Amino lipid compound as well as preparation method and application thereof

Abstract

The present application relates to amino lipid compounds having the following structural formula (I) or pharmaceutically acceptable salts or stereoisomers thereof, and their use as components of lipid nanoparticle formulations for the delivery of therapeutic agents. The present application also relates to compositions, in particular lipid nanoparticles, comprising said amino lipid compounds, and uses thereof. # imgabs0 #

Description

Amino lipid compound, preparation method and application thereof Technical Field

[0001] The present invention relates to the pharmaceutical field, and in particular to an amino lipid compound, a preparation method and application thereof. Background Art

[0002] Gene therapy involves artificially delivering genes containing specific genetic information to target cells. The expressed target protein can regulate, treat, or even cure conditions caused by congenital or acquired genetic defects. Alternatively, the gene sequence can interfere with or regulate the expression of related genes, achieving clinical therapeutic effects. Nucleic acids and cell membranes both have negative charges, making naked nucleic acids difficult to directly introduce into cells and easily degraded by nucleases in the cytoplasm. Therefore, gene delivery requires the use of external forces or carriers.

[0003] Gene vectors are generally categorized as viral and non-viral. While viral vectors offer high transfection efficiencies both in vivo and in vitro, they also have numerous drawbacks, including high toxicity, strong immune responses, small gene storage capacity, poor targeting, and complex preparation processes. Non-viral vectors, on the other hand, are attracting increasing research and development investment due to their ease of preparation, transportation, and storage, as well as their safety, efficacy, and lack of immunogenicity.

[0004] Lipid nanoparticles (LNPs) are currently the mainstream non-viral carriers, commonly used in vaccines due to their easy uptake by antigen-presenting cells. Numerous compounds for use in lipid nanoparticles have been reported in the prior art. However, in actual use, these compounds have been found to suffer from low delivery efficiency and poor drug stability when used for vaccine delivery. Furthermore, they suffer from issues such as a long elimination half-life, high toxicity, and low safety, making them unsuitable for clinical application and failing to meet the demands of modern vaccine formulations.

[0005] For example, the commercially available cationic liposome compound MC3, when actually used for vaccine delivery, has insufficient protein expression levels and causes the body to produce low levels of antibodies; in addition, the document "Pharmacokinetics of Patisiran, the First Approved RNA Interference Therapy in Patients With Hereditary Transthyretin-Mediated Amyloidosis" by Xiaoping Zhang et al., The Journal of Clinical Pharmacology 2020, 60(5)573–585, reported that in a Phase II clinical study of Patisiran, the elimination phase half-life of cationic liposome MC3 was as long as 14.6 to 28.7 days.

[0006] Therefore, there is an urgent need to develop substances that have strong delivery capabilities, good stability, moderate elimination phase half-life and / or high safety (ie, low incidence of adverse reactions) that can be used for intracellular delivery of therapeutic agents.

[0007] Summary of the Invention

[0008] Through extensive research, the inventors of the present disclosure unexpectedly discovered that amino lipid compounds having a cycloalkyl structural unit and an additional substituent at the meta position of the amine substituent of the cycloalkyl group achieve the following functions: good delivery ability, good stability, and high safety, and can be used to deliver bioactive agents (such as nucleic acids) into cells to increase their protein expression levels.

[0009] The present disclosure therefore provides an amino lipid compound having a cycloalkyl structural unit and further having an additional substituent at the meta position of the amine substituent of the cycloalkyl group. The amino lipid compound of the present disclosure remains stable during in vivo circulation, can be rapidly degraded in endosomes / lysosomes, and has significantly enhanced delivery efficiency.

[0010] The cycloalkyl structural unit in the amino lipid compound disclosed herein and the additional substituent at the meta position of the amine substituent on the cycloalkyl group enable the compound or lipid nanoparticles containing the compound to have good biological activity, high protein expression level, significant immune activity, and good stability when used as a vaccine carrier. The compound can be stored, transported and used at room temperature with a low incidence of adverse reactions.

[0011] The present disclosure also provides a method for preparing the amino lipid compound or a pharmaceutically acceptable salt or stereoisomer thereof, which has readily available raw materials, mild reaction conditions, good reaction selectivity, high yield, low instrument and equipment requirements, and simple operation.

[0012] The present disclosure also provides compositions, such as lipid nanoparticles, comprising the amino lipid compound or a pharmaceutically acceptable salt or stereoisomer thereof.

[0013] In one aspect, the present disclosure provides a compound represented by the following structural formula I or a pharmaceutically acceptable salt or stereoisomer thereof:

[0014] in:

[0015] L 1 and L 2 The same or different, each independently selected from C1-C 12 Alkylene, C2-C 12 Alkenylene or C 2- C 12 Alkyne; preferably, L 1 and L 2 The same or different, each independently selected from C3-C 10 Alkylene, C3-C 10 Alkenylene or C 3- C 10 Alkyne; further preferably, L 1 and L 2 The same or different, each independently selected from C3-C 10 Alkylene; most preferably, L 1 and L 2 The same or different, each independently selected from C5-C8 alkylene;

[0016] G 1 or G 2 The same or different, each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S-, -SC(=O)-; preferably, G 1 or G 2 The same or different, each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-; most preferably, G 1 or G 2 the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-;

[0017] R 1 and R 2 The same or different, each independently selected from C5-C 27 Alkyl, C5-C 27 Alkenyl; preferably, R 1 and R 2The same or different, each independently selected from C8-C 20 Alkyl, C8-C 20 Alkenyl; further preferably, R 1 and R 2 The same or different, each independently selected from C9-C 17 Alkyl, C9-C 18 Alkenyl; most preferably, R 1 and R 2 The same or different, each independently selected from

[0018] R 3 is selected from halogen, hydroxy, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; preferably, R 3 is selected from halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; further preferably, R 3 is selected from halogen, hydroxy, 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;

[0019] n is selected from 1, 2, and 3.

[0020] Furthermore, the present disclosure provides a compound represented by the above structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0021] L 1 and L 2 The same or different, each independently selected from C3-C 10 Alkylene, C3-C 10 Alkenylene or C 3- C 10 Alkynylidene;

[0022] G 1 or G 2 the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-;

[0023] R 1 and R 2The same or different, each independently selected from C8-C 20 Alkyl, C8-C 20 alkenyl;

[0024] R 3 is selected from halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino;

[0025] n is selected from 1, 2, and 3.

[0026] Furthermore, the present disclosure provides a compound represented by the above structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0027] L 1 and L 2 The same or different, each independently selected from C3-C 10 alkylene;

[0028] G 1 or G 2 the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-;

[0029] R 1 and R 2 The same or different, each independently selected from C9-C 17 Alkyl, C9-C 18 alkenyl;

[0030] R 3 is selected from halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 alkylcarbonylamino;

[0031] n is selected from 1, 2, and 3.

[0032] Furthermore, the present disclosure provides a compound represented by the above structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0033] L 1 and L 2 The same or different, each independently selected from C5-C8 alkylene;

[0034] G 1 or G 2 the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-;

[0035] R1 and R 2 The same or different, each independently selected from

[0036] R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido;

[0037] n is selected from 1, 2, and 3.

[0038] Further, the present disclosure provides a compound represented by the above structural formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is selected from:

[0039] In another aspect, the present disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[0040] In another aspect, the present disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[0041] Furthermore, the present disclosure provides a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof:

[0042] In another aspect, the present disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[0043] Furthermore, the present disclosure provides a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof:

[0044] In another aspect, the present disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[0045] Furthermore, the present disclosure provides a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof:

[0046] In another aspect, the present disclosure provides a compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

[0047] Furthermore, the present disclosure provides a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof:

[0048] In one aspect, the present disclosure provides lipid nanoparticles (LNPs), which contain a compound represented by structural formula (I) of the present disclosure according to any of the above embodiments, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0049] In some embodiments, lipid nanoparticles refer to particles having a nanometer scale (eg, 1 nm to 1000 nm) that include one or more lipids.

[0050] In some embodiments, the average diameter of the lipid nanoparticles is 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, 40nm~100nm, 50nm~800nm, 50nm~600nm, 50nm~500nm, 50nm~500nm, 50nm~400nm, 50nm~500nm, 50nm~400nm, 50nm~300nm, 50nm~200nm, 50nm~100nm, 60nm~800nm, 60nm~600nm, 60nm~500nm, 60nm~400nm, 60nm~300nm, 60nm~200nm or 60nm~100nm. In some optional specific examples, the average diameter of the lipid nanoparticles is 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. Herein, the average diameter of lipid nanoparticles can be expressed as the z-average value determined by dynamic light scattering.

[0051] In some embodiments, the lipid nanoparticles further contain one or more of the following: helper lipids, structural lipids, and polymer-lipids (eg, polyethylene glycol-lipids).

[0052] In some embodiments, the lipid nanoparticles contain (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.

[0053] In some embodiments, the helper lipids of the lipid nanoparticles include phospholipids. Phospholipids are typically semi-synthetic, but can also be naturally derived or chemically modified. In an alternative specific example, the helper lipids of the lipid nanoparticles are phospholipids. In some embodiments, the phospholipids of the lipid nanoparticles include one or more of the following: DSPC (distearoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoylphosphatidylcholine), DOPS (dioleoylphosphatidylserine), DSPG (1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DPPG (dipalmitoylphosphatidylglycerol), DPPC (dipalmitoylphosphatidylcholine), DGTS (1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)homoserine), and lysophospholipids. In some embodiments, the helper lipids of the lipid nanoparticles are one or more selected from the following: DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid of the lipid nanoparticle is DSPC and / or DOPE.

[0054] In some embodiments, the structural lipids of the lipid nanoparticles include sterols. In an alternative specific example, the structural lipids of the lipid nanoparticles are sterols. In some embodiments, the sterols of the lipid nanoparticles include one or more of the following: 20α-hydroxycholesterol, cholesterol, cholesterol esters, steroid hormones, steroid vitamins, bile acids, cholesterol, ergosterol, β-sitosterol, and oxidized cholesterol derivatives. In some embodiments, the structural lipids of the lipid nanoparticles include 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 an alternative specific example, the structural lipid of the lipid nanoparticles is high-purity cholesterol, particularly injection-grade high-purity cholesterol, such as CHO-HP (produced by AVT). In other embodiments, the structural lipid is 20α-hydroxycholesterol.

[0055] In some embodiments, the polymer-lipid of the lipid nanoparticle refers to a conjugate comprising a polymer and a lipid coupled to the polymer. The polymer-lipid (e.g., polyethylene glycol-lipid) in the lipid nanoparticle can improve the stability of the lipid nanoparticle in vivo.

[0056] In some embodiments, the lipids of the polymer-lipid used to form lipid nanoparticles include one or more of the following: 1,2-dimyristoyl-sn-glycerol (DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipids, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG) and 1,2-dipalmitoyl-rac-glycero (DPG).

[0057] In some embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticles includes one or both of the following: a hydrophilic polymer and an amphiphilic polymer.

[0058] In some embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticles is a hydrophilic polymer. In other embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticles is an amphoteric polymer.

[0059] In some embodiments, the hydrophilic polymer comprises one or more of the following: polyethylene glycol (PEG), polyoxazolines (POX), polyglycerols (PGs), polyhydroxypropyl methacrylate (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polyvinylpyrrolidone (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloyl morpholine) (PAcM), polyaminoacids, glycosaminoglycans (GAGs), heparin, hyaluronic acid (HA), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond acid (HA), polysialic acid (PSA), elastin-like polypeptide (ELPs), serum albumin and CD47. In an alternative embodiment, the hydrophilic polymer includes polyethylene glycol.

[0060] Correspondingly, polymer-lipids include one or more of the following: polyethylene glycol-lipid (PEG-lipid), polyoxazoline-lipid, polyglycerol-lipid, polyhydroxypropyl methacrylate-lipid, polymethacrylate-2-hydroxyethyl ester-lipid, poly N-(2-hydroxypropyl) methacrylamide-lipid, polyvinyl pyrrolidone-lipid, poly N,N-dimethylacrylamide-lipid, poly N acryloylmorpholine-lipid, glycosaminoglycan-lipid, heparin-lipid, hyaluronic acid-lipid, polysialic acid-lipid, elastin-lipid, serum albumin-lipid and CD47-lipid. It should be noted that "PEG-lipid" is a conjugate of polyethylene glycol and lipid, "polyoxazoline-lipid" refers to a conjugate formed by coupling polyoxazoline and lipid, and "polyglycerol-lipid" refers to a conjugate formed by coupling polyglycerol and lipid. The same applies to other polymer-lipids.

[0061] In some embodiments, the polymer-lipid includes a PEG-lipid. In an optional specific example, the polymer-lipid is a PEG-lipid. In some embodiments, the PEG-lipid includes one or more of the following: PEG-myristoyl diglycerol (PEG-DMG), PEG-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-diacylglycerol (PEG-DAG), PEG-dialkyloxypropyl (PEG-DAA), PEG-phospholipids, 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 the following: PEG-DMG, PEG-DSG and PEG-DPG. PEG-DMG is a polyethylene glycol derivative of 1,2-dimyristoylglycerol. In some embodiments, the average molecular weight of the PEG in the PEG-lipid is about 2000 to 5000. In an alternative embodiment, the average molecular weight of PEG in the PEG-lipid is about 2000. In some embodiments, the PEG-lipid is PEG2000-DMG.

[0062] In some embodiments, the amphoteric polymer comprises one or more of the following: poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-based polymers, and phosphorylcholine polymers. In some embodiments, the amphoteric polymer includes one or more of the following: poly(carboxybetaine acrylamide, pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylpyridine.

[0063] Correspondingly, the polymer-lipid comprises one or more of the following: polyhydroxybetaine-lipid, polysulfobetaine-lipid, phosphobetaine-based polymer-lipid and phosphorylcholine polymer-lipid. In some embodiments, the polymer-lipid comprises one or more of the following: poly(carboxybetaine acrylamide)-lipid, poly(carboxybetaine methacrylate)-lipid, poly(sulfobetaine methacrylate)-lipid, poly(methacryloyloxyethylphosphorylcholine)-lipid, poly(vinylpyridylpropanesulfonate)-lipid, polyvinylimidazolylbetaine-lipid, polyvinylimidazolylsulfobetaine-lipid, polyvinylpyridylsulfobetaine-lipid.

[0064] In addition, in some embodiments, the polymers used in nanoparticles in "The Importance of Poly(ethylene glycol) Alternatives for Overcoming PEG Immunogenicity in Drug Delivery and Bioconjugation." Polymers vol. 12, 2 298 by Hoang Thi, Thai Thanh et al. are also incorporated herein.

[0065] 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. 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 lipid nanoparticles comprise the compound represented by structural formula (I) of the present disclosure, or a pharmaceutically acceptable salt or stereoisomer thereof, in the following amounts (in molar percentage): about 25% to 75%, for example, about 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%-75%, 45%-46.3%, 46.3%-48%, 48%-49.5%, 49.5%-50%, 50%-55% or 60%-65%.

[0066] In some embodiments, the lipid nanoparticles comprise: (1) an auxiliary 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. 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 auxiliary lipid, the structural lipid, and the PEG-lipid, the lipid nanoparticles comprise the following amounts (in molar percentage): Lipid: 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%.

[0067] 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. 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 lipid nanoparticles comprise the following amount (in mole percentage) of the structural lipid: 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%, 39.5%-40.5%, 40.5%-41.5%, 41.5%-45%, 45%-46.5%, 46.5%-50%, 15.5%-18.5%, 18.5%-22.5%, 22.5%-23.5%, 23.5%-28.5%, 28.5%-33.5%, 33.5%-35%, 36.5%-38%, 38%-38.5%, 38.5%-39%, 39%-39.5%, 41.5%-42.5%, 42.5%-42.7%, 42.7%-43%, 43%-43.5%, 43.5%-45% or 46.5%-48.5%.

[0068] 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, wherein the lipid nanoparticles comprise the following amount (molar percentage) of the PEG-lipid, 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: about 0.5% to 5%, for example, 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%.

[0069] In some embodiments as described above, the lipid nanoparticles comprise: (1) an auxiliary 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: auxiliary 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.

[0070] 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, and 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 approximately (28-60): (5-42): (15.5-53.5): (0.5-3.5).

[0071] 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: auxiliary lipid: structural lipid: PEG-lipid is approximately 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.

[0072] 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 approximately (35-60): (5-40): (18.5-53.5): (1.5-3).

[0073] 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: 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, and 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, 40:1 5:43.5:1.5, 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.

[0074] In some embodiments, the lipid nanoparticles comprise: (1) a helper lipid, (2) a structural lipid, (3) a PEG-lipid, (4) a compound represented by structural 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).

[0075] In some embodiments, the lipid nanoparticles comprise: (1) a helper lipid, (2) a structural lipid, (3) a PEG-lipid, (4) a compound represented by structural 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.

[0076] In another aspect, the present disclosure provides a pharmaceutical composition comprising 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 long non-polar residues, the resulting compounds all have hydrophobic properties and, due to the amino group, also have hydrophilic properties. This amphiphilic property can be used to form lipid nanoparticles, such as lipid bilayers, micelles, liposomes, and the like.

[0077] In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier, diluent or excipient, and one of the following: a compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, and the lipid nanoparticles of the present disclosure.

[0078] In some embodiments, the pharmaceutical composition is a lipid nanoparticle.

[0079] The compound represented by structural formula (I) of the present disclosure or its pharmaceutically acceptable salt or stereoisomer can make the lipid nanoparticle containing it have excellent performance of encapsulating biologically active ingredients. The lipid nanoparticles of the present disclosure containing biologically active ingredients can be used to deliver any one or more of a variety of therapeutic agents to cells, tissues or organs. The present disclosure provides a use of the lipid nanoparticles of the present disclosure in delivering biologically active ingredients (such as DNA, RNA, etc.) to cells, tissues or organs. In addition, a lipid nanoparticle or pharmaceutical composition containing biologically active ingredients (such as DNA, RNA, etc.) is provided for producing polypeptides and / or proteins of interest, for use in preparing drugs, for use in preparing drugs for nucleic acid transfer, and for use in preventing and / or treating diseases.

[0080] The present disclosure provides a use of a compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, or lipid nanoparticles in delivering biologically active ingredients (such as DNA, RNA, etc.) to cells, tissues or organs.

[0081] In some embodiments, the use comprises the step of contacting cells, tissues or organs with lipid nanoparticles comprising a biologically active component, such as a nucleic acid (eg, mRNA) encoding a polypeptide and / or protein of interest.

[0082] In some embodiments, the tissue or organ is selected from the group consisting of spleen, liver, kidney, lung, femur, eye tissue, vascular endothelium in blood vessels, lymphatic, and tumor tissue.

[0083] In some embodiments, the cell is a mammalian cell. As used herein, a mammalian cell can be a cell of any mammal. In some embodiments, mammals include one or more of the following: humans, mice, rats, pigs, cats, dogs, horses, goats, cows, and monkeys. In some embodiments, the mammal is a human.

[0084] In some embodiments, the mammalian cell is in a mammal.

[0085] The present disclosure provides a method for producing a polypeptide and / or protein of interest, comprising the step of contacting a cell, tissue or organ with a lipid nanoparticle or pharmaceutical composition of the present disclosure comprising a biologically active component, such as a nucleic acid (e.g., mRNA) encoding the polypeptide and / or protein of interest.

[0086] In some embodiments, after the cell is contacted with the lipid nanoparticle or pharmaceutical composition, the nucleic acid can be taken up into the cell, and the nucleic acid taken up into the cell is utilized by the cell to produce the polypeptide and / or protein of interest.

[0087] In some embodiments, the description of the use of the compound represented by structural formula (I) disclosed above or its pharmaceutically acceptable salt or stereoisomer, or lipid nanoparticles in delivering biologically active ingredients (such as DNA, RNA, etc.) to cells, tissues or organs is not repeated here.

[0088] In some embodiments, the method of producing a polypeptide and / or protein of interest is performed in vivo or partially in vivo.

[0089] In yet another aspect, the present disclosure further provides a 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 preparation of a drug.

[0090] In some embodiments, the medicament is for the treatment and / or prevention of a disease.

[0091] In some embodiments, the disease is selected from the group consisting of rare diseases, infectious diseases, cancer, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases.

[0092] In some embodiments, the cancer is selected from one or more of 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 selected from one or more of hemophilia, thalassemia, and Gaucher's disease.

[0093] In some embodiments, the rare disease comprises one or more of the following: Brittle Bone Disease, Wilson Disease, Spinal Muscular Atrophy (SMA), Huntington'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 (GSDIII), Crigler-Najjar Syndrome, Ornithine Transcarbamylase Deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine transcarbamylase deficiency (OTD), ornithine The following rare diseases are included in this article: 1) OTCD, 2) propionic acidemia (PA), 2) phenylketonuria (PKU), 3) hemophilia A, 4) hemophilia B, β-thalassemia, 5) Lafora disease, 6) Dravet syndrome (DS), 7) Alexander disease, 8) Leber's congenital amaurosis (LCA), 9) myelodysplastic syndrome (MDS), and 10) homocystinuria due to CBS deficiency. In addition, the rare diseases listed at www.orpha.net / consor / cgi-bin / Disease_Search_List.php and rarediseases.info.nih.gov / diseases are also included in this article.

[0094] In some embodiments, the medicament is used, for example, in gene therapy, protein replacement therapy, antisense therapy, or therapy by interfering RNA, or genetic vaccination.

[0095] In some embodiments, the drug is a nucleic acid drug. In some embodiments, the nucleic acid in the nucleic acid drug includes at least one of the following: RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA) and cas9mRNA.

[0096] 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.

[0097] In some embodiments, the drug is a genetic vaccine. In some embodiments, the drug is a nucleic acid vaccine. In some embodiments, the drug is an mRNA vaccine.

[0098] In some embodiments, the genetic vaccination is used to treat and / or prevent one or more of cancer, allergy, toxicity, and pathogen infection.

[0099] In some embodiments, the pathogen is selected from one or more of a virus, a bacterium, or a fungus.

[0100] In some embodiments, the drug is a gene therapy agent. In some embodiments, the drug is used to produce a protein associated with a genetic disease.

[0101] In some embodiments, the medicament is for the production of antibodies, such as scFV or nanobodies.

[0102] In another aspect, the present disclosure further provides a 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 preparation of a drug for nucleic acid transfer.

[0103] In some embodiments, the nucleic acid in the drug for nucleic acid transfer is selected from RNA, antisense oligonucleotide, and DNA.

[0104] In some embodiments, the RNA is selected from messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single-stranded guide RNA (sgRNA), cas9 mRNA, or a mixture thereof.

[0105] In some embodiments, the DNA is a plasmid.

[0106] In addition, the present disclosure also provides a drug comprising the compound represented by structural formula (I) of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof or lipid nanoparticles.

[0107] In addition, the present disclosure also provides a method for preventing and / or treating a disease, comprising administering to a subject in need thereof a lipid nanoparticle, a pharmaceutical composition, or a drug 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 encoding a polypeptide and / or protein of interest (e.g., mRNA)).

[0108] In some embodiments, the diseases and conditions are described in the description of the use of the compound represented by structural formula (I) or its pharmaceutically acceptable salt or stereoisomer, lipid nanoparticle or pharmaceutical composition in the preparation of a medicament disclosed above.

[0109] In another aspect, the present disclosure provides the use of the pharmaceutical composition, in particular the lipid nanoparticles, in the preparation of a medicament for use in, for example, gene therapy, genetic vaccination, antisense therapy, or treatment by interfering RNA. Preferably, the gene therapy can be used to treat cancer and genetic diseases. The cancer is preferably selected from one or more of lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, or prostate cancer; the genetic disease is preferably selected from one or more of hemophilia, thalassemia, and Gaucher's disease. The gene vaccine is preferably used for the treatment of cancer, allergies, toxicity, and pathogen infection. The pathogen is preferably selected from one or more of viruses, bacteria, or fungi.

[0110] In another aspect, the present disclosure relates to the use of the compound or a pharmaceutically acceptable salt or stereoisomer thereof, and lipid nanoparticles containing the compound or a pharmaceutically acceptable salt or stereoisomer thereof, in the preparation of a medicament for nucleic acid delivery. Preferably, the nucleic acid is selected from RNA, DNA, and antisense oligonucleotides; preferably, the RNA is selected from messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA), and small nuclear RNA (snRNA); preferably, the DNA is a plasmid.

[0111] In another aspect, the present disclosure also provides a method for preparing the compound of formula (I) described above. The general procedure of the method is as follows:

[0112] General procedure 1:

[0113] in,

[0114] n, L1, L2, G1, G2, R1, R2, and R3 have the same meanings as those defined above for the compounds of formula (I) of the present disclosure;

[0115] X is selected from halogen; preferably, X is selected from bromine;

[0116] Specifically, the compound of formula (I) of the present disclosure is obtained by subjecting the intermediate compound (II) to a substitution reaction with the intermediate compound (III) in an organic solvent at room temperature in the presence or absence of an acid-binding agent and iodide.

[0117] The organic solvent is selected from nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE); 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; and the iodide is such as potassium iodide.

[0118] General Procedure 2

[0119] in,

[0120] n, L1, L2, G1, G2, R1, R2, and R3 have the same meanings as those defined above for the compounds of formula (I) of the present disclosure;

[0121] Y is (=O);

[0122] Specifically, the compound of formula (I) of the present disclosure is obtained by subjecting the intermediate compound (II) to a reduction reaction with the intermediate compound (V) in an organic solvent in the presence of a reducing agent at room temperature.

[0123] The organic solvent is selected from nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE); and the reducing agent is selected from, for example, sodium triacetoxyborohydride.

[0124] Further, when the groups L1 and L2 in the compound of formula (I) of the present disclosure 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 is prepared by the following general procedure 3:

[0125] General Procedure 3

[0126] in,

[0127] n, L1, L2, G1, G2, R1, R2, R3 have the same meanings as those defined above for the compounds of formula (I) of the present disclosure; and L=L1=L2, G=G1=G2, R=R1=R2;

[0128] Specifically, the compound of formula (I) of the present disclosure is obtained by subjecting the intermediate compound (IV) to a reduction reaction with the intermediate compound (VI) in an organic solvent in the presence of a reducing agent at room temperature.

[0129] The organic solvent is selected from nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE); and the reducing agent is selected from, for example, sodium triacetoxyborohydride.

[0130] In another aspect, the present disclosure also provides an intermediate compound (II) for preparing the compound of formula (I) of the present disclosure, whose structural formula is as follows:

[0131] in:

[0132] 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; further preferably, L1 is selected from C3-C10 alkylene; most preferably, L1 is selected from C5-C8 alkylene;

[0133] 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-;

[0134] R1 is selected from C5-C27 alkyl groups connected via any carbon atom, C5-C27 alkenyl groups containing one or more double bonds; preferably, R1 is selected from C8-C20 alkyl groups connected via any carbon atom, C8-C20 alkenyl groups containing one or more double bonds; further preferably, R1 is selected from C9-C17 alkyl groups connected via any carbon atom, C9-C18 alkenyl groups containing one or two double bonds; most preferably, R1 is selected from

[0135] R3 is selected from halogen, hydroxyl, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; preferably, R3 is selected from halogen, hydroxyl, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; further preferably, R3 is selected from halogen, hydroxyl, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 alkylcarbonylamino; most preferably, R3 is selected from fluorine, hydroxyl, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido;

[0136] n is selected from 1, 2, and 3.

[0137] Preferably, the present disclosure provides the intermediate compound (II) as described above or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0138] L1 is selected from C3-C10 alkylene, C3-C10 alkenylene or C3-C10 alkynylene;

[0139] G1 is selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-;

[0140] R1 is selected from C8-C20 alkyl groups connected via any one of the carbon atoms, and C8-C20 alkenyl groups containing one or more double bonds;

[0141] R3 is selected from halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino;

[0142] n is selected from 1, 2, and 3.

[0143] Further preferably, the present disclosure provides the intermediate compound (II) as described above or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0144] L1 is selected from C3-C10 alkylene;

[0145] G1 is selected from -O-(C=O)-, -(C=O)-O-;

[0146] R1 is selected from C9-C17 alkyl groups connected via any one of the carbon atoms, and C9-C18 alkenyl groups containing one or two double bonds;

[0147] R3 is selected from halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 alkylcarbonylamino;

[0148] n is selected from 1, 2, and 3.

[0149] More preferably, the present disclosure provides the intermediate compound (II) as described above or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0150] L1 is selected from C5-C8 alkylene;

[0151] G1 is selected from -O-(C=O)-, -(C=O)-O-;

[0152] R1 is selected from

[0153] R3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido;

[0154] n is selected from 1, 2, and 3.

[0155] Most preferably, the intermediate compound (II) is selected from:

[0156] Furthermore, the present disclosure also provides a preparation method for the intermediate compound (II), the general reaction procedure of which is as follows:

[0157] wherein R1, R3, G1, L1, and n have the same meanings as those defined above for the intermediate compound (II); and X is a halogen, preferably bromine.

[0158] Specifically, intermediate compound (IV) and intermediate compound (VII) are reacted at room temperature in an organic solvent in the presence of an acid-binding agent to obtain intermediate compound (II). The organic solvent is selected from nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE); 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.

[0159] In another aspect, the present disclosure provides a method for preparing an optical isomer (X) of the compound of formula (I) described in the present disclosure. The general reaction procedure of the method is as follows:

[0160] wherein n, L1, L2, G1, G2, R1, R2, and R3 have the same meanings as those defined above for the compound of formula (I); and X is a halogen, preferably bromine.

[0161] Specifically, first, compound (VIII) and intermediate compound (VII) are reacted in an organic solvent in the presence of an acid-binding agent through a substitution reaction to obtain intermediate compound (VIIII), wherein the organic solvent is selected from nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE); 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. Then, the intermediate compound (VIIII) reacts with the intermediate compound (III) in the presence or absence of an acid-binding agent and an iodide to obtain the optical isomer (X) of the compound (I), wherein the organic solvent is selected from nitriles, alcohols, halogenated hydrocarbons, amides, and aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE); 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; and the iodide is such as KI.

[0162] Furthermore, the present disclosure provides a method for preparing an optical isomer (I-6) of compound 6 described in the present disclosure. The general reaction procedure of the method is as follows:

[0163] Wherein, X is a halogen, preferably bromine.

[0164] Specifically, the optical isomer (I-6) of compound 6 described in the present disclosure is prepared by the following steps:

[0165] In a solvent such as a nitrile, alcohol, halogenated hydrocarbon, amide, or aromatic hydrocarbon solvent, specifically acetonitrile, methanol, ethanol, dichloromethane, or dichloroethane (DCE), compound (6-X) and compound (6-VI) are subjected to an N-alkylation reaction at 30-50° C. to prepare compound (6-VII);

[0166] In a solvent such as nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, and ether solvents, specifically acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE), cyclopentane methyl ether, and methyl tert-butyl ether, compound (6-VII) is reacted with 8-halogenated octanoic acid nonyl ester at 60-110° C. in the presence or absence of an acid-binding agent and a catalyst to produce compound (I-6), wherein the acid-binding agent is selected from an organic base or an inorganic base, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA; and the catalyst is an iodide, preferably KI.

[0167] Furthermore, according to the above method, when the compound (6-X) used is (1S, 3R)-3-aminocyclohexanol and the compound (6-VII) is represented by the structural formula (6-VII-I):

[0168] Obtain the optical isomer of compound 6, compound (I-6-II):

[0169] When the compound (6-X) used is (1S,3S)-3-aminocyclohexanol and the compound (6-VII) is represented by the structural formula (6-VII-II):

[0170] Obtain the optical isomer of compound 6, compound (I-6-III):

[0171] When the compound (6-X) used is (1R, 3R)-3-aminocyclohexanol and the compound (6-VII) is represented by the structural formula (6-VII-III):

[0172] Obtain the optical isomer of compound 6, compound (I-6-IV):

[0173] When the compound (6-X) used is (1R, 3S)-3-aminocyclohexanol and the compound (6-VII) is represented by the structural formula (6-VII-IV):

[0174] Obtain the optical isomer of compound 6, compound (I-6-V):

[0175] In another aspect, the present disclosure provides an intermediate compound (6-VII) for preparing the optical isomer (I-6) of the above-mentioned compound 6, whose structure is:

[0176] Specifically, the intermediate compound (6-VII) is selected from: BRIEF DESCRIPTION OF THE DRAWINGS

[0177] FIG1 shows the humoral antibody titers generated by subcutaneous administration of a representative amino lipid compound for delivery of OVA mRNA in Experiment 2 of the present disclosure, for example, in the biological assay.

[0178] FIG2 shows the humoral antibody titers generated by intramuscular administration of a representative amino lipid compound for delivering influenza mRNA vaccine in Experiment 3 of the biological experiment disclosed herein.

[0179] FIG3 is an HPLC spectrum of a mixture of compounds (I-6-II), (I-6-III), (I-6-IV) and (I-6-V).

[0180] FIG4 is an HPLC spectrum of compound (I-6-II).

[0181] FIG5 is the HPLC spectrum of compound (I-6-III).

[0182] FIG6 is the HPLC spectrum of compound (I-6-IV).

[0183] FIG7 is an HPLC spectrum of compound (I-6-V).

[0184] Figure 8 shows the fluorescence intensity of the liver after targeted test and intramuscular injection.

[0185] Figure 9 is a test of adverse reactions to intramuscular injection, showing statistics of severe swelling and moderate lameness at the injection site.

[0186] Various exemplary embodiments of the disclosed amino lipid compounds, compositions containing the same, lipid nanoparticles, and their use for delivering bioactive agents, such as nucleic acids, into cells are described in further detail below. DETAILED DESCRIPTION

[0187] definition

[0188] Unless the context requires otherwise, in this specification and claims, the word "comprise" and variations such as "include" and "containing" are to be construed in an open and inclusive sense, that is, as meaning "including, but not limited to."

[0189] Throughout this specification, reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0190] Unless defined otherwise, 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 disclosure belongs. As used in this specification and claims, unless the context clearly dictates otherwise.

[0191] The notation "Cx-Cy" used in this disclosure represents a range of carbon atom numbers, wherein x and y are both integers, for example, C3-C8 cycloalkyl represents a cycloalkyl group having 3-8 carbon atoms, and C0-C2 alkyl represents an alkyl group having 0-2 carbon atoms, wherein C0 alkyl refers to a chemical single bond.

[0192] In the present disclosure, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched chain groups of 1 to 30 carbon atoms, for example, straight and branched chain groups of 1 to 6 carbon atoms, 5 to 27 carbon atoms, 8 to 20 carbon atoms, 9 to 17 carbon atoms. Non-limiting examples include n-nonyl, undecyl, 7-pentadecyl, 9-heptadecyl and various isomers thereof.

[0193] In the present disclosure, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon group comprising 3 to 12 ring atoms, for example, 3 to 12, 3 to 10, 3 to 8, or 3 to 6 ring atoms, or a 3-, 4-, 5-, or 6-membered ring. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0194] In the present disclosure, the term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group having at least one double bond, including straight-chain and branched alkene groups of 1 to 30 carbon atoms, for example, straight-chain and branched alkene 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.

[0195] In the present disclosure, the term "alkynyl" refers to an unsaturated aliphatic hydrocarbon group having at least one triple bond, including straight-chain and branched alkyne groups of 1 to 30 carbon atoms, for example, straight-chain and branched alkyne 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-decynyl, and various isomers thereof.

[0196] In the present disclosure, the term "alkylene" refers to a substituted or unsubstituted alkyl group having two terminal monovalent radical cores, which is generated by removing one hydrogen atom from each of the two terminal atoms; said alkyl group has the meaning described above. Non-limiting examples of "alkylene" include C3-C10 alkylene, C5-C8 alkylene, etc.

[0197] In the present disclosure, the term "alkenylene" refers to a substituted or unsubstituted alkenyl group having two terminal monovalent radical cores, which is generated by removing one hydrogen atom from each of the two terminal atoms; the alkenyl group has the meaning described above. Non-limiting examples of "alkenylene" include C3-C10 alkenylene, etc.

[0198] In the present disclosure, the term "alkynylene" refers to a substituted or unsubstituted alkynyl group having two terminal monovalent radical cores, which are generated by removing one hydrogen atom from each of the two terminal atoms; the alkynyl group has the meaning described above. Non-limiting examples of "alkynylene" include C3-C10 alkynylene, etc.

[0199] In this disclosure, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0200] In this disclosure, the term "alkoxy" refers to an alkyl-oxy group, wherein the alkyl group has the meaning as described above. Preferably, the alkoxy group is a C1-C10 alkoxy group; more preferably, the alkoxy group is a C1-C6 alkoxy group; further preferably, the alkoxy group is a C1-C4 alkoxy group; most preferably, the alkoxy group is a methoxy group.

[0201] In the present disclosure, the term "alkylcarbonyloxy" refers to an alkyl-C(O)O group, wherein the alkyl group has the meaning as described above. Preferably, the alkylcarbonyloxy group is a C1-C10 alkylcarbonyloxy group; more preferably, the alkylcarbonyloxy group is a C1-C6 alkylcarbonyloxy group; further preferably, the alkylcarbonyloxy group is a C1-C4 alkylcarbonyloxy group; most preferably, the alkylcarbonyloxy group is an acetoxy group.

[0202] In this disclosure, the term "alkoxycarbonyl" refers to an alkyl-OC(O)- group, wherein the alkyl group has the meaning as described above. Preferably, the alkoxycarbonyl group is a C1-C10 alkoxycarbonyl group; more preferably, the alkoxycarbonyl group is a C1-C6 alkoxycarbonyl group; further preferably, the alkoxycarbonyl group is a C1-C4 alkoxycarbonyl group; most preferably, the alkoxycarbonyl group is a methoxycarbonyl group.

[0203] In the present disclosure, the term "alkylcarbonylamino" refers to an alkyl-C(O)NH- group, wherein the alkyl group has the meaning as described above. Preferably, the alkylcarbonylamino group is a C1-C10 alkylcarbonylamino group; more preferably, the alkylcarbonylamino group is a C1-C6 alkylcarbonylamino group; further preferably, the alkylcarbonylamino group is a C1-C4 alkylcarbonylamino group; most preferably, the alkylcarbonylamino group is an acetylamino group.

[0204] In the present disclosure, the term "alkylaminocarbonyl" refers to an alkyl-NHC(O)- group, wherein the alkyl group has the meaning as described above. Preferably, the alkylaminocarbonyl group is a C1-C10 alkylaminocarbonyl group; more preferably, the alkylaminocarbonyl group is a C1-C6 alkylaminocarbonyl group; further preferably, the alkylaminocarbonyl group is a C1-C4 alkylaminocarbonyl group; most preferably, the alkylaminocarbonyl group is a butylaminocarbonyl group.

[0205] In the present disclosure, the term "optionally substituted" means that one or more hydrogen atoms attached to an atom or group are independently unsubstituted or substituted with one or more (e.g., 1, 2, 3, or 4) substituents. The substituents may be independently selected from, but are not limited to, 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 (e.g., -OH), ester (e.g., -C(=O)ORi or -OC(=O)Ri), aldehyde (e.g., -C(=O)H), carbonyl (e.g., -C(=O)Ri, or represented by C=O), acyl halide (e.g., -C(=O)X, wherein X is selected from bromine, fluorine, chlorine, or iodine), carbonate (e.g., -OC(=O)ORi), alkoxy (e.g., -ORi), acetal (e.g., -C(=O) i) 2Ri, wherein each ORi is the same or different alkoxy), phosphate (e.g., P(=O)43-), thiol (e.g., -SH), sulfoxide (e.g., -S(=O)Ri), sulfinic acid (e.g., -S(=O)OH), sulfonic acid (e.g., -S(=O)2OH), thialdehyde (e.g., -C(=S)H), sulfate (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), azido (e.g., -N3), nitro (e.g., -NO2), cyano alkyl (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(=O)Ri), amino (e.g., -N(Ri)2, -N(Ri)H, or -NH2), carbamoyl (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, cycloalkyl (e.g., cycloalkyl, cycloalkenyl or cycloalkynyl), heterocycloalkyl (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 fused ring), heteroaryl (e.g., 8-10 membered bicyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur), -C(=O)SRi, -C(=N-CN)N(Ri)2, -C(=NO-CH3)N(Ri)2, -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(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, R1 is hydrogen, or alkyl, or alkenyl, or alkynyl, or heteroalkyl, or heteroalkenyl, or heteroalkynyl as defined herein. In some embodiments, R is hydrogen, as defined herein, or C1-C12 alkyl, or C1-C12 alkenyl, or C1-C12 alkynyl, or C1-C12 heteroalkyl, or C1-C12 heteroalkenyl, or C1-C12 heteroalkynyl. 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 substituents as defined herein. For example, a C1-C6 alkyl group as a substituent may be further substituted with one or more substituents as described herein.

[0206] Unless otherwise specifically stated, the alkyl, cycloalkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, alkoxy, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonylamino, and alkylaminocarbonyl groups described herein are optionally substituted.

[0207] The "pharmaceutically acceptable salts" of the present disclosure are discussed in Berge, et al., "Pharmaceutically acceptable salts", J. Pharm. Sci., 66, 1-19 (1977), and are obvious to pharmaceutical chemists. The salts are substantially non-toxic and can provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion.

[0208] Examples of "pharmaceutically acceptable salts" as used herein include acid addition salts or base addition salts of the compounds of the present disclosure, which retain the biological effectiveness and properties of the compounds of the present disclosure and are generally not biologically or otherwise undesirable. In many cases, the compounds of the present disclosure are capable of forming acid and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto.

[0209] Pharmaceutically acceptable acid addition salts can be formed with inorganic and / or organic acids with the compounds of the present disclosure, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; such as, but 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, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic 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, galactose, benzoic ... Diacid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, 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, 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 and undecylenic acid, etc.

[0210] Pharmaceutically acceptable base addition salts can be formed with inorganic bases 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 and 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, 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, denanol, 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, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0211] The pharmaceutically acceptable salts disclosed herein can be synthesized by general chemical methods.

[0212] Generally, salts can be prepared by reacting a free base or acid with an equal chemical equivalent or excess of an acid (inorganic or organic) or base in a suitable solvent or solvent combination.

[0213] In this disclosure, the term "meta" refers to a position on a cycloalkyl structural unit that is one carbon atom away from its amine substituent.

[0214] In this disclosure, the term "compound" encompasses all compounds that have been isotopically labeled by replacing one or more atoms with atoms having a different atomic weight or mass number. "Isotopes" refer to atoms with the same atomic number but different mass numbers due to the number of neutrons in their nuclei. For example, isotopes of hydrogen include tritium and deuterium.

[0215] The compounds of the present invention or their pharmaceutically acceptable salts may contain one or more asymmetric centers and thus may produce enantiomers, diastereomers and other stereoisomeric forms, such as amino acids, which can be defined as (R)- or (S)-, or as (D)- or (L)-, based on absolute stereochemistry. The present disclosure is intended to include all of these possible isomers, as well as racemic and optically pure forms thereof. 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 crystallization. Conventional techniques for preparing / isolating single enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0216] As used herein, the term "stereoisomer" refers to a compound composed of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interconvertible. "Optical isomer" refers to two or more stereoisomers that exhibit different optical properties due to differences in 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 proportion is referred to as a "racemic" mixture. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0217] "Stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds described herein are isolated as E or Z isomers. In other embodiments, the compounds described herein are mixtures of E and Z isomers.

[0218] "Tautomers" refers to isomeric forms of a compound that are in equilibrium with each other. The concentration of the isomeric forms will depend on the environment in which the compound is found, and may differ depending on, for example, whether the compound is a solid or in organic or aqueous solution.

[0219] In the present disclosure, the term "lipid nanoparticle" refers to a nanometer-sized substance (lipid nanoparticle) prepared by adding an amino lipid compound to an aqueous solution, and these particles are particularly lipid bilayer vesicles (liposomes), multilamellar vesicles or micelles. In a preferred embodiment, the lipid nanoparticle is a liposome containing an amino lipid compound of the present disclosure.

[0220] In the present disclosure, the term "liposome" means a microvesicle composed of a bilayer of lipid amphiphilic molecules enclosing an aqueous compartment. The formation of liposomes is not a spontaneous process. Lipid vesicles are first formed when lipids are placed in water, thereby forming a bilayer or a series of bilayers, each separated by water molecules. Liposomes can be formed by ultrasonicating lipid vesicles in water.

[0221] In the present disclosure, the term "lipid bilayer" means a thin membrane formed by two layers of lipid molecules.

[0222] In this disclosure, the term "micelle" means an aggregate of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solution form aggregates upon contact with water with the hydrophilic head region sequestering the hydrophobic single tail region in the center of the micelle.

[0223] In the present disclosure, the term "cell" has the meaning known in the art and includes cultured single cells, tissues, organs, insect cells, avian cells, fish cells, amphibian cells, mammalian cells, primary cells, continuous cell lines, stem cells and / or genetically engineered cells (such as 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).

[0224] In a preferred embodiment, the lipid nanoparticles or liposomes of the present disclosure further contain 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, the non-cationic lipid can contain a cationic functional group (e.g., an ammonium group), but should contain an anionic functional group to at least neutralize the molecule. The overall composition of all functional groups in the lipid molecule should be 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 an even more preferred embodiment, the non-cationic lipid is DOPE or DSPC.

[0225] In a further preferred embodiment, the lipid nanoparticles or liposomes of the present disclosure further comprise a sterol. Sterols, such as cholesterol, are natural components of cell membranes and can serve to stabilize the particles and aid integration with the cell membrane.

[0226] 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 response or inflammatory response, by exerting enzymatic activity, or by complementing a mutation, and bioactive agents are particularly nucleic acids, peptides, proteins, antibodies, and small molecules. When liposomes are used to encapsulate a drug within the lipid bilayer or within the internal aqueous space of the liposome, the term "lipid nanoparticle drug" can be used.

[0227] In a preferred embodiment, the bioactive agent is a nucleic acid. In another preferred embodiment, the bioactive agent is a member selected from antitumor agents, antibiotics, immunomodulators, anti-inflammatory agents, agents acting on the central nervous system, polypeptides or polypeptoids.

[0228] In another embodiment, lipid nanoparticle or liposome further contain at least one polyethylene glycol (PEG)-lipid.PEG lipid helps to protect particle and inclusion thereof from external or internal degradation.In addition, PEG forms a protective layer on the liposome surface and improves internal circulation time.It can be used for liposome drug delivery (PEG-liposome).Preferably, the polyethylene glycol lipid is PEG2000-DMG.

[0229] Lipid nanoparticles or liposomes containing a bioactive agent can be used to deliver any of a variety of therapeutic agents into cells. The present disclosure includes the use of lipid nanoparticles (especially liposomes) as described above for delivering a bioactive agent into a cell.

[0230] Preferably, the bioactive agent is a nucleic acid, including but not limited to RNA, DNA, antisense oligonucleotides; wherein RNA includes but is not limited to messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA); DNA includes but is not limited to plasmids. The bioactive agent can also be an anti-tumor agent, an antibiotic, an immunomodulator, an anti-inflammatory agent, an agent acting on the central nervous system, an antigen or a fragment thereof, a protein, a peptide, a polypeptide, a vaccine and a small molecule, or a mixture thereof. As shown above, lipid nanoparticles or liposomes containing the amino lipid compound defined in the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof are suitable for delivering the bioactive agent into cells.

[0231] A variety of different amino lipid compounds synthesized by general synthetic methods can be screened for specific characteristics conferred on liposomes for specific applications, such as transfection efficiency, cytotoxicity, adhesion of the agent to be delivered to the cell, liposome stability, liposome size, etc.

[0232] The lipid nanoparticles or liposomes disclosed herein can be used to transfect multiple cellular tissues or organs, which provides new therapeutic treatment possibilities for patients.

[0233] According to the present disclosure, the patient can be any mammal, preferably selected from humans, mice, rats, pigs, cats, dogs, horses, goats, cows and monkeys and / or others. Most preferably, the patient is a human.

[0234] A preferred embodiment of the present disclosure relates to the use of lipid nanoparticles or liposomes containing the amino lipid compound of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof as a medicament.

[0235] In particular, the lipid nanoparticles or liposomes can be administered to patients for gene therapy, gene vaccination, antisense therapy, or treatment via interfering RNA. Specific applications include, but are not limited to:

[0236] (1) The lipid nanoparticles disclosed herein can deliver nucleic acids for gene therapy. Exogenous genes are introduced into target cells through the amino lipids disclosed herein to correct or compensate for diseases caused by defective and abnormal genes to achieve therapeutic purposes. This also includes technical applications in areas such as transgenics, that is, inserting exogenous genes into appropriate recipient cells of the patient through gene transfer technology so that the products produced by the exogenous genes can treat certain diseases, such as common lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, prostate cancer, etc. Gene-edited nucleic acid substances can also be introduced for the treatment of various genetic diseases, such as hemophilia, thalassemia, Gaucher disease, etc.

[0237] (2) The lipid nanoparticles of the present disclosure can be used in 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 elicit immune responses against various antigens used to treat and / or prevent a variety of conditions, such as cancer, allergies, toxicity, and infection with pathogens (e.g., viruses, bacteria, fungi, and other pathogenic organisms).

[0238] In another preferred embodiment, the lipid nanoparticles disclosed herein can be used to prepare drugs for nucleic acid transfer, preferably, the nucleic acid is RNA, DNA, antisense oligonucleotide; preferably, the RNA is selected from messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA); preferably, the DNA is a plasmid.

[0239] Example

[0240] In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with specific examples. Obviously, the described examples are part of the implementation scheme of the present disclosure. Based on the examples in the present disclosure, all other examples obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure. The experimental methods for which specific conditions are not specified in the following examples shall be selected according to conventional methods and conditions, or according to the product instructions.

[0241] The structures of all compounds disclosed herein can be identified by nuclear magnetic resonance (1H NMR) and / or mass spectrometry (MS).

[0242] 1H NMR chemical shifts (δ) are reported in parts per million (PPM). NMR spectrometry was performed on a Bruker AVANCE III-400 MHz spectrometer. Suitable solvents were selected from deuterated chloroform (CDCl3), deuterated methanol (CD3OD), deuterated dimethyl sulfoxide (DMSO-d6), and the like, with tetramethylsilane as the internal standard (TMS).

[0243] Low-resolution mass spectra (MS) were measured on an Agilent 1260 Infinity II-G6125C mass spectrometer.

[0244] The specific rotation measurement method disclosed in the present invention is as follows: take a sample, accurately weigh it, dissolve it in anhydrous ethanol, and quantitatively dilute it to make a solution containing approximately 10 mg per 1 ml, and measure it according to the optical rotation method 0621 of the fourth general rule of the 2020 edition of the "Pharmacopoeia of the People's Republic of China".

[0245] The known starting materials disclosed herein can be synthesized using or according to methods known in the art, or can be commercially obtained.

[0246] The column chromatography eluent system used for the purified compound includes A: dichloromethane and methanol system (20:1 to 5:1); B: n-hexane and ethyl acetate system (10:1 to 2:1). The volume ratio of the solvent is adjusted according to the polarity of the compound.

[0247] Unless otherwise specified in the examples, the reaction temperature is room temperature, which ranges from 15°C to 30°C.

[0248] The HPLC analysis method disclosed in the present invention: HPLC-CAD method.

[0249] Table 1: HPLC analysis method conditions of the present disclosure.

[0250] Example 1: Synthesis of Compound 1

[0251] Step 1): Synthesis of 1-octylnonyl 8-bromooctanoate

[0252] In a 250 mL reaction bottle, 8-bromooctanoic acid (22.3 g, 100 mmol), 9-heptadecanol (25.6 g, 100 mmol), and 100 mL of dichloromethane were added in sequence. After stirring and dissolving, 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 h. After washing with water three times, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (n-hexane: ethyl acetate = 10:1 to 2:1) to obtain 8-bromooctanoic acid-1-octylnonyl ester (41.5 g, 90%).

[0253] Step 2): Synthesis of 8-((3-hydroxycyclobutyl)amino)nonyl octanoate (intermediate compound II-1)

[0254] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.50 g, 10 mmol), 3-aminocyclobutanol (8.7 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 8-((3-hydroxycyclobutyl)amino)octanoic acid nonyl ester (2.38 g, 67%).

[0255] 1H NMR (600MHz, 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).

[0256] LCMS: 356.3 [M+H] +.

[0257] Step 3): Synthesis of Compound 1

[0258] In a 100 mL reaction flask, 8-((3-hydroxycyclobutyl)amino)octanoic acid nonyl ester (355 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The reaction was carried out at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain compound 1 (501 mg, 68%).

[0259] 1H NMR (600MHz, CDCl3) δ: 4.94-4.78 (m, 1H), 4.42-4.37 (m, 0.5H), 4.05 (t, 2H) ),3.99(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).

[0260] LCMS: 737.2 [M+H] +.

[0261] Example 2: Synthesis of Compound 2

[0262] Step 1): Synthesis of 6-((3-hydroxycyclobutyl)amino)hexanoic acid undecyl ester (intermediate compound II-2)

[0263] In a 100 mL reaction flask, undecyl 8-bromohexanoate (3.50 g, 10 mmol), 3-aminocyclobutanol (8.7 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain undecyl 6-((3-hydroxycyclobutyl)amino)hexanoate (2.38 g, 67%).

[0264] 1H NMR (600MHz, 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).

[0265] LCMS: 356.3 [M+H] +.

[0266] Step 2): Synthesis of Compound 2

[0267] Compound 2 was synthesized in a similar manner to step 3) of Example 1, except that undecyl 6-((3-hydroxycyclobutyl)amino)hexanoate (intermediate compound II-2) was used instead of nonyl 8-((3-hydroxycyclobutyl)amino)octanoate.

[0268] 1H NMR (600MHz, 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),2.5 6(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).

[0269] LCMS: 737.2 [M+H] +.

[0270] Example 3: Synthesis of Compound 6

[0271] Step 1): Synthesis of 8-((3-hydroxycyclohexyl)amino)nonyl octanoate (intermediate compound II-6)

[0272] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.50 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (2.34 g, 61%).

[0273] 1H NMR (600MHz, CDCl3) δ4.20-4.13(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).

[0274] LCMS: 384.3 [M+H] +.

[0275] Step 2): Synthesis of compound 6

[0276] To a 100 mL reaction flask, 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The reaction was carried out at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain compound 6 (596 mg, 78%).

[0277] 1H NMR (600MHz, CDCl3) δ4.95-4.81(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).

[0278] LCMS: 765.3 [M+H] +.

[0279] Example 4: Synthesis of Compound 7

[0280] Step 1): Synthesis of 6-((3-hydroxycyclohexyl)amino)hexanoic acid undecyl ester (intermediate compound II-7)

[0281] In a 100 mL reaction flask, undecyl 6-bromohexanoate (3.50 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain undecyl 6-((3-hydroxycyclohexyl)amino)hexanoate (2.53 g, 66%).

[0282] 1H NMR (600MHz, CDCl3) δ4.20(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).

[0283] LCMS: 384.4 [M+H] +.

[0284] Step 2): Synthesis of compound 7

[0285] In a 100 mL reaction flask, 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 in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The mixture was reacted at room temperature for 24 h, and 100 mL of dichloromethane was added. After washing with water three times, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain compound 7 (572 mg, 75%).

[0286] 1H NMR (600MHz, CDCl3) δ4.93-4.81(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),2.50-2.3 6(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).

[0287] LCMS: 765.0 [M+H] +.

[0288] Example 5: Synthesis of Compound 8

[0289] Step 1) Synthesis of 7-pentadecyl 6-oxohexanoate

[0290] In a 250 mL reaction flask, 6-((tert-butyldimethylsilyl)oxy)hexanoic acid (2.46 g, 10 mmol), pentadecane-7-ol (2.28 g, 10 mol), and 100 mL of dichloromethane were added in sequence. After stirring and dissolving, dicyclohexylcarbodiimide (2.47 g, 12 mmol) and 4-dimethylaminopyridine (0.06 g, 0.5 mmol) were added. The mixture was reacted at room temperature for 2 h, washed with water 3 times, dried over anhydrous sodium sulfate, and concentrated to dryness. Then, 50 mL of tetrahydrofuran and tetrabutylammonium fluoride ( The product was reacted at room temperature for 1 hour, concentrated to near dryness, dissolved in 100 mL of dichloromethane, washed three times with water, dried over anhydrous sodium sulfate, and then added with Dess-Martin periodinane (5.09 g, 12 mmol). The product was stirred at room temperature for 12 hours, washed three times with a saturated sodium bicarbonate solution, washed once with water, dried over anhydrous sodium sulfate, and purified using a flash column chromatography system (n-hexane: ethyl acetate = 10:1 to 5:1) to obtain 7-pentadecyl 6-oxohexanoate (2.76 g, 81%).

[0291] Step 2): Synthesis of Compound 8

[0292] In a 250 mL reaction flask, 7-pentadecyl 6-oxohexanoate (3.41 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added in sequence, and then 3-aminocyclohexanol (0.57 g, 5 mmol) was added. The reaction was carried out at room temperature for 24 h. After washing with water three times, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain compound 8 (2.83 g, 74%).

[0293] 1H NMR (600MHz, CDCl3) δ4.93-4.80(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).

[0294] LCMS: 765.1 [M+H] +.

[0295] Example 6: Synthesis of Compound 9

[0296] Step 1): Synthesis of 2-hexyldecanoate (6-oxohexyl)

[0297] In a 250 mL reaction flask, 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 in sequence. After stirring and dissolving, dicyclohexylcarbodiimide (20.6 g, 100 mmol) and 4-dimethylaminopyridine (0.61 g, 5 mmol) were added. The mixture was reacted at room temperature for 2 h. After washing with water 3 times, the mixture was dried over anhydrous sodium sulfate and purified using a flash column chromatography system (n-hexane: ethyl acetate). To the reaction mixture, 100 mL of dichloromethane and Dess-Martin periodinane (50.9 g, 120 mmol) were added, followed by stirring at room temperature for 12 h. The mixture was washed three times with a saturated sodium bicarbonate solution, washed once with water, and dried over anhydrous sodium sulfate. The mixture was purified by flash column chromatography (n-hexane:ethyl acetate = 10:1 to 5:1) to obtain 7-hydroxyheptyl 2-hexyldecanoate (19.1 g, 54%).

[0298] Step 2): Synthesis of compound 9

[0299] In a 250 mL reaction flask, 2-hexyldecanoate (6-oxohexyl) ester (3.55 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added in sequence, and then 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (3.83 g, 10 mmol) was added. The mixture was reacted at room temperature for 24 h. After washing with water three times, it was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain compound 9 (4.98 g, 69%).

[0300] 1H NMR (600MHz, CDCl3) δ4.26-4.19(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).

[0301] LCMS: 723.1[M+H]+.

[0302] Example 7: Synthesis of Compound 10

[0303] Step 1): Synthesis of 8-bromooctyl oleate

[0304] In a 250 mL reaction flask, 8-bromooctanol (20.9 g, 100 mmol), oleic acid (28.5 g, 100 mmol), and 100 mL of dichloromethane were added in sequence. After stirring and dissolving, 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 h. After washing with water three times, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (n-hexane: ethyl acetate = 10:1 to 3:1) to obtain 8-bromooctyl oleate (37.4 g, 79%).

[0305] Step 2): Synthesis of 8-((3-hydroxycyclohexyl)amino)octyl oleate (Intermediate Compound II-10)

[0306] In a 100 mL reaction flask, 8-bromononanyl oleate (4.73 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 8-((3-hydroxycyclohexyl)amino)octyl oleate (3.2 g, 63%).

[0307] 1H NMR (600MHz, CDCl3) δ5.41-5.29(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).

[0308] LCMS: 508.7 [M+H] +.

[0309] Step 3): Synthesis of compound 10

[0310] In a 250 mL reaction flask, 2-hexyldecanoate (6-oxohexyl) ester (3.55 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added sequentially, and then 8-((3-hydroxycyclohexyl)amino)octyl oleate (5.08 g, 10 mmol) was added. The mixture was reacted at room temperature for 24 h. After washing with water three times, it was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to give compound 10 (6.26 g, 74%).

[0311] 1H NMR (600MHz, CDCl3) δ5.41-5.31(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).

[0312] LCMS: 847.3 [M+H] +.

[0313] Example 8: Synthesis of Compound 12

[0314] Step 1): Synthesis of (6Z,9Z)-diene-octadecane-8-((3-hydroxycyclohexyl)amino)octyl ester (intermediate compound II-12)

[0315] In a 100 mL reaction bottle, (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 in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain (6Z,9Z)-diene-octadecane-8-((3-hydroxycyclohexyl)amino)octanoate (3.3 g, 66%).

[0316] 1H NMR (600MHz, CDCl3) δ5.43-5.28(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),2.8 2-2.69(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).

[0317] LCMS: 506.7 [M+H] +.

[0318] Step 2): Synthesis of compound 12

[0319] Compound 12 was synthesized in a similar manner to step 3) of Example 7, except that (6Z,9Z)-diene-octadecane-8-((3-hydroxycyclohexyl)amino)octyl ester was used instead of the original intermediate compound oleic acid 8-((3-hydroxycyclohexyl)amino)octyl ester.

[0320] 1H NMR (600MHz, CDCl3) δ5.43-5.28(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).

[0321] LCMS: 845.1 [M+H] +.

[0322] Example 9: Synthesis of Compound 14

[0323] In a 250 mL reaction flask, 2-hexyldecanoate (6-oxohexyl) ester (3.55 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added in sequence, and then 3-aminocyclohexanol (0.57 g, 5 mmol) was added. The reaction was carried out at room temperature for 24 h. After washing with water three times, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain compound 14 (2.81 g, 71%).

[0324] 1H NMR (600MHz, CDCl3) δ4.32-4.20(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).

[0325] LCMS: 793.2 [M+H] +.

[0326] Example 10: Synthesis of Compound 15

[0327] Compound 15 was synthesized in a similar manner to Example 7, step 3), except that 7-pentadecyl 6-((3-hydroxycyclohexyl)amino)hexanoate was used instead of the original intermediate compound 8-((3-hydroxycyclohexyl)amino)octyl oleate.

[0328] 1H NMR (600MHz, CDCl3) δ4.94-4.80(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).

[0329] LCMS: 779.2 [M+H] +.

[0330] Example 11: Synthesis of Compound 16

[0331] Step 1): Synthesis of 8-((3-methoxycyclohexyl)amino)nonyl octanoate (intermediate compound II-16)

[0332] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.49 g, 10 mmol), 3-methoxycyclohexylamine (12.9 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 8-((3-methoxycyclohexyl)amino)octanoic acid nonyl ester (2.35 g, 59%).

[0333] 1H NMR(600MHz, CDCl3)δ4.07(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),3.0 0-2.89(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).

[0334] LCMS: 398.5 [M+H] +.

[0335] Step 2): Synthesis of compound 16

[0336] In a 100 mL reaction flask, 8-((3-methoxycyclohexyl)amino)octanoic acid nonyl ester (400 mg, 1 mmol), 8-bromooctanoic acid-1-octyl nonyl ester (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The reaction was carried out at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times. It was then dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain compound 16 (552 mg, 71%).

[0337] 1H NMR (600MHz, CDCl3) δ4.93-4.77(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),2.4 3(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).

[0338] LCMS: 779.1 [M+H] +.

[0339] Example 12: Synthesis of Compound 19

[0340] Step 1): Synthesis of 8-((3-acetoxycyclohexyl)amino)nonyl octanoate (intermediate compound II-19)

[0341] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.49 g, 10 mmol), 3-acetoxycyclohexylamine (15.7 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 8-((3-acetoxycyclohexyl)amino)octanoic acid nonyl ester (2.76 g, 65%).

[0342] 1H NMR(600MHz, CDCl3)δ5.23(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).

[0343] LCMS: 426.4 [M+H] +.

[0344] Step 2): Synthesis of compound 19

[0345] Compound 19 was synthesized in a similar manner to Example 11, step 2), except that 8-((3-acetoxycyclohexyl)amino)octanoic acid nonyl ester was used instead of the original intermediate compound 8-((3-methoxycyclohexyl)amino)octanoic acid nonyl ester.

[0346] 1H NMR (600MHz, CDCl3) δ5.16(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).

[0347] LCMS: 806.7 [M+H] +.

[0348] Example 13: Synthesis of Compound 20

[0349] Step 1): Synthesis of methyl 3-((8-(nonyloxy)-8-oxooctyl)amino)cyclohexane-1-carboxylate (intermediate compound II-20)

[0350] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.49 g, 10 mmol), 3-aminocyclohexanecarboxylic acid methyl ester (15.7 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 3-((8-(nonyloxy)-8-oxooctyl)amino)cyclohexane-1-carboxylic acid methyl ester (2.85 g, 67%).

[0351] 1H NMR(600MHz, CDCl3)δ4.05(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).

[0352] LCMS: 426.4 [M+H] +.

[0353] Step 2): Synthesis of compound 20

[0354] Compound 20 was synthesized in a similar manner to Example 11, step 2), except that methyl 3-((8-(nonyloxy)-8-oxooctyl)amino)cyclohexane-1-carboxylate was used instead of the original intermediate compound 8-((3-methoxycyclohexyl)amino)octanoate.

[0355] 1H NMR (600MHz, CDCl3) δ4.95-4.78(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,5 H),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).

[0356] LCMS: 806.8 [M+H] +.

[0357] Example 14 Synthesis of Compound (I-6-II)

[0358] To a 250ml single-necked flask, 8g (17.3mmol) of 9-heptadecyl-8-bromooctanoate, 10g (76.5mmol) of (1S,3R)-3-aminocyclohexanol, and 100ml of ethanol were added and reacted at 50°C for 15h. The 9-heptadecyl-8-bromooctanoate reacted completely. After removing the solvent by rotary evaporation, the crude product was added to 200ml of EA and washed twice with 100ml of water to thoroughly remove the (1S,3R)-3-aminocyclohexanol. The EA phase was then dried to yield 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 yield 6.77g of compound (6-VII-I) with a purity of 99.2% and a yield of 78.6%.

[0359] NMR data:

[0360] 1H NMR (600MHz, CDCl3): δ4.91-4.81(m,1H),3.86-3.82(m,1H),2.84-2.81(m,1H),2.69-2.54(qt,J=11. 2,7.3Hz,2H),2.29-2.26(t,J=7.5Hz,2H),1.93-1.86(m,1H),1.77-1.74(d,J=12.0Hz,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=7.0Hz,6H).

[0361] LCMS: 496.7 [M+H] +.

[0362] 6 g (12.1 mmol) of the above compound (6-VII-I) was placed in a 250 ml single-necked bottle, 90 ml of acetonitrile and 60 ml of cyclopentane methyl ether were added, and then 6.7 g of potassium carbonate powder (48.4 mmol), 2 g of potassium iodide (12.1 mmol) and 5 g of 8-bromooctanoic acid nonyl ester (17.5 mmol) were added. The mixture was reacted at 90 ° C for 24 h. The raw material compound (6-VII-I) was reacted completely. The oil bath was removed and the reaction was allowed to cool to room temperature. The solid was removed by filtration and the filtrate was dried to obtain a crude product. Finally, it 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%.

[0363] NMR data:

[0364] 1H NMR (600MHz, CDCl3): δ4.93-4.84(m,1H),4.09(t,J=6.8Hz,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= 7.4Hz,4H),1.97(m,1H),1.91-1.78(m,2H),1.71-1.61(m,7H),1.54(d,J=6.0Hz,4H),1.43-1.37(m,4H),1.39-1.23(m,52H),0.91(m,9H).

[0365] Specific rotation: +4.3°

[0366] LCMS: 765.2 [M+H] +.

[0367] Example 15 Synthesis of Compound (I-6-III)

[0368] To a 100ml single-necked flask, 2g of 9-heptadecyl-8-bromooctanoate (4.3mmol), 2.5g of (1S,3S)-3-aminocyclohexanol (20.2mmol), and 20ml of ethanol were added and reacted at 50°C for 15h. The 9-heptadecyl-8-bromooctanoate reacted completely. After removing the solvent by rotary evaporation, the crude product was added with 50ml of EA and washed twice with 25ml of water to thoroughly remove the (1S,3S)-3-aminocyclohexanol. The EA phase was then dried to yield 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 yield 1.63g of compound (6-VII-II) with a purity of 98.5% and a yield of 75.5%.

[0369] NMR data:

[0370] 1H NMR (600MHz, CDCl3): δ4.91-4.82(m,1H),4.16-4.10(m,1H),2.96-2.87(m,1H),2.65-2.55(qt,J=11.2,7.3Hz,2H),2.28-2.26(t,J =7.5Hz,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=7.0Hz,6H).

[0371] LCMS: 496.7 [M+H] +.

[0372] 1.5 g (3.0 mmol) of the above compound (6-VII-II) was placed in a 100 ml single-necked bottle, 25 ml of acetonitrile and 15 ml of cyclopentane methyl ether were added, and then 1.67 g of potassium carbonate powder (12 mmol), 0.5 g of potassium iodide (3 mmol) and 1.26 g of 8-bromooctanoic acid nonyl ester (3.6 mmol) were added. The reaction was placed at 90 ° C. for 24 h. The raw material compound (6-VII-II) was reacted completely. The oil bath was removed and the reaction was allowed to cool to room temperature. After the solid was removed by filtration, the filtrate was dried to obtain a crude product. Finally, it 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%.

[0373] NMR data:

[0374] 1H NMR (600MHz, CDCl3): δ4.91-4.80(m,1H),4.28-4.22(m,1H),4.06-4.04(t,J=6.8Hz,2H ),3.03-2.97(m,1H),2.47-2.41(m,4H),2.33-2.25(q,J=7.5Hz,4H),1.89-1.85(d,J=12 .1Hz,1H),1.80-1.77(t,J=12.2Hz,1H),1.73-1.65(m,2H),1.64-1.59(m,6H),1.54-1.4 8(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).

[0375] Specific rotation: -12.9°

[0376] LCMS: 765.2 [M+H] +.

[0377] Example 16 Synthesis of Compound (I-6-IV)

[0378] To a 100ml single-necked flask, 2g of 9-heptadecyl-8-bromooctanoate (4.3mmol), 2.5g of (1R,3R)-3-aminocyclohexanol (20.2mmol), and 20ml of ethanol were added and reacted at 50°C for 15h. The 9-heptadecyl-8-bromooctanoate reacted completely. After removing the solvent by rotary evaporation, the crude product was added to 50ml of EA and washed twice with 25ml of water to thoroughly remove the (1R,3R)-3-aminocyclohexanol. The EA phase was then dried to yield 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 yield 1.58g of compound (6-VII-III) with a purity of 99.2% and a yield of 73.2%.

[0379] NMR data:

[0380] 1H NMR (600MHz, CDCl3): δ4.91-4.81(m,1H),4.17-4.08(m,1H),2.94-2.84(m,1H),2.64-2.5(qt,J=11.2,7.3Hz,2H),2.28-2.26(t,J =7.5Hz,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=7.0Hz,6H).

[0381] LCMS: 496.7 [M+H] +.

[0382] 1.5 g (3.0 mmol) of the above compound (6-VII-III) was placed in a 100 ml single-necked bottle, 25 ml of acetonitrile and 15 ml of cyclopentane methyl ether were added, and then 1.67 g of potassium carbonate powder (12 mmol), 0.5 g of potassium iodide (3 mmol) and 1.26 g of 8-bromooctanoic acid nonyl ester (3.6 mmol) were added. The reaction was placed at 90 ° C. for 24 h. The raw material compound (6-VII-III) was reacted completely. The oil bath was removed and the reaction mixture was allowed to cool to room temperature. After that, the solid was removed by filtration and the filtrate was dried to obtain a crude product. Finally, it 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%.

[0383] NMR data:

[0384] 1H NMR (600MHz, CDCl3): δ4.89-4.82(m,1H),4.24-4.22(m,1H),4.06-4.04(t,J=6.8Hz,2H ),3.00-2.95(m,1H),2.47-2.36(m,4H),2.30-2.26(q,J=7.5Hz,4H),1.86-1.84(d,J=12 .1Hz,1H),1.79-1.77(t,J=12.2Hz,1H),1.70-1.66(m,2H),1.65-1.57(m,6H),1.53-1.4 8(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).

[0385] Specific rotation: +12.6°

[0386] LCMS: 765.2 [M+H] +.

[0387] Example 17 Synthesis of Compound (I-6-V)

[0388] To a 250ml single-necked flask, 8g of 9-heptadecyl-8-bromooctanoate (17.3mmol), 10g of (1R,3S)-3-aminocyclohexanol (76.5mmol), and 100ml of ethanol were added and reacted at 50°C for 15h. The 9-heptadecyl-8-bromooctanoate reacted completely. The solvent was removed by rotary evaporation, and the crude product was added to 200ml of EA and washed twice with 100ml of water to thoroughly remove the (1R,3S)-3-aminocyclohexanol. The EA phase was then dried to afford 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 yield 6.4g of compound (6-VII-IV) with a purity of 98.5% and a yield of 74.8%.

[0389] NMR data:

[0390] 1H NMR (600MHz, CDCl3): δ4.90-4.83(m,1H),3.86-3.82(m,1H),2.86-2.82(m,1H) ),2.69-2.58(qt,J=11.2,7.3Hz,2H),2.29-2.26(t,J=7.5Hz,2H),1.94-1.86( m,1H),1.79-1.77(d,J=12.0Hz,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=7.0Hz,6H).

[0391] LCMS: 496.7 [M+H] +.

[0392] Take 6g of the above compound (6-VII-IV) product (12.1mmol) in a 250ml single-necked bottle, add 90ml of acetonitrile and 60ml of cyclopentane methyl ether, then add 6.7g of potassium carbonate powder (48.4mmol), 2g of potassium iodide (12.1mmol) and 5g of 8-bromooctanoic acid nonyl ester (17.4), and place it at 90°C to react for 24h. The raw material compound (6-VII-IV) reacts completely. After the oil bath is removed and the reaction is allowed to cool to room temperature, the solid is removed by filtration and the filtrate is dried to obtain a crude product. Finally, it is purified by column chromatography (dichloromethane: methanol = 20:1 to 5:1) to obtain 5.35g of compound (I-6-V) with a purity of 99.1% and a yield of 57.9%.

[0393] NMR data:

[0394] 1H NMR (600MHz, CDCl3): δ4.90-4.82(m,1H),4.05(t,J=6.8Hz,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= 7.5Hz,4H),1.95(m,1H),1.91-1.77(m,2H),1.69-1.56(m,7H),1.50(d,J=6.0Hz,4H),1.41-1.37(m,4H),1.34-1.18(m,52H),0.88(m,9H).

[0395] Specific rotation: -4.5°

[0396] LCMS: 765.2 [M+H] +.

[0397] Example 18 Synthesis of Compound 4

[0398] Compound 4 was synthesized in a similar manner to step 3) of Example 7. Compound 4 (152 mg, yield 40.75%) was synthesized from intermediate II-4 (184 mg, 0.50 mmol) and 9-heptadecan-8-oxooctanoate (296 mg).

[0399] 1H NMR (600MHz, CDCl3) δ4.89-4.84(m,1H),4.44-4.38(m,0.5H),4.27-4.19(m,0 .5H),4.05(t,J=6.8Hz,2H),3.53-3.41(m,0.5H),3.24-3.14(m,0.5H),2.76( t,J=6.0Hz,2H),2.72-2.63(t,J=6.0Hz,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).

[0400] LC-MS: 751.2 [M+H]+.

[0401] Example 19 Synthesis of Compound 5

[0402] Compound 5 was synthesized in a similar manner to step 3) of Example 7. From compound intermediate II-5 (150 mg, 0.38 mmol) and 9-heptadecan-8-oxooctanoate (225 mg), 250 mg of compound 5 was obtained with a yield of 81.9%.

[0403] 1H NMR (600MHz, CDCl3) δ4.93-4.81(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).

[0404] LC-MS: 751.0 [M+H] +.

[0405] Example 20 Synthesis of Compound 11

[0406] Compound 11 was synthesized in a similar manner to step 3) of Example 7. From compound intermediate II-11 (140 mg, 0.28 mmol) and 9-heptadecanyl-8-oxooctanoate (146 mg), 96 mg of compound 5 was obtained in a yield of 42.5%.

[0407] 1H NMR (600MHz, CDCl3) δ5.37-5.29(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),2.2 9-2.25(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).

[0408] LC-MS: 847.3[M+H]+.

[0409] Example 21 Synthesis of Compound 18

[0410] Compound 18 was synthesized in a similar manner to step 3) of Example 7. From compound intermediate II-18 (120 mg, 0.31 mmol) and 9-heptadecan-8-oxooctanoate (185 mg), 177 mg of compound 18 was obtained in a yield of 74.4%.

[0411] 1H NMR(600MHz, CDCl3)δ5.04-5.02(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=6.8Hz,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).

[0412] LC-MS: 767.3[M+H]+.

[0413] Example 22 Synthesis of Compound 21

[0414] Compound 21 was synthesized in a similar manner to step 3) of Example 7. From compound intermediate II-21 (400 mg, 0.86 mmol) and 9-heptadecan-8-oxooctanoate (510 mg), 100 mg of compound 18 was obtained in a yield of 13.8%.

[0415] 1H NMR (600MHz, CDCl3) δ4.90-4.81(m,1H),4.06-4.04(t,J=6.8Hz,2H),3.26-3.22(dq,J=8.9,6 .9Hz,2H),2.66-2.55(m,1H),2.53-2.35(m,4H),2.30-2.26(q,J=7.5Hz,4H),2.12-2.04(m,1 H),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=6.0Hz,3H),0.88-0.86(m,9H).

[0416] LC-MS: 848.3 [M+H] +.

[0417] Example 23 Synthesis of Compound 22

[0418] Compound 22 was synthesized in a similar manner to step 3) of Example 7. From compound intermediate II-22 (300 mg, 0.71 mmol) and 9-heptadecan-8-oxooctanoate (422 mg), 266 mg of compound 18 was obtained in a yield of 46.8%.

[0419] 1H NMR (600MHz, CDCl3) δ4.884.80 (m, 1H), 4.03-3.98 (t, J = 6.8Hz, 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).

[0420] LC-MS: 806.1[M+H]+.

[0421] Comparative Example:

[0422] Comparative Example 1: Synthesis of Comparative Compound 1

[0423] Step 1): Synthesis of 8-((2-hydroxycyclopentyl)amino)nonyl octanoate

[0424] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.50 g, 10 mmol), 2-aminocyclopentanol (10.12 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 8-((2-hydroxycyclopentyl)amino)octanoic acid nonyl ester (2.29 g, 62%).

[0425] 1H NMR(600MHz,CDCl3)δ4.07(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).

[0426] LCMS: 370.3 [M+H] +.

[0427] Step 2): Synthesis of comparative compound 1

[0428] Comparative Compound 1 was synthesized in a similar manner to Step 3) of Example 1, except that the intermediate compound 8-((2-hydroxycyclopentyl)amino)nonyloctanoate was used in place of the original intermediate compound 8-((3-hydroxycyclobutyl)amino)nonyloctanoate.

[0429] 1H NMR (600MHz, CDCl3) δ4.91-4.80(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).

[0430] LCMS: 750.9 [M+H] +.

[0431] Comparative Example 2: Synthesis of Comparative Compound 2

[0432] Comparative Compound 2

[0433] Comparative compound 2 was prepared according to the synthesis method of compound 22 described in Chinese patent CN110520409A.

[0434] 1H NMR (600MHz, 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).

[0435] LCMS: 765.0 [M+H] +.

[0436] Comparative Example 3: Synthesis of Comparative Compound 3

[0437] Step 1): Synthesis of 6-((2-hydroxycyclohexyl)amino)hexanoic acid undecyl ester

[0438] In a 100 mL reaction flask, undecyl 6-bromohexanoate (3.50 g, 10 mmol), 2-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain undecyl 6-((2-hydroxycyclohexyl)amino)hexanoate (2.57 g, 67%).

[0439] 1H NMR (600MHz, CDCl3) δ4.05(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).

[0440] LCMS: 384.4 [M+H] +.

[0441] Step 2): Synthesis of comparative compound 3

[0442] In a 100 mL reaction flask, 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 in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The mixture was reacted at room temperature for 24 h, and 100 mL of dichloromethane was added. After washing with water three times, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain comparative compound 3 (550 mg, 72%).

[0443] 1H NMR(600MHz, CDCl3)δ4.86(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).

[0444] LCMS: 765.0 [M+H] +.

[0445] Comparative Example 4: Synthesis of Comparative Compound 4

[0446] In a 250 mL reaction flask, 7-pentadecyl 6-oxohexanoate (3.40 g, 10 mmol), 100 mL of dichloroethane, and sodium triacetoxyborohydride (3.18 g, 15 mmol) were added in sequence, and then 2-aminocyclohexanol (0.57 g, 5 mmol) was added. The reaction was carried out at room temperature for 24 h. After washing with water three times, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain comparative compound 4 (2.67 g, 70%).

[0447] 1H NMR (600MHz, CDCl3) δ4.91-4.80(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).

[0448] LCMS: 765.2 [M+H] +.

[0449] Comparative Example 5: Synthesis of Comparative Compound 5 (SM102)

[0450] Comparative compound 5 was prepared according to the synthesis method of compound 25 described in Chinese patent CN110520409A.

[0451] 1H NMR (600MHz, CDCl3) δ4.86-4.74(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).

[0452] LCMS: 711.0 [M+H]+.

[0453] Comparative Example 6: Synthesis of Comparative Compound 6

[0454] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.50 g, 10 mmol), p-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 8-((4-hydroxycyclohexyl)amino)hexanoic acid nonyl ester (2.65 g, 69%).

[0455] 1H NMR (600MHz, CDCl3) δ4.05(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).

[0456] LCMS: 384.4 [M+H] +.

[0457] Step 2): Synthesis of comparative compound 6

[0458] In a 100 mL reaction flask, 8-((4-hydroxycyclohexyl)amino)hexanoic acid nonyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octylnonyl ester (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The mixture was reacted at room temperature for 24 h, and 100 mL of dichloromethane was added. After washing with water three times, it was dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain comparative compound 6 (535 mg, 70%).

[0459] 1H NMR(600MHz, CDCl3)δ4.90-4.82(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).

[0460] LCMS: 765.0 [M+H] +.

[0461] Comparative Example 7: Synthesis of Comparative Compound 7

[0462] Step 1): Synthesis of 6-((4-hydroxycyclohexyl)amino)hexanoic acid undecyl ester

[0463] In a 100 mL reaction flask, undecyl 6-bromohexanoate (3.50 g, 10 mmol), p-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain undecyl 6-((4-hydroxycyclohexyl)amino)hexanoate (2.57 g, 67%).

[0464] 1H NMR (600MHz, CDCl3) δ4.05(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).

[0465] LCMS: 384.4 [M+H] +.

[0466] Step 2): Synthesis of comparative compound 7

[0467] To a 100 mL reaction flask, 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 in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The reaction was carried out at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times and dried over anhydrous sodium sulfate. After concentration, it was purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain comparative compound 7 (550 mg, 72%).

[0468] 1H NMR (600MHz, CDCl3) δ4.93-4.83(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).

[0469] LCMS: 765.2 [M+H] +.

[0470] Biological tests

[0471] The structures of Lipids used in the biological experiments disclosed herein: MC3, SM102, and ALC-0315 are shown below:

[0472] Among them, MC3 and ALC-0315 are commercially available or can be prepared according to techniques known in the art.

[0473] Preparation of lipid nanoparticles:

[0474] Preparation Method 1: The amino lipid compound described in this disclosure was mixed with DOPE, cholesterol, and PEG2000-DMG at a molar ratio of 45:10:42.5:2.5 and dissolved in anhydrous ethanol. Using a microsyringe pump, the mixture was adjusted to a 1:3 ratio of ethanol solution to sodium acetate solution (50 mM, pH 4.0). A crude lipid nanoparticle solution was prepared in a microfluidic chip. The solution was then dialyzed against 1X PBS at 4°C for 6 h using a dialysis cassette (Fisher, MWCO 20,000). The solution was then filtered through a 0.22 μm microporous membrane before use.

[0475] Preparation method 2: The molar ratio of the amino lipid compound to DSPC, cholesterol, and PEG2000-DMG is 50:10:38.5:1.5, and the preparation method is the same as method 1.

[0476] The lipid nanoparticles prepared according to the above-mentioned preparation methods 1 or 2 were used in the biological experiments described below to evaluate the in vivo delivery performance of luciferase mRNA (Fluc mRNA). The lipid nanoparticles obtained in the preparation method 1 had a mass ratio of the amino lipid compound to the luciferase mRNA of approximately 10:1 and were administered subcutaneously; the lipid nanoparticles obtained in the preparation method 2 were administered via the tail vein and intramuscularly.

[0477] Experimental Example 1: Evaluation of the in vivo delivery performance of luciferase mRNA using lipid nanoparticles prepared from the amino lipid compounds described in the present disclosure

[0478] Animal preparation: Six-week-old female BALB / c mice weighing approximately 20 g were selected and housed in an SPF-grade enclosure. Animal experiments were conducted in strict accordance with the guidelines of national health agencies and animal ethics requirements.

[0479] In vivo delivery: Nine mice were randomly selected from each group and injected with lipid nanoparticles at a dose of 0.5 mg / kg using three different routes: subcutaneous, intramuscular, and tail vein injection (three mice per route). Twelve hours later, each mouse was injected with 200 μL of 10 mg / mL D-luciferin potassium salt via the tail vein. Ten minutes later, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed and photographed. The expression intensity of Fluc mRNA delivered by representative amino lipid compounds using the three administration routes is shown in Tables 2-4. MC3 served as a control. SM102 served as a comparative compound.

[0480] Table 2: Expression intensity of Fluc mRNA delivered by subcutaneous administration of representative amino lipid compounds.

[0481] Table 3: Expression intensity of Fluc mRNA delivered by intramuscular injection of representative amino lipid compounds.

[0482] Table 4: Expression intensity of Fluc mRNA delivered by tail vein administration of representative amino lipid compounds.

[0483] Experimental Example 2: In vivo delivery of ovalbumin mRNA and immune performance evaluation of lipid nanoparticles prepared from the amino lipid compound described in the present disclosure

[0484] Preparation of lipid nanoparticles:

[0485] Preparation method: The amino lipid compound of formula (I) disclosed herein was mixed with DOPE, cholesterol, and PEG2000-DMG at a molar ratio of 50:10:38.5:1.5 and dissolved in anhydrous ethanol. Ovalbumin mRNA (OVA mRNA) was dissolved in a 50 mM sodium acetate solution (pH = 4.0). Using a microsyringe pump, the ratio of ethanol solution to sodium acetate solution (50 mM, pH = 4.0) was controlled to be 1:3. A crude lipid nanoparticle solution was prepared in a microfluidic chip. The solution was then dialyzed in 1X PBS at 4°C for 6 h using a dialysis cassette (Fisher, MWCO 20,000). The solution was then filtered through a 0.22 μm microporous filter membrane before use.

[0486] The mass ratio of the amino lipid compound to ovalbumin mRNA (OVA mRNA) in the obtained lipid nanoparticles was approximately 10:1.

[0487] Animal preparation: Six-week-old female BALB / c mice weighing approximately 20 g were selected and housed in an SPF-grade enclosure. Animal experiments were conducted in strict accordance with the guidelines of national health agencies and animal ethics requirements.

[0488] In vivo delivery: Three mice were randomly selected from each group and injected intramuscularly with lipid nanoparticles at a dose of 0.5 mg / kg (Day 0). Seven days later, a booster injection with the same dose was performed (Day 7). Serological analysis was performed by collecting blood from the tail vein on Day 21. MC3 mice served as a control.

[0489] Enzyme-linked immunosorbent assay (ELISA): Flat-bottom 96-well plates (Nunc) were precoated with 0.5 μg of OVA protein per well in 50 mM carbonate buffer (pH 9.6) overnight at 4°C and then blocked with 5% glycine. Antisera obtained from immunized animals were diluted from 102 to 106 protein in PBS-0.05% Tween (PBS-T), pH 7.4, added to the wells and incubated at 37°C for 1 hour. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was labeled at a dilution of 1:10,000 in PBS-T-1% BSA. After addition of the HRP substrate, the optical density was determined at a wavelength of 450 nm using an ELISA plate reader (Bio-Rad).

[0490] As shown in Figure 1, Lipid2 (Compound 2) produced comparable IgG antibody titers to MC3 and SM102, while the other compounds produced significantly higher IgG antibody titers than the control group. It should be noted that Lipid7 (Compound 7) produced protein at levels comparable to SM102, yet evoked a stronger immune response.

[0491] It can be seen that the amino lipid compound provided by the present disclosure has excellent immunological activity and a strong adjuvant effect.

[0492] Experimental Example 3: In vivo delivery and immune performance evaluation of influenza mRNA vaccine using lipid nanoparticles prepared from the amino lipid compound described in the present disclosure

[0493] Preparation method: The amino lipid compound of formula (I) disclosed herein is mixed with DSPC, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5 and dissolved in anhydrous ethanol. The influenza mRNA is dissolved in a sodium acetate solution (50mM, pH = 4.0). Using a microinjection pump, the ratio of ethanol solution to sodium acetate solution (50mM, pH = 4.0) is controlled to be 1:3. A crude solution of lipid nanoparticles is prepared in a microfluidic chip, and then dialyzed using a dialysis cassette (Fisher, MWCO 20,000) in 1X PBS at 4°C for 6 hours. Filter with a 0.22μm microporous membrane before use.

[0494] The mass ratio of amino lipid compound to influenza mRNA (OVA mRNA) in the obtained lipid nanoparticles was approximately 10:1.

[0495] Animal preparation: Six-week-old female BALB / c mice weighing approximately 20 g were selected and housed in an SPF-grade enclosure. Animal experiments were conducted in strict accordance with the guidelines of national health agencies and animal ethics requirements.

[0496] In vivo delivery: Three mice were randomly selected from each group and administered lipid nanoparticles subcutaneously on the back at a dose of 0.5 mg / kg (Day 0). Seven days later, a booster dose of the same dose was administered (Day 7). Serological analysis was performed by collecting blood from the tail vein on Day 21. MC3 mice served as a control.

[0497] Enzyme-linked immunosorbent assay (ELISA): The assay method is the same as in Experimental Example 2.

[0498] The test results are shown in Figure 2, where the IgG antibody titer of the MC3 control group was the lowest, the IgG antibody titer produced by Lipid10 (Compound 10) and SM102 was comparable, and the IgG antibody titer of other compounds was significantly better than that of the control group, which also shows that the amino lipid compounds provided by the present disclosure have excellent immune activity and a strong adjuvant effect.

[0499] Experimental Example 4: Evaluation of the Stability of Lipid Nanoparticles Prepared Using the Amino Lipid Compound Described in the Present Disclosure Using Formulation Method 1

[0500] Preparation of lipid nanoparticles: LNPs were prepared using formulation method 1 for subcutaneous administration.

[0501] 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 for particle size measurement and the reaction was repeated three times, with each cycle lasting 30 seconds.

[0502] The particle size was measured on the day of preparation (week 0), after storage at 25°C for one week (week 1), two weeks (week 2), and four weeks (week 4). The expression intensity of Fluc mRNA delivered subcutaneously was measured on the day of preparation (week 0) and four weeks (week 4). The test results are shown in Table 5.

[0503] Table 5: DLS characterization and subcutaneous expression of Fluc-mRNA of LNPs prepared from representative amino lipid compounds of the present disclosure.

[0504] As can be seen from Table 5, the test temperature was 25°C. In terms of particle size, on the same day (week 0), the representative compounds 5, 6, 7, 9 and SM102 of the present disclosure were all relatively uniform nanoparticles with a particle size of about 100 nm and a PDI of less than 0.1. After four weeks of storage, the particle size increased. Among them, the particle size of SM102 more than doubled from 120 to 245, while the particle size of the representative compounds 5, 6, 7, and 9 of the present disclosure did not show a significant increase. In terms of PDI, after four weeks, the PDI of the representative compounds 5, 6, 7, and 9 of the present disclosure were all less than 0.1, while the PDI of SM102 increased from 0.05 on the same day (week 0) to 0.1 on the same day (week 1). 4) 0.28; In terms of Fluc expression, after four weeks, the Fluc expression of the representative compounds 5, 6, 7, and 9 of the present disclosure did not decrease significantly, while the Fluc expression of SM102 decreased significantly from 2.1E+07 to 3.6E+06. This shows that the amino lipid compounds described in the present disclosure have excellent stability.

[0505] Experimental Example 5: Evaluation of the activity performance of lipid nanoparticles prepared using the amino lipid compound described in the present disclosure using the second formulation method

[0506] Preparation of lipid nanoparticles: LNPs were prepared using formulation method 2 for intramuscular administration.

[0507] The test method is the same as that of Test Example 1. The test results are shown in Table 6:

[0508] Table 6: Expression intensity of Fluc mRNA delivered by intramuscular injection of representative amino lipid compounds of the present disclosure.

[0509] As can be seen from Table 6, when other structural units are the same, the expression intensity of Fluc mRNA is higher when the hydroxyl group on the cycloalkyl structural unit is located at the meta position of the amine substituent compared to when the hydroxyl group on the cycloalkyl structural unit is located at other substitution positions of the amine substituent. Specifically, for example, the expression intensity of Fluc mRNA of representative compound 6 of the present disclosure is 1.2E+08; in contrast, the expression intensity of Fluc mRNA of 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) is 2.8E+06; further, the expression intensity of Fluc mRNA of 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) is 1.9E+07. The same conclusion can be seen, for example, between representative compound 7 of the present disclosure and comparative compound 3 and comparative compound 7, and between representative compound 8 of the present disclosure and comparative compound 4 and comparative compound 8, etc. This shows that, when other structural units are the same, lipid nanoparticles containing amino lipid compounds in which the substituent on the cycloalkyl structural unit is located at the meta position of the amine substituent have the highest activity performance.

[0510] Test Example 6: Targeting Test of Aminolipid Compounds

[0511] Preparation of mRNA lipid nanoparticle (LNP) formulations: Lipid was mixed with DSPC, CHO-HP, and M-DMG2000 (i.e., PEG2000-DMG) at a molar ratio of 50:10:38.5:1.5 and dissolved in anhydrous ethanol. mRNA was dissolved in sodium acetate solution (0.2 M, pH 5.0). Each formulation was prepared using a microfluidic device (MPE-L2) and chip (SN.000035) at a flow rate of 9 ml / min for the aqueous phase and 3 ml / min for the alcohol phase. The two phases were injected into the microfluidic chip according to the interface requirements and mixed. The drug solutions were then loaded into 100 kDa dialysis bags and immersed in a beaker containing 1 L of dialysis solution. The beaker was wrapped with aluminum foil and dialyzed at 100 rpm for 1 hour at room temperature. The dialysate was then replaced and dialysis continued for another hour.

[0512] Dialysate preparation: 1×PBS + 8% sucrose solution: Take 2 packets of 1×PBS powder injection and place them in a beaker. Dissolve and mix thoroughly with 2L of DEPC water. Add 160g of sucrose and mix thoroughly to obtain 1×PBS + 8% sucrose solution.

[0513] The mass ratio of mRNA:Lipid in the obtained lipid nanoparticles was approximately 1:10.

[0514] The preparation method of the aforementioned mRNA lipid nanoparticle (LNP) preparation is adopted, wherein the mRNA is selected as mRNA encoding fluorescent protein, and the Lipid is selected as compound (I-6-II), MC3 and SM102, respectively, to prepare mRNA-LNP preparations encoding fluorescent protein containing compound (I-6-II), MC3 or SM102, respectively.

[0515] Test method:

[0516] Each test preparation was administered to mice by intramuscular injection, with each animal receiving 15ug of mRNA preparation sample. Six hours after sample injection, the mice were anesthetized using isoflurane inhalation anesthesia and intraperitoneally injected with 200μL of D-Luciferin (10mg / ml) luciferase development substrate. The animals were placed in a supine position, and 10 minutes after substrate injection, the signal distribution and expression intensity of luciferase in the mice were observed under the IVIS in vivo imaging system. After in vivo imaging of the animals injected intramuscularly, they were immediately dissected, and the fluorescence of the liver was quickly observed after dissection.

[0517] The specific fluorescence intensity values ​​are as follows:

[0518] mRNA-LNP formulation encoding fluorescent protein containing MC3: 1.23E+05

[0519] mRNA-LNP formulation encoding fluorescent protein containing SM102: 3.23E+06

[0520] mRNA-LNP formulation encoding fluorescent protein containing compound (I-6-II): 2.49E+05

[0521] The test results are shown in Figure 8. When administered by intramuscular injection, the liver fluorescence intensity of the compound (I-6-II) group was significantly lower than that of SM102. The organ distribution of the mRNA encoding the fluorescent protein in the liver, an important organ, was relatively low, showing a good local targeting effect and effectively reducing the risk of liver toxicity.

[0522] Test Example 7 Adverse reaction test of amino lipid compound intramuscular injection

[0523] Preparation of mRNA-LNP preparation encoding novel coronavirus S protein:

[0524] The preparation method of the mRNA lipid nanoparticle (LNP) preparation in Experimental Example 6 was adopted, wherein the mRNA was selected to encode the novel coronavirus S protein mRNA, and Lipid was selected from compound 6, compound (I-6-II), ALC-0315, and SM102, respectively, to prepare mRNA-LNP preparations encoding the novel coronavirus S protein containing compound 6, compound (I-6-II), ALC-0315 or SM102, respectively.

[0525] The sequence encoding the novel coronavirus S protein mRNA is obtained by replacing all uracil (u) in SEQ ID NO.1 with N1-methylpseudouridine. It should be noted that according to WIPO Standard ST.26 for nucleotide or amino acid sequence listings, the t (thymine) in the RNA sequence SEQ ID NO.1 in the sequence listing is actually u (uracil).

[0526] Test method:

[0527] Sprague-Dawley rats were randomly divided into four groups, half male and half female, to receive a high-dose compound 6 group (100 μg mRNA / rat), a high-dose compound (I-6-II) group (100 μg mRNA / rat), a high-dose ALC-0315 group (the cationic lipid in the Pfizer vaccine BNT162-b2) group (100 μg mRNA / rat), and a high-dose SM-102 group (the cationic lipid in the Moderna vaccine mRNA-1273) group (100 μg mRNA / rat), with three rats per sex per group. The rats were administered intramuscularly once weekly for three consecutive weeks. Abnormalities such as swelling at the injection site were observed clinically.

[0528] The test results are shown in Figure 9. As can be seen from Figure 9, compared with the cationic lipid component of the same level of mRNA vaccine already on the market, compound (I-6-II) has a significant reduction or decrease in the number of severe swelling and moderate lameness at the injection site, and can therefore be considered to have higher safety.

[0529] Test Example 8 Toxicokinetics

[0530] Preparation of empty liposome preparation:

[0531] 1.9078g of compound I-6-II, 0.3985g of DSPC, 0.7575g of CHO-HP, and 0.1972g of PEG-DMG (average molecular weight, 2000) were weighed in a molar ratio of 49.5:10:39:1.5, dissolved in anhydrous ethanol, and the volume was adjusted to 237.5ml to obtain an alcohol phase. Hybrid encapsulation was performed using a microfluidic device (MPE-L2) with acetic acid-sodium acetate buffer (0.2mol / L, pH=5) as the aqueous phase and a volume ratio of 3:1 between the aqueous phase and the alcohol phase. The encapsulated drug solution was ultrafiltered at a TMP of 0.2 bar and an inlet flow rate of 300 ml / min. The dialysate used for the exchange contained 8 mg / ml sodium chloride, 0.2 mg / ml potassium chloride, 0.2 mg / ml potassium dihydrogen phosphate, 1.15 mg / ml sodium dihydrogen phosphate dihydrate, and 80 mg / ml sucrose. After exchange, the solution was sterile filtered to obtain the I-6-II empty liposome preparation.

[0532] Test method:

[0533] A total of 5 groups (5 animals / sex / group) were included in the animal experiment. The groups and dosages were: negative control group (0 doses / animal), empty liposome low-dose group (1 dose / animal) and empty liposome high-dose group (4 doses / animal). Each group of animals was given intramuscular injection once every 2 weeks, for a total of 3 doses, namely D1 (the day of the first dose), D15 (the 15th day after the dose), and D29 (the 29th day after the dose).

[0534] Five groups (5 animals / sex / group) were included in the animal experiment. The groups and dosages were as follows: negative control group (0 μg compound I-6-II / animal), empty liposome low-dose group (300 μg compound I-6-II / animal) and empty liposome high-dose group (1200 μg compound I-6-II / animal). Each group of animals was administered intramuscularly once every two weeks, for a total of three times, namely, on D1, D15 and D29.

[0535] Blood samples were collected from the negative control group before the first (D1) and final (D29) doses and 4 hours after the drug administration. Blood samples were collected from the empty liposome group before the first (D1) and final (D29) doses and 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 32 hours, and 48 hours after the drug administration. The plasma levels of compound I-6-II were measured in cynomolgus monkeys to investigate the exposure of compound I-6-II to the cynomolgus monkeys after drug administration.

[0536] The main TK parameters of compound I-6-II in the animals of the empty liposome low- and high-dose groups after the first administration (D1) and the last administration (D29) are shown in Table 7 below:

[0537] Table 7 Main TK parameters of compound I-6-II in animals of empty liposome low and high dose groups

[0538] Test data show that:

[0539] No I-6-II liposomes were detected in the first (D1) and last (D29) plasma samples of animals in the negative control group.

[0540] As can be seen from Table 7, compound I-6-II can be rapidly cleared in cynomolgus monkeys, and its plasma drug concentration half-life is 4.68-6.45 hours; compared with the first dose (D1) of the animals after the last dose (D29), the Cmax ratio (D29 / D1) of compound I-6-II in each empty liposome group was between 0.58 and 0.84, and the AUClast ratio (D29 / D1) was between 0.68 and 1.02, indicating that after 4 weeks of continuous administration (3 times in total), compound I-6-II did not accumulate in cynomolgus monkeys.

[0541] In summary, the amino lipid compound provided by the present disclosure, which has a cycloalkyl structural unit and further has an additional substituent at the meta position of the amine substituent of the cycloalkyl group, can be used to deliver bioactive agents into cells. Compared with the amino lipid compounds of similar structures known in the prior art, it has one or more of the following advantages: good delivery ability, good stability, and high safety. It can be used to deliver bioactive agents (such as nucleic acids) into cells, increase their protein expression levels, and more effectively induce the body to produce an immune response; it has excellent targeting; and can be stored, transported, and used under room temperature conditions.

[0542] List of abbreviations

[0543] DIPEA N,N-Diisopropylethylamine

[0544] DNA deoxyribonucleic acid

[0545] RNA ribonucleic acid

[0546] DOPE Dioleoylphosphatidylethanolamine

[0547] DSPC Distearoylphosphatidylcholine

[0548] PEG2000-DMG 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000

[0549] kD kilodaltons

[0550] PBS Phosphate-buffered saline.

[0551] It will be apparent to those skilled in the art that the present disclosure is not limited to the foregoing illustrative embodiments and that it may be embodied in other specific forms without departing from the essential characteristics of the present disclosure. It is therefore intended that these embodiments be considered in all respects as illustrative and non-restrictive, and that reference should be made to the appended claims rather than to the foregoing embodiments, and that all variations coming within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. A compound represented by the following structural formula I or a pharmaceutically acceptable salt or stereoisomer thereof: in: L 1 and L 2 The same or different, each independently selected from C1-C 12 Alkylene, C2-C 12 Alkenylene or C 2- C 12 Alkyne; preferably, L 1 and L 2 The same or different, each independently selected from C3-C 10 Alkylene, C3-C 10 Alkenylene or C 3- C 10 Alkyne; further preferably, L 1 and L 2 The same or different, each independently selected from C3-C 10 Alkylene; most preferably, L 1 and L 2 The same or different, each independently selected from C5-C8 alkylene; G 1 or G 2 The same or different, each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S-, -SC(=O)-; preferably, G 1 or G 2 The same or different, each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-; most preferably, G 1 or G 2 the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-; R 1 and R 2 The same or different, each independently selected from C5-C 27 Alkyl, C5-C 27 Alkenyl; preferably, R 1 and R 2 The same or different, each independently selected from C8-C 20 Alkyl or C8-C 20 Alkenyl; further preferably, R 1 and R 2 The same or different, each independently selected from C9-C 17 Alkyl or C9-C 18 Alkenyl; most preferably, R 1 and R 2 The same or different, each independently selected from R 3 is selected from halogen, hydroxy, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; preferably, R 3 is selected from halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; further preferably, R 3 is selected from halogen, hydroxy, 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.

2. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 and L 2 The same or different, each independently selected from C3-C 10 Alkylene, C3-C 10 Alkenylene or C 3- C 10 Alkynylidene; G 1 or G 2 the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-; R 1 and R 2 The same or different, each independently selected from C8-C 20 Alkyl or C8-C 20 alkenyl; R 3 is selected from halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; n is selected from 1, 2, and 3.

3. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 and L 2 The same or different, each independently selected from C3-C 10 alkylene; G 1 or G 2 the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-; R 1 and R 2 The same or different, each independently selected from C9-C 17 Alkyl or C9-C 18 alkenyl; R 3 is selected from halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 alkylcarbonylamino; n is selected from 1, 2, and 3.

4. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 and L 2 The same or different, each independently selected from C5-C8 alkylene; G 1 or G 2 the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-; R 1 and R 2 The same or different, each independently selected from R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido; n is selected from 1, 2, and 3.

5. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is selected from:

6. A compound having the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

7. A compound having a structure selected from the group consisting of:

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

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

10. A pharmaceutical composition comprising the 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 the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt or stereoisomer thereof, the lipid nanoparticle according to claim 9 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy or treatment by interfering RNA.

12. The use according to claim 11, wherein the gene therapy can be used for the treatment of cancer and genetic diseases.

13. The method according to claim 12, wherein the cancer is selected from one or more of 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 selected from one or more of hemophilia, thalassemia and Gaucher disease.

14. The use according to claim 11, wherein the genetic vaccination is used to treat cancer, allergy, toxicity and pathogen infection.

15. The use according to claim 14, wherein the pathogen is selected from one or more of viruses, bacteria or fungi.

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

17. A compound represented by the following structural formula II or a pharmaceutically acceptable salt or stereoisomer thereof: in: L 1 Selected from C1-C 12 Alkylene, C2-C 12 Alkenylene or C 2- C 12 Alkyne; preferably, L 1 Selected from C3-C 10 Alkylene, C3-C 10 Alkenylene or C 3- C 10 Alkyne; further preferably, L 1 Selected from C3-C 10 Alkylene; most preferably, L 1 Selected from C5-C8 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 Selected from -O-(C=O)-, -(C=O)-O-; R 1 Selected from C5-C 27 Alkyl, C5-C 27 Alkenyl; preferably, R 1 Selected from C8-C 20 Alkyl, C8-C 20 Alkenyl; further preferably, R 1 Selected from C9-C 17 Alkyl, C9-C 18 Alkenyl; most preferably, R 1 Selected from R 3 Selected from halogen, hydroxy, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonyl amino group; preferably, R 3 is selected from halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; further preferably, R 3 is selected from halogen, hydroxy, 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.

18. The compound according to claim 17 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 Selected from C3-C 10 Alkylene, C3-C 10 Alkenylene or C 3- C 10 Alkynylidene; G 1 Selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-; R 1 Selected from C8-C 20 Alkyl, C8-C 20 alkenyl; R 3 is selected from halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl, C1-C6 alkylcarbonylamino; n is selected from 1, 2, and 3.

19. The compound according to claim 17 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 Selected from C3-C 10 alkylene; G 1 Selected from -O-(C=O)-, -(C=O)-O-; R 1 Selected from C9-C 17 Alkyl, C9-C 18 alkenyl; R 3 is selected from halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 alkylcarbonylamino; n is selected from 1, 2, and 3.

20. The compound according to claim 17, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 Selected from C5-C8 alkylene; G 1 Selected from -O-(C=O)-, -(C=O)-O-; R 1 Selected from R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetamido; n is selected from 1, 2, and 3.

21. The compound according to claim 17 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is selected from:

22. The compound according to claim 17 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

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

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