presence of a reducing agent in an organic solvent at room temperature.

[0097] Here, the organic solvent may be selected from nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE); and the reducing agent may be selected from sodium triacetoxyborohydride.

[0098] In yet another aspect, the present disclosure also provides an intermediate compound (II) for preparing the compound of formula (I) of the present disclosure, which is represented by the following structural formula: [ka] [In formula: L1 is selected from C1-C12 alkylene, C2-C12 alkenylene, or C2-C12 alkynylene; preferably, L1 is selected from C3-C10 alkylene, C3-C10 alkenylene, or C3-C10 alkynylene; more preferably, L1 is selected from C3-C10 alkylene; most preferably, L1 is selected from C5-C8 alkylene; G1 is selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S-, -SC(=O)-; preferably, G1 is selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-; most preferably, G1 is selected from -O-(C=O)-, -(C=O)-O-; R1 is selected from C5-C27 alkyl bonded through any carbon, C5-C27 alkenyl containing one or more double bonds; preferably, R1 is selected from C8-C20 alkyl bonded through any carbon, C8-C20 alkenyl containing one or more double bonds; more preferably, R1 is selected from C9-C17 alkyl bonded through any carbon, C9-C18 alkenyl containing one or two double bonds; most preferably, R1 is [ka] Selected from; R3 is selected from halogen, hydroxyl, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, and C1-C6 alkylcarbonylamino; preferably, R3 is selected from hydroxyl, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, and C1-C6 alkylcarbonylamino; more preferably, R3 is selected from hydroxyl, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl, and C1-C4 alkylcarbonylamino; most preferably, R3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl, and acetamido; n is selected from 1, 2, and 3. Preferably, the present disclosure provides the above intermediate compound (II) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L1 is selected from C3-C10 alkylene, C3-C10 alkenylene, or C3-C10 alkynylene; G1 is selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-; R1 is selected from C8-C20 alkyl bonded through any carbon, C8-C20 alkenyl containing one or more double bonds; R3 is selected from halogen, hydroxyl, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, and C1-C6 alkylcarbonylamino; n is selected from 1, 2, and 3.

[0099] More preferably, the present disclosure provides the above intermediate compound (II) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L1 is selected from C3-C10 alkylene; G1 is selected from -O-(C=O)-, -(C=O)-O-; R1 is selected from C9-C17 alkyl bonded through any carbon, C9-C18 alkenyl containing one or two double bonds; R3 is selected from halogen, hydroxyl, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl, and C1-C4 alkylcarbonylamino; n is selected from 1, 2, and 3.

[0100] More preferably, the present disclosure provides the above intermediate compound (II) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L1 is selected from C5-C8 alkylene; G1 is selected from -O-(C=O)-, -(C=O)-O-; R1 is [ka] Selected from; R3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido; n is selected from 1, 2, and 3.

[0101] Most preferably, said intermediate compound (II) is selected from the following compounds: [Table 4] [Table 5] [Table 6]

[0102] Furthermore, the present disclosure also provides a process for preparing said intermediate compound (II), wherein the general reaction scheme is as follows: [ka] wherein R1, R3, G1, L1, and n have the same meanings as defined earlier in this disclosure for intermediate compound (II); and X is a halogen, preferably bromine.

[0103] Specifically, intermediate compound (IV) is subjected to a substitution reaction with intermediate compound (VII) in the presence of an acid-binding agent in an organic solvent at room temperature to obtain intermediate compound (II), where the organic solvent is selected from nitriles, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE), alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons; and the acid-binding agent is selected from organic bases and inorganic bases, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA.

[0104] In yet another aspect, the present disclosure provides a method for preparing an optical isomer (X) of a compound of formula (I) according to the present disclosure, the method having the following general reaction scheme: [ka] wherein n, L1, L2, G1, G2, R1, R2, and R3 have the same meanings as defined earlier in this disclosure for compounds of formula (I); and X is a halogen, preferably bromine.

[0105] Specifically, compound (VIII) and intermediate compound (VII) are first subjected to a substitution reaction in an organic solvent in the presence of an acid binder to produce intermediate compound (VII). Here, the organic solvent is selected from nitriles, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE), alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, and the acid binder is selected from organic bases and inorganic bases, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA. Next, intermediate compound (VII) is reacted with intermediate compound (III) in the presence or absence of an acid binder and iodide to produce optical isomer (X) of compound (I). Wherein, the organic solvent is selected from nitriles, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE), alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons; the acid binder is selected from organic bases and inorganic bases, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA; and the iodide is, for example, potassium iodide.

[0106] Furthermore, the present disclosure provides a manufacturing method for producing the optical isomer (I-6) of compound 6 described in the present disclosure, the manufacturing method having the following general reaction scheme: [ka] However, X is a halogen, preferably bromine.

[0107] Specifically, the optical isomer (I-6) of compound 6 described in the present disclosure was prepared by the following steps: Compound (6-X) and compound (6-VI) are subjected to an N-alkylation reaction in a solvent at 30-50°C to produce compound (6-VII). Examples of the solvent include nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbon solvents, and specific examples include acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE). Compound (6-VII) is N-alkylated with 8-halooctanoate nonyl in a solvent at 60-110°C in the presence or absence of an acid binder and a catalyst to produce compound (I-6). The solvent may be, for example, a nitrile, an alcohol, a halogenated hydrocarbon, an amide, an aromatic hydrocarbon, or an ether solvent, such as acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE), cyclopentyl methyl ether, or methyl tert-butyl ether. The acid binder may be an organic base or an inorganic base, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, or DIPEA. The catalyst may be an iodide, preferably potassium iodide.

[0108] Furthermore, according to the above method, when (1S,3R)-3-aminocyclohexanol is used as compound (6-X) and compound (6-VII) is represented by structural formula (6-VII-I), [ka]

[0109] Compound (I-6-II), an optical isomer of Compound 6, is produced. [ka]

[0110] When (1S,3S)-3-aminocyclohexanol is used as compound (6-X) and compound (6-VII) is represented by structural formula (6-VII-II), [ka]

[0111] Compound (I-6-III), which is an optical isomer of Compound 6, is produced. [ka]

[0112] When (1R,3R)-3-aminocyclohexanol is used as compound (6-X) and compound (6-VII) is represented by structural formula (6-VII-III), [ka]

[0113] Compound (I-6-IV), an optical isomer of Compound 6, is produced. [ka]

[0114] When (1R,3S)-3-aminocyclohexanol is used as compound (6-X) and compound (6-VII) is represented by structural formula (6-VII-IV): [ka]

[0115] Compound (I-6-V), an optical isomer of Compound 6, is produced. [ka]

[0116] In yet another aspect, the present disclosure provides an intermediate compound (6-VII) for producing the optical isomer (I-6) of compound 6 above, which has the following structure: [ka]

[0117] In particular, the intermediate compound (6-VII) is selected from: [ka] [Brief explanation of the drawings]

[0118] [Figure 1] FIG. 1 shows antibody titers in body fluids when OVA mRNA was subcutaneously administered using a representative amino lipid compound in assay 2 of the bioassay in the present disclosure. [Figure 2] FIG. 2 shows antibody titers in body fluids following intramuscular administration of an influenza mRNA vaccine using a representative amino lipid compound in assay 3 of the bioassay in this disclosure. [Figure 3] FIG. 3 shows the HPLC profile of the mixture of compounds (I-6-II), (I-6-III), (I-6-IV) and (I-6-V). [Figure 4] FIG. 4 shows the HPLC profile of compound (I-6-II). [Figure 5] FIG. 5 shows the HPLC profile of compound (I-6-III). [Figure 6] FIG. 6 shows the HPLC profile of compound (I-6-IV). [Figure 7] FIG. 7 shows the HPLC profile of compound (I-6-V). [Figure 8] FIG. 8 shows the fluorescence intensity of the liver after intramuscular injection in a targeting test. [Figure 9] FIG. 9 shows the results of a poor response study to intramuscular injection, with statistics of severe swelling at the injection site and moderate lameness.

[0119] Various exemplary embodiments of the amino lipid compounds described in this disclosure, compositions containing same, lipid nanoparticles, and their use for delivering bioactive agents, such as nucleic acids, to cells are described in further detail below. DETAILED DESCRIPTION OF THE INVENTION

[0120] definition Unless the context specifically requires otherwise, throughout this specification and claims, the word "comprises" and variations thereof, such as "include" and "comprising," are to be interpreted in an open, inclusive sense, i.e., "including, but not limited to."

[0121] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with this embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0122] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in the specification and claims, unless the context clearly dictates otherwise, this is the case.

[0123] As used herein, the expression "Cx-Cy," when used, represents a range of numbers of carbon atoms, where x and y are both integers. For example, C3-C8 cycloalkyl represents a cycloalkyl having 3 to 8 carbon atoms, and C0-C2 alkyl represents an alkyl group having 0 to 2 carbon atoms, where C0 alkyl represents a single chemical bond.

[0124] In this disclosure, the term "alkyl" refers to saturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 30 carbon atoms, such as 1 to 6 carbon atoms, 5 to 27 carbon atoms, 8 to 20 carbon atoms, and 9 to 17 carbon atoms, including, but not limited to, n-nonyl, undecyl, 7-pentadecyl, 9-heptadecyl, and various isomers thereof.

[0125] In the present disclosure, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon group containing 3 to 12 ring atoms, e.g., 3 to 12, 3 to 10, 3 to 8, or 3 to 6 ring atoms, or it may be a 3-, 4-, 5-, or 6-membered ring. Examples of monocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0126] In this disclosure, the term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group having at least one double bond, including straight-chain and branched alkenyl groups of 1 to 30 carbon atoms, such as straight-chain and branched alkenyl groups of 5 to 27 carbon atoms, 8 to 20 carbon atoms, or 9 to 18 carbon atoms, having one or two double bonds. Non-limiting examples include 8-heptadecenyl, 12-octadecadienyl, and various isomers thereof.

[0127] In this disclosure, the term "alkynyl" refers to an unsaturated aliphatic hydrocarbon group having at least one triple bond, including straight-chain and branched alkynyl groups of 1 to 30 carbon atoms, such as straight-chain and branched alkynyl groups of 5 to 27 carbon atoms, 5 to 15 carbon atoms, or 8 to 10 carbon atoms, having one or two triple bonds. Non-limiting examples include 2-nonynyl, 3-decanyl, and isomers thereof.

[0128] In this disclosure, the term "alkylene" refers to a substituted or unsubstituted alkyl having two terminal monovalent radical cores, which are formed by removing one hydrogen atom from each of the two terminal carbon atoms, said alkyl having the same meaning as above. Non-limiting examples include C3-C10 alkylene, C5-C8 alkylene, etc.

[0129] In this disclosure, the term "alkenylene" refers to a substituted or unsubstituted alkenyl having two terminal monovalent radical cores, which are formed by removing one hydrogen atom from each of the two terminal carbon atoms, said alkenyl having the same meaning as above. Non-limiting examples include C3-C10 alkenylene, etc.

[0130] In this disclosure, the term "alkynylene" refers to a substituted or unsubstituted alkynyl having two terminal monovalent radical cores, which are formed by removing one hydrogen atom from each of the two terminal carbon atoms, said alkynyl having the same meaning as above. Non-limiting examples include C3-C10 alkynylene, etc.

[0131] In this disclosure, the term "halogen" means fluorine, chlorine, bromine or iodine.

[0132] In the present disclosure, the term "alkoxy" refers to alkyl-oxy, wherein the alkyl group has the meaning as defined above. Preferably, the alkoxy is a C1-C10 alkoxy; more preferably, the alkoxy is a C1-C6 alkoxy; even more preferably, the alkoxy is a C1-C4 alkoxy; and most preferably, the alkoxy group is a methoxy group.

[0133] In the present disclosure, the term "alkylcarbonyloxy" refers to an alkyl-C(O)O group, wherein the alkyl group has the meaning as defined above. Preferably, the alkylcarbonyloxy is a C1-C10 alkylcarbonyloxy; more preferably, the alkylcarbonyloxy is a C1-C6 alkylcarbonyloxy; even more preferably, the alkylcarbonyloxy is a C1-C4 alkylcarbonyloxy; and most preferably, the alkylcarbonyloxy group is an acetoxy group.

[0134] In the present disclosure, the term "alkoxycarbonyl" refers to an alkyl-OC(O)- group, wherein the alkyl group has the meaning as defined above. Preferably, the alkoxycarbonyl is a C1-C10 alkoxycarbonyl; more preferably, the alkoxycarbonyl is a C1-C6 alkoxycarbonyl; even more preferably, the alkoxycarbonyl is a C1-C4 alkoxycarbonyl; and most preferably, the alkoxycarbonyl is a methoxycarbonyl.

[0135] In the present disclosure, the term "alkylcarbonylamino" refers to an alkyl-C(O)NH- group, where the alkyl group has the meaning as defined above. Preferably, the alkylcarbonylamino group is a C1-C10 alkylcarbonylamino group; more preferably, the alkylcarbonylamino group is a C1-C6 alkylcarbonylamino group; even more preferably, the alkylcarbonylamino group is a C1-C4 alkylcarbonylamino group; and most preferably, the alkylcarbonylamino group is an acetamido group.

[0136] In the present disclosure, the term "alkylaminocarbonyl" refers to alkyl-NHC(O)-yl, where the alkyl group has the meaning as defined above. Preferably, the alkylaminocarbonyl group is a C1-C10 alkylaminocarbonyl group; more preferably, the alkylaminocarbonyl group is a C1-C6 alkylaminocarbonyl group; even more preferably, the alkylaminocarbonyl group is a C1-C4 alkylaminocarbonyl group; and most preferably, the alkylaminocarbonyl group is a butylaminocarbonyl group.

[0137] In this disclosure, the term "optionally substituted" means that one or more hydrogen atoms bonded to an atom or group are independently unsubstituted or substituted with one or more substituents, e.g., 1, 2, 3, or 4 substituents. The substituents are independently selected from halogen (e.g., chlorine, bromine, fluorine, or iodine), deuterium (D), tritium (T), carboxylic acid (e.g., -C(=O)OH), oxygen (e.g., =O), sulfur (e.g., =S), hydroxyl group (e.g., -OH), ester group (e.g., -C(=O)ORi or -OC(=O)Ri), aldehyde group (e.g., -C(=O)H), carbonyl group (e.g., represented by -C(=O)Ri or C=O), acylhalo (e.g., -C(=O)X, where wherein X is selected from bromine, fluorine, chlorine, or iodine), a carbonate group (e.g., —OC(═O)ORi), an alkoxy group (e.g., —ORi), an acetal group (e.g., —C(ORi)Ri, where each ORi is the same or different alkoxy group), a phosphate group (e.g., P(═O)43—), a mercapto group (e.g., —SH), a sulfoxide group (e.g., —S(═O)Ri), a sulfinic acid (e.g., —S(═O)OH), a sulfonic acid (e.g., —S(═O) 2OH), thioaldehyde (e.g., -C(=S)H), sulfate ion (e.g., S(=O)42-), sulfonyl (e.g., -S(=O)2Ri), sulfinyl (e.g., -S(=O)Ri), amide (e.g., -C(=O)N(Ri)2 or -N(Ri)C(=O)Ri), azide (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(=O)Ri), amino (e.g., -N(Ri)2, -N(Ri)H, or -NH2), aminocarbonyl (e.g., -OC(=O)N(Ri)2, -OC(=O)N(Ri)H, or -OC(=O)NH2), sulfonamide (e.g., -S(=O)2N(Ri)2, -S(=O)2N(Ri)H, -S(=O)2NH2, -N(Ri)S(=O)2Ri, -N(H)S(=O)2Ri, -N(Ri)S(=O)2H or -N(H)S(=O)2H), alkyl, alkenyl, alkynyl, cyclohydrocarbyl (e.g., cycloalkyl,cycloalkenyl or cycloalkynyl), heterocyclohydrocarbyl (e.g., heterocycloalkyl containing one or more heteroatoms selected from S, N, and O, or heterocycloalkenyl containing one or more heteroatoms selected from S, N, and O), aryl (e.g., phenyl, or a fused ring group), heteroaryl (e.g., an 8-10 membered bicyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur), -C(=O)SRi, -C(=N-CN)N(Ri), -C(=NO-CH3)N(Ri), -C(=N-SO2-NH2)N(Ri)2, -C(=CH-NO2)N(Ri)2, -OC(=O)N(Ri)2, -CHN(Ri)N(Ri)2, -C(=O)N(Ri)ORi, -N(Ri)2C(=O)ORi, -OP(=O)(ORi)2, -P(=O)(ORi)2, -N( Selected from ORi)C(=O)Ri, -N(ORi)S(=O)2Ri, -N(ORi)C(=O)ORi, -N(ORi)C(=O)N(Ri)2, -N(ORi)C(=S)N(Ri)2, -N(ORi)C(NRi)N(Ri)2, -N(ORi)C(CHRi)N(Ri)2. In any of the foregoing, R is hydrogen, or alkyl, or alkenyl, or alkynyl, or heteroalkyl, or heteroalkenyl, or heteroalkynyl, as defined herein. In some embodiments, R is hydrogen, or C1-C12 alkyl, or C1-C12 alkenyl, or C1-C12 alkynyl, or C1-C12 heteroalkyl, or C1-C12 heteroalkenyl, or C1-C12 heteroalkynyl, as defined herein. When an atom or group is substituted with multiple substituents, the multiple substituents may be the same or different. In some embodiments, the substituent itself may be further substituted, for example, with one or more of the substituents defined herein. For example, a C1-C6 alkyl group as a substituent may be further substituted with one or more of the substituents described herein.

[0138] Unless otherwise specified, alkyl, cycloalkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, alkoxy, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonylamino, alkylaminocarbonyl described herein may be optionally substituted.

[0139] The term "pharmaceutically acceptable salts" in the present disclosure is defined in Berge et al., "Pharmaceutically acceptable salts," J. Pharm. Sci., 66, 1-19 (1977), and would be obvious to a medicinal chemist. Salts are substantially non-toxic and provide desirable pharmacokinetic properties, palatability, absorption, distribution, metabolism, excretion, etc.

[0140] Examples of "pharmaceutically acceptable salts" according to the present disclosure include acid addition salts or base addition salts of the compounds of the present disclosure. Such salts refer to salts that retain the biological effectiveness and properties of the compounds of the present invention and are typically not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0141] Pharmaceutically acceptable salts are salts formed from inorganic and / or organic acids with the compounds of the present disclosure. The inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and the organic acids include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, 10-camphorsulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, Formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid acid), propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0142] Pharmaceutically acceptable salts are salts formed from inorganic and / or organic bases and the compounds of the present disclosure. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0143] The pharmaceutically acceptable salts of the present disclosure can be synthesized by conventional chemical methods.

[0144] Generally, salts can be prepared by reacting the free base or acid with an equistochemical equivalent or an excess of an acid (inorganic or organic) or base in a suitable solvent or solvent composition.

[0145] In this disclosure, the term "meta" refers to a position on a cycloalkyl structural unit that is one carbon atom away from the amine substituent.

[0146] In this disclosure, the term "compound" includes compounds that are isotopically labeled by replacing one or more atoms with atoms having different atomic masses or mass numbers. "Isotopes" refer to atoms with the same atomic number but different mass numbers due to the different number of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0147] The compounds of the present disclosure, or their pharmaceutically acceptable salts, may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. For example, amino acids can be defined in terms of absolute stereochemistry as (R)- or (S)-, or (D)- or (L)-. The present disclosure is intended to encompass all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional recrystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from appropriate optically pure precursors, or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0148] As used herein, the term "stereoisomer" refers to compounds composed of the same atoms bonded by the same bonds but with different, non-interchangeable three-dimensional structures. "Optical isomers" refer to two or more stereoisomers that exhibit different optical activity due to their different configuration. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any ratio may be known as a "racemic" mixture. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0149] "Stereoisomer" can also include E and Z isomers, or mixtures thereof, and cis and trans isomers, or mixtures thereof. In certain embodiments, the compounds described herein are isolated as either the E or Z isomer. In certain embodiments, the compounds described herein are a mixture of E or Z isomers.

[0150] "Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The concentrations of isomeric forms depend on the environment in which the compound is found and may differ depending on whether the compound is a solid or in an organic or aqueous solution.

[0151] In the present disclosure, the term "lipid nanoparticle" refers to nano-sized substances prepared by disposing amino lipid compounds in aqueous solution.These particles are bilayer lipid vesicles (liposomes), multilayer vesicles or micelles.In a preferred embodiment, the lipid nanoparticle is a liposome containing the amino lipid compounds of the present disclosure.

[0152] In this disclosure, the term "liposome" refers to a microscopic vesicle consisting of a bilayer of lipid amphiphilic molecules surrounding an aqueous compartment, and liposome formation is not a natural process. Lipid vesicles are formed by first adding lipid to water, forming a bilayer or series of bilayers, each separated by water molecules. Liposomes can be formed by sonicating lipid vesicles in water.

[0153] In this disclosure, the term "lipid bilayer" refers to a thin film formed from two layers of lipid molecules.

[0154] In this disclosure, the term "micelle" refers to an aggregate of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solution, upon contact with water, form associations with the hydrophilic head region, which chelates the single hydrophobic tail region in the center of the micelle.

[0155] As used herein, "cell" has the meaning known in the art, including, for example, individual cells, tissues, organs, insect cells, avian cells, fish cells, amphibian cells, mammalian cells, primary cells, continuous cell lines, stem cells, and / or cultures of genetically engineered cells (recombinant cells expressing heterologous polypeptides or proteins). Recombinant cells include, for example, cells expressing heterologous polypeptides or proteins, such as growth factors or blood factors.

[0156] In a preferred embodiment, the lipid nanoparticles or liposomes of the present disclosure further comprise a helper lipid. In a preferred embodiment, the helper lipid is a non-cationic lipid. In a more preferred embodiment, the helper lipid is a non-cationic phospholipid. Within the scope of the present disclosure, non-cationic lipids may contain cationic functional groups (e.g., ammonium), but it is desirable to contain at least anionic functional groups to neutralize the molecule. It is desirable that the sum of all functional groups contained in the lipid molecule is non-cationic. Liposomes composed of a mixture of cationic amino lipids and non-cationic (neutral) phospholipids are most effective for delivering nucleic acids into cells. In a more preferred embodiment, the non-cationic lipid is DOPE or DSPC.

[0157] In a further preferred embodiment, the lipid nanoparticles or liposomes of the present disclosure further comprise a sterol. The sterol, cholesterol, is a natural component of cell membranes and is useful for stabilizing the particles and aiding in their integration into the cell membrane.

[0158] In another embodiment, the lipid nanoparticles or liposomes of the present disclosure further contain a bioactive agent. Within the scope of the present disclosure, a bioactive agent is a substance that has a biological effect when introduced into a cell or host, for example, by stimulating an immune or inflammatory response, by exerting enzymatic activity, or by complementary mutation. Bioactive substances are particularly nucleic acids, peptides, proteins, antibodies, and small molecules. In addition, when a drug is encapsulated in a lipid bilayer membrane or in the aqueous space within a liposome, the term "lipid nanoparticle drug" can be used.

[0159] In a preferred embodiment, the bioactive agent is a nucleic acid. In another preferred embodiment, the bioactive agent is selected from the group consisting of an antitumor agent, an antibiotic, an immunomodulatory agent, an anti-inflammatory agent, an agent acting on the central nervous system, a polypeptide, and a polypeptoid.

[0160] In another embodiment, the lipid nanoparticles or liposomes further comprise at least one polyethylene glycol (PEG) lipid. The PEG lipid contributes to protecting the particles and their contents from degradation in vivo or outside the body. Furthermore, PEG forms a protective layer on the surface of the liposomes, increasing their circulation time in the body. It can be used for the delivery of liposomal drugs (PEG-liposomes). Preferably, the polyethylene glycol-lipid is PEG2000-DMG.

[0161] Lipid nanoparticles or liposomes containing bioactive agents can be used to deliver any of a variety of therapeutic agents into cells. The present disclosure includes the use of such lipid nanoparticles (particularly liposomes) to deliver bioactive substances into cells.

[0162] Preferably, the bioactive agent includes, but is not limited to, RNA, DNA, and antisense oligonucleotides. Here, RNA includes, but is not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), and small nuclear RNA (snRNA). DNA includes, but is not limited to, plasmids. The bioactive agent is selected from the group consisting of antitumor agents, antibiotics, immunomodulators, anti-inflammatory agents, agents acting on the central nervous system, antigens or fragments thereof, proteins, peptides, polypeptides, vaccines, and small molecules, and mixtures thereof. As described above, lipid nanoparticles or liposomes containing the amino lipid compounds described in the present disclosure or pharmaceutically acceptable salts or stereoisomers thereof are useful for delivering bioactive agents to cells.

[0163] Various amino lipid compounds synthesized by common synthetic methods can be used to screen liposomes for specific properties, such as transfection efficiency, cytotoxicity, adhesion of drugs to be delivered into cells, liposome stability, and liposome size.

[0164] The lipid nanoparticles or liposomes of the present disclosure can be used to transfect multicellular tissues or organs, thereby offering new therapeutic treatment possibilities for patients.

[0165] According to the present disclosure, the patient can be selected from any mammal, preferably humans, mice, rats, pigs, cats, dogs, horses, goats, cows, and monkeys and / or others. Most preferably, the patient is a human.

[0166] A preferred embodiment of the present disclosure relates to the use of lipid nanoparticles or liposomes containing the amino lipid compounds of the present disclosure, or pharmaceutically acceptable salts or stereoisomers thereof, as pharmaceuticals.

[0167] In particular, the lipid nanoparticles or liposomes can be administered to patients for gene therapy, gene vaccination, antisense therapy, or RNA interference therapy. Specific uses include, but are not limited to:

[0168] (1) The lipid nanoparticles of the present disclosure can deliver nucleic acids for gene therapy. The amino lipids of the present disclosure can be used to introduce foreign genes into target cells, thereby repairing and treating diseases caused by defective or abnormal genes, achieving therapeutic goals. This includes the application of genetic engineering, i.e., gene transfer technology, to insert foreign genes into appropriate recipient cells in a patient so that the products produced by the foreign genes can treat specific diseases, such as common lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain tumors, lymphoma, blood cancer, and prostate cancer. Gene-edited nucleic acid materials can also be introduced to treat various genetic disorders, such as hemophilia, thalassemia, and Gaucher disease.

[0169] (2) The lipid nanoparticles of the present disclosure can be used for vaccination. The lipid nanoparticles or liposomes of the present disclosure can be used to deliver antigens or nucleic acids encoding antigens. The lipid nanoparticles of the present disclosure can also be used to induce immune responses to various antigens used in the treatment and / or prevention of various conditions, such as cancer, allergies, toxicity, and infections by pathogens (e.g., viruses, bacteria, fungi, and other pathogenic organisms).

[0170] In another preferred embodiment, the lipid nanoparticles of the present disclosure can be used to manufacture pharmaceuticals for nucleic acid transfer, preferably wherein the nucleic acid is RNA, DNA, or antisense oligonucleotide; preferably, the RNA is selected from messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), and small nuclear RNA (snRNA); preferably, the DNA is a plasmid. [Example]

[0171] In order to clarify the objectives, technical solutions and advantages of the embodiments of the present disclosure, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below with reference to the drawings. The described embodiments are only some of the embodiments of the present disclosure. Based on the described embodiments of the present disclosure, all other embodiments that can be obtained by those skilled in the art without creative efforts fall within the scope of protection of the present invention. In the following examples, experimental methods without specific conditions are selected according to conventional methods and conditions or product specifications.

[0172] The structures of all compounds in this disclosure are shown by nuclear magnetic resonance ( 1 The identity can be determined by H-NMR and / or mass spectrometry (MS).

[0173] 1H-NMR chemical shifts (δ) are reported in ppm (parts per million). NMR was performed on a Bruke RAVANCE III-400 MHz spectrometer. Suitable solvents were selected from deuterated chloroform (CDCl), deuterated methanol (CD3OD), deuterated dimethyl sulfoxide (DMSO-d6), etc., and tetramethylsilane (TMS) was used as the internal standard.

[0174] Low-resolution mass spectrometry (MS) was performed using an Agilent 1260 Infinity II-G6125C mass spectrometer.

[0175] The specific rotation measurement method of the present disclosure: A sample is collected, precisely weighed, dissolved in absolute ethanol, and diluted quantitatively to prepare a solution of approximately 10 mg per ml. The measurement is performed in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part 4 General Provisions 0621, Optical Rotation Measurement Method.

[0176] Known starting materials of the present disclosure may be synthesized by adopting or according to methods known in the art, or may be commercially available.

[0177] The eluent for column chromatography used to purify the compounds is A: dichloromethane and methanol (20:1-5:1); B: n-hexane and ethyl acetate (10:1-2:1). The volume ratio of the solvents is adjusted depending on the polarity of the compounds.

[0178] In the examples, unless otherwise specified, the reaction temperature is room temperature, in the range of 15°C to 30°C.

[0179] The HPLC analysis method of the present disclosure is the HPLC-CAD method.

[0180] [Table 7]

[0181] Example 1: Synthesis of Compound 1 Step 1): Synthesis of 1-octylnonyl 8-bromooctanoate [ka] 8-Bromooctanoic acid (22.3 g, 100 mmol), 9-heptadecanol (25.6 g, 100 mmol), and 100 mL of dichloromethane were added sequentially to a 250 mL reaction bottle. After stirring to dissolve, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.0 g, 120 mmol), 4-dimethylaminopyridine (0.61 g, 5 mmol), and N,N-diisopropylethylamine (25.8 g, 200 mmol) were added and reacted at room temperature for 2 hours. The mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (n-hexane:ethyl acetate = 10:1 to 2:1) to obtain 1-octylnonyl 8-bromooctanoate (41.5 g, 90%).

[0182] Step 2: Synthesis of nonyl 8-((3-hydroxycyclobutyl)amino)octanoate (intermediate compound II-1) [ka] Nonyl 8-bromooctanoate (3.50 g, 10 mmol), 3-aminocyclobutanol (8.7 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and then purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain nonyl 8-((3-hydroxycyclobutyl)amino)octanoate (2.38 g, 67%). 1H NMR(600 MHz, CDCl3)δ:4.66-4.55 (m, 0.5H), 4.14-4.06 (m, 2H), 3.61-3.51 (m, 0.5H), 3.01 (m, 0.5H), 2.82-2.71 (m, 2H), 2.71-2.63 (m, 2H), 2.58 (m, 0.5H), 2.31 (t, 2H), 2.27-2.01 (m, 2H), 1.72-1.53 (m, 6H), 1.44-1.21 (m, 18H), 0.91 (t, 3H). LCMS: 356.3 [M+H]+.

[0183] Step 3): Synthesis of Compound 1 [ka] Nonyl 8-((3-hydroxycyclobutyl)amino)octanoate (355 mg, 1 mmol), 1-octylnonyl 8-bromooctanoate (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, and the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 1 (501 mg, 68%). 1H NMR (600 MHz, CDCl3)δ:4.94-4.78(m, 1H), 4.42-4.37(m, 0.5H), 4.05(t, 2H), 3.9 9(m, 0.5H), 3.61-3.45(m, 0.5H), 2.75-2.65(s, 0.5H), 2.60-2.52(m, 2H) , 2.50-2.45(m, 3H), 2.40(m, 1H), 2.28(m, 4H), 1.99(m, 2H), 1.67-1.57(m , 6H), 1.57-1.45(m, 4H), 1.43-1.38(m, 4H), (m, 49H), 0.89-0.83(m, 9H). LCMS: 737.2 [M+H]+.

[0184] Example 2: Synthesis of Compound 2 Step 1): Synthesis of undecyl 6-((3-hydroxycyclobutyl)amino)hexanoate (intermediate compound II-2) [ka] Undecyl 8-bromohexanoate (3.50 g, 10 mmol), 3-aminocyclobutanol (8.7 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain undecyl 6-((3-hydroxycyclobutyl)amino)hexanoate (2.38 g, 67%). 1H NMR(600 MHz, CDCl3)δ:4.66-4.57 (m, 0.5H), 4.06-4.04 (t, 2H), 3.62-3.54 (m, 0.5H), 3.13-2.96 (m, 0.5H), 2.82-2.73 (m, 2H), 2.71-2.66 (m, 2H), 2.66-2.59 (m, 0.5H), 2.31 (t, 2H), 2.28-2.10 (m, 2H), 1.73-1.55 (m, 6H), 1.43-1.21 (m, 18H), 0.88 (t, 3H). LCMS: 356.3 [M+H]+.

[0185] Step 2): Synthesis of Compound 2 [ka] Compound 2 was synthesized in the same manner as in step 3) of Example 1, except that the intermediate compound nonyl 8-((3-hydroxycyclobutyl)amino)octanoate was replaced with undecyl 6-((3-hydroxycyclobutyl)amino)hexanoate (intermediate compound II-2). 1H NMR(600 MHz, CDCl3)δ:4.95-4.77 (m, 1H), 4.42 (t, 0.5H), 4.08 (t, 2H), 4.02-3.94 (m, 0.5H), 3.51-3.42 (m, 0.5H), 2.72-2.62 (m, 0.5H), 2.56 (m, 2H), 2.50-2.44 (m, 3H), 2.42 (s, 1H), 2.38-2.29 (m, 4H), 1.94 (s, 2H), 1.71-1.58 (m, 6H), 1.53 (m, 4H), 1.44 (m, 4H), 1.40-1.21 (m, 49H), 0.90 (m, 9H). LCMS: 737.2 [M+H]+.

[0186] Example 3: Synthesis of Compound 6 Step 1): Synthesis of nonyl 8-((3-hydroxycyclohexyl)amino)octanoate (intermediate compound II-6) [ka] Nonyl 8-bromooctanoate (3.50 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain nonyl 8-((3-hydroxycyclohexyl)amino)octanoate (2.34 g, 61%). 1H NMR(600 MHz, CDCl3)δ 4.20-4.13 (m, 0.5H), 4.05 (t, 2H), 3.83 (m, 0.5H), 3.09 (m, 0.5H), 2.87 (m, 0.5H), 2.76-2.59 (m, 2H), 2.29 (t, 2H), 2.00 (m, 0.5H), 1.93-1.78 (m, 1.5H), 1.78-1.65 (m, 2H), 1.65-1.46 (m, 8H), 1.40-1.18 (m, 20H), 0.88 (t, 3H). LCMS: 384.3 [M+H]+.

[0187] Step 2): Synthesis of Compound 6 [ka] Nonyl 8-((3-hydroxycyclohexyl)amino)octanoate (383 mg, 1 mmol), 1-octylnonyl 8-bromooctanoate (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, and the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give compound 6 (596 mg, 78%). 1H NMR(600 MHz, CDCl3)δ 4.95-4.81 (m, 1H), 4.30-4.12 (m, 0.5H), 4.07 (t, 2H), 3.72-3.61 (m, 0.5H), 2.97 (m, 0.5H), 2.64-2.52 (m, 0.5H), 2.48-2.37 (m, 4H), 2.30 (q, 4H), 1.93-1.81 (m, 2H), 1.72-1.57 (m, 8H), 1.51 (t, 4H), 1.43-1.18 (m, 56H), 0.89 (m, 9H). LCMS: 765.3 [M+H]+.

[0188] Example 4: Synthesis of Compound 7 Step 1): Synthesis of undecyl 6-((3-hydroxycyclohexyl)amino)hexanoate (intermediate compound II-7) [ka] Undecyl 6-bromohexanoate (3.50 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain undecyl 6-((3-hydroxycyclohexyl)amino)hexanoate (2.53 g, 66%). 1H NMR(600 MHz, CDCl3)δ 4.20 (m, 0.5H), 4.05 (t, 2H), 3.93 (m, 0.5H), 3.21 (m, 0.5H), 3.03 (m, 0.5H), 2.84-2.68 (m, 2H), 2.35-2.25 (m, 2H), 2.12-2.06 (m, 0.5H), 1.91 (m, 1.5H), 1.94-1.82 (m, 2H), 1.77-1.56 (m, 8H), 1.43-1.19 (m, 20H), 0.88 (t, 3H). LCMS: 384.4 [M+H]+.

[0189] Step 2): Synthesis of Compound 7 [ka] Undecyl 6-((3-hydroxycyclohexyl)amino)hexanoate (383 mg, 1 mmol), 1-octylnonyl 8-bromooctanoate (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, and the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give compound 7 (572 mg, 75%). 1H NMR(600 MHz, CDCl3)δ 4.93-4.81 (m, 1H), 4.24 (m, 0.5H), 4.05 (t, 2H), 3.68-3.61 (m, 0.5H), 2.98 (m, 0.5H), 2.56 (m, 0.5H), 2.50-2.36 (m, 4H), 2.35-2.22 (m, 4H), 1.97-1.73 (m, 3H), 1.69-1.55 (m, 7H), 1.48 (m, 4H), 1.46-1.36 (m, 4H), 1.36-1.18 (m, 52H), 0.88 (m, 9H). LCMS: 765.0 [M+H]+.

[0190] Example 5: Synthesis of Compound 8 Step 1) Synthesis of 7-pentadecyl 6-oxohexanoate [ka] 6-((tert-butyldimethylsilyl)oxy)hexanoic acid (2.46 g, 10 mmol), pentadecan-7-ol (2.28 g, 10 mol), and 100 mL of dichloromethane were added sequentially to a 250 mL reaction bottle and stirred to dissolve. After that, dicyclohexylcarbodiimide (2.47 g, 12 mmol) and 4-dimethylaminopyridine (0.06 g, 0.5 mmol) were added and reacted at room temperature for 2 hours. The mixture was washed with water three times, dried over anhydrous sodium sulfate, and concentrated to dryness. 50 mL of tetrahydrofuran and tetrabutylammonium fluoride (2.75 g, 10.5 mmol) were added and reacted at room temperature for 1 hour. The mixture was concentrated to dryness and dissolved in 100 mL of dichloromethane. The mixture was washed with water three times, dried over anhydrous sodium sulfate, and then added with Dess-Martin oxidant (5.09 g, 12 mmol). After adding 1.2 mmol) and reacting with stirring at room temperature for 12 hours, the mixture was washed three times with saturated sodium bicarbonate solution, then washed once with water, dried over anhydrous sodium sulfate, and purified using flash column chromatography (n-hexane:ethyl acetate = 10:1 to 5:1) to obtain 7-pentadecyl 6-oxohexanoate (2.76 g, 81%).

[0191] Step 2): Synthesis of Compound 8 [ka] 7-Pentadecyl 6-oxohexanoate (3.41 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added sequentially to a 250 mL reaction bottle, and 3-aminocyclohexanol (0.57 g, 5 mmol) was then added. The mixture was allowed to react at room temperature for 24 hours, washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give compound 8 (2.83 g, 74%). 1H NMR(600 MHz, CDCl3)δ 4.93-4.80 (m, 2H), 4.24 (m, 0.5H), 3.66-3.58 (m, 0.5H), 3.05 (m, 0.5H), 2.54 (m, 0.5H), 2.42 (m, 4H), 2.30 (m, 4H), 1.96-1.74 (m, 3H), 1.72-1.56 (m, 6H), 1.50 (m, 11H), 1.35-1.19 (m, 48H), 0.88 (t, 12H). LCMS: 765.1 [M+H]+.

[0192] Example 6: Synthesis of Compound 9 Step 1): Synthesis of 2-hexyldecanoic acid-(6-oxohexyl) ester [ka] 2-n-Hexyldecanoic acid (25.6 g, 100 mmol), 1,6-hexanediol (59.0 g, 0.5 mol), and 150 mL of dichloromethane were added sequentially to a 250 mL reaction bottle and stirred to dissolve. Then, dicyclohexylcarbodiimide (20.6 g, 100 mmol) and 4-dimethylaminopyridine (0.61 g, 5 mmol) were added and the mixture was allowed to react at room temperature for 2 hours. The mixture was washed with water three times, dried over anhydrous sodium sulfate, and purified using flash column chromatography (n-hexane:ethyl acetate = 5:1 to 1:1) to obtain 2-hexyldecanoate-7-hydroxyheptyl. 100 mL of dichloromethane and Dess-Martin oxidant (50.9 g, 120 mmol) were added sequentially, and the mixture was stirred at room temperature for 12 hours. After that, the mixture was washed three times with saturated sodium bicarbonate solution, washed once with water, dried over anhydrous sodium sulfate, and purified using flash column chromatography (n-hexane:ethyl acetate = 10:1 to 5:1) to obtain 2-hexyldecanoic acid-(6-oxohexyl) ester (19.1 g, 54%).

[0193] Step 2): Synthesis of Compound 9 [ka] 2-Hexyldecanoic acid-(6-oxohexyl) ester (3.55 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added sequentially to a 250 mL reaction bottle, and nonyl 8-((3-hydroxycyclohexyl)amino)octanoate (3.83 g, 10 mmol) was then added. The mixture was allowed to react at room temperature for 24 hours, washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 9 (4.98 g, 69%). 1H NMR(600 MHz, CDCl3)δ 4.26-4.19 (m, 0.5H), 4.12 (m, 2H), 4.09-4.02 (m, 2H), 3.65 (m, 0.5H), 2.98 (m, 0.5H), 2.59 (m, 0.5H), 2.51-2.35 (m, 4H), 2.30 (m, 3H), 1.98-1.89 (m, 1H), 1.89-1.72 (m, 3H), 1.72-1.53 (m, 9H), 1.51-1.34 (m, 10H), 1.34-1.16 (m, 42H), 0.92-0.79 (m, 9H). LCMS: 723.1 [M+H]+.

[0194] Example 7: Synthesis of Compound 10 Step 1): Synthesis of 8-bromooctyl oleate [ka] 8-Bromooctanol (20.9 g, 100 mmol), oleic acid (28.5 g, 100 mmol), and dichloromethane (100 mL) were added sequentially to a 250 mL reaction bottle. After stirring to dissolve, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.0 g, 120 mmol), 4-dimethylaminopyridine (0.61 g, 5 mmol), and N,N-diisopropylethylamine (25.8 g, 200 mmol) were added and reacted at room temperature for 2 hours. The mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified by flash column chromatography (n-hexane:ethyl acetate = 10:1 to 3:1) to give 8-bromooctyl oleate (37.4 g, 79%).

[0195] Step 2): Synthesis of 8-((3-hydroxycyclohexyl)amino)octyl oleate (intermediate compound II-10) [ka] 8-Bromonoyl oleate (4.73 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give 8-((3-hydroxycyclohexyl)amino)octyl oleate (3.2 g, 63%). 1H NMR(600 MHz, CDCl3)δ 5.41-5.29 (m, 2H), 4.19 (m, 0.5H), 4.05 (t, 2H), 3.92 (m, 0.5H), 3.20 (m, 0.5H), 3.00 (m, 0.5H), 2.81-2.67 (m, 2H), 2.29 (t, 2H), 2.02 (m, 3H), 1.93-1.79 (m, 2H), 1.69-1.56 (m, 11H), 1.36-1.20 (m, 30H), 0.88 (t, 3H). LCMS: 508.7 [M+H]+.

[0196] Step 3): Synthesis of Compound 10 [ka] 2-Hexyldecanoic acid-(6-oxohexyl) ester (3.55 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added sequentially to a 250 mL reaction bottle. 8-((3-hydroxycyclohexyl)amino)octyl oleate (5.08 g, 10 mmol) was then added and reacted at room temperature for 24 hours. The mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give compound 10 (6.26 g, 74%). 1H NMR(600 MHz, CDCl3)δ 5.41-5.31 (m, 2H), 4.24 (m, 0.5H), 4.06 (m, 4H), 3.70-3.60 (m, 0.5H), 3.03-2.92 (m, 0.5H), 2.57 (m, 0.5H), 2.49-2.36 (m, 4H), 2.30 (m, 3H), 2.01 (m, 3H), 1.98-1.90 (m, 2H), 1.85 (m, 1H), 1.82-1.74 (m, 1H), 1.61 (m, 9H), 1.45-1.38 (m, 6H), 1.33-1.20 (m, 56H), 0.88 (m, 9H). LCMS: 847.3 [M+H]+.

[0197] Example 8: Synthesis of Compound 12 Step 1: Synthesis of (6Z,9Z)-diene-octadecane-8-((3-hydroxycyclohexyl)amino)octanoate (intermediate compound II-12) [ka] (6Z,9Z)-diene-octadecane-8-bromooctanoate (4.71 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give (6Z,9Z)-diene-octadecane-8-((3-hydroxycyclohexyl)amino)octanoate (3.3 g, 66%). 1H NMR(600 MHz, CDCl3)δ 5.43-5.28 (m, 4H), 4.18 (m, 0.5H), 4.07-4.03 (m, 2H), 3.95 (m, 0.5H), 3.22 (m, 0.5H), 3.04 (m, 0.5H), 2.82-2.69 (m, 4H), 2.31-2.23 (m, 2H), 2.09-2.01 (m, 4H), 1.85 (m, 1H), 1.76-1.56 (m, 10H), 1.40-1.26 (m, 23H), 0.92-0.84 (t, 3H). LCMS: 506.7 [M+H]+.

[0198] Step 2): Synthesis of Compound 12 [ka] Compound 12 was synthesized in the same manner as in step 3) of Example 7, except that the intermediate compound, 8-((3-hydroxycyclohexyl)amino)octyl oleate, was replaced with (6Z,9Z)-diene-octadecane-8-((3-hydroxycyclohexyl)amino)octanoate. 1H NMR(600 MHz, CDCl3)δ 5.43-5.28 (m, 4H), 4.24 (m, 0.5H), 4.06 (m, 4H), 3.63 (m, 0.5H), 2.98 (m, 0.5H), 2.77 (t, 2H), 2.55 (m, 0.5H), 2.45 (m, 4H), 2.30 (m, 3H), 2.10-2.01 (m, 4H), 1.88-1.73 (m, 3H), 1.66-1.53 (m, 10H), 1.35-1.16 (m, 55H), 0.88 (m, 9H). LCMS: 845.1 [M+H]+.

[0199] Example 9: Synthesis of Compound 14 [ka] 2-Hexyldecanoic acid-(6-oxohexyl) ester (3.55 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added sequentially to a 250 mL reaction bottle, and then 3-aminocyclohexanol (0.57 g, 5 mmol) was added. The mixture was reacted at room temperature for 24 hours, washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give compound 14 (2.81 g, 71%). 1H NMR(600 MHz, CDCl3)δ 4.32-4.20 (m, 0.5H), 4.13-4.03 (m, 4H), 3.71-3.61 (m, 0.5H), 3.02 (m, 0.5H), 2.59 (m, 0.5H), 2.48 (m, 4H), 2.37-2.29 (m, 2H), 1.91-1.75 (m, 2H), 1.74-1.56 (m, 10H), 1.51-1.36 (m, 14H), 1.36-1.21 (m, 46H), 0.90 (m, 12H). LCMS: 793.2 [M+H]+.

[0200] Example 10: Synthesis of Compound 15 [ka] Compound 15 was synthesized in the same manner as in step 3) of Example 7, except that the intermediate compound, 8-((3-hydroxycyclohexyl)amino)octyl oleate, was changed to 7-pentadecyl 6-((3-hydroxycyclohexyl)amino)hexanoate. 1H NMR(600 MHz, CDCl3)δ 4.94-4.80 (m, 1H), 4.23 (m, 0.5H), 4.08 (t, 2H), 3.72-3.62 (m, 0.5H), 3.00 (m, 0.5H), 2.55 (m, 0.5H), 2.50-2.37 (m, 4H), 2.35-2.25 (m, 3H), 1.90-1.75 (m, 2H), 1.73-1.57 (m, 9H), 1.51 (m, 4H), 1.44 (m, 7H), 1.39 (m, 2H), 1.36-1.21 (m, 46H), 0.90 (m, 12H). LCMS: 779.2 [M+H]+.

[0201] Example 11: Synthesis of Compound 16 Step 1): Synthesis of nonyl 8-((3-methoxycyclohexyl)amino)octanoate (intermediate compound II-16) [ka] Nonyl 8-bromooctanoate (3.49 g, 10 mmol), 3-methoxycyclohexylamine (12.9 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain nonyl 8-((3-methoxycyclohexyl)amino)octanoate (2.35 g, 59%). 1H NMR(600 MHz, CDCl3)δ 4.07 (t, 2H), 3.64 (s, 0.5H), 3.36 (s, 1.5H), 3.35 (m, 0.5H), 3.29 (s, 1.5H), 3.28-3.23 (m, 0.5H), 3.18 (s, 0.5H), 3.00-2.89 (m, 2H), 2.30 (t, 2H), 2.07-2.02 (m, 4H), 1.92-1.84 (m, 2H), 1.69 (m, 3H), 1.67-1.58 (m, 4H), 1.40-1.23 (m, 18H), 0.94-0.82 (m, 3H). LCMS: 398.5 [M+H]+.

[0202] Step 2): Synthesis of Compound 16 [ka]

[0203] Nonyl 8-((3-methoxycyclohexyl)amino)octanoate (400 mg, 1 mmol), 1-octylnonyl 8-bromooctanoate (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give compound 16 (552 mg, 71%). 1H NMR(600 MHz, CDCl3)δ 4.93-4.77 (m, 1H), 4.05 (t, 2H), 3.62 (m, 0.5H), 3.36 (s, 1.5H), 3.29 (s, 1.5H), 3.15-3.07 (m, 0.5H), 2.86 (m, 0.5H), 2.55 (t, 0.5H), 2.43 (m, 4H), 2.32-2.24 (m, 4H), 2.07-1.99 (m, 1H), 1.85-1.69 (m, 2H), 1.62 (m, 6H), 1.53-1.48 (m, 4H), 1.45-1.39 (m, 4H), 1.36-1.24 (m, 54H), 0.90-0.85 (m, 9H). LCMS: 779.1 [M+H]+.

[0204] Example 12: Synthesis of Compound 19 Step 1): Synthesis of nonyl 8-((3-acetoxycyclohexyl)amino)octanoate (intermediate compound II-19) [ka] To a 100 mL reaction bottle, nonyl 8-bromooctanoate (3.49 g, 10 mmol), 3-acetoxycyclohexylamine (15.7 g, 100 mmol), and 30 mL of ethanol were added in that order. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, and the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and then purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain nonyl 8-((3-acetoxycyclohexyl)amino)octanoate (2.76 g, 65%). 1H NMR(600 MHz, CDCl3)δ 5.23 (m, 0.5H), 4.76-4.67 (m, 0.5H), 4.09 (t, 2H), 3.30 (m, 0.5H), 3.05 (m, 0.5H), 3.01-2.86 (m, 3H), 2.45-2.38 (m, 1H), 2.32 (t, 2H), 2.12 (m, 1H), 2.10 (s, 1.5H), 2.08 (s, 1.5H), 2.05 (m, 1H), 1.97-1.91 (m, 1H), 1.77 (m, 3H), 1.71 (m, 3H), 1.68-1.60 (m, 5H), 1.41-1.22 (m, 16H), 0.92 (t, 3H). LCMS: 426.4 [M+H]+.

[0205] Step 2): Synthesis of Compound 19 [ka] Compound 19 was synthesized in the same manner as in Step 2) of Example 11, except that the intermediate compound nonyl 8-((3-methoxycyclohexyl)amino)octanoate was changed to nonyl 8-((3-acetoxycyclohexyl)amino)octanoate. 1H NMR(600 MHz, CDCl3)δ 5.16 (m, 0.5H), 4.91-4.81 (m, 1H), 4.69 (m, 0.5H), 4.05 (t, 2H), 2.86 (m 0.5H), 2.64-2.50 (m, 0.5H), 2.45-2.32 (m, 4H), 2.32-2.22 (m, 4H), 2.04 (s, 1.5H), 2.03 (s, 1.5H), 1.93 (m, 1H), 1.84-1.75 (m, 1H), 1.74-1.69 (m, 1H), 1.66-1.58 (m, 7H), 1.49 (m, 4H), 1.37 (m, 4H), 1.34-1.21 (m, 52H), 0.90-0.85 (m, 9H). LCMS: 806.7 [M+H]+.

[0206] Example 13: Synthesis of Compound 20 Step 1): Synthesis of methyl 3-((8-(nonyloxy)-8-oxooctyl)amino)cyclohexane-1-carboxylate (intermediate compound II-20) [ka] Nonyl 8-bromooctanoate (3.49 g, 10 mmol), methyl 3-aminocyclohexanecarboxylate (15.7 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and then purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give methyl 3-((8-(nonyloxy)-8-oxooctyl)amino)cyclohexane-1-carboxylate (2.85 g, 67%). 1H NMR(600 MHz, CDCl3)δ 4.05 (t, 2H), 3.70 (m, 2H), 3.67 (s, 3H), 3.00-2.80 (m, 3H), 2.41 (m, 1H), 2.35 (m, 1H), 2.32-2.26 (t, 2H), 2.23-2.15 (m, 1H), 2.01 (m, 1H), 1.94-1.89 (m, 1H), 1.86-1.75 (m, 2H), 1.69-1.57 (m, 3H), 1.40-1.19 (m, 18H), 0.88 (t, 3H). LCMS: 426.4 [M+H]+.

[0207] Step 2): Synthesis of Compound 20 [ka] Compound 20 was synthesized in the same manner as in step 2) of Example 11, except that the intermediate compound nonyl 8-((3-methoxycyclohexyl)amino)octanoate was replaced with methyl 3-((8-(nonyloxy)-8-oxooctyl)amino)cyclohexane-1-carboxylate. 1H NMR(600 MHz, CDCl3)δ 4.95-4.78 (m, 1H), 4.05 (t, 2H), 3.67 (s, 3H), 2.55-2.45 (m, 1H), 2.45-2.34 (m, 4H), 2.32-2.24 (m, 5H), 1.99 (m, 1H), 1.94-1.82 (m, 2H), 1.74 (m, 1H), 1.65-1.58 (m, 6H), 1.57-1.45 (m, 4H), 1.43-1.19 (m, 56H), 0.88 (m, 9H). LCMS: 806.8 [M+H]+.

[0208] Example 14 Synthesis of Compound (I-6-II) [ka] A 250 mL single-neck flask was charged with 8 g (17.3 mmol) of 9-heptadecyl-8-bromooctanoate, 10 g (76.5 mmol) of (1S,3R)-3-aminocyclohexanol, and 100 mL of ethanol, and the resulting mixture was reacted at 50°C for 15 hours to completely convert the starting material, 9-heptadecyl-8-bromooctanoate. After distilling off the solvent, 200 mL of EA was added, and the mixture was washed twice with 100 mL of water to completely convert the (1S,3R)-3-aminocyclohexanol. The EA phase was then dried on a rotary evaporator to obtain crude compound (6-VII-I), 9-heptadecyl-8-(((1R,3S)-3-hydroxycyclohexyl)amino)octanoate. The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 6.77 g of compound (6-VII-I) with a purity of 99.2% and a yield of 78.6%. 1H NMR(600 MHz, CDCl3): δ 4.91-4.81 (m, 1H), 3.86-3.82 (m, 1H), 2.84-2.81 (m, 1H), 2.69-2.54 (qt, J = 11.2, 7.3 Hz, 2H), 2.29-2.26 (t, J = 7.5 Hz, 2H), 1.93-1.86 (m, 1H), 1.77-1.74(d, J = 12.0 Hz, 1H), 1.72-1.57 (m, 6H), 1.54-1.45 (m, 6H), 1.37-1.30 (m, 8H), 1.30-1.22 (m, 24H), 0.88 (t, J = 7.0 Hz, 6H). LCMS: 496.7 [M+H]+.

[0209] 6 g (12.1 mmol) of the above compound (6-VII-I) was placed in a 250 ml single-neck flask, 90 ml of acetonitrile and 60 ml of cyclopentyl methyl ether were added, and 6.7 g (48.4 mmol) of potassium carbonate powder, 2 g (12.1 mmol) of potassium iodide, and 5 g (17.5 mmol) of nonyl 8-bromooctanoate were added. The mixture was reacted at 90 ° C for 24 hours to completely react the raw material compound (6-VII-I). The reaction solution was removed from the oil bath and cooled to room temperature. The solid was then removed by suction filtration, and the filtrate was dried on a rotary evaporator to obtain a crude product. Finally, the crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 5.67 g of compound (I-6-II) with a purity of 98.6% and a yield of 61.4%. 1H NMR(600 MHz, CDCl3): δ 4.93-4.84 (m, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.68-3.61 (m, 1H), 2.62-2.54 (m, 1H), 2.53-2.41 (m, 4H), 2.31-2.26 (q, J = 7.4 Hz, 4H), 1.97 (m, 1H), 1.91-1.78 (m, 2H), 1.71-1.61 (m, 7H), 1.54 (d, J = 6.0 Hz, 4H), 1.43-1.37 (m, 4H), 1.39-1.23 (m, 52H), 0.91 (m, 9H). Specific rotation: +4.3° LCMS: 765.2 [M+H]+.

[0210] Example 15 Synthesis of Compound (I-6-III) [ka] A 100 mL single-neck flask was charged with 2 g (4.3 mmol) of 9-heptadecyl-8-bromooctanoate, 2.5 g (20.2 mmol) of (1S,3S)-3-aminocyclohexanol, and 20 mL of ethanol, and the resulting mixture was reacted at 50 °C for 15 hours to completely convert the starting material, 9-heptadecyl-8-bromooctanoate. After distilling off the solvent, 50 mL of EA was added, and the mixture was washed twice with 25 mL of water to completely convert the (1S,3S)-3-aminocyclohexanol. The EA phase was then dried on a rotary evaporator to obtain crude compound (6-VII-II), 9-heptadecyl-8-(((1S,3S)-3-hydroxycyclohexyl)amino)octanoate. The crude product was purified by column chromatography (dichloromethane:methanol=20:1 to 5:1) to obtain 1.63 g of compound (6-VII-II) with a purity of 98.5% and a yield of 75.5%. 1H NMR(600 MHz, CDCl3): δ 4.91-4.82 (m, 1H), 4.16-4.10 (m, 1H), 2.96-2.87 (m, 1H), 2.65-2.55 (qt, J = 11.2, 7.3 Hz, 2H), 2.28-2.26 (t, J = 7.5 Hz, 2H), 1.93-1.77 (m, 4H), 1.67-1.55 (m, 6H), 1.54-1.45 (m, 6H), 1.33-1.31 (m, 6H), 1.30-1.22 (m, 24H), 0.88 (t, J = 7.0 Hz, 6H). LCMS: 496.7 [M+H]+.

[0211] 1.5 g (3.0 mmol) of the above compound (6-VII-II) was placed in a 100 ml one-neck flask, 25 ml of acetonitrile and 15 ml of cyclopentyl methyl ether were added, 1.67 g of potassium carbonate powder (12 mmol), 0.5 g of potassium iodide (3 mmol), and 1.26 g of nonyl 8-bromooctanoate (3.6 mmol) were added, and the mixture was reacted at 90 ° C for 24 hours to completely react the raw material compound (6-VII-II). The reaction solution was removed from the oil bath and cooled to room temperature. The solid was then removed by suction filtration, and the filtrate was dried on a rotary evaporator to obtain a crude product. Finally, the crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 1.37 g of compound (I-6-III) with a purity of 99.3% and a yield of 59.5%. 1H NMR(600 MHz, CDCl3): δ 4.91-4.80 (m, 1H), 4.28-4.22 (m, 1H), 4.06-4.04 (t, J = 6.8 Hz, 2H), 3.03-2.97 (m, 1H), 2.47-2.41 (m, 4H), 2.33-2.25 (q, J = 7.5 Hz, 4H), 1.89-1.85 (d, J = 12.1 Hz, 1H), 1.80-1.77 (t, J = 12.2 Hz, 1H), 1.73-1.65 (m, 2H), 1.64-1.59 (m, 6H), 1.54-1.48 (m, 4H), 1.48-1.39 (m, 4H), 1.34-1.29 (m, 14H), 1.29-1.23 (m, 38H), 0.89-0.87 (m, 9H). Specific rotation: -12.9° LCMS: 765.2 [M+H]+.

[0212] Example 16 Synthesis of Compound (I-6-IV) [ka] 2 g (4.3 mmol) of 9-heptadecyl-8-bromooctanoate, 2.5 g (20.2 mmol) of (1R,3R)-3-aminocyclohexanol, and 20 mL of ethanol were added to a 100 mL single-neck flask and reacted at 50 °C for 15 hours to completely convert the starting material, 9-heptadecyl-8-bromooctanoate. After distilling off the solvent, 50 mL of EA was added, and the mixture was washed twice with 25 mL of water to completely convert the (1R,3R)-3-aminocyclohexanol. The EA phase was dried on a rotary evaporator to obtain crude compound (6-VII-III), 9-heptadecyl-8-(((1R,3R)-3-hydroxycyclohexyl)amino)octanoate. The crude product was purified by column chromatography (dichloromethane:methanol=20:1 to 5:1) to obtain 1.58 g of compound (6-VII-III) with a purity of 99.2% and a yield of 73.2%. 1H NMR(600 MHz, CDCl3): δ 4.91-4.81 (m, 1H), 4.17-4.08 (m, 1H), 2.94-2.84 (m, 1H), 2.64-2.5 (qt, J = 11.2, 7.3 Hz, 2H), 2.28-2.26 (t, J = 7.5 Hz, 2H), 1.88-1.76 (m, 2H), 1.75-1.54 (m, 8H), 1.54-1.40 (m, 6H), 1.33-1.3 (m, 6H), 1.30-1.23 (m, 24H), 0.88 (t, J = 7.0 Hz, 6H). LCMS: 496.7 [M+H]+.

[0213] 1.5 g (3.0 mmol) of the above compound (6-VII-III) was placed in a 100 ml single-neck flask, 25 ml of acetonitrile and 15 ml of cyclopentyl methyl ether were added, 1.67 g of potassium carbonate powder (12 mmol), 0.5 g of potassium iodide (3 mmol), and 1.26 g of nonyl 8-bromooctanoate (3.6 mmol) were added, and the mixture was reacted at 90 ° C for 24 hours to completely react the raw material compound (6-VII-III). The reaction solution was removed from the oil bath and cooled to room temperature. The solid was then removed by suction filtration, and the filtrate was dried using a rotary evaporator to obtain a crude product. Finally, the crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 1.29 g of compound (I-6-IV) with a purity of 98.6% and a yield of 55.8%. 1H NMR(600 MHz, CDCl3): δ 4.89-4.82 (m, 1H), 4.24-4.22 (m, 1H), 4.06-4.04 (t, J = 6.8 Hz, 2H), 3.00-2.95 (m, 1H), 2.47-2.36 (m, 4H), 2.30-2.26 (q, J = 7.5 Hz, 4H), 1.86-1.84 (d, J = 12.1 Hz, 1H), 1.79-1.77 (t, J = 12.2 Hz, 1H), 1.70-1.66 (m, 2H), 1.65-1.57 (m, 6H), 1.53-1.48 (m, 4H), 1.45-1.38 (m, 4H), 1.35-1.30 (m, 14H), 1.29-1.22 (m, 38H), 0.89-0.87 (m, 9H). Specific rotation: +12.6° LCMS: 765.2 [M+H]+.

[0214] Example 17 Synthesis of Compound (I-6-V) [ka] A 250 mL single-neck flask was charged with 8 g (17.3 mmol) of 9-heptadecyl-8-bromooctanoate, 10 g (76.5 mmol) of (1R,3S)-3-aminocyclohexanol, and 100 mL of ethanol, and the resulting mixture was reacted at 50°C for 15 hours to completely convert the starting material, 9-heptadecyl-8-bromooctanoate. After distilling off the solvent, 200 mL of EA was added, and the mixture was washed twice with 100 mL of water to completely convert the (1R,3S)-3-aminocyclohexanol. The EA phase was then dried on a rotary evaporator to obtain crude compound (6-VII-IV), 9-heptadecyl-8-(((1S,3R)-3-hydroxycyclohexyl)amino)octanoate. The crude product was purified by column chromatography (dichloromethane:methanol=20:1 to 5:1) to obtain 6.4 g of compound (6-VII-IV) with a purity of 98.5% and a yield of 74.8%. 1H NMR(600 MHz, CDCl3): δ 4.90-4.83 (m, 1H), 3.86-3.82 (m, 1H), 2.86-2.82 (m, 1H), 2.69-2.58 (qt, J = 11.2, 7.3 Hz, 2H), 2.29-2.26 (t, J = 7.5 Hz, 2H), 1.94-1.86 (m, 1H), 1.79-1.77 (d, J = 12.0 Hz, 1H), 1.74-1.64 (m, 3H), 1.64-1.59 (m, 3H), 1.51-1.46 (m, 6H), 1.36-1.30 (m, 8H), 1.30-1.22 (m, 24H), 0.88 (t, J = 7.0 Hz, 6H). LCMS: 496.7 [M+H]+.

[0215] 6 g of the above compound (6-VII-IV) (12.1 mmol) was placed in a 250 ml single-neck flask, 90 ml of acetonitrile and 60 ml of cyclopentyl methyl ether were added, 6.7 g of potassium carbonate powder (48.4 mmol), 2 g of potassium iodide (12.1 mmol), and 5 g of nonyl 8-bromooctanoate (17.4 mmol) were added, and the mixture was reacted at 90 ° C for 24 hours to completely react the raw material compound (6-VII-IV). The reaction solution was removed from the oil bath and cooled to room temperature. The solid was then removed by suction filtration, and the filtrate was dried on a rotary evaporator to obtain a crude product. Finally, the crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 5.35 g of compound (I-6-V) with a purity of 99.1% and a yield of 57.9%.

[0216] 1H NMR(600 MHz, CDCl3): δ 4.90-4.82 (m, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.70-3.60 (m, 1H), 2.64-2.54 (m, 1H), 2.51-2.41 (m, 4H), 2.30-2.26 (q, J = 7.5 Hz, 4H), 1.95 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.56 (m, 7H), 1.50 (d, J = 6.0 Hz, 4H), 1.41-1.37 (m, 4H), 1.34-1.18 (m, 52H), 0.88 (m, 9H). Specific rotation: -4.5° LCMS: 765.2 [M+H]+

[0217] Example 18 Synthesis of Compound 4 [ka] Compound 4 was synthesized in the same manner as in Step 3 of Example 7. Compound 4 (152 mg, yield 40.75%) was synthesized from compound intermediate I-4 (184 mg, 0.50 mmol) and 9-heptadecyl-8-oxooctanoate (296 mg). 1H NMR(600 MHz, CDCl3)δ 4.89-4.84 (m, 1H), 4.44-4.38 (m, 0.5H), 4.27-4.19 (m, 0.5H), 4.05 (t, J = 6.8 Hz, 2H), 3.53-3.41 (m, 0.5H), 3.24-3.14 (m, 0.5H), 2.76 (t, J = 6.0 Hz, 2H), 2.72-2.63 (t, J = 6.0 Hz, 2H), 2.33-2.24 (m, 4H), 2.21-2.00 (m, 2H), 1.91-1.74 (m, 2H), 1.72-1.45 (m, 15H), 1.39-1.19 (m, 49H), 0.88 (m, 9H). LC-MS: 751.2 [M+H]+.

[0218] Example 19: Synthesis of Compound 5 [ka] Compound 5 was synthesized in the same manner as in Step 3 of Example 7. Compound 5 (250 mg, yield 81.9%) was synthesized from compound intermediate II-5 (150 mg, 0.38 mmol) and 9-heptadecyl-8-oxooctanoate (225 mg). 1H NMR(600 MHz, CDCl3)δ 4.93-4.81 (m, 1H), 4.44-4.36 (m, 0.5H), 4.29-4.20 (m, 0.5H), 4.10-4.01 (m, 2H), 3.54-3.42 (m, 0.5H), 3.24-3.08 (m, 0.5H), 2.73 (m, 3H), 2.36-2.25 (m, 4H), 2.17-2.08 (m, 1H), 2.08-2.00 (m, 1H), 1.95-1.75 (m, 2H), 1.71-1.57 (m, 8H), 1.57-1.42 (m, 8H), 1.40-1.18 (m, 49H), 0.88 (m, 9H). LC-MS: 751.0 [M+H]+.

[0219] Example 20: Synthesis of Compound 11 [ka] Compound 11 was synthesized in the same manner as in Step 3) of Example 7. Compound 11 (96 mg, yield 42.5%) was synthesized from compound intermediate II-11 (140 mg, 0.28 mmol) and 9-heptadecyl-8-oxooctanoate (146 mg). 1H NMR(600 MHz, CDCl3)δ 5.37-5.29 (m, 2H), 4.21-4.19 (m, 0.5H), 4.02-4.00 (m, 4H), 3.67-3.60 (m, 0.5H), 2.99-2.91 (m, 0.5H), 2.57-2.53 (m, 0.5H), 2.46-2.34 (m, 4H), 2.29-2.25 (m, 3H), 2.01 -1.97(m, 3H), 1.96-1.88 (m, 2H), 1.83-1.80 (m, 1H), 1.79-1.72 (m, 1H), 1.58-1.52 (m, 9H), 1.43-1.35 (m, 6H), 1.31-1.18 (m, 56H), 0.87-0.84 (m, 9H). LC-MS: 847.3 [M+H]+.

[0220] Example 21: Synthesis of Compound 18 [ka] Compound 18 was synthesized in the same manner as in Step 3) of Example 7. Compound 18 (177 mg, yield 74.4%) was synthesized from compound intermediate II-18 (120 mg, 0.31 mmol) and 9-heptadecyl-8-oxooctanoate (185 mg). 1H NMR(600 MHz, CDCl3)δ 5.04-5.02 (m, 0.5H), 4.96-5.04 (m, 0.5H), 4.95-4.81 (m, 1H), 4.30-4.12 (m, 0.5H), 4.08-4.06 (t, J = 6.8 Hz, 2H), 3.72-3.61 (m, 0.5H), 2.98-2.96 (m, 0.5H), 2.64-2.52 (m, 0.5H), 2.48-2.37 (m, 4H), 2.31-2.29 (m, 4H), 1.93-1.81 (m, 2H), 1.72-1.57 (m, 8H), 1.52-1.50 (m, 4H), 1.43-1.18 (m, 55H), 0.91-0.87 (m, 9H). LC-MS: 767.3[M+H]+.

[0221] Example 22 Synthesis of Compound 21 [ka] Compound 21 was synthesized in the same manner as in Step 3) of Example 7. Compound 21 (100 mg, yield 13.8%) was synthesized from compound intermediate II-21 (400 mg, 0.86 mmol) and 9-heptadecyl-8-oxooctanoate (510 mg). 1H NMR(600 MHz, CDCl3)δ 4.90-4.81 (m, 1H), 4.06-4.04 (t, J = 6.8 Hz, 2H), 3.26-3.22 (dq, J = 8.9, 6.9 Hz, 2H), 2.66-2.55 (m, 1H), 2.53-2.35 (m, 4H), 2.30-2.26 (q, J = 7.5 Hz, 4H), 2.12-2.04 (m, 1H), 1.95-1.93 (m, 1H), 1.90-1.85 (m, 1H), 1.78-1.70 (m, 2H), 1.66-1.57 (m, 6H), 1.55-1.45 (m, 6H), 1.43-1.38 (m, 5H), 1.37-1.20 (m, 54H), 0.94-0.90 (t, J = 6.0 Hz, 3H), 0.88-0.86 (m, 9H). LC-MS: 848.3[M+H]+.

[0222] Example 23: Synthesis of Compound 22 [ka] Compound 22 was synthesized in the same manner as in Step 3) of Example 7. Compound 22 (266 mg, yield 46.8%) was synthesized from compound intermediate II-22 (300 mg, 0.71 mmol) and 9-heptadecyl-8-oxooctanoate (422 mg). 1H NMR(600 MHz, CDCl3)δ 4.884.80 (m, 1H), 4.03-3.98 (t, J = 6.8 Hz, 2H), 3.96-3.94(m, 1H), 2.49-2.32 (m, 4H), 2.28-2.23 (m, 4H), 2.09-2.01 (m, 1H), 1.93-1.91 (m, 1H), 1.88-1.83 (m, 1H), 1.76-1.68 (m, 2H), 1.64-1.53 (m, 6H), 1.53-1.43 (m, 6H), 1.40-1.35 (m, 5H), 1.34-1.16 (m, 52H), 0.84-0.81 (m, 9H). LC-MS: 806.1[M+H]+.

[0223] Comparative Example Comparative Example 1: Synthesis of Comparative Compound 1 Step 1): Synthesis of nonyl 8-((2-hydroxycyclopentyl)amino)octanoate [ka] Nonyl 8-bromooctanoate (3.50 g, 10 mmol), 2-aminocyclopentanol (10.12 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain nonyl 8-((2-hydroxycyclopentyl)amino)octanoate (2.29 g, 62%). 1H NMR(600 MHz, CDCl3)δ 4.07 (t, 2H), 4.01-3.94 (q, 1H), 2.91 (q, 1H), 2.72 (m, 1H), 2.63 (m, 1H), 2.31 (t, 2H), 2.09-1.97 (m, 2H), 1.72 (m, 2H), 1.67-1.59 (m, 4H), 1.56 (m, 3H), 1.45-1.38 (m, 1H), 1.38-1.22 (m, 18H), 0.90 (t, 3H). LCMS: 370.3 [M+H]+.

[0224] Step 2): Synthesis of comparative compound 1 [ka] Comparative compound 1 was synthesized in the same manner as in step 3) of Example 1, except that the intermediate compound nonyl 8-((3-hydroxycyclobutyl)amino)octanoate was changed to the intermediate compound nonyl 8-((2-hydroxycyclopentyl)amino)octanoate. 1H NMR(600 MHz, CDCl3)δ 4.91-4.80 (q, 1H), 4.05 (t, 2H), 3.92 (q, 1H), 2.85 (q, 1H), 2.50 (m, 2H), 2.44-2.35 (m, 2H), 2.28 (q, 4H), 1.95-1.87 (m, 2H), 1.83 (br, 2H), 1.75-1.66 (m, 2H), 1.64-1.56 (m, 6H), 1.55-1.47 (m, 5H), 1.47-1.38 (m, 5H), 1.36-1.18 (m, 46H), 0.88 (m, 9H). LCMS: 750.9 [M+H]+.

[0225] Comparative Example 2: Synthesis of Comparative Compound 2 [ka]

[0226] Comparative compound 2 Comparative compound 2 was prepared according to the synthesis method of compound 22 described in Chinese patent CN110520409A. 1H NMR(600 MHz, CDCl3)δ4.10- 4.07 (t, 2H), 3.26-3.20 (m, 1H), 2.85-2.79 (m, 1H), 2.54-2.48 (m, 1H), 2.34-2.30 (t, 2H), 2.28-2.19 (m, 1H), 2.15-2.01 (m, 2H), 1.75 (m, 2H), 1.69-1.58 (m, 4H), 1.58-1.46 (m, 2H), 1.41-1.25 (m, 20H), 0.91 (t, 3H). LCMS: 765.0 [M+H]+.

[0227] Comparative Example 3: Synthesis of Comparative Compound 3 Step 1): Synthesis of undecyl 6-((2-hydroxycyclohexyl)amino)hexanoate [ka] Undecyl 6-bromohexanoate (3.50 g, 10 mmol), 2-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain undecyl 6-((2-hydroxycyclohexyl)amino)hexanoate (2.57 g, 67%). 1H NMR(600 MHz, CDCl3)δ 4.05 (t, 2H), 3.31-3.20 (m, 1H), 2.90-2.75 (m, 1H), 2.53 (m, 1H), 2.35 -2.26 (m, 3H), 2.13-2.01 (m, 2H), 1.80-1.68 (m, 2H), 1.67-1.58 (m, 4H), 1.57-1.46 (m, 2H), 1.44-1.34 (m, 2H), 1.34-1.15 (m, 20H), 0.88 (t, 3H). LCMS: 384.4 [M+H]+.

[0228] Step 2): Synthesis of comparative compound 3 [ka] Undecyl 6-((2-hydroxycyclohexyl)amino)hexanoate (383 mg, 1 mmol), 1-octylnonyl 8-bromooctanoate (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, and the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain comparative compound 3 (550 mg, 72%). 1H NMR(600 MHz, CDCl3)δ 4.86 (m, 1H), 4.05 (t, 2H), 3.39-3.21 (m, 1H), 2.55-2.43 (m, 2H), 2.35- 2.23 (m, 6H), 2.10 (m, 1H), 1.76 (m, 2H), 1.70 (m, 1H), 1.67-1.57 (m, 6H), 1.49 (m, 6H), 1.38-1.19 (m, 55H), 0.88 (m, 9H). LCMS: 765.0 [M+H]+.

[0229] Comparative Example 4: Synthesis of Comparative Compound 4 [ka] 7-Pentadecyl 6-oxohexanoate (3.40 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added sequentially to a 250 mL reaction bottle, and 2-aminocyclohexanol (0.57 g, 5 mmol) was then added. The mixture was allowed to react at room temperature for 24 hours, washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain comparative compound 4 (2.67 g, 70%). 1H NMR(600 MHz, CDCl3)δ 4.91-4.80 (m, 2H), 3.28 (m, 1H), 2.48 (m, 2H), 2.30 (m, 6H), 2.24 (m, 1H), 2.11 (m, 1H), 1.75 (m, 2H), 1.68 (m, 1H), 1.66-1.60 (m, 4H), 1.48 (m, 10H), 1.44-1.07 (m, 50H), 0.88 (t, 12H). LCMS: 765.2 [M+H]+.

[0230] Comparative Example 5: Synthesis of Comparative Compound 5 (SM102) [ka] Comparative compound 5 was prepared according to the synthesis method of compound 25 described in Chinese patent CN110520409A. 1H NMR(600 MHz, CDCl3)δ 4.86-4.74 (m, 1H), 3.99 (t, 2H), 3.46 (t, 2H), 2.51 (t, 2H), 2.39 (q, 4H), 2.26-2.18 (m, 4H), 1.62-1.52 (m, 6H), 1.47-1.36 (m, 8H), 1.28-1.14 (m, 48H), 0.81 (m, 9H). LCMS: 711.0 [M+H]+.

[0231] Comparative Example 6: Synthesis of Comparative Compound 6 [ka] Nonyl 8-bromooctanoate (3.50 g, 10 mmol), p-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to give nonyl 8-((4-hydroxycyclohexyl)amino)hexanoate (2.65 g, 69%). 1H NMR(600 MHz, CDCl3)δ 4.05 (t, 2H), 3.96-3.90 (m, 0.5H), 3.67-3.57 (m, 0.5H), 2.75-2.67 (t, 2H), 2.66 (m, 0.5H), 2.54 (m, 0.5H), 2.28 (t, 2H), 2.03-1.96 (m, 1H), 1.86-1.78 (m, 1H), 1.78-1.71 (m, 3H), 1.66-1.50 (m, 7H), 1.40-1.20 (m, 20H), 0.88 (t, 3H). LCMS: 384.4 [M+H]+.

[0232] Step 2): Synthesis of comparative compound 6 [ka] To a 100 mL reaction bottle, nonyl 8-((4-hydroxycyclohexyl)amino)hexanoate (383 mg, 1 mmol), 1-octylnonyl 8-bromooctanoate (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added in that order, and after stirring to dissolve, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, and the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and then purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain comparative compound 6 (535 mg, 70%). 1H NMR(600 MHz, CDCl3)δ 4.90-4.82 (m, 1H), 4.05 (t, 2H), 3.99 (m, 0.5H), 3.59-3.51 (m, 0.5H), 2.47 (m, 0.5H), 2.45-2.42 (m, 3H), 2.39-2.32 (m, 1.5H), 2.28 (m, 4H), 2.01 (m, 1H), 1.82 (m, 1H), 1.76 (m, 1H), 1.68-1.58 (m, 7H), 1.52 (m, 4H), 1.43-1.36 (m, 4H), 1.36-1.20 (m, 52H), 0.88 (m, 9H). LCMS: 765.0 [M+H]+.

[0233] Comparative Example 7: Synthesis of Comparative Compound 7 Step 1): Synthesis of undecyl 6-((4-hydroxycyclohexyl)amino)hexanoate [ka] Undecyl 6-bromohexanoate (3.50 g, 10 mmol), p-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain undecyl 6-((4-hydroxycyclohexyl)amino)hexanoate (2.57 g, 67%). 1H NMR(600 MHz, CDCl3)δ 4.05 (t, 2H), 3.94 (m, 0.5H), 3.62 (m, 0.5H), 2.73 (t, 2H), 2.70-2.65 (m, 0.5H), 2.59-2.51 (m, 0.5H), 2.30 (t, 2H), 1.99 (m, 1H), 1.85-1.80 (m, 1H), 1.80-1.73 (m, 3H), 1.66 -1.50 (m, 7H), 1.39-1.21 (m, 20H), 0.88 (t, 3H). LCMS: 384.4 [M+H]+.

[0234] Step 2): Synthesis of comparative compound 7 [ka] Undecyl 6-((4-hydroxycyclohexyl)amino)hexanoate (383 mg, 1 mmol), 1-octylnonyl 8-bromooctanoate (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added sequentially to a 100 mL reaction bottle. After stirring to dissolve, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added and the mixture was allowed to react at room temperature for 24 hours. 100 mL of dichloromethane was added, and the mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain comparative compound 7 (550 mg, 72%). 1H NMR(600 MHz, CDCl3)δ 4.93-4.83 (m, 1H), 4.07 (t, 2H), 3.99 (m, 0.5H), 3.63-3.49 (m, 0.5H), 2.52 (m, 0.5H), 2.49-2.44 (m, 3H), 2.40 (m, 1.5H), 2.30 (m, 4H), 2.02 (m, 1H), 1.85 (m, 1H), 1.78 (m, 1H), 1.69-1.60 (m, 7H), 1.53 (m, 4H), 1.46-1.39 (m, 4H), 1.35-1.23 (m, 52H), 0.90 (m, 9H). LCMS: 765.2 [M+H]+.

[0235] Bioassay The structures of Lipid:MC3, SM102, and ALC-0315 used in the bioassays of this disclosure are shown below. [ka] Of these, MC3 and ALC-0315 are commercially available and may be prepared according to techniques known in the art.

[0236] Preparation of lipid nanoparticles: Formulation Method 1: The amino lipid compound described in this disclosure was mixed with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 and dissolved in absolute ethanol. A microinjection pump was used to mix the ethanol solution with a sodium acetate solution (50 mM, pH 4.0) in a 1:3 ratio within a microchannel chip to prepare a crude lipid nanoparticle solution. This crude lipid nanoparticle solution was dialyzed in a dialysis box (Fisher, MWCO 20,000) against 1X PBS at 4°C for 6 hours and filtered through a 0.22 μm microporous membrane before use.

[0237] Formulation method 2: The amino lipid compound, DSPC, cholesterol, and PEG2000-DMG were prepared in the same manner as in preparation method 1, using a molar ratio of 50:10:38.5:1.5.

[0238] The lipid nanoparticles prepared by the above-mentioned formulation method 1 or 2 were used in the bioassay described below to evaluate the in vivo delivery performance of luciferase mRNA (Fluc mRNA). The mass ratio of the amino lipid compound to luciferase mRNA in the lipid nanoparticles obtained by formulation method 1 was approximately 10:1, and they were administered subcutaneously. The lipid nanoparticles obtained in formulation example 2 were administered by tail vein injection and intramuscular injection.

[0239] Test Example 1: Evaluation of the in vivo delivery performance of luciferase mRNA by lipid nanoparticles prepared from the amino lipid compounds described in the present disclosure Animal preparation: Six-week-old female BALB / c mice weighing approximately 20 g were selected and housed in an SPF-grade feeding room. Animal experiments were conducted in strict accordance with the guidelines and animal ethics requirements of the National Institute of Health and Medical Care.

[0240] In vivo delivery: Nine mice were randomly assigned to each group and injected with lipid nanoparticles at a dose of 0.5 mg / kg via three routes: subcutaneous, intramuscular, and tail vein injection (three mice per route). Twelve hours later, 200 μL of 10 mg / mL LD-fluorescein potassium salt was injected into each mouse via the tail vein. Ten minutes later, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen) to observe the total fluorescence intensity of each mouse and take photographs. The Fluc mRNA expression intensity of representative amino lipid compounds delivered via the three routes of administration is shown in Tables 2-4. SM102 was used as a control compound.

[0241] [Table 8]

[0242] [Table 9]

[0243] [Table 10]

[0244] [Table 11]

[0245] [Table 12]

[0246] Test Example 2: In vivo delivery of ovalbumin mRNA and evaluation of immune activity of lipid nanoparticles prepared from amino lipid compounds described herein

[0247] Preparation of lipid nanoparticles: Formulation Method: The amino lipid compound of Formula (I) of the present disclosure was mixed with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 50:10:38.5:1.5 and dissolved in absolute ethanol. Ovalbumin mRNA (OVA mRNA) was dissolved in sodium acetate solution (50 mM, pH = 4.0). Using a microinjection pump, the ethanol solution and sodium acetate solution (50 mM, pH = 4.0) were mixed in a 1:3 ratio in a microchannel chip to prepare a crude lipid nanoparticle solution. The crude lipid nanoparticle solution was dialyzed in 1X PBS at 4°C for 6 hours using a dialysis box (Fisher, MWCO 20,000) and filtered through a 0.22 μm microporous filter membrane before use.

[0248] The mass ratio of the amino lipid compound to ovalbumin mRNA (OVA mRNA) in the obtained lipid nanoparticles was approximately 10:1.

[0249] Animal preparation: Six-week-old female BALB / c mice weighing approximately 20 g were selected and housed in an SPF-grade feeding room. Animal experiments were conducted in strict accordance with the guidelines and animal ethics requirements of the National Institute of Health and Medical Care.

[0250] In vivo delivery: Three mice were randomly assigned to each group and received intramuscular injections of lipid nanoparticles at a dose of 0.5 mg / kg in the leg (Day 0). Seven days later, the same dose was administered once more (Day 7). On Day 21, blood was collected from the tail vein for serological analysis. MC3 was used as a control.

[0251] Enzyme-linked immunosorbent assay (ELISA): Flat-bottom 96-well plates (Nunc) were precoated with OVA protein at a concentration of 0.5 μg per well in 50 mM carbonate buffer (pH 9.6), incubated overnight at 4°C, and encapsulated with 5% glycine. Antisera from immunized animals were diluted 102 to 106 in PBS-0.05% Tween (PBS-T), pH 7.4, added to the wells, incubated at room temperature, and then incubated at 37°C for 1 hour. Goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) was labeled at a 1:10,000 dilution in PBS-T with 1% BSA. After addition of HRP substrate, absorbance at 450 nm was measured using an ELISA microplate reader (Bio-Rad).

[0252] The results of the study are shown in Figure 1. Lipid 2 (compound 2) induced IgG antibody titers equivalent to those induced by MC3 and SM102, but the other compounds induced significantly greater IgG antibody titers than the control group. Lipid 7 (compound 7) had protein expression levels equivalent to those of SM102, but was shown to induce a stronger immune response.

[0253] Thus, it was found that the amino lipid compounds provided by the present disclosure have excellent immune activity as well as a strong adjuvant effect.

[0254] Test Example 3: In vivo delivery and immune performance evaluation of influenza mRNA vaccines in lipid nanoparticles prepared from amino lipid compounds described in this disclosure Formulation method: The amino lipid compound of formula (I) of the present disclosure was mixed with DSPC, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 and dissolved in absolute ethanol. Influenza-expressing mRNA was dissolved in sodium acetate solution (50 mM, pH = 4.0). Using a microinjection pump, the ethanol solution and sodium acetate solution (50 mM, pH = 4.0) were mixed in a ratio of 1:3 in a microchannel chip to prepare a crude lipid nanoparticle solution. The crude lipid nanoparticle solution was dialyzed in 1X PBS at 4°C for 6 hours using a dialysis box (Fisher, MWCO 20,000) and filtered through a 0.22 μm microporous filter membrane before use. The mass ratio of the amino lipid compound to influenza mRNA (OVA mRNA) in the obtained lipid nanoparticles was approximately 10:1.

[0255] Animal preparation: Six-week-old female BALB / c mice weighing approximately 20 g were selected and housed in an SPF-grade feeding room. Animal experiments were conducted in strict accordance with the guidelines and animal ethics requirements of the National Institute of Health and Medical Care.

[0256] In vivo delivery: Three mice were randomly assigned to each group and received lipid nanoparticles subcutaneously in the back at a dose of 0.5 mg / kg (Day 0). Seven days later, the same dose was administered once more (Day 7). On Day 21, blood was collected from the tail vein for serological analysis. MC3 was used as a control.

[0257] Enzyme-linked immunosorbent assay (ELISA): The same measurement method as in Test Example 2.

[0258] The test results are shown in Figure 2. The MC3 control group had the lowest IgG antibody titer, Lipid10 (compound 10) had an IgG antibody titer equivalent to that of SM102, and the other compounds showed IgG antibody titers significantly superior to those of the control group. Similarly, it was found that the amino lipid compounds provided by the present disclosure have excellent immune activity as well as a strong adjuvant effect.

[0259] Test Example 4: Evaluation of stability of lipid nanoparticles prepared from the amino lipid compound according to the present disclosure by formulation method 1 Preparation of lipid nanoparticles: LNPs were prepared using formulation method 1 and administered subcutaneously.

[0260] Characterization of lipid nanoparticles: The particle size and PDI of the prepared lipid nanoparticles were measured using a Nano-ZSZEN3600 (Malvern). 40 μL of the LNP solution was taken, particle size measurement was performed, and the solution was circulated three times for 30 s each.

[0261] On the day of preparation (week 0), the pellets were stored at 25°C for 1 week (week 1), 2 weeks (week 2), and 4 weeks (week 4), and the particle size was measured for each. The expression intensity of Fluc mRNA delivered by subcutaneous administration on the day (week 0) and 4 weeks (week 4) was also measured. The results are shown in Table 5.

[0262] Table 5: DLS identification and Fluc-mRNA subcutaneous expression of LNPs prepared from representative amino lipid compounds of the present disclosure. [Table 13]

[0263] As can be seen from Table 5, the test temperature was 25°C. Regarding particle size, representative compounds 5, 6, 7, 9, and SM102 of the present disclosure all formed relatively uniform nanoparticles with particle sizes of about 100 nm. The PDIs for all three were less than 0.1. After 4 weeks of storage, the particle sizes of all three compounds increased. The particle size of SM102 increased by more than 1-fold, from 120 to 245, while the particle sizes of representative compounds 5, 6, 7, and 9 of the present disclosure did not show a significant increase. Regarding PDI, the PDIs of representative compounds 5, 6, 7, and 9 of the present disclosure were less than 0.1 after 4 weeks, and the PDI of SM102 increased from 0.05 on the day (week 0) to 0.28 (week 4). The Fluc expression of representative compounds 5, 6, 7, and 9 of the present disclosure was not significantly reduced after 4 weeks, but the Fluc expression of SM102 was significantly reduced, from 2.1E +07 to 3.6E +06. Therefore, it was found that the amino lipid compounds described in the present disclosure have excellent stability.

[0264] Test Example 5: Evaluation of the activity of lipid nanoparticles prepared from the amino lipid compound according to the present disclosure by formulation method 2 Preparation of lipid nanoparticles: LNPs were produced using formulation method 2 and administered intramuscularly.

[0265] The test method was the same as in Test Example 1, and the test results are shown in Table 6: Table 6: Expression intensity of Fluc mRNA delivered by intramuscular administration of representative amino lipid compounds of the present disclosure. [Table 14] [Table 15]

[0266] Table 6 reveals that, with other structural units remaining the same, when the hydroxyl group on the cycloalkyl structural unit is located meta to the amine substituent, the Fluc mRNA expression intensity is higher than when the hydroxyl group on the cycloalkyl structural unit is located at any other substitution position of the amine substituent. Specifically, for example, in the present disclosure, compound 6 had a Fluc mRNA expression intensity of 1.2E+08, whereas comparative compound 2 (which differs from representative compound 6 of the present disclosure only in that the hydroxyl group on the cycloalkyl structural unit is located adjacent to the amine substituent) had a Fluc mRNA expression intensity of 2.8E+06. Furthermore, comparative compound 6 (which differs from representative compound 6 of the present disclosure only in that the hydroxyl group on the cycloalkyl structural unit is located two carbon atoms away from the amine substituent) had a Fluc mRNA expression intensity of 1.9E+07. Similar conclusions were also observed when comparing representative compound 7 of the present disclosure with comparative compounds 3 and 7, and representative compound 8 of the present disclosure with comparative compounds 4 and 8. Thus, it was found that, when other structural units are identical, lipid nanoparticles containing amino lipid compounds in which the substituent of the cycloalkyl structural unit is in the meta position relative to the amine substituent have the highest activity.

[0267] Test Example 6 Targeting Assay of Aminolipid Compounds mRNA lipid nanoparticle (LNP) formulation method: Lipid, DSPC, CHO-HP, and M-DMG2000 (i.e., PEG2000-DMG) were dissolved in absolute ethanol at a molar ratio of 50:10:38.5:1.5 and mixed. mRNA was dissolved in sodium acetate solution (0.2 M, pH = 5.0). Using a microfluidic device (MPE-L2) and chip (SN.000035), each formulation was prepared at a flow rate of 9 ml / min for the aqueous phase and 3 ml / min for the alcohol phase. The two phases were then mixed by injecting them into the microfluidic chip via the interface. Each drug solution was then placed in a 100KD dialysis bag and immersed in a beaker containing 1 L of dialysis solution. The beaker was wrapped in aluminum foil and dialyzed at room temperature for 1 hour at 100 rpm. The dialysate was then renewed and dialysis continued for another hour.

[0268] Preparation of dialysis solution: 1x PBS + 8% sucrose solution: Two packets of 1x PBS powder were placed in a beaker and dissolved and mixed with 2 L of DEPC water. 160 g of sucrose was then added and mixed well to obtain a 1x PBS + 8% sucrose solution.

[0269] The mass ratio of mRNA:Lipid in the obtained lipid nanoparticles was approximately 1:10.

[0270] Using the above-mentioned method for preparing mRNA lipid nanoparticle (LNP) formulations, mRNA encoding a fluorescent protein was selected, and compound (I-6-II), MC3, and SM102 were selected as lipids, respectively, to prepare mRNA-LNP formulations encoding fluorescent proteins containing compound (I-6-II), MC3, or SM102, respectively.

[0271] Test Method: Each test formulation was administered intramuscularly to mice, with 15 μg of mRNA formulation sample injected per mouse. Six hours after sample injection, the mice were anesthetized with isoflurane inhalation anesthesia and 200 μL of luciferase substrate, luciferin (concentration 10 mg / ml), was injected intraperitoneally. The animals were placed in a supine position, and 10 minutes after the substrate injection, the luciferase signal distribution and expression intensity in the mice were observed using an IVIS in vivo imaging system. After imaging, the animals that received the intramuscular injection were immediately dissected, and liver fluorescence was immediately observed after dissection.

[0272] The specific fluorescence intensity values were as follows:

[0273] MC3-containing fluorescent protein-encoding mRNA-LNP formulation: 1.23E +05 SM102-containing fluorescent protein-encoding mRNA-LNP formulation: 3.23E +06 mRNA-LNP formulation encoding a fluorescent protein containing compound (I-6-II): 2.49E +05 The experimental results are shown in Figure 8. When administered intramuscularly, the liver fluorescence intensity of compound (I-6-II) was significantly lower than that of SM102, and the distribution of mRNA encoding fluorescent proteins in the liver, a vital organ, was relatively low, demonstrating good local targeting effect and effectively reducing the risk of liver toxicity.

[0274] Test Example 7: Adverse Reaction Test of Aminolipid Compounds Following Intramuscular Injection Preparation of mRNA-LNP formulations encoding novel coronavirus S protein: The mRNA lipid nanoparticle (LNP) preparation method of Test Example 6 was employed. Here, mRNA encoding the novel coronavirus S protein was selected as the mRNA, and Compound 6, Compound (I-6-II), ALC-0315, or SM102 was selected as the lipid, respectively, to prepare mRNA-LNP preparations encoding the novel coronavirus S protein containing Compound 6, Compound (I-6-II), ALC-0315, or SM102, respectively.

[0275] Here, the sequence encoding the mRNA of the novel coronavirus S protein was the sequence obtained after substituting all uracils (u) with N1-methylpseudouridine in SEQ ID NO: 1. Note that, according to the WIPO Standard for Nucleotide Sequences or Amino Acid Sequence Listings (ST.26), the t (thymine) in SEQ ID NO: 1 of the sequence listing RNA was actually u (uracil).

[0276] Test Method: SD rats were randomly assigned to four groups, half male and half female, each receiving a high dose of Compound 6 (100 μG mRNA / rat), a high dose of Compound (I-6-II) (100 μG mRNA / rat), a high dose of ALC-0315 (Pfizer vaccine BNT162-b2 cationic lipid) (100 μG mRNA / rat), or a high dose of SM-102 (Moderna vaccine mRNA-1273 cationic lipid) (100 μG mRNA / rat). Three rats per sex were administered intramuscularly once weekly for three consecutive weeks. Clinical observations were performed for swelling and other abnormalities at the injection site.

[0277] The test results are shown in Figure 9. As is clear from Figure 9, compared to the cationic lipid component of the commercially available mRNA vaccine at the same level, compound (I-6-II) showed a significant reduction or decrease in severe swelling at the injection site and the number of moderate lameness episodes, and was considered to have a higher safety profile.

[0278] Test Example 8 Toxicokinetics Preparation of blank liposome formulation: Compound I-6-II (1.9078 g), DSPC (0.3985 g), CHO-HP (0.7575 g), and PEG-DMG (0.1972 g, average molecular weight 2000) were weighed in a molar ratio of 49.5:10:39:1.5, dissolved in absolute ethanol, and the volume was adjusted to 237.5 mL to obtain an alcohol phase. Acetic acid-sodium acetate buffer (0.2 mol / L, pH = 5) was used as the aqueous phase. The aqueous phase:alcohol phase was mixed at a volume ratio of 3:1 using a microfluidic device (MPE-L2). The encapsulated drug solution was subjected to ultrafiltration under the parameters of TMP 0.2 bar and a supply flow rate of 300 ml / min. The dialysate used contained 8 mg / ml sodium chloride, 0.2 mg / ml potassium chloride, 0.2 mg / ml potassium dihydrogen phosphate, 1.15 mg / ml disodium hydrogen phosphate dihydrate, and 80 mg / ml sucrose. After ultrafiltration, the solution was sterile filtered to obtain the I-6-II blank liposome formulation.

[0279] Test Method: In the animal testing, five groups (five animals / sex / group) were set up, and the group divisions and dosages were as follows: negative control group (0 agents / group), blank low-dose liposome group (1 agent / group), and blank high-dose liposome group (4 agents / group). Animals in each group received intramuscular administration once every two weeks on D1 (the day of the first administration), D15 (15 days after administration), and D29 (29 days after administration), for a total of three times.

[0280] In the animal study, five groups (five animals / sex / group) were established, and the group division and dosage were as follows: negative control group (0 μg of compound I-6-II / group), blank liposome low-dose group (300 μg of compound I-6-II / group), and blank liposome high-dose group (1200 μg of compound I-6-II / group). Animals in each group were intramuscularly administered once every two weeks, a total of three times on Days 1, 15, and 29.

[0281] Blood samples were collected from animals in the negative control group before and 4 hours after the first (D1) and last (D29) doses. Blood samples were collected from animals in the blank liposome group before and 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 32 hours, and 48 hours after the first (D1) and last (D29) doses, respectively. The content of Compound I-6-II in cynomolgus monkey plasma was measured, and the exposure of Compound I-6-II to cynomolgus monkeys in each group was examined after administration.

[0282] The main TK parameters of compound I-6-II in animals of the blank liposome low dose group and the high dose group after the first dose (D1) and the last dose (D29) are shown in Table 7 below.

[0283] Table 7: Main TK parameters of compounds I-6-II in animals of blank liposome low and high dose groups [Table 16]

[0284] Test data shows: Animals in the negative control group did not detect I-6-II liposomes in either the first (D1) or last (D29) plasma samples.

[0285] As can be seen from Table 7, compound I-6-II can be rapidly cleared in cynomolgus monkeys, with a plasma drug concentration half-life of 4.68 to 6.45 hours. The Cmax ratio (D29 / D1) of compound I-6-II in each blank liposome group was 0.58 to 0.84 compared with the first administration (D1) of the animals at the final administration (D29). The AUClast ratio (D29 / D1) was 0.68 to 1.02, indicating that compound I-6-II did not accumulate in the body of cynomolgus monkeys after 4 weeks of continuous administration (a total of 3 times).

[0286] As described above, the present disclosure provides amino lipid compounds having a cycloalkyl structural unit and further having another substituent at the meta position of the amine substituent of the cycloalkyl group, which are useful for delivering bioactive agents to cells and have one or more of the following advantages over amino lipid compounds of similar structure known in the prior art: good delivery capacity, stability, safety, useful for delivering bioactive agents (e.g., nucleic acids) to cells, and also enhances protein expression levels, more effectively generates an immune response, has excellent targeting properties, and can be stored, transported, and used under ambient temperature conditions.

[0287] List of Abbreviations DIPEA N,N-Diisopropylethylamine DNA deoxyribonucleic acid RNA ribonucleic acid DOPE dioleylphosphatidylethanolamine DSPC distearoylphosphatidylcholine PEG2000-DMG 1,2-Dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 kD kilodaltons PBS phosphate buffer

[0288] It is clear to those skilled in the art that the present disclosure is not limited to the above examples, and can be embodied in other specific forms without departing from the essential characteristics of the present disclosure. Therefore, these embodiments are expected to be illustrative and non-limiting in all respects, and reference should be made to the scope of the claims that are not the above embodiments, and therefore, the scope of the claims and all equivalent modifications thereof are included therein.

Claims

1. A compound represented by the following structural formula I, or a pharmaceutically acceptable salt or stereoisomer thereof: 【Chemical 1】 [In the formula: L 1 and L 2 are the same or different, and each independently represents C 1 -C 12 Alkylene, C 2 -C 12 Alkenylene or C 2- C 12 alkynylene; preferably, L 1 and L 2 are the same or different, and each independently represents C 3 -C 10 Alkylene, C 3 -C 10 Alkenylene or C 3- C 10 alkynylene; more preferably, L 1 and L 2 are the same or different, and each independently represents C 3 -C 10 alkylene; most preferably, L 1 and L 2 are the same or different, and each independently represents C 5 -C 8 alkylene; G 1 or G 2 are the same or different and are each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S-, and -SC(=O)-; preferably, G 1 or G 2 are the same or different and are each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, and -O-; most preferably, G 1 or G 2 are the same or different and are each independently selected from —O—(C═O)—, —(C═O)—O—; R 1 and R 2 are the same or different, and each independently represents a C bonded via any carbon atom. 5 -C 27 Alkyl, C containing one or more double bonds 5 -C 27 alkenyl; preferably, R 1 and R 2 are the same or different, and each independently represents a C bonded via any carbon atom. 8 -C 20 Alkyl or C containing one or more double bonds 8 -C 20 alkenyl; more preferably, R 1 and R 2 are the same or different, and each independently represents a C bonded via any carbon atom. 9 -C 17 Alkyl or C containing one or two double bonds 9 -C 18 alkenyl; most preferably, R 1 and R 2 are the same or different and are each independently 【Chemistry 2】 Selected from: R 3 is halogen, hydroxyl, cyano, C 1 -C 6 Alkyl, Nitro, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylcarbonyloxy, C 1 -C 6 Alkoxycarbonyl, C 1 -C 6 Alkylaminocarbonyl, C 1 -C 6 alkylcarbonylamino; preferably, R 3 is halogen, hydroxyl, cyano, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylcarbonyloxy, C 1 -C 6 Alkoxycarbonyl, C 1 -C 6 Alkylaminocarbonyl, C 1 -C 6 alkylcarbonylamino; more preferably, R 3 is halogen, hydroxyl, cyano, C 1 -C 4 Alkoxy, C 1 -C 4 Alkylcarbonyloxy, C 1 -C 4 Alkoxycarbonyl, C 1 -C 4 Alkylaminocarbonyl, C 1 -C 4 alkylcarbonylamino; most preferably R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido; n is selected from 1, 2, and 3.

2. 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 and L 2 are the same or different, and each independently represents C 3 -C 10 Alkylene, C 3 -C 10 Alkenylene or C 3- C 10 alkynylene; G 1 or G 2 are the same or different and each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, and -O-; R 1 and R 2 are the same or different, and each independently represents a C bonded via any carbon atom. 8 -C 20 Alkyl or C containing one or more double bonds 8 -C 20 alkenyl; R 3 is halogen, hydroxyl, cyano, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylcarbonyloxy, C 1 -C 6 Alkoxycarbonyl, C 1 -C 6 Alkylaminocarbonyl, C 1 -C 6 alkylcarbonylamino; n is selected from 1, 2, and 3; 2. The compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof.

3. 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 and L 2 are the same or different, and each independently represents C 3 -C 10 alkylene; G 1 or G 2 are the same or different and are each independently selected from —O—(C═O)—, —(C═O)—O—; R 1 and R 2 are the same or different, and each independently represents a C bonded via any carbon atom. 9 -C 17 Alkyl or C containing one or two double bonds 9 -C 18 alkenyl; R 3 is halogen, hydroxyl, cyano, C 1 -C 4 Alkoxy, C 1 -C 4 Alkylcarbonyloxy, C 1 -C 4 Alkoxycarbonyl, C 1 -C 4 Alkylaminocarbonyl, C 1 -C 4 alkylcarbonylamino; n is selected from 1, 2, and 3; 2. The compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof.

4. 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 and L 2 are the same or different, and each independently represents C 5 -C 8 alkylene; G 1 or G 2 are the same or different and are each independently selected from —O—(C═O)—, —(C═O)—O—; R 1 and R 2 are the same or different and are each independently 【Chemistry 3】 Selected from: R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido; n is selected from 1, 2, and 3; 2. The compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof.

5. 2. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, selected from the following compounds: 【Table 1】 【Table 2】 【Table 3】

6. A compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: 【Chemistry 4】 。

7. A compound having the following structure or a pharmaceutically acceptable salt thereof: 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 。

8. A compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: 【Chemistry 8】 。

9. Lipid nanoparticles containing the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt or stereoisomer thereof.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

11. Use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt or stereoisomer thereof, a lipid nanoparticle according to claim 9, or a pharmaceutical composition according to claim 10, in the manufacture of a medicament for gene therapy, gene vaccination, antisense therapy, or therapy using interfering RNA.

12. The use according to claim 11, wherein the gene therapy is applied to the treatment of cancer and genetic diseases.

13. 13. The use of claim 12, wherein the cancer is one or more selected from lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, or prostate cancer; and the genetic disease is one or more selected from hemophilia, thalassemia, and Gaucher disease.

14. The use according to claim 11, wherein said genetic vaccination is for the treatment of cancer, allergies, toxicity and pathogen infections.

15. The use according to claim 14, wherein the pathogen is one or more selected from a virus, a bacterium, or a fungus.

16. Use of the lipid nanoparticles of claim 9, or the compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt or stereoisomer thereof, in the manufacture of a pharmaceutical for the transfer of nucleic acids, wherein the nucleic acid is RNA, DNA, or an antisense oligonucleotide; preferably, the RNA is selected from messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), and small nuclear RNA (snRNA); and preferably, the DNA is a plasmid.

17. A compound represented by the following structural formula II, or a pharmaceutically acceptable salt or stereoisomer thereof: 【Chemistry 9】 [In the formula: L 1 is C 1 -C 12 Alkylene, C 2 -C 12 Alkenylene or C 2- C 12 alkynylene; preferably, L 1 is C 3 -C 10 Alkylene, C 3 -C 10 Alkenylene or C 3- C 10 alkynylene; more preferably, L 1 is C 3 -C 10 alkylene; most preferably, L 1 is C 5 -C 8 alkylene; G 1 is selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S-, -SC(=O)-; preferably, G 1 is selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-; most preferably, G 1 is selected from -O-(C=O)-, -(C=O)-O-; R 1 is bonded through any carbon C 5 -C 27 Alkyl, C containing one or more double bonds 5 -C 27 alkenyl; preferably, R 1 is bonded through any carbon C 8 -C 20 Alkyl, C containing one or more double bonds 8 -C 20 alkenyl; more preferably, R 1 is bonded through any carbon C 9 -C 17 Alkyl, C containing one or two double bonds 9 -C 18 alkenyl; most preferably, R 1 teeth, 【Chemistry 10】 Selected from; R 3 is halogen, hydroxyl, cyano, C 1 -C 6 Alkyl, Nitro, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylcarbonyloxy, C 1 -C 6 Alkoxycarbonyl, C 1 -C 6 Alkylaminocarbonyl, C 1 -C 6 alkylcarbonylamino; preferably, R 3 is halogen, hydroxyl, cyano, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylcarbonyloxy, C 1 -C 6 Alkoxycarbonyl, C 1 -C 6 Alkylaminocarbonyl, C 1 -C 6 alkylcarbonylamino; more preferably, R 3 is halogen, hydroxyl, cyano, C 1 -C 4 Alkoxy, C 1 -C 4 Alkylcarbonyloxy, C 1 -C 4 Alkoxycarbonyl, C 1 -C 4 Alkylaminocarbonyl, C 1 -C 4 alkylcarbonylamino; most preferably R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido; n is selected from 1, 2, and 3.

18. 18. The compound of claim 17, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 is C 3 -C 10 Alkylene, C 3 -C 10 Alkenylene or C 3- C 10 alkynylene; G 1 is selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-; R 1 is bonded through any carbon C 8 -C 20 Alkyl, C containing one or more double bonds 8 -C 20 alkenyl; R 3 is halogen, hydroxyl, cyano, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylcarbonyloxy, C 1 -C 6 Alkoxycarbonyl, C 1 -C 6 Alkylaminocarbonyl, C 1 -C 6 alkylcarbonylamino; n is selected from 1, 2, and 3; 18. The compound of claim 17, or a pharmaceutically acceptable salt or stereoisomer thereof.

19. 18. The compound of claim 17, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 is C 3 -C 10 alkylene; G 1 is selected from -O-(C=O)-, -(C=O)-O-; R 1 is bonded through any carbon C 9 -C 17 Alkyl, C containing one or two double bonds 9 -C 18 alkenyl; R 3 is halogen, hydroxyl, cyano, C 1 -C 4 Alkoxy, C 1 -C 4 Alkylcarbonyloxy, C 1 -C 4 Alkoxycarbonyl, C 1 -C 4 Alkylaminocarbonyl, C 1 -C 4 alkylcarbonylamino; n is selected from 1, 2, and 3; 18. The compound of claim 17, or a pharmaceutically acceptable salt or stereoisomer thereof.

20. 18. The compound of claim 17, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 is C 5 -C 8 alkylene; G 1 is selected from -O-(C=O)-, -(C=O)-O-; R 1 teeth 【Chemistry 11】 Selected from; R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido; n is selected from 1, 2, and 3; 18. The compound of claim 17, or a pharmaceutically acceptable salt or stereoisomer thereof.

21. 18. The compound of claim 17, wherein the compound is selected from the following structure: or a pharmaceutically acceptable salt or stereoisomer thereof. 【Table 4】 【Table 5】 【Table 6】

22. 18. The compound of claim 17, selected from the following compounds, or a pharmaceutically acceptable salt thereof: 【Chemistry 12】 【Chemistry 13】 。

23. Use of a compound according to any one of claims 17 to 22 for the preparation of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt or stereoisomer thereof.

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