Amino lipid compounds, methods of making and using the same

Abstract

The present application relates to an aminolipid compound having the following structural Formula I or a pharmaceutically acceptable salt or stereoisomer thereof, and its use as a component of a lipid nanoparticle formulation for the delivery of a therapeutic agent. The present application also relates to compositions, in particular lipid nanoparticles, comprising said aminolipid compound, and their use.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and particularly relates to an amino lipid compound, a preparation method and application thereof. BACKGROUND

[0002] Gene drugs are to deliver genes with specific genetic information to target cells by artificial means, and the expressed target proteins have the effect of adjusting, treating or even curing the diseases caused by congenital or acquired genetic defects, or the gene series can interfere or regulate the expression of related genes to achieve clinical therapeutic effect. Nucleic acids and cell membranes both have negative charges, and naked nucleic acids are difficult to be directly introduced into cells and are easily degraded by nucleic acid degradation enzymes in the cytoplasm, so that the effect of gene introduction and gene therapy cannot be achieved, and therefore external force or carriers are needed to realize gene delivery.

[0003] Gene vectors are generally divided into viral vectors and non-viral vectors. Viral vectors have high transfection efficiency in vivo and in vitro, but they also have many defects, such as high toxicity, strong immune response, small gene capacity, poor targeting, complex preparation process, etc. Non-viral vectors have attracted more and more companies to invest in research and development due to their easy preparation, transportation, storage, safety, effectiveness, and non-immunogenicity.

[0004] Lipid nanoparticles (LNP) are currently the mainstream non-viral vectors, and are often used in vaccines because they are easily absorbed by antigen-presenting cells. In the prior art, a variety of compounds for lipid nanoparticles have been reported, but in actual use, it is found that when these compounds are used for vaccine delivery, the delivery efficiency is low, the drug stability is poor, and there are also problems of long elimination phase half-life, high toxicity, low safety, etc., which are not conducive to clinical application and cannot meet the needs of modern vaccine preparations.

[0005] For example, the commercially available cationic liposome compound MC3 has insufficient protein expression level and low antibody production level when actually used for vaccine delivery. In addition, the literature "Pharmacokinetics of Patisiran, the First Approved RNA Interference Therapy in Patients With Hereditary Transthyretin-Mediated Amyloidosis" Xiaoping Zhang et al., The Journal of Clinical Pharmacology 2020, 60(5) 573-585, reports that the elimination phase half-life of Patisiran in the clinical phase II study is as long as 14.6 to 28.7 days.

[0006]

[0007] Therefore, it is urgent to develop a substance having a strong delivery capacity, good stability, a moderate elimination half-life of the phase and / or high safety (i.e. a low incidence of adverse reactions) for use in intracellular delivery of a therapeutic agent. SUMMARY

[0008] Through extensive research, the inventors of the present application surprisingly found that an amino lipid compound having a cycloalkyl structural unit and further having another substituent at the meta position of the amine substituent of the cycloalkyl group achieves the following functions: good delivery capacity, good stability, high safety, and can be used to deliver a bioactive agent (e.g. a nucleic acid) into a cell to increase the protein expression level thereof.

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

[0010] The cycloalkyl structural unit and the other substituent at the meta position of the amine substituent of the cycloalkyl group in the amino lipid compound according to the present application allow the compound or a lipid nanoparticle containing the same to have good bioactivity, a high protein expression level, significant immunological activity, and good stability when used as a vaccine carrier, and can be stored, transported and used at room temperature with a low incidence of adverse reactions.

[0011] The present application 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 requirements for equipment and simple operation.

[0012] The present application also provides a composition, such as a lipid nanoparticle, comprising the amino lipid compound or a pharmaceutically acceptable salt or stereoisomer thereof.

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

[0014]

[0015] wherein:

[0016] L 1 and L 2 are the same or different, each independently selected from C1-C 12 alkylene, C2-C 12 alkenylene or C2- C 12 alkynylene; preferably, L 1 and L 2 are the same or different, each independently selected from C3-C 10 alkylene, C3-C 10 alkenylene or C 3- C 10 alkynylene; further preferably, L 1 and L 2 are the same or different, each independently selected from C3-C 10 alkylene; most preferably, L 1 and L 2 are the same or different, each independently selected from C5-C8alkylene;

[0017] G 1 or G 2 are the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S-, -S-C(=O)-; preferably, G 1 or G 2 are the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-; most preferably, G 1 or G 2 are the same or different, each independently selected from -O-(C=O)-, -(C=O)-O-;

[0018] R 1 and R 2 are the same or different, each independently selected from C5-C 27 alkyl attached via any one of the carbons, C5-C 27 alkenyl containing one or more double bonds; preferably, R 1 and R 2 are the same or different, each independently selected from C8-C 20 alkyl attached via any one of the carbons, C8-C 20 alkenyl containing one or more double bonds; further preferably, R 1 and R 2 are the same or different, each independently selected from C9-C 17 alkyl attached via any one of the carbons, C9-C 18 alkenyl containing one or two double bonds; most preferably, R 1 and R 2 are the same or different, each independently selected from

[0019]

[0020] R3 selected from halogen, hydroxy, cyano, Ci-C6alkyl, nitro, Ci-C6alkyloxy, Ci-C6alkylcarbonyloxy, Ci-C6alkyloxycarbonyl, Ci-C6alkylaminocarbonyl, Ci-C6alkylcarbonylamino; preferably, R 3 selected from halogen, hydroxy, cyano, Ci-C6alkyloxy, Ci-C6alkylcarbonyloxy, Ci-C6alkyloxycarbonyl, Ci-C6alkylaminocarbonyl, Ci-C6alkylcarbonylamino; further preferably, R 3 selected from halogen, hydroxy, cyano, Ci-C4alkyloxy, Ci-C4alkylcarbonyloxy, Ci-C4alkyloxycarbonyl, Ci-C4alkylaminocarbonyl, Ci-C4alkylcarbonylamino; most preferably, R 3 selected from fluorine, hydroxy, cyano, methyloxy, acetyloxy, methyloxycarbonyl, butylaminocarbonyl and acetylamino;

[0021] n is selected from 1, 2, 3.

[0022] Further, the present application provides a compound represented by structural formula (I) as described above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0023] L 1 and L 2 are the same or different, each independently selected from C3-C 10 alkylene, C3-C 10 alkenylene or C 3- C 10 alkynylene;

[0024] G 1 or G 2 are the same or different, each independently selected from -0-(C=0)-, -(C=0)-0-, -C(=0)-, -0-;

[0025] R 1 and R 2 are the same or different, each independently selected from C8-C 20 alkyl attached through any one of the carbons, C8-C 20 alkenyl containing one or more double bonds;

[0026] R 3 selected from halogen, hydroxy, cyano, Ci-C6alkyloxy, Ci-C6alkylcarbonyloxy, Ci-C6alkyloxycarbonyl, Ci-C6alkylaminocarbonyl, Ci-C6alkylcarbonylamino;

[0027] n is selected from 1, 2, 3.

[0028] Further, the present application provides a compound represented by structural formula (I) as described above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0029] L 1 and L 2 are the same or different, each independently selected from C3-C 10 alkylene;

[0030] G 1 or G 2 are the same or different, each independently selected from -0-(C=0)-, -(C=0)-0-;

[0031] R 1 and R 2 are the same or different, each independently selected from C9-C 17 alkyl, C9-C 18 alkenyl containing one or two double bonds, connected through any one of the carbons;

[0032] R 3 is selected from halogen, hydroxy, cyano, C1-C4alkoxy, C1-C4alkylcarbonyloxy, C1-C4alkoxycarbonyl, C1-C4alkylaminocarbonyl, C1-C4alkylcarbonylamino;

[0033] n is selected from 1, 2, 3.

[0034] Further, the present application provides a compound represented by structural formula (I) as described above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0035] L 1 and L 2 are the same or different, each independently selected from C5-C8alkylene;

[0036] G 1 or G 2 are the same or different, each independently selected from -0-(C=0)-, -(C=0)-0-;

[0037] R 1 and R 2 are the same or different, each independently selected from

[0038]

[0039] R 3 is selected from fluoro, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetylamino; n is selected from 1, 2, 3.

[0040] Still further, the present application provides a compound represented by the following structural formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof:

[0041]

[0042]

[0043]

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

[0045]

[0046] Further, the present application provides a compound represented by the following structural formula (I) or a pharmaceutically acceptable salt thereof:

[0047]

[0048]

[0049] In another aspect, the present application provides a pharmaceutical composition containing a compound of the present application or a pharmaceutically acceptable salt or a stereoisomer thereof and a pharmaceutically acceptable carrier, diluent or excipient. Preferably, the composition is a lipid nanoparticle. Since the amino lipid compound of the present application or a pharmaceutically acceptable salt or a stereoisomer thereof contains a long non-polar residue, the resulting compound has a hydrophobic characteristic in its entirety, and has a hydrophilic characteristic due to the amino group at the same time. This amphiphilic characteristic can be used to form a lipid nanoparticle such as a lipid bilayer, a micelle, a liposome, etc.

[0050] In still another aspect, the present application provides use of the pharmaceutical composition, particularly a lipid nanoparticle, in the manufacture of a medicament for, for example, gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA. Preferably, the gene therapy is used for the treatment of cancer and genetic disease. The cancer is preferably selected from one or more of lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphatic cancer, blood cancer or prostate cancer; the genetic disease is preferably selected from one or more of hemophilia, thalassemia, Gaucher's disease. The gene vaccine is preferably used for the treatment of cancer, allergy, toxicity and pathogen infection. The pathogen is preferably selected from one or more of virus, bacteria or fungus.

[0051] In yet another aspect, the present application relates to the use of said compound or a pharmaceutically acceptable salt or stereoisomer thereof and a lipid nanoparticle containing said compound or a pharmaceutically acceptable salt or stereoisomer thereof for the preparation of a medicament for nucleic acid transfer. Preferably, said nucleic acid is selected from the group consisting of RNA, DNA, antisense oligonucleotide; preferably, said 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, said DNA is a plasmid.

[0052] In yet another aspect, the present application also provides a process for the preparation of the compound of formula (I) as described hereinbefore, the general procedure of which is as follows:

[0053] General procedure 1 :

[0054]

[0055] wherein,

[0056] n, L 1 , L 2 , G 1 , G 2 , R 1 , R 2 , R 3 have the same meaning as defined hereinbefore for the compound of formula (I) of the present application;

[0057] X is selected from the group consisting of halogen; preferably, X is selected from the group consisting of bromine;

[0058] In particular, the compound of formula (I) of the present application is obtained by performing a substitution reaction of intermediate compound (II) with intermediate compound (III) in an organic solvent at room temperature, in the presence or absence of an acid binding agent and an iodide.

[0059] wherein said organic solvent is selected from the group consisting of nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE); said acid binding agent is selected from the group consisting of organic bases, inorganic bases, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, DIPEA; said iodide is, for example, potassium iodide.

[0060] General procedure 2

[0061]

[0062] wherein,

[0063] n, L 1 , L 2 , G 1 , G2 , R 1 , R 2 , R 3 have the same meaning as defined previously for the compounds of formula (I) according to the application;

[0064] Y is (=0);

[0065] In particular, the compounds of formula (I) according to the application are obtained by submitting intermediate compound (II) to a reduction reaction with intermediate compound (V) in the presence of a reducing agent in an organic solvent at room temperature.

[0066] wherein the organic solvent is selected from nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE); and the reducing agent is selected from, for example, sodium triacetoxyborohydride.

[0067] Further, when the groups L 1 and L 2 are identical, R 1 and R 2 are identical, and G 1 and G 2 are identical, the compounds of formula (I) according to the application are prepared by the following general procedure 3:

[0068] General procedure 3

[0069]

[0070] wherein,

[0071] n, L 1 , L 2 , G 1 , G 2 , R 1 , R 2 , R 3 have the same meaning as defined previously for the compounds of formula (I) according to the application; and L = L 1 = L 2 , G = G 1 = G 2 , R = R 1 = R 2 ;

[0072] In particular, the compounds of formula (I) according to the application are obtained by submitting intermediate compound (IV) to a reduction reaction with intermediate compound (VI) in the presence of a reducing agent in an organic solvent at room temperature.

[0073] wherein said organic solvent is selected from nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE); said reducing agent is selected from, for example, sodium triacetoxyborohydride.

[0074] In yet another aspect, the present application also provides an intermediate compound (II) for the preparation of a compound of formula (I) of the present application, having the following structural formula:

[0075]

[0076] wherein:

[0077] L 1 is selected from C1-C 12 alkylene, C2-C 12 alkenylene or C 2- C 12 alkynylene; preferably, L 1 is selected from C3-C 10 alkylene, C3-C 10 alkenylene or C 3- C 10 alkynylene; further preferably, L 1 is selected from C3-C 10 alkylene; most preferably, L 1 is selected from C5-C8alkylene;

[0078] G 1 is selected from -0-(C=0)-, -(C=0)-0-, -C(=0)-, -0-, -C(=0)-S-, -S-C(=0)-; preferably, G 1 is selected from -0-(C=0)-, -(C=0)-0-, -C(=0)-, -0-; most preferably, G 1 is selected from -0-(C=0)-, -(C=0)-0-;

[0079] R 1 is selected from C5-C 27 alkyl, C5-C 27 alkenyl containing one or more double bonds; preferably, R 1 is selected from C8-C 20 alkyl, C8-C 20 alkenyl containing one or more double bonds; further preferably, R 1 is selected from C9-C 17 alkyl, C9-C 18 alkenyl containing one or two double bonds; most preferably, R 1 is selected from

[0080]

[0081] R 3 selected from halogen, hydroxy, cyano, Ci-C6alkyl, nitro, Ci-C6alkyloxy, Ci-C6alkylcarbonyloxy, Ci-C6alkyloxycarbonyl, Ci-C6alkylaminocarbonyl, Ci-C6alkylcarbonylamino; preferably, R 3 selected from halogen, hydroxy, cyano, Ci-C6alkyloxy, Ci-C6alkylcarbonyloxy, Ci-C6alkyloxycarbonyl, Ci-C6alkylaminocarbonyl, Ci-C6alkylcarbonylamino; further preferably, R 3 selected from halogen, hydroxy, cyano, Ci-C4alkyloxy, Ci-C4alkylcarbonyloxy, Ci-C4alkyloxycarbonyl, Ci-C4alkylaminocarbonyl, Ci-C4alkylcarbonylamino; most preferably, R 3 selected from fluorine, hydroxy, cyano, methoxy, acetyloxy, methoxycarbonyl, butylaminocarbonyl and acetylamino;

[0082] n is selected from 1, 2, 3.

[0083] Preferably, the present application provides an intermediate compound (II) as described above, or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein:

[0084] L 1 selected from C3-C 10 alkylene, C3-C 10 alkenylene or C 3- C 10 alkynylene;

[0085] G 1 selected from -0-(C=0)-, -(C=0)-0-, -C(=0)-, -0-;

[0086] R 1 selected from C8-C 20 alkyl, C8-C 20 alkenyl containing one or more double bonds, connected via any one of the carbons;

[0087] R 3 selected from halogen, hydroxy, cyano, Ci-C6alkyloxy, Ci-C6alkylcarbonyloxy, Ci-C6alkyloxycarbonyl, Ci-C6alkylaminocarbonyl, Ci-C6alkylcarbonylamino;

[0088] n is selected from 1, 2, 3.

[0089] Further preferably, the present application provides an intermediate compound (II) as described above, or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein:

[0090] L1 selected from C3-C 10 alkylene;

[0091] G 1 selected from -0-(C=0)-, -(C=0)-0-;

[0092] R 1 selected from C9-C 17 alkyl, C9-C 18 alkenyl;

[0093] R 3 selected from halogen, hydroxy, cyano, C1-C4alkoxy, C1-C4alkylcarbonyloxy, C1-C4alkoxycarbonyl, C1-C4alkylaminocarbonyl, C1-C4alkylcarbonylamino;

[0094] n is selected from 1, 2, 3.

[0095] Further preferably, the present application provides an intermediate compound (II) as described above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0096] L 1 selected from C5-C8alkylene;

[0097] G 1 selected from -0-(C=0)-, -(C=0)-0-;

[0098] R 1 selected from

[0099]

[0100] R 3 selected from fluorine, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl and acetylamino; n is selected from 1, 2, 3.

[0101] Most preferably, the intermediate compound (II) is selected from:

[0102]

[0103]

[0104]

[0105] Further, the present application also provides a preparation method for preparing said intermediate compound (II), the general reaction procedure of which is as follows:

[0106]

[0107] wherein R1 R 3 G 1 L 1 n has the same meaning as defined above for the intermediate compound (II); X is halogen, preferably bromine.

[0108] Specifically, the intermediate compound (IV) is reacted with the intermediate compound (V) in an organic solvent at room temperature in the presence of an acid binding agent to obtain the intermediate compound (II) by substitution reaction. Wherein, the organic solvent is selected from nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, such as acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE); the acid binding agent is selected from organic bases, inorganic bases, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, DIPEA.

[0109] In another aspect, the present application provides a method for preparing the optical isomer (I-6) of the compound 6, the general reaction procedure of the method is as follows:

[0110]

[0111]

[0112] Wherein, X is halogen, preferably bromine.

[0113] Specifically, the optical isomer (I-6) of the compound 6 is prepared by the following steps:

[0114] In a solvent, such as nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, specifically acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE), etc., the compound (6-X) is reacted with the compound (6-VI) at 30-50°C to prepare the compound (6-VII) by N-alkylation reaction;

[0115] In a solvent, such as nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, ethers, specifically acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE), cyclopentyl methyl ether, methyl tert-butyl ether, etc., the 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 prepare the compound (I-6), wherein the acid binding agent is selected from organic bases, inorganic bases, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, DIPEA; the catalyst is iodide, preferably KI.

[0116] Further, according to the above method, when the compound (6-X) employed is (1S,3R)-3-aminocyclohexanol, and the compound (6-VII) is as shown in the structural formula (6-VII-I):

[0117]

[0118] to obtain the optical isomer of compound 6, compound (I-6-II):

[0119]

[0120] When the compound (6-X) employed is (1S,3S)-3-aminocyclohexanol, and the compound (6-VII) is as shown in the structural formula (6-VII-II):

[0121]

[0122] to obtain the optical isomer of compound 6, compound (I-6-III):

[0123]

[0124] When the compound (6-X) employed is (1R,3R)-3-aminocyclohexanol, and the compound (6-VII) is as shown in the structural formula (6-VII-III):

[0125]

[0126] to obtain the optical isomer of compound 6, compound (I-6-IV):

[0127]

[0128] When the compound (6-X) employed is (1R,3S)-3-aminocyclohexanol, and the compound (6-VII) is as shown in the structural formula (6-VII-IV):

[0129]

[0130] to obtain the optical isomer of compound 6, compound (I-6-V):

[0131]

[0132] In still another aspect, the present application provides an intermediate compound (6-VII) for preparing the optical isomer (I-6) of compound 6 as described above, which has the structure:

[0133]

[0134] Specifically, the intermediate compound (6-VII) is selected from:

[0135] BRIEF DESCRIPTION OF DRAWINGS

[0136] Figure 1 is the humoral antibody titer generated by subcutaneous administration of a representative amino lipid compound of the present invention, e.g., in Experiment 2 of the biological testing.

[0137] Figure 2 is the humoral antibody titer generated by intramuscular administration of a representative amino lipid compound of the present invention, e.g., in Experiment 3 of the biological testing.

[0138] Figure 3 is the HPLC profile of a mixture of compounds (I-6-II), (I-6-III), (I-6-IV), and (I-6-V).

[0139] Figure 4 is the HPLC profile of compound (I-6-II).

[0140] Figure 5 is the HPLC profile of compound (I-6-III).

[0141] Figure 6 is the HPLC profile of compound (I-6-IV).

[0142] Figure 7 is the HPLC profile of compound (I-6-V).

[0143] Figure 8 is the fluorescence intensity of the liver after intramuscular injection in a targeted test.

[0144] Figure 9 is the severe swelling of the injection site, moderate lameness statistics in an intramuscular injection adverse reaction test.

[0145] Various exemplary embodiments of the amino lipid compounds of the present invention, compositions containing the same, lipid nanoparticles, and their use in delivering biologically active agents, such as nucleic acids, into cells are described in further detail below. DETAILED DESCRIPTION

[0146] DEFINITIONS

[0147] Unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be construed in an open, inclusive sense, that is, as "including but not limited to." The word "comprise" and variations such as "comprises" and "comprising" will be construed in an open, inclusive sense, that is, "including, but not limited to," unless otherwise noted to the context.

[0148] In this specification, a 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 application. The appearance of the phrase "in one embodiment" or "in an embodiment" in various places in the specification is therefore not necessarily referring to the same embodiment. In addition, the described particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0149] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the specification and the claims will be interpreted as being consistent with the ordinary meanings used by those skilled in the art unless otherwise specified.

[0150] In the present application, the expression "Cx-Cy" means a range of the number of carbon atoms, wherein x and y are each an integer, for example, C3-C8 cycloalkyl means a cycloalkyl group having 3 to 8 carbon atoms, C0-C2 alkyl means an alkyl group having 0 to 2 carbon atoms, wherein C0 alkyl means a chemical single bond.

[0151] In the present application, the term "alkyl" refers to saturated aliphatic hydrocarbon groups, including straight chain and branched chain groups of 1 to 30 carbon atoms, for example, 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, and the like.

[0152] In the present application, the term "cycloalkyl" refers to saturated monocyclic or polycyclic cyclic hydrocarbon groups, including 3 to 12 ring atoms, for example, 3 to 12, 3 to 10, 3 to 8, or 3 to 6 ring atoms, or can be 3, 4, 5, 6 membered rings. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0153] In the present application, the term "alkenyl" refers to unsaturated aliphatic hydrocarbon groups having at least one double bond, including straight chain and branched chain alkenyl groups of 1 to 30 carbon atoms, for example, 5 to 27 carbon atoms, 8 to 20 carbon atoms, 9 to 18 carbon atoms having one or two double bonds. Non-limiting examples include 8-heptadecenyl, 12-octadecadienyl, and various isomers thereof, and the like.

[0154] In the present application, the term "alkynyl" refers to an unsaturated aliphatic hydrocarbon group having at least one triple bond, including straight-chain and branched-chain alkynyl groups of 1 to 30 carbon atoms, for example, straight-chain and branched-chain alkynyl groups of 5 to 27 carbon atoms, 5 to 15 carbon atoms, 8 to 10 carbon atoms, which can have one or two triple bonds. Non-limiting examples include 2-nonyl, 3-decynyl, various isomers thereof, and the like.

[0155] In the present application, the term "alkylene" refers to a substituted or unsubstituted alkyl group having a core of two terminal monovalent radicals, resulting from the removal of one hydrogen atom from each of the two terminal atoms of the alkyl group, which has the meaning previously described. Non-limiting examples of "alkylene" include C3-C 10 alkylene, C5-C8 alkylene, and the like.

[0156] In the present application, the term "alkenylene" refers to a substituted or unsubstituted alkenyl group having a core of two terminal monovalent radicals, resulting from the removal of one hydrogen atom from each of the two terminal atoms of the alkenyl group, which has the meaning previously described. Non-limiting examples of "alkenylene" include C3-C 10 alkenylene, and the like.

[0157] In the present application, the term "alkynylene" refers to a substituted or unsubstituted alkynyl group having a core of two terminal monovalent radicals, resulting from the removal of one hydrogen atom from each of the two terminal atoms of the alkynyl group, which has the meaning previously described. Non-limiting examples of "alkynylene" include C3-C 10 alkynylene, and the like.

[0158] In the present application, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0159] In the present application, the term "alkoxy" means an alkyl-oxy group, the alkyl group having the meaning previously described. Preferably, the alkoxy group is a C1-C 10 alkoxy; more preferably, the alkoxy group is a C1-C6 alkoxy; further preferably, the alkoxy group is a C1-C4 alkoxy; most preferably, the alkoxy group is methoxy.

[0160] In the present application, the term "alkylcarbonyloxy" means an alkyl-C(O)O group, the alkyl group having the meaning previously described. Preferably, the alkylcarbonyloxy group is a C1-C 10 alkylcarbonyloxy; more preferably, the alkylcarbonyloxy group is a C1-C6 alkylcarbonyloxy; further preferably, the alkylcarbonyloxy group is a C1-C4 alkylcarbonyloxy; most preferably, the alkylcarbonyloxy group is acetoxy.

[0161] In the present application, the term "alkylcarbonyl" means an alkyl-C(O)- group, the alkyl group having the previously described meaning. Preferably, the alkylcarbonyl group is a C1-C6 alkylcarbonyl group; more preferably, the alkylcarbonyl group is a C1-C4 alkylcarbonyl group; most preferably, the alkylcarbonyl group is a propionyl group. 10 In the present application, the term "alkylcarbonyl" means an alkyl-C(O)- group, the alkyl group having the previously described meaning. Preferably, the alkylcarbonyl group is a C1-C6 alkylcarbonyl group; more preferably, the alkylcarbonyl group is a C1-C4 alkylcarbonyl group; most preferably, the alkylcarbonyl group is a propionyl group.

[0162] In the present application, the term "alkylcarbonyl" means an alkyl-C(O)- group, the alkyl group having the previously described meaning. Preferably, the alkylcarbonyl group is a C1-C6 alkylcarbonyl group; more preferably, the alkylcarbonyl group is a C1-C4 alkylcarbonyl group; most preferably, the alkylcarbonyl group is a propionyl group. 10 In the present application, the term "alkylcarbonyl" means an alkyl-C(O)- group, the alkyl group having the previously described meaning. Preferably, the alkylcarbonyl group is a C1-C6 alkylcarbonyl group; more preferably, the alkylcarbonyl group is a C1-C4 alkylcarbonyl group; most preferably, the alkylcarbonyl group is a propionyl group.

[0163] In the present application, the term "alkylcarbonyl" means an alkyl-C(O)- group, the alkyl group having the previously described meaning. Preferably, the alkylcarbonyl group is a C1-C6 alkylcarbonyl group; more preferably, the alkylcarbonyl group is a C1-C4 alkylcarbonyl group; most preferably, the alkylcarbonyl group is a propionyl group. 10 In the present application, the term "alkylcarbonyl" means an alkyl-C(O)- group, the alkyl group having the previously described meaning. Preferably, the alkylcarbonyl group is a C1-C6 alkylcarbonyl group; more preferably, the alkylcarbonyl group is a C1-C4 alkylcarbonyl group; most preferably, the alkylcarbonyl group is a propionyl group.

[0164] The "pharmaceutically acceptable salts" according to the application are discussed in Berge, et al., "Pharmaceutically acceptable salts", J. Pharm. Sci., 66, 1-19 (1977) and are apparent to a person skilled in medicinal chemistry, which salts are substantially non-toxic and capable of providing the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion.

[0165] The pharmaceutically acceptable salts according to the application can be synthesized by standard chemical methods.

[0166] In general, the salts can be prepared by reaction of the free base or acid with an equivalent chemical amount or an excess of the acid (mineral or organic) or base in a suitable solvent or combination of solvents.

[0167] In the present application, the term "meta" means the position on the cycloalkyl structural element which is separated by one carbon atom from its amine substituent.

[0168] In the present application, the term "compound" encompasses all compounds that are isotopically labeled by the substitution of one or more atoms for an atom having the same atomic number but different atomic weight or mass number. An "isotope" refers to an atom having the same atomic number but a different mass number due to a different number of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0169] In the present application, the term "lipid nanoparticle" means a nanometer-sized material (lipid nanoparticle) made by adding an aminolipid compound to an aqueous solution, which particles are particularly lipid bilayer vesicles (liposomes), multilamellar vesicles, or micelles. In a preferred embodiment, the lipid nanoparticle is a liposome containing an aminolipid compound of the present application.

[0170] In the present application, the term "liposome" means a microvesicle composed of a bilayer of lipid amphiphilic (amphiphilic) molecules that enclose an aqueous compartment. The formation of liposomes is not a spontaneous process. When lipids are placed in water, lipid vesicles are first formed, thus forming a bilayer or a series of bilayers, each separated by water molecules. Liposomes can be formed by sonicating lipid vesicles in water.

[0171] In the present application, the term "lipid bilayer" means a thin film formed of two layers of lipid molecules.

[0172] In the present application, the term "micelle" means an aggregate of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solution aggregate with the hydrophilic head region in contact with water, sequestering the hydrophobic single-tail region in the center of the micelle.

[0173] In the present application, 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 that express a heterologous polypeptide or protein). Recombinant cells include, for example, cells that express a heterologous polypeptide or protein (such as a growth factor or a blood factor).

[0174] In a preferred embodiment, the lipid nanoparticle or liposome of the present application further contains 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 application, a 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 body of functional groups in the lipid molecule should be non-cationic. Liposomes composed of a mixture of cationic aminolipids 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.

[0175] In a further preferred embodiment, the lipid nanoparticle or liposome of the application further comprises a solid alcohol. Solid alcohols, such as cholesterol, are natural components in cell membranes, which can be used to stabilize the particle and help integration with the cell membrane.

[0176] In another embodiment, the lipid nanoparticle or liposome of the application further contains a biologically active agent. Within the scope of the application, a biologically active agent is a substance that has a biological effect when introduced into a cell or host, for example, by stimulating an immune or inflammatory response, by exerting an enzymatic activity or by complementing a mutation, etc. The biologically active agent is in particular a nucleic acid, a peptide, a protein, an antibody and a small molecule. The term "lipid nanoparticle drug" can be used both when the liposome is used to enclose a drug within the lipid bilayer or in the internal aqueous space of the liposome.

[0177] In a preferred embodiment, the biologically active agent is a nucleic acid. In another preferred embodiment, the biologically active agent is a member selected from the group consisting of an antineoplastic agent, an antibiotic, an immunomodulatory agent, an anti-inflammatory agent, an agent acting on the central nervous system, a polypeptide or a polypeptoid.

[0178] In another embodiment, the lipid nanoparticle or liposome further contains at least one polyethylene glycol (PEG)-lipid. PEG lipids help to protect the particle and its contents from degradation in vitro or in vivo. In addition, PEG forms a protective layer on the surface of the liposome and improves the circulation time in vivo. It can be used in liposome drug delivery (PEG-liposomes). Preferably, the polyethylene glycol lipid is PEG2000-DMG.

[0179] The lipid nanoparticle or liposome containing a biologically active agent can be used to deliver any of a variety of therapeutic agents into a cell. The application includes the use of a lipid nanoparticle (especially a liposome) as described above for delivering a biologically active agent into a cell.

[0180] Preferably, the biologically active agent is a nucleic acid, including but not limited to, RNA, DNA, antisense oligonucleotide; 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, plasmid. The biologically active agent can also be an antitumor agent, an antibiotic, an immunomodulator, an anti-inflammatory agent, a central nervous system acting agent, an antigen or fragment thereof, a protein, a peptide, a polypeptide, a vaccine and a small molecule, or a mixture thereof. As shown above, the lipid nanoparticle or liposome containing the amino lipid compound defined in the present application or a pharmaceutically acceptable salt or stereoisomer thereof is suitable for delivering the biologically active agent into a cell.

[0181] Various different amino lipid compounds synthesized by the general synthetic method can be screened for specific characteristics of liposomes for specific applications. The characteristics are, for example, transfection efficiency, cytotoxicity, adhesion of an agent to be delivered into a cell, stability of the liposome, size of the liposome, etc.

[0182] The lipid nanoparticle or liposome of the present application can be used to transfect a multi-cellular tissue or organ. This provides a new therapeutic treatment possibility for a patient.

[0183] According to the present application, the patient can be any mammal, preferably selected from the group consisting of human, mouse, rat, pig, cat, dog, horse, goat, cow and monkey and / or others. Most preferably, the patient is a human.

[0184] A preferred embodiment of the present application relates to the use of the lipid nanoparticle or liposome containing the amino lipid compound of the present application or a pharmaceutically acceptable salt or stereoisomer thereof as a medicament.

[0185] In particular, the lipid nanoparticle or liposome can be administered to a patient for use in gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA. The specific application ranges include but are not limited to:

[0186] (1) The lipid nanoparticle of the present application can deliver nucleic acid for gene therapy. By introducing an exogenous gene into a target cell through the amino lipid of the present application, the purpose of treatment is achieved to correct or compensate for diseases caused by defective and abnormal genes. This also includes technical applications in the field of transgenesis, that is, the exogenous gene is inserted into the appropriate recipient cells of the patient through gene transfer technology, so that the product produced by the exogenous gene 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. Genetically edited nucleic acid substances can also be introduced for the treatment of various genetic diseases, such as hemophilia, thalassemia, Gaucher's disease, etc.

[0187] (2) The lipid nanoparticles of the present application can be used in vaccination. The lipid nanoparticles or liposomes of the present application can be used to deliver antigens or nucleic acids encoding antigens. The lipid nanoparticles of the present application can also be used to elicit immune responses against various antigens for the treatment and / or prevention of a variety of conditions, such as cancer, allergies, toxicity, and infection by pathogenic organisms (e.g., viruses, bacteria, fungi, and other disease-causing organisms).

[0188] In another preferred embodiment, the lipid nanoparticles of the present application can be used for the preparation of a medicament for the transfer of nucleic acids, preferably RNA, DNA, antisense oligonucleotides; 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.

[0189] Examples

[0190] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application. The experimental methods not specified in the following embodiments are selected according to conventional methods and conditions or according to the instructions of the commodity.

[0191] The structures of all the compounds of the present application can be identified by nuclear magnetic resonance (1H NMR) and / or mass spectrometry (MS).

[0192] 1 The H NMR chemical shift (δ) is recorded in PPM (parts per million). NMR is performed by a Bruker AVANCE III-400MHz spectrometer. The appropriate solvent is selected from deuterated chloroform (CDCl3), deuterated methanol (CD3OD), deuterated dimethyl sulfoxide (DMSO-d6), etc., and tetramethylsilane is used as an internal standard (TMS).

[0193] Low-resolution mass spectrometry (MS) is determined by an Agilent 1260 Infinity II-G6125C mass spectrometer.

[0194] The specific rotation measurement method of the present application: take the sample, accurately weigh and determine, dissolve in anhydrous ethanol and quantitatively dilute to prepare a solution containing about 10 mg per 1 ml, and determine according to the Determination of Optical Rotation in the Fourth Part of the Pharmacopoeia of the People's Republic of China 2020 edition 0621.

[0195] Known starting materials of the present application can be synthesized or purchased according to methods known in the art.

[0196] The eluent system of column chromatography used for purifying the compounds 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 solvents is adjusted according to the polarity of the compound.

[0197] Unless otherwise specified, the reaction temperature in the examples is room temperature, and the temperature range is 15-30°C.

[0198] HPLC analysis method of the present application: HPLC-CAD method

[0199]

[0200] Example 1: Synthesis of Compound 1

[0201] Step 1): Synthesis of 8-bromooctanoic acid-1-octylnonyl ester

[0202]

[0203] In a 250 mL reaction bottle, 8-bromooctanoic acid (22.3 g, 100 mmol), 9- heptadecanol (25.6 g, 100 mmol), dichloromethane 100 mL, after stirring and dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.0 g, 120 mmol), 4-dimethylaminopyridine (0.61 g, 5 mmol), N,N-diisopropylethylamine (25.8 g, 200 mmol) were added in sequence, and the reaction was carried out at room temperature for 2 h. After washing with water 3 times, drying with anhydrous sodium sulfate, and concentrating, 8-bromooctanoic acid-1-octylnonyl ester (41.5 g, 90%) was obtained by purification using a flash column chromatography system (n-hexane: ethyl acetate = 10:1 to 2:1).

[0204] Step 2): Synthesis of 8-((3-hydroxycyclobutyl)amino)octanoic acid n- butyl ester (intermediate compound II-1)

[0205] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.50 g, 10 mmol), 3-aminocyclobutanol (8.7 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with 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%).

[0206] 1 H NMR (600 MHz, CDC13) 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).

[0207] LCMS: 356.3 [M+H] + .

[0208] Step 3): Synthesis of Compound 1

[0209]

[0210] 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), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with 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%).

[0211] 1H NMR (600 MHz, CDC13) 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).

[0212] LCMS: 737.2 [M+H] + .

[0213] Example 2: Synthesis of compound 2

[0214] Step 1): Synthesis of 6-((3-hydroxycyclobutyl)amino)undecanoic acid (Intermediate compound II-2)

[0215]

[0216] In a 100 mL reaction bottle, 8-undecanoyl bromide (3.50 g, 10 mmol), 3- aminocyclobutanol (8.7 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N, N-diisopropyl ethylamine (2.58 g, 20 mmol) was added, and the reaction was carried out at room temperature for 24 h, 100 mL of dichloromethane was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified with a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain 6-((3- hydroxycyclobutyl)amino)undecanoic acid (2.38 g, 67%).

[0217] 1 H NMR (600 MHz, CDC13) 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).

[0218] LCMS: 356.3 [M+H] + .

[0219] Step 2): Synthesis of compound 2

[0220]

[0221] Compound 2 was synthesized in a similar manner as in Example 1, Step 3) except that 6-((3-hydroxycyclobutyl)amino)hexanoic acid undecyl ester (Intermediate Compound II-2) was used instead of intermediate compound 8-((3- hydroxycyclobutyl)amino)octanoic acid nonyl ester.

[0222] 1 H NMR (600 MHz, CDC13) δ: 4.95-4.77 (m, 1H), 4.42 (t, 0.5H), 4.08 (t, 2H), 4.02-3.94 (m, 0.5H), 3.51-3.42 (m, 0.5H), 2.72-2.62 (m, 0.5H), 2.56 (m, 2H), 2.50-2.44 (m, 3H), 2.42 (s, 1H), 2.38-2.29 (m, 4H), 1.94 (s, 2H), 1.71-1.58 (m, 6H), 1.53 (m, 4H), 1.44 (m, 4H), 1.40-1.21 (m, 49H), 0.90 (m, 9H).

[0223] LCMS: 737.2 [M+H] + .

[0224] Example 3: Synthesis of compound 6

[0225] Step 1): Synthesis of 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (Intermediate Compound II-6)

[0226]

[0227] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.50 g, 10 mmol), 3- aminocyclohexanol (11.5 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N,N- diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was carried out at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with 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%).

[0228] 1H NMR (600 MHz, CDC13) δ 4.20-4.13 (m, 0.5H), 4.05 (t, 2H), 3.83 (m, 0.5H), 3.09 (m, 0.5H), 2.87 (m, 0.5H), 2.76-2.59 (m, 2H), 2.29 (t, 2H), 2.00 (m, 0.5H), 1.93-1.78 (m, 1.5H), 1.78-1.65 (m, 2H), 1.65-1.46 (m, 8H), 1.40-1.18 (m, 20H), 0.88 (t, 3H).

[0229] LCMS: 384.3 [M+H] + .

[0230] Step 2): Synthesis of compound 6

[0231]

[0232] In a 100 mL reaction bottle, 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) were added, and the reaction was carried out at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with 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%).

[0233] 1 H NMR (600 MHz, CDC13) δ 4.20-4.13 (m, 0.5H), 4.05 (t, 2H), 3.83 (m, 0.5H), 3.09 (m, 0.5H), 2.87 (m, 0.5H), 2.76-2.59 (m, 2H), 2.29 (t, 2H), 2.00 (m, 0.5H), 1.93-1.78 (m, 1.5H), 1.78-1.65 (m, 2H), 1.65-1.46 (m, 8H), 1.40-1.18 (m, 20H), 0.88 (t, 3H).

[0234] LCMS: 765.3 [M+H] + .

[0235] Example 4: Synthesis of compound 7

[0236] Step 1): Synthesis of 6-((3-hydroxycyclohexyl)amino)hexanoic acid undecyl ester (intermediate compound II-7)

[0237]

[0238] In a 100 mL reaction flask, 6-bromohexanoic acid undecyl ester (3.50 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried using anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain 6-((3-hydroxycyclohexyl)amino)hexanoic acid undecyl ester (2.53 g, 66%).

[0239] 1 H NMR (600 MHz, CDC13) δ 4.20 (m, 0.5H), 4.05 (t, 2H), 3.93 (m, 0.5H), 3.21 (m, 0.5H), 3.03 (m, 0.5H), 2.84-2.68 (m, 2H), 2.35-2.25 (m, 2H), 2.12-2.06 (m, 0.5H), 1.91 (m, 1.5H), 1.94-1.82 (m, 2H), 1.77-1.56 (m, 8H), 1.43-1.19 (m, 20H), 0.88 (t, 3H).

[0240] LCMS: 384.4 [M+H] + .

[0241] Step 2): Synthesis of compound 7

[0242]

[0243] In a 100 mL reaction flask, 6-((3-hydroxycyclohexyl)amino)hexanoic acid undecyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried using 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%).

[0244] 1H NMR (600 MHz, CDC13) δ 4.93-4.81 (m, 1H), 4.24 (m, 0.5H), 4.05 (t, 2H), 3.68-3.61 (m, 0.5H), 2.98 (m, 0.5H), 2.56 (m, 0.5H), 2.50-2.36 (m, 4H), 2.35-2.22 (m, 4H), 1.97-1.73 (m, 3H), 1.69-1.55 (m, 7H), 1.48 (m, 4H), 1.46-1.36 (m, 4H), 1.36-1.18 (m, 52H), 0.88 (m, 9H).

[0245] LCMS: 765.0 [M+H] + .

[0246] Example 5: Synthesis of compound 8

[0247] Step 1): Synthesis of 6-oxohexanoic acid-7-pentadecyl ester

[0248]

[0249] In a 250 mL reaction bottle, 6-((tert-butyldimethylsilyl)oxy)hexanoic acid (2.46 g, 10 mmol), pentadecan-7-ol (2.28 g, 10 mol), dichloromethane 100 mL, after stirring and dissolving, adding dicyclohexyl carbodiimide (2.47 g, 12 mmol), 4-dimethylaminopyridine (0.06 g, 0.5 mmol), reacting at room temperature for 2 h, washing with water 3 times, drying with anhydrous sodium sulfate and concentrating to dryness, adding tetrahydrofuran 50 mL, tetrabutylammonium fluoride (2.75 g, 10.5 mmol), reacting at room temperature for 1 h, dissolving in dichloromethane 100 mL after concentrating to near dryness, washing with water 3 times, drying with anhydrous sodium sulfate, adding des-martin periodinane (5.09 g, 12 mmol), stirring and reacting at room temperature for 12 h, washing with saturated sodium bicarbonate solution 3 times, washing with water once, drying with anhydrous sodium sulfate, and purifying using a flash column chromatography system (n-hexane: ethyl acetate = 10:1 to 5:1) to obtain 6-oxohexanoic acid-7-pentadecyl ester (2.76 g, 81%).

[0250] Step 2): Synthesis of compound 8

[0251]

[0252] In a 250 mL reaction flask, 6-oxohexanoic acid-7-pentadecyl ester (3.41 g, 10 mmol) was added, followed by dichloroethane 100 mL, sodium triacetoxyborohydride (3.18 g, 15 mmol), and then 3-aminocyclohexanol (0.57 g, 5 mmol). The reaction was stirred at room temperature for 24 h. After washing with water three times, drying with anhydrous sodium sulfate, and concentration, compound 8 (2.83 g, 74%) was obtained by purification using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1).

[0253] 1 H NMR (600 MHz, CDC13) δ 4.93-4.80 (m, 2H), 4.24 (m, 0.5H), 3.66-3.58 (m, 0.5H), 3.05 (m, 0.5H), 2.54 (m, 0.5H), 2.42 (m, 4H), 2.30 (m, 4H), 1.96-1.74 (m, 3H), 1.72-1.56 (m, 6H), 1.50 (m, 11H), 1.35-1.19 (m, 48H), 0.88 (t, 12H).

[0254] LCMS: 765.1 [M+H] + .

[0255] Example 6: Synthesis of compound 9

[0256] Step 1): Synthesis of 2-hexyl decanoic acid-(6-oxohexyl) ester

[0257]

[0258] In a 250 mL reaction flask, 2-n-hexyl decanoic acid (25.6 g, 100 mmol) was added, followed by 1,6-hexanediol (59.0 g, 0.5 mol), dichloromethane 150 mL, and stirring and dissolving. Then, dicyclohexyl carbodiimide (20.6 g, 100 mmol) and 4-dimethylaminopyridine (0.61 g, 5 mmol) were added, and the reaction was stirred at room temperature for 2 h. After washing with water three times, drying with anhydrous sodium sulfate, and purification using a flash column chromatography system (n-hexane: ethyl acetate = 5:1 to 1:1), 2-hexyl decanoic acid-7-hydroxyheptyl ester was obtained. Then, dichloromethane 100 mL and Dess-Martin oxidant (50.9 g, 120 mmol) were added, and the reaction was stirred at room temperature for 12 h. After washing with a saturated sodium bicarbonate solution three times and water once, drying with anhydrous sodium sulfate, and purification using a flash column chromatography system (n-hexane: ethyl acetate = 10:1 to 5:1), 2-hexyl decanoic acid-(6-oxohexyl) ester (19.1 g, 54%) was obtained.

[0259] Step 2): Synthesis of compound 9

[0260]

[0261] In a 250 mL reaction flask, 2-hexyl decanoic acid-(6-oxohexyl) ester (3.55 g, 10 mmol), dichloromethane 100 mL, sodium triacetoxyborohydride (3.18 g, 15 mmol) were added in sequence, then 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (3.83 g, 10 mmol) was added, and the reaction was carried out at room temperature for 24 h. After washing with water for 3 times, drying with anhydrous sodium sulfate, and concentration, compound 9 (4.98 g, 69%) was obtained by purification using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1).

[0262] 1 H NMR (600 MHz, CDC13) δ 4.26-4.19 (m, 0.5H), 4.12 (m, 2H), 4.09-4.02 (m, 2H), 3.65 (m, 0.5H), 2.98 (m, 0.5H), 2.59 (m, 0.5H), 2.51-2.35 (m, 4H), 2.30 (m, 3H), 1.98-1.89 (m, 1H), 1.89-1.72 (m, 3H), 1.72-1.53 (m, 9H), 1.51-1.34 (m, 10H), 1.34-1.16 (m, 42H), 0.92-0.79 (m, 9H). LCMS: 723.1 [M+H] + .

[0263] Example 7: Synthesis of compound 10

[0264] Step 1): Synthesis of oleic acid-8-bromooctyl ester

[0265]

[0266] In a 250 mL reaction flask, 8-bromooctan-1-ol (20.9 g, 100 mmol), oleic acid (28.5 g, 100 mmol), dichloromethane 100 mL were added in sequence, and after stirring and dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.0 g, 120 mmol), 4-dimethylaminopyridine (0.61 g, 5 mmol), N,N-diisopropylethylamine (25.8 g, 200 mmol) were added, and the reaction was carried out at room temperature for 2 h. After washing with water for 3 times, drying with anhydrous sodium sulfate, and concentration, oleic acid-8-bromooctyl ester (37.4 g, 79%) was obtained by purification using a flash column chromatography system (n-hexane: ethyl acetate = 10:1 to 3:1).

[0267] Step 2): Synthesis of oleic acid-8-((3-hydroxycyclohexyl)amino)octyl ester (intermediate compound II-10)

[0268]

[0269] In a 100 mL reaction flask, oleic acid-8-bromononylester (4.73 g, 10 mmol), 3- aminocyclohexanol (11.5 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. After adding dichloromethane 100 mL, washing with water 3 times, drying using anhydrous sodium sulfate, and concentrating, oleic acid-8-((3-hydroxycyclohexyl)amino)octyl ester (3.2 g, 63%) was obtained by purification using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1).

[0270] 1 H NMR (600 MHz, CDC13) δ 5.41-5.29 (m, 2H), 4.19 (m, 0.5H), 4.05 (t, 2H), 3.92 (m, 0.5H), 3.20 (m, 0.5H), 3.00 (m, 0.5H), 2.81-2.67 (m, 2H), 2.29 (t, 2H), 2.02 (m, 3H), 1.93-1.79 (m, 2H), 1.69-1.56 (m, 11H), 1.36-1.20 (m, 30H), 0.88 (t, 3H).

[0271] LCMS: 508.7 [M+H] + .

[0272] Step 3): Synthesis of compound 10

[0273]

[0274] In a 250 mL reaction flask, 2-hexyldecanoic acid-(6-oxohexyl) ester (3.55 g, 10 mmol), dichloroethane 100 mL, sodium triacetoxyborohydride (3.18 g, 15 mmol), and oleic acid-8-((3-hydroxycyclohexyl)amino)octyl ester (5.08 g, 10 mmol) were sequentially added, and the reaction was allowed to proceed at room temperature for 24 h. After washing with water 3 times, drying using anhydrous sodium sulfate, and concentrating, compound 10 (6.26 g, 74%) was obtained by purification using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1).

[0275] 1H NMR (600 MHz, CDC13) δ 5.41-5.31 (m, 2H), 4.24 (m, 0.5H), 4.06 (m, 4H), 3.70-3.60 (m, 0.5H), 3.03-2.92 (m, 0.5H), 2.57 (m, 0.5H), 2.49-2.36 (m, 4H), 2.30 (m, 3H), 2.01 (m, 3H), 1.98-1.90 (m, 2H), 1.85 (m, 1H), 1.82-1.74 (m, 1H), 1.61 (m, 9H), 1.45-1.38 (m, 6H), 1.33-1.20 (m, 56H), 0.88 (m, 9H).

[0276] LCMS: 847.3 [M+H] + .

[0277] Example 8: Synthesis of compound 12

[0278] Step 1): Synthesis of (6Z,9Z)-dien-octadecan-8-((3-hydroxycyclohexyl)amino) octanoate (intermediate compound II-12)

[0279]

[0280] In a 100 mL reaction bottle, (6Z,9Z)-dien-octadecan-8-bromooctanoate (4.71 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N, N-diisopropyl ethylamine (2.58 g, 20 mmol) was added, and the reaction was carried out at room temperature for 24 h, dichloromethane 100 mL was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified with a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain (6Z,9Z)-dien-octadecan-8-((3-hydroxycyclohexyl)amino) octanoate (3.3 g, 66%).

[0281] 1 H NMR (600 MHz, CDC13) δ 5.43-5.28 (m, 4H), 4.18 (m, 0.5H), 4.07-4.03 (m, 2H), 3.95 (m, 0.5H), 3.22 (m, 0.5H), 3.04 (m, 0.5H), 2.82-2.69 (m, 4H), 2.31-2.23 (m, 2H), 2.09-2.01 (m, 4H), 1.85 (m, 1H), 1.76-1.56 (m, 10H), 1.40-1.26 (m, 23H), 0.92-0.84 (t, 3H).

[0282] LCMS: 506.7 [M+H] + .

[0283] Step 2): Synthesis of compound 12

[0284]

[0285] Compound 12 was synthesized in a similar manner as in Example 7, Step 3) except that (6Z,9Z)-dien-octadecan-8-((3-hydroxycyclohexyl)amino)octyl ester was used instead of the original intermediate compound oleic acid-8-((3-hydroxycyclohexyl)amino)octyl ester.

[0286] 1 H NMR (600 MHz, CDC13) δ 5.43-5.28 (m, 4H), 4.24 (m, 0.5H), 4.06 (m, 4H), 3.63 (m, 0.5H), 2.98 (m, 0.5H), 2.77 (t, 2H), 2.55 (m, 0.5H), 2.45 (m, 4H), 2.30 (m, 3H), 2.10-2.01 (m, 4H), 1.88-1.73 (m, 3H), 1.66-1.53 (m, 10H), 1.35-1.16 (m, 55H), 0.88 (m, 9H).

[0287] LCMS: 845.1 [M+H] + .

[0288] Example 9: Synthesis of compound 14

[0289]

[0290] In a 250 mL reaction flask, 2-hexyl decanoic acid-(6-oxohexyl) ester (3.55 g, 10 mmol), dichloroethane 100 mL, sodium triacetoxyborohydride (3.18 g, 15 mmol) were added sequentially, followed by 3-aminocyclohexanol (0.57 g, 5 mmol), and the reaction was allowed to proceed at room temperature for 24 h. After washing with water 3 times, drying with anhydrous sodium sulfate, and concentrating, compound 14 (2.81 g, 71%) was purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1).

[0291] 1H NMR (600 MHz, CDC13) δ 4.32-4.20 (m, 0.5H), 4.13-4.03 (m, 4H), 3.71-3.61 (m, 0.5H), 3.02 (m, 0.5H), 2.59 (m, 0.5H), 2.48 (m, 4H), 2.37-2.29 (m, 2H), 1.91-1.75 (m, 2H), 1.74-1.56 (m, 10H), 1.51-1.36 (m, 14H), 1.36-1.21 (m, 46H), 0.90 (m, 12H).

[0292] LCMS: 793.2 [M+H] + .

[0293] Example 10: Synthesis of compound 15

[0294]

[0295] Compound 15 was synthesized in a similar manner as in Example 7, step 3) except using 7-pentadecyl 6-((3-hydroxycyclohexyl)amino)hexanoate instead of oleic acid-8-((3-hydroxycyclohexyl)amino)octyl ester in the original intermediate compound.

[0296] 1 H NMR (600 MHz, CDC13) δ 4.94-4.80 (m, 1H), 4.23 (m, 0.5H), 4.08 (t, 2H), 3.72-3.62 (m, 0.5H), 3.00 (m, 0.5H), 2.55 (m, 0.5H), 2.50-2.37 (m, 4H), 2.35-2.25 (m, 3H), 1.90-1.75 (m, 2H), 1.73-1.57 (m, 9H), 1.51 (m, 4H), 1.44 (m, 7H), 1.39 (m, 2H), 1.36-1.21 (m, 46H), 0.90 (m, 12H). LCMS: 779.2 [M+H] + .

[0297] Example 11: Synthesis of compound 16

[0298] Step 1): Synthesis of nonyl 8-((3-methoxycyclohexyl)amino)octanoate (intermediate compound II-16)

[0299] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.49 g, 10 mmol), 3-methoxycyclohexylamine (12.9 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried using 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%).

[0300] 1 H NMR (600 MHz, CDC13) δ 4.07 (t, 2H), 3.64 (s, 0.5H), 3.36 (s, 1.5H), 3.35 (m, 0.5H), 3.29 (s, 1.5H), 3.28-3.23 (m, 0.5H), 3.18 (s, 0.5H), 3.00-2.89 (m, 2H), 2.30 (t, 2H), 2.07-2.02 (m, 4H), 1.92-1.84 (m, 2H), 1.69 (m, 3H), 1.67-1.58 (m, 4H), 1.40-1.23 (m, 18H), 0.94-0.82 (m, 3H). LCMS: 398.5 [M+H] + .

[0301] Step 2): Synthesis of compound 16

[0302]

[0303] 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), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) were added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried using 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%).

[0304] 1H NMR (600 MHz, CDC13) δ 4.93-4.77 (m, 1H), 4.05 (t, 2H), 3.62 (m, 0.5H), 3.36 (s, 1.5H), 3.29 (s, 1.5H), 3.15-3.07 (m, 0.5H), 2.86 (m, 0.5H), 2.55 (t, 0.5H), 2.43 (m, 4H), 2.32-2.24 (m, 4H), 2.07-1.99 (m, 1H), 1.85-1.69 (m, 2H), 1.62 (m, 6H), 1.53-1.48 (m, 4H), 1.45-1.39 (m, 4H), 1.36-1.24 (m, 54H), 0.90-0.85 (m, 9H).

[0305] LCMS: 779.1 [M+H] + .

[0306] Example 12: Synthesis of compound 19

[0307] Step 1): Synthesis of nonyl 8-((3-acetyloxycyclohexyl)amino)octanoate (intermediate compound II-19)

[0308]

[0309] In a 100 mL reaction flask, 8-bromononyl octanoate (3.49 g, 10 mmol), 3- acetyloxycyclohexylamine (15.7 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was carried out at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified with a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain nonyl 8-((3-acetyloxycyclohexyl)amino)octanoate (2.76 g, 65%).

[0310] 1 H NMR (600 MHz, CDC13) δ 5.23 (m, 0.5H), 4.76-4.67 (m, 0.5H), 4.09 (t, 2H), 3.30 (m, 0.5H), 3.05 (m, 0.5H), 3.01-2.86 (m, 3H), 2.45-2.38 (m, 1H), 2.32 (t, 2H), 2.12 (m, 1H), 2.10 (s, 1.5H), 2.08 (s, 1.5H), 2.05 (m, 1H), 1.97-1.91 (m, 1H), 1.77 (m, 3H), 1.71 (m, 3H), 1.68-1.60 (m, 5H), 1.41-1.22 (m, 16H), 0.92 (t, 3H).

[0311] LCMS: 426.4 [M+H] + .

[0312] Step 2): Synthesis of compound 19

[0313]

[0314] Compound 19 was synthesized in a similar manner as in Example 11, Step 2) except that 8-((3-acetyloxycyclohexyl)amino)nonanoic acid was used instead of intermediate compound 8-((3-methoxycyclohexyl)amino)nonanoic acid.

[0315] 1 H NMR (600 MHz, CDC13) δ 5.16 (m, 0.5H), 4.91-4.81 (m, 1H), 4.69 (m, 0.5H), 4.05 (t, 2H), 2.86 (m 0.5H), 2.64-2.50 (m, 0.5H), 2.45-2.32 (m, 4H), 2.32-2.22 (m, 4H), 2.04 (s, 1.5H), 2.03 (s, 1.5H), 1.93 (m, 1H), 1.84-1.75 (m, 1H), 1.74-1.69 (m, 1H), 1.66-1.58 (m, 7H), 1.49 (m, 4H), 1.37 (m, 4H), 1.34-1.21 (m, 52H), 0.90-0.85 (m, 9H).

[0316] LCMS: 806.7 [M+H] + .

[0317] Example 13: Synthesis of compound 20

[0318] Step 1): Synthesis of methyl 3-((8-(nonyloxy)-8-oxooctyl)amino)cyclohexane-1- carboxylate (intermediate compound II-20)

[0319]

[0320] 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), ethanol 30 mL, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. After adding dichloromethane 100 mL, washing with water 3 times, drying with anhydrous sodium sulfate, and concentrating, 3-((8-(nonyloxy)-8-oxooctyl)amino)cyclohexane-1-carboxylic acid methyl ester (2.85 g, 67%) was obtained by purification using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1).

[0321] 1 H NMR (600 MHz, CDC13) δ 4.05 (t, 2H), 3.70 (m, 2H), 3.67 (s, 3H), 3.00-2.80 (m, 3H), 2.41 (m, 1H), 2.35 (m, 1H), 2.32-2.26 (t, 2H), 2.23-2.15 (m, 1H), 2.01 (m, 1H), 1.94-1.89 (m, 1H), 1.86-1.75 (m, 2H), 1.69-1.57 (m, 3H), 1.40-1.19 (m, 18H), 0.88 (t, 3H).

[0322] LCMS: 426.4 [M+H] + .

[0323] Step 2): Synthesis of compound 20

[0324]

[0325] Compound 20 was synthesized in a similar manner to Example 11, Step 2), except that 3-((8-(nonyloxy)-8-oxooctyl)amino)cyclohexane-1-carboxylic acid methyl ester was used instead of intermediate compound 8-((3-methoxycyclohexyl)amino)octanoic acid nonyl ester.

[0326] 1 H NMR (600 MHz, CDC13) δ 4.05 (t, 2H), 3.70 (m, 2H), 3.67 (s, 3H), 3.00-2.80 (m, 3H), 2.41 (m, 1H), 2.35 (m, 1H), 2.32-2.26 (t, 2H), 2.23-2.15 (m, 1H), 2.01 (m, 1H), 1.94-1.89 (m, 1H), 1.86-1.75 (m, 2H), 1.69-1.57 (m, 3H), 1.40-1.19 (m, 18H), 0.88 (t, 3H).

[0327] LCMS: 806.8 [M+H] + .

[0328] Preparation of compound (I-6-II) of Example 14

[0329]

[0330] Into a 250 ml single-necked flask was added 9-heptadecyl-8-bromooctanoate 8 g (17.3 mmol), (1S,3R)-3-aminocyclohexanol 10 g (76.5 mmol) and 100 ml of ethanol, and the reaction was carried out at 50°C for 15 h, and the starting material 9-heptadecyl-8-bromooctanoate was completely reacted. After the solvent was removed by rotary evaporation, the obtained crude product was added to 200 ml of EA, washed with 100 ml of water twice, and (1S,3R)-3-aminocyclohexanol was completely washed off, and the EA phase was dried by rotary evaporation to obtain 9-heptadecyl-8-(((1R,3S)-3-hydroxycyclohexyl)amino)octanoate as a crude product of compound (6-VII-I). The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 6.77 g of compound (6-VII-I) with a purity of 99.2% and a yield of 78.6%.

[0331] NMR data:

[0332] 1 H NMR (600 MHz, CDCl3): δ 4.91-4.81 (m, 1H), 3.86-3.82 (m, 1H), 2.84-2.81 (m, 1H), 2.69-2.54 (qt, J = 11.2, 7.3 Hz, 2H), 2.29-2.26 (t, J = 7.5 Hz, 2H), 1.93-1.86 (m, 1H), 1.77-1.74 (d, J = 12.0 Hz, 1H), 1.72-1.57 (m, 6H), 1.54-1.45 (m, 6H), 1.37-1.30 (m, 8H), 1.30-1.22 (m, 24H), 0.88 (t, J = 7.0 Hz, 6H).

[0333] LCMS: 496.7 [M+H] + .

[0334] To the above compound (6-VII-I) 6 g (12.1 mmol) in a 250 ml single neck flask, 90 ml of acetonitrile and 60 ml of cyclopentyl methyl ether were added, followed by the addition of 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 nonylester (17.5 mmol), and the reaction was carried out at 90°C for 24 hours. When the reaction of the starting compound (6-VII-I) was completed, the solid was removed by suction filtration after the reaction was allowed to cool to room temperature, and the filtrate was spin-dried to obtain a crude product. Finally, column chromatography (dichloromethane:methanol = 20:1 to 5:1) was performed to obtain 5.67 g of compound (I-6-II) with a purity of 98.6% and a yield of 61.4%.

[0335] NMR data:

[0336] 1 H NMR (600 MHz, CDC13): δ 4.93-4.84 (m, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.68-3.61 (m, 1H), 2.62-2.54 (m, 1H), 2.53-2.41 (m, 4H), 2.31-2.26 (q, J = 7.4 Hz, 4H), 1.97 (m, 1H), 1.91-1.78 (m, 2H), 1.71-1.61 (m, 7H), 1.54 (d, J = 6.0 Hz, 4H), 1.43-1.37 (m, 4H), 1.39-1.23 (m, 52H), 0.91 (m, 9H).

[0337] Specific rotation: +4.3°

[0338] LCMS: 765.2 [M+H] + .

[0339] Preparation of compound (I-6-III) of Example 15

[0340]

[0341] Into a 100 ml single neck flask, 2 g of 9-heptadecyl-8-bromooctanoate (4.3 mmol), (1S,3S)-3-aminocyclohexanol 2.5 g (20.2 mmol) and 20 ml of ethanol were added, and the reaction was carried out at 50°C for 15 h, and the raw material 9-heptadecyl-8-bromooctanoate was completely reacted. After the solvent was removed by rotary evaporation, the obtained crude product was added with 50 ml of EA, and washed twice with 25 ml of water to completely wash away (1S,3S)-3-aminocyclohexanol, and the EA phase was rotary evaporated to obtain 1.63 g of compound (6-VII-II) 9-heptadecyl-8-(((1S,3S)-3-hydroxycyclohexyl)amino)octanoate crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 1.63 g of compound (6-VII-II) with a purity of 98.5% and a yield of 75.5%.

[0342] NMR data:

[0343] 1 H NMR (600 MHz, CDCl3): δ 4.91-4.82 (m, 1H), 4.16-4.10 (m, 1H), 2.96-2.87 (m, 1H), 2.65-2.55 (qt, J = 11.2, 7.3 Hz, 2H), 2.28-2.26 (t, J = 7.5 Hz, 2H), 1.93-1.77 (m, 4H), 1.67-1.55 (m, 6H), 1.54-1.45 (m, 6H), 1.33-1.31 (m, 6H), 1.30-1.22 (m, 24H), 0.88 (t, J = 7.0 Hz, 6H).

[0344] LCMS: 496.7 [M+H] + .

[0345] Into a 100 ml single neck flask, 2 g of 9-heptadecyl-8-bromooctanoate (4.3 mmol), (1S,3S)-3-aminocyclohexanol 2.5 g (20.2 mmol) and 20 ml of ethanol were added, and the reaction was carried out at 50°C for 15 h, and the raw material 9-heptadecyl-8-bromooctanoate was completely reacted. After the solvent was removed by rotary evaporation, the obtained crude product was added with 50 ml of EA, and washed twice with 25 ml of water to completely wash away (1S,3S)-3-aminocyclohexanol, and the EA phase was rotary evaporated to obtain 1.63 g of compound (6-VII-II) 9-heptadecyl-8-(((1S,3S)-3-hydroxycyclohexyl)amino)octanoate crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 1.63 g of compound (6-VII-II) with a purity of 98.5% and a yield of 75.5%.

[0346] NMR data:

[0347] 1H NMR (600 MHz, CDC13): δ 4.91-4.80 (m, 1H), 4.28-4.22 (m, 1H), 4.06-4.04 (t, J = 6.8 Hz, 2H), 3.03-2.97 (m, 1H), 2.47-2.41 (m, 4H), 2.33-2.25 (q, J = 7.5 Hz, 4H), 1.89-1.85 (d, J = 12.1 Hz, 1H), 1.80-1.77 (t, J = 12.2 Hz, 1H), 1.73-1.65 (m, 2H), 1.64-1.59 (m, 6H), 1.54-1.48 (m, 4H), 1.48-1.39 (m, 4H), 1.34-1.29 (m, 14H), 1.29-1.23 (m, 38H), 0.89-0.87 (m, 9H).

[0348] Specific rotation: -12.9°

[0349] LCMS: 765.2 [M+H] + .

[0350] Preparation of compound (I-6-IV) of Example 16

[0351]

[0352] Into a 100 ml single necked flask, 2 g of 9-heptadecyl-8-bromooctanoate (4.3 mmol), (1R,3R)-3-aminocyclohexanol 2.5 g (20.2 mmol) and 20 ml of ethanol were added and the reaction was carried out at 50 °C for 15 h. The starting material 9-heptadecyl-8-bromooctanoate was completely reacted. After the solvent was removed by rotary evaporation, the obtained crude product was added with 50 ml of EA and washed with 25 ml of water twice to completely wash away (1R,3R)-3-aminocyclohexanol, and the EA phase was spin-dried to obtain 1.58 g of compound (6-VII-III) 9-heptadecyl-8-(((1R,3R)-3-hydroxycyclohexyl)amino)octanoate crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 1.58 g of compound (6-VII-III) with a purity of 99.2% and a yield of 73.2%.

[0353] NMR data:

[0354] 1H NMR (600 MHz, CDC13): δ 4.89-4.82 (m, 1H), 4.24-4.22 (m, 1H), 4.06-4.04 (t, J = 6.8 Hz, 2H), 3.00-2.95 (m, 1H), 2.47-2.36 (m, 4H), 2.30-2.26 (q, J = 7.5 Hz, 4H), 1.86-1.84 (d, J = 12.1 Hz, 1H), 1.79-1.77 (t, J = 12.2 Hz, 1H), 1.70-1.66 (m, 2H), 1.65-1.57 (m, 6H), 1.53-1.48 (m, 4H), 1.45-1.38 (m, 4H), 1.35-1.30 (m, 14H), 1.29-1.22 (m, 38H), 0.89-0.87 (m, 9H).

[0355] LCMS: 496.7 [M+H] + .

[0356] To the above compound (6-VII-III) 1.5 g (3.0 mmol) in a 100 ml single-necked flask, 25 ml of acetonitrile and 15 ml of cyclopentyl methyl ether were added, followed by the addition of 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), and the reaction was carried out at 90°C for 24 h. After the reaction of the starting compound (6-VII-III) was completed, the solid was removed by suction filtration after the reaction was allowed to cool to room temperature, and the filtrate was rotary-evaporated to obtain a crude product. Finally, column chromatography (dichloromethane:methanol = 20:1 to 5:1) was performed to obtain 1.29 g of compound (I-6-IV) with a purity of 98.6% and a yield of 55.8%.

[0357] NMR data:

[0358] 1 H NMR (600 MHz, CDC13): δ 4.89-4.82 (m, 1H), 4.24-4.22 (m, 1H), 4.06-4.04 (t, J = 6.8 Hz, 2H), 3.00-2.95 (m, 1H), 2.47-2.36 (m, 4H), 2.30-2.26 (q, J = 7.5 Hz, 4H), 1.86-1.84 (d, J = 12.1 Hz, 1H), 1.79-1.77 (t, J = 12.2 Hz, 1H), 1.70-1.66 (m, 2H), 1.65-1.57 (m, 6H), 1.53-1.48 (m, 4H), 1.45-1.38 (m, 4H), 1.35-1.30 (m, 14H), 1.29-1.22 (m, 38H), 0.89-0.87 (m, 9H).

[0359] Specific rotation: +12.6°

[0360] LCMS: 765.2 [M+H] + .

[0361] Preparation of compound (I-6-V)

[0362]

[0363] Into a 250 ml single necked flask, 8 g of 9-heptadecyl-8-bromooctanoate (17.3 mmol), (1R,3S)-3-aminocyclohexanol 10 g (76.5 mmol) and 100 ml of ethanol were added, and the reaction was carried out at 50 °C for 15 h, and the raw material 9-heptadecyl-8-bromooctanoate was completely reacted. After the solvent was removed by rotary evaporation, the obtained crude product was added with 200 ml of EA, washed with 100 ml of water twice, and (1R,3S)-3-aminocyclohexanol was completely washed away, and the EA phase was spin-dried to obtain 9-heptadecyl-8-(((1S,3R)-3-hydroxycyclohexyl)amino)octanoate crude product of compound (6-VII-IV), which was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain 6.4 g of compound (6-VII-IV) with a purity of 98.5% and a yield of 74.8%.

[0364] NMR data:

[0365] 1 H NMR (600 MHz, CDCl3): δ 4.90-4.83 (m, 1H), 3.86-3.82 (m, 1H), 2.86-2.82 (m, 1H), 2.69-2.58 (qt, J = 11.2, 7.3 Hz, 2H), 2.29-2.26 (t, J = 7.5 Hz, 2H), 1.94-1.86 (m, 1H), 1.79-1.77 (d, J = 12.0 Hz, 1H), 1.74-1.64 (m, 3H), 1.64-1.59 (m, 3H), 1.51-1.46 (m, 6H), 1.36-1.30 (m, 8H), 1.30-1.22 (m, 24H), 0.88 (t, J = 7.0 Hz, 6H).

[0366] LCMS: 496.7 [M+H] + .

[0367] To the above compound (6-VII-IV) 6 g product (12.1 mmol) in a 250 ml single neck flask, 90 ml of acetonitrile and 60 ml of cyclopentyl methyl ether were added, followed by the addition of 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 nonylester (17.4), and the mixture was placed in a 90°C oil bath for 24 hours. When the reaction of the starting compound (6-VII-IV) was completed, the solid was removed by suction filtration after the reaction was allowed to cool to room temperature, and the filtrate was spin-dried to obtain a crude product. Finally, column chromatography (dichloromethane:methanol = 20:1 to 5:1) was performed to obtain 5.35 g of compound (I-6-V) with a purity of 99.1% and a yield of 57.9%.

[0368] NMR data:

[0369] 1 H NMR (600 MHz, CDC13): δ 4.90-4.82 (m, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.70-3.60 (m, 1H), 2.64-2.54 (m, 1H), 2.51-2.41 (m, 4H), 2.30-2.26 (q, J = 7.5 Hz, 4H), 1.95 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.56 (m, 7H), 1.50 (d, J = 6.0 Hz, 4H), 1.41-1.37 (m, 4H), 1.34-1.18 (m, 52H), 0.88 (m, 9H).

[0370] Specific rotation: -4.5°

[0371] LCMS: 765.2 [M+H] + .

[0372] Comparative Example:

[0373] Comparative Example 1: Synthesis of Comparative Compound 1

[0374] Step 1): Synthesis of 8-((2-hydroxycyclopentyl)amino)octanoic acid nonylester

[0375]

[0376] In a 100 mL reaction flask, 8-bromononyl octanoate (3.50 g, 10 mmol), 2- aminocyclopentanol (10.12 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. After adding dichloromethane 100 mL, washing with water 3 times, drying with anhydrous sodium sulfate, and concentrating, purification was performed using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain 8-((2-hydroxycyclopentyl)amino)nonyl octanoate (2.29 g, 62%).

[0377] 1 H NMR (600 MHz, CDC13) δ 4.07 (t, 2H), 4.01-3.94 (q, 1H), 2.91 (q, 1H), 2.72 (m, 1H), 2.63 (m, 1H), 2.31 (t, 2H), 2.09-1.97 (m, 2H), 1.72 (m, 2H), 1.67-1.59 (m, 4H), 1.56 (m, 3H), 1.45-1.38 (m, 1H), 1.38-1.22 (m, 18H), 0.90 (t, 3H).

[0378] LCMS: 370.3 [M+H] + .

[0379] Step 2) Synthesis of Comparative Compound 1

[0380]

[0381] Comparative Compound 1 was synthesized in a similar manner to Example 1, Step 3), except that intermediate compound 8-((2-hydroxycyclopentyl)amino)nonyl octanoate was used instead of the original intermediate compound 8-((3-hydroxycyclobutyl)amino)nonyl octanoate.

[0382] 1 H NMR (600 MHz, CDC13) δ 4.91-4.80 (q, 1H), 4.05 (t, 2H), 3.92 (q, 1H), 2.85 (q, 1H), 2.50 (m, 2H), 2.44-2.35 (m, 2H), 2.28 (q, 4H), 1.95-1.87 (m, 2H), 1.83 (br, 2H), 1.75-1.66 (m, 2H), 1.64-1.56 (m, 6H), 1.55-1.47 (m, 5H), 1.47-1.38 (m, 5H), 1.36-1.18 (m, 46H), 0.88 (m, 9H). LCMS: 750.9 [M+H] + .

[0383] Comparative Example 2: Synthesis of Comparative Compound 2

[0384]

[0385] Comparative Compound 2

[0386] Comparative Compound 2 was prepared according to the synthetic method of compound 22 described in Chinese patent CN110520409A.

[0387] 1 H NMR (600 MHz, CDC13) δ 4.10-4.07 (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).

[0388] LCMS: 765.0 [M+H] + .

[0389] Comparative Example 3: Synthesis of Comparative Compound 3

[0390] Step 1): Synthesis of 6-((2-hydroxycyclohexyl)amino)undecyl hexanoate

[0391]

[0392] In a 100 mL reaction bottle, 6-bromoundecyl hexanoate (3.50 g, 10 mmol), 2- aminocyclohexanol (11.5 g, 100 mmol), ethanol 30 mL were added in turn, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was carried out at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain 6-((2-hydroxycyclohexyl)amino)undecyl hexanoate (2.57 g, 67%).

[0393] 1H NMR (600 MHz, CDC13) δ 4.05 (t, 2H), 3.31-3.20 (m, 1H), 2.90-2.75 (m, 1H), 2.53 (m, 1H), 2.35-2.26 (m, 3H), 2.13-2.01 (m, 2H), 1.80-1.68 (m, 2H), 1.67-1.58 (m, 4H), 1.57-1.46 (m, 2H), 1.44-1.34 (m, 2H), 1.34-1.15 (m, 20H), 0.88 (t, 3H).

[0394] LCMS: 384.4 [M+H] + .

[0395] Step 2): Synthesis of Comparative Compound 3

[0396]

[0397] In a 100 mL reaction bottle, 6-((2-hydroxycyclohexyl)amino)hexanoic acid undecyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) were added, and the reaction was carried out at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with 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%).

[0398] 1 H NMR (600 MHz, CDC13) δ 4.86 (m, 1H), 4.05 (t, 2H), 3.39-3.21 (m, 1H), 2.55-2.43 (m, 2H), 2.35-2.23 (m, 6H), 2.10 (m, 1H), 1.76 (m, 2H), 1.70 (m, 1H), 1.67-1.57 (m, 6H), 1.49 (m, 6H), 1.38-1.19 (m, 55H), 0.88 (m, 9H).

[0399] LCMS: 765.0 [M+H] + .

[0400] Comparative Example 4: Synthesis of Comparative Compound 4

[0401]

[0402] In a 250 mL reaction flask, 6-oxohexanoic acid-7-pentadecyl ester (3.40 g, 10 mmol) was added, followed by dichloroethane 100 mL, sodium triacetoxyborohydride (3.18 g, 15 mmol), and then 2-aminocyclohexanol (0.57 g, 5 mmol). The reaction was stirred at room temperature for 24 h. After washing with water for 3 times, drying with anhydrous sodium sulfate, and concentrating, the product was purified by flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to give comparative compound 4 (2.67 g, 70%).

[0403] 1 H NMR (600 MHz, CDC13) δ 4.91-4.80 (m, 2H), 3.28 (m, 1H), 2.48 (m, 2H), 2.30 (m, 6H), 2.24 (m, 1H), 2.11 (m, 1H), 1.75 (m, 2H), 1.68 (m, 1H), 1.66-1.60 (m, 4H), 1.48 (m, 10H), 1.44-1.07 (m, 50H), 0.88 (t, 12H).

[0404] LCMS: 765.2 [M+H] + .

[0405] Comparative Example 5: Synthesis of comparative compound 5 (SM102)

[0406]

[0407] Comparative compound 5 was prepared according to the method described in the synthesis of compound 25 in Chinese patent CN110520409A.

[0408] 1 H NMR (600 MHz, CDC13) δ 4.86-4.74 (m, 1H), 3.99 (t, 2H), 3.46 (t, 2H), 2.51 (t, 2H), 2.39 (q, 4H), 2.26-2.18 (m, 4H), 1.62-1.52 (m, 6H), 1.47-1.36 (m, 8H), 1.28-1.14 (m, 48H), 0.81 (m, 9H).

[0409] LCMS: 711.0 [M+H] + .

[0410] Comparative Example 6: Synthesis of comparative compound 6

[0411]

[0412] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.50 g, 10 mmol), p-aminocyclohexanol (11.5 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried using 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%).

[0413] 1 H NMR (600 MHz, CDC13) δ 4.05 (t, 2H), 3.96-3.90 (m, 0.5H), 3.67-3.57 (m, 0.5H), 2.75-2.67 (t, 2H), 2.66 (m, 0.5H), 2.54 (m, 0.5H), 2.28 (t, 2H), 2.03-1.96 (m, 1H), 1.86-1.78 (m, 1H), 1.78-1.71 (m, 3H), 1.66-1.50 (m, 7H), 1.40-1.20 (m, 20H), 0.88 (t, 3H).

[0414] LCMS: 384.4 [M+H] + .

[0415] Step 2): Synthesis of Comparative Compound 6

[0416]

[0417] In a 100 mL reaction flask, 8-((4-hydroxycyclohexyl)amino)hexanoic acid nonyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) were added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried using 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%).

[0418] 1H NMR (600 MHz, CDC13) δ 4.90-4.82 (m, 1H), 4.05 (t, 2H), 3.99 (m, 0.5H), 3.59-3.51 (m, 0.5H), 2.47 (m, 0.5H), 2.45-2.42 (m, 3H), 2.39-2.32 (m, 1.5H), 2.28 (m, 4H), 2.01 (m, 1H), 1.82 (m, 1H), 1.76 (m, 1H), 1.68-1.58 (m, 7H), 1.52 (m, 4H), 1.43-1.36 (m, 4H), 1.36-1.20 (m, 52H), 0.88 (m, 9H).

[0419] LCMS: 765.0 [M+H] + .

[0420] Synthesis of Comparative Compound 7

[0421] Step 1): Synthesis of 6-((4-hydroxycyclohexyl)amino)undecyl hexanoate

[0422]

[0423] In a 100 mL reaction flask, 6-bromoundecyl hexanoate (3.50 g, 10 mmol), p-aminocyclohexanol (11.5 g, 100 mmol), ethanol 30 mL, after stirring and dissolving, N,N-diisopropyl ethylamine (2.58 g, 20 mmol) was added, and the reaction was carried out at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain 6-((4-hydroxycyclohexyl)amino)undecyl hexanoate (2.57 g, 67%).

[0424] 1 H NMR (600 MHz, CDC13) δ 4.90-4.82 (m, 1H), 4.05 (t, 2H), 3.99 (m, 0.5H), 3.59-3.51 (m, 0.5H), 2.47 (m, 0.5H), 2.45-2.42 (m, 3H), 2.39-2.32 (m, 1.5H), 2.28 (m, 4H), 2.01 (m, 1H), 1.82 (m, 1H), 1.76 (m, 1H), 1.68-1.58 (m, 7H), 1.52 (m, 4H), 1.43-1.36 (m, 4H), 1.36-1.20 (m, 52H), 0.88 (m, 9H).

[0425] LCMS: 384.4 [M+H] + .

[0426] Step 2): Synthesis of Comparative Compound 7

[0427]

[0428] In a 100 mL reaction flask, 6-((4-hydroxycyclohexyl)amino)hexanoic acid undecyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) were added, and the reaction was carried out at room temperature for 24 h. Then, dichloromethane 100 mL was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain Comparative Compound 7 (550 mg, 72%).

[0429] 1 H NMR (600 MHz, CDC13) δ 4.93-4.83 (m, 1H), 4.07 (t, 2H), 3.99 (m, 0.5H), 3.63-3.49 (m, 0.5H), 2.52 (m, 0.5H), 2.49-2.44 (m, 3H), 2.40 (m, 1.5H), 2.30 (m, 4H), 2.02 (m, 1H), 1.85 (m, 1H), 1.78 (m, 1H), 1.69-1.60 (m, 7H), 1.53 (m, 4H), 1.46-1.39 (m, 4H), 1.35-1.23 (m, 52H), 0.90 (m, 9H).

[0430] LCMS: 765.2 [M+H] + .

[0431] Biological test

[0432] The lipids used in the biological experiments of the present application are MC3, SM102, and ALC-0315, the structures of which are as follows:

[0433]

[0434] Among them, MC3 and ALC-0315 can be purchased or prepared according to the known technology in the art.

[0435] Preparation of lipid nanoparticles:

[0436] Preparation method one: the amino lipid compound described in the present application is mixed with DOPE, cholesterol, and PEG2000-DMG in a molar ratio of 45:10:42.5:2.5, and dissolved in anhydrous ethanol. A microsyringe pump is used to control the ratio of the ethanol solution to a sodium acetate solution (50 mM, pH = 4.0) to be 1:3, and a crude solution of the lipid nanoparticles is prepared in a microfluidic chip. The solution is then dialyzed in a dialysis box (Fisher, MWCO 20,000) in 1X PBS at 4°C for 6 h, and filtered through a 0.22 μm microporous filter before use.

[0437] Preparation method two: the amino lipid compound is mixed with DSPC, cholesterol, and PEG2000-DMG in a molar ratio of 50:10:38.5:1.5, and the preparation method is the same as method one.

[0438] The lipid nanoparticles prepared according to the preparation method one or two described above are used in the biological experiments for evaluating the in vivo delivery performance of luciferase mRNA (Fluc mRNA) described below. The mass ratio of the amino lipid compound in the lipid nanoparticles obtained in the preparation method one to the luciferase mRNA is about 10:1, and the nanoparticles are administered subcutaneously. The lipid nanoparticles obtained in the preparation method two are administered by tail vein and intramuscular injection.

[0439] Test example 1: evaluation of the in vivo delivery performance of luciferase mRNA of the lipid nanoparticles prepared from the amino lipid compound described in the present application Animal preparation: 6-week-old female BALB / c mice weighing about 20 g are selected, and the mice are raised in a SPF-level feeding room. The animal experiments are strictly conducted in accordance with the guidelines of the national health organization and the requirements of animal ethics.

[0440] In vivo delivery: 9 mice are randomly selected in each group, and the mice are injected with the lipid nanoparticles at a dose of 0.5 mg / kg by subcutaneous injection, intramuscular injection, and tail vein injection (three mice for each administration method). After 12 h, 200 μL of 10 mg / mL D-luciferin potassium salt is injected into each mouse through the tail vein, and the mice are placed under an in vivo imaging system (IVIS-200, Xenogen) after 10 min. The total fluorescence intensity of each mouse is observed, and a photograph is taken. The expression intensity of Fluc mRNA delivered by the representative amino lipid compound by the three administration methods is shown in Tables 1-3. MC3 is used as a control. SM102 is a comparative compound.

[0441] Table 1: expression intensity of Fluc mRNA delivered by subcutaneous administration of the representative amino lipid compound

[0442]

[0443]

[0444] Table 2: Expression intensity of Fluc mRNA delivered by representative aminolipid compounds via intramuscular injection.

[0445]

[0446]

[0447] Table 3: Expression intensity of Fluc mRNA delivered by representative aminolipid compounds via tail vein injection.

[0448]

[0449]

[0450] Test Example 2: Ovalbumin mRNA in vivo delivery and immune performance evaluation of lipid nanoparticles prepared from aminolipid compounds according to the present application

[0451] Preparation of lipid nanoparticles:

[0452] Preparation method: The aminolipid compound of formula (I) according to the present application was mixed with DOPE, cholesterol and PEG2000-DMG in a molar ratio of 50:10:38.5:1.5 and dissolved in anhydrous ethanol. Ovalbumin mRNA (OVA mRNA) was dissolved in sodium acetate solution (50 mM, pH = 4.0). Using a microsyringe pump, the ratio of ethanol solution to sodium acetate solution (50 mM, pH = 4.0) was controlled at 1:3, and the crude solution of lipid nanoparticles was prepared in a microfluidic chip. Then, dialysis was performed using a dialysis cassette (Fisher, MWCO 20,000) in 1X PBS at 4°C for 6 h, and the lipid nanoparticles were filtered using a 0.22 μm microporous filter before use.

[0453] The mass ratio of aminolipid compound to ovalbumin mRNA (OVA mRNA) in the obtained lipid nanoparticles was about 10:1.

[0454] Animal preparation: 6-week-old female BALB / c mice weighing about 20 g were selected, and the animal experiment was carried out in a SPF-grade feeding room in strict accordance with the guidelines of the national health agency and the requirements of animal ethics.

[0455] In vivo delivery: 3 mice were randomly selected from each group, and the lipid nanoparticles were injected into the leg muscles at a dose of 0.5 mg / kg (Day 0). After 7 days, the same amount was used for reinforcement (Day 7). On day 21, the blood was taken from the tail vein for serological analysis. MC3 was used as a control.

[0456] Enzyme-linked immunosorbent assay (ELISA): Flat-bottom 96-well plates (Nunc) were pre-coated with 0.5 μg of protein per well of OVA protein in 50 mM carbonate buffer (pH 9.6) overnight at 4°C, and then blocked with 5% glycine. Antisera were obtained from immunized animals and proteins were diluted to 10 2 diluted to 10 6 PBS-0.05% Tween (PBS-T), pH 7.4, and added to the wells and incubated at room temperature for 1 hour at 37°C. 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 one wavelength and the absorbance at 450 nm was measured in an ELISA reader (Bio-Rad).

[0457] The results of the test are shown in Table 1. Figure 1 It is noted that Lipid 7 (compound 7) has a protein expression level comparable to that of SM102, but can elicit a stronger immune response.

[0458] It can be seen that the amino lipid compound provided by the present application has excellent immune activity and also has a strong adjuvant effect.

[0459] Test Example 3: In vivo delivery and immune performance evaluation of an influenza mRNA vaccine prepared from the amino lipid compound of the present application Preparation method: The amino lipid compound of formula (I) of the present application, DSPC, cholesterol, and PEG2000-DMG were mixed and dissolved in anhydrous ethanol at a molar ratio of 45:10:42.5:2.5. The mRNA expressing influenza was dissolved in a sodium acetate solution (50 mM, pH = 4.0). Using a microsyringe pump, the ratio of the ethanol solution to the sodium acetate solution (50 mM, pH = 4.0) was controlled to be 1:3, and a crude solution of the lipid nanoparticles was prepared in a microfluidic chip. Then, the solution was dialyzed in a dialysis box (Fisher, MWCO 20,000) in 1X PBS at a controlled temperature of 4°C for 6 h, and filtered with a 0.22 μm microporous filter before use.

[0460] The mass ratio of the amino lipid compound to the influenza mRNA (OVA mRNA) in the obtained lipid nanoparticles was about 10:1.

[0461] Animal preparation: 6-week-old female BALB / c mice weighing about 20 g were selected, and the animal test was strictly performed in accordance with the guidelines of the national health organization and the requirements of animal ethics.

[0462] In vivo delivery: Three mice were randomly selected from each group and administered lipid nanoparticles subcutaneously via the back at a dose of 0.5 mg / kg (Day 0). A booster dose of the same amount was administered 7 days later (Day 7). Serological analysis was performed on tail vein blood samples on day 21. MC3 served as a control.

[0463] Enzyme-linked immunosorbent assay (ELISA): The assay method is as described in Example 2.

[0464] The test results are as follows Figure 2 As shown, the IgG antibody titer of the MC3 control group was the lowest, the IgG antibody titer of Lipid10 (compound 10) was comparable to that of SM102, while the IgG antibody titers of other compounds were significantly better than those of the control group. This also shows that the aminolipid compounds provided by the present invention have excellent immunomodulatory activity and a strong adjuvant effect.

[0465] Experimental Example 4: Stability Evaluation of Lipid Nanoparticles Prepared from the Aminolipid Compounds of the Present Invention Using Formulation Method 1

[0466] Preparation of lipid nanoparticles: LNPs were prepared using formulation method one for subcutaneous administration.

[0467] Characterization of lipid nanoparticles: The particle size and PDI of the prepared lipid nanoparticles were determined using a Nano-ZSZEN3600 (Malvern) microscope. 40 μL of LNP solution was used for particle size measurement, with three cycles of 30 s each.

[0468] 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 experimental results are shown in Table 4.

[0469] Table 4: DLS characterization and subcutaneous expression of Fluc-mRNA of LNPs prepared from representative aminolipid compounds of the present invention.

[0470]

[0471] As can be seen from Table 4, at the test temperature of 25°C, in terms of particle size, the representative compounds 5, 6, 7, 9 and SM102 of the present application were all relatively uniform nanoparticles with a particle size of about 100 nm and a PDI of less than 0.1 on the day (week 0), and after 4 weeks of storage, the particle size of each of them increased, among which the particle size of SM102 increased by more than one time, from 120 to 245, while the particle size of the representative compounds 5, 6, 7, 9 of the present application did not increase significantly; in terms of PDI, after 4 weeks, the PDI of the representative compounds 5, 6, 7, 9 of the present application was less than 0.1, while the PDI of SM102 increased from 0.05 on the day (week 0) to 0.28 (week 4); in terms of Fluc expression, after 4 weeks, the Fluc expression of the representative compounds 5, 6, 7, 9 of the present application did not decrease significantly, while the Fluc expression of SM102 decreased significantly, from 2.1E+07 to 3.6E+06, thus it can be seen that the amino lipid compound described in the present application has excellent stability.

[0472] Test Example 5: Evaluation of the activity of the lipid nanoparticles prepared from the amino lipid compound described in the present application using preparation method two

[0473] Preparation of the lipid nanoparticles: LNP was prepared using preparation method two, and was administered by intramuscular injection.

[0474] The test method was the same as that of Test Example 1, and the test results are shown in Table 5:

[0475] Table 5: Expression intensity of Fluc mRNA delivered by intramuscular administration of the representative amino lipid compounds of the present application.

[0476]

[0477]

[0478] As can be seen from Table 5, in the case of the same other structural units, 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 than 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 application 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 application only in that the hydroxyl group on the cycloalkyl structural unit is located at the ortho position of 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 application only in that the hydroxyl group on the cycloalkyl structural unit is located at a position two carbon atoms apart from the amine substituent) is 1.9E+07. The same conclusion can be seen between, for example, representative compound 7 of the present application and comparative compound 3 and comparative compound 7, between representative compound 8 of the present application and comparative compound 4 and comparative compound 8, and the like. Thus, in the case of the same other structural units, the activity performance of the lipid nanoparticle of the amino lipid compound containing a substituent on the cycloalkyl structural unit at the meta position of the amine substituent is the highest.

[0479] Test Example 6 Amino lipid compound targeting test mRNA lipid nanoparticle (LNP) preparation method: Lipid was mixed with DSPC, CHO-HP, M-DMG2000 in a molar ratio of 50:10:38.5:1.5 in anhydrous ethanol. The mRNA was dissolved in a sodium acetate solution (0.2M, pH=5.0). The preparation of each prescription sample was carried out using a microfluidic device (MPE-L2) and a chip (SN.000035) with a water phase: alcohol phase = 9ml / min: 3ml / min. The two phases were injected into the microfluidic chip according to the interface requirements for mixing, and the drug solution was then loaded into a 100KD dialysis bag, which was immersed in a beaker containing 1L of dialysis solution. The beaker was wrapped with aluminum foil and dialyzed at room temperature at a speed of 100rpm for 1h, and then the dialysis solution was replaced and dialyzed for another 1h.

[0480] Dialysis solution preparation: 1xPBS+8% sucrose solution: take 2 bags of 1xPBS powder into a beaker, dissolve with 2L DEPC water, continue to add 160g sucrose, mix well to get 1xPBS+8% sucrose solution.

[0481] The mass ratio of mRNA to lipid in the obtained lipid nanoparticles is about 1:10.

[0482] The preparation method of the foregoing mRNA lipid nanoparticle (LNP) preparation is adopted, wherein the mRNA is selected from an mRNA encoding a fluorescent protein, the Lipid is selected from compound (I-6-II), MC3 and SM102 respectively, and mRNA-LNP preparations containing compound (I-6-II), MC3 or SM102 respectively for encoding a fluorescent protein are prepared.

[0483] Test method:

[0484] Each test preparation is administered to mice by intramuscular injection, with 15ug of mRNA preparation sample injected into each animal. After 6 hours of injection, the mice are anesthetized by isoflurane inhalation, and 200ul of D-Luciferin (concentration 10mg / ml) luciferase imaging substrate is injected intraperitoneally. The animals are placed in a supine position, and at 10 minutes after injection of the substrate, the distribution and expression intensity of Luciferase in the mice are observed under the IVIS live imaging system. After live imaging of the animals injected intramuscularly, the animals are immediately dissected, and the fluorescence of the liver is observed rapidly after dissection.

[0485] The specific fluorescence intensity values are as follows:

[0486] mRNA-LNP preparation containing MC3 for encoding a fluorescent protein: 1.23E+05

[0487] mRNA-LNP preparation containing SM102 for encoding a fluorescent protein: 3.23E+06

[0488] mRNA-LNP preparation containing compound (I-6-II) for encoding a fluorescent protein: 2.49E+05

[0489] The test results are shown in Table 1. Figure 8 As shown in Table 1, when administered by intramuscular injection, the liver fluorescence intensity of the compound (I-6-II) group is significantly lower than that of SM102, the organ distribution of the mRNA encoding a fluorescent protein in the important organ liver is relatively low, and the local targeting effect is good, effectively reducing the risk of liver toxicity.

[0490] Test Example 7: Test of adverse reactions of amino lipid compounds by intramuscular injection

[0491] Preparation of mRNA-LNP preparation encoding S protein of novel coronavirus:

[0492] The mRNA lipid nanoparticle (LNP) formulation was prepared using the method described in Experimental Example 6, wherein the mRNA was selected to encode the novel coronavirus S protein mRNA, and the lipids were selected from compound 6, compound (I-6-II), ALC-0315, and SM102, respectively, to prepare novel coronavirus S protein mRNA-LNP formulations containing compound 6, compound (I-6-II), ALC-0315, or SM102, respectively.

[0493] The sequence encoding the novel coronavirus S protein mRNA is obtained by replacing all uracil (u) in SEQ ID NO.1 with N1-methylpseuuridine. It should be noted that, according to WIPO standard ST.26 for nucleotide or amino acid sequence listings, t (thymine) in the RNA sequence SEQ ID NO.1 is actually u (uracil).

[0494] Test method:

[0495] SD rats were randomly divided into four groups (half male and half female): a high-dose group of compound 6 (100 μg mRNA / rat), a high-dose group of compound (I-6-II) (100 μg mRNA / rat), a high-dose group of ALC-0315 (cationic lipids from Pfizer vaccine BNT162-b2) (100 μg mRNA / rat), and a high-dose group of SM-102 (cationic lipids from Moderna vaccine mRNA-1273) (100 μg mRNA / rat). Three rats per group (sex) were administered the drugs via intramuscular injection once a week for three consecutive weeks. Clinical observation was conducted to detect abnormalities such as swelling at the injection site.

[0496] The test results are as follows Figure 9 As shown, from Figure 9 It can be seen that, compared with the cationic lipid components of commercially available mRNA vaccines at the same level, compound (I-6-II) significantly reduced or decreased the number of severe swelling and moderate claudication at the injection site, and therefore can be considered to have higher safety.

[0497] Experimental Example 8: Toxicity Kinetics

[0498] Preparation of empty liposome formulations:

[0499] The alcohol phase was obtained by taking 1.9078 g of compound I-6-II, 0.3985 g of DSPC, 0.7575 g of CH0-HP and 0.1972 g of PEG-DMG (average molecular weight 2000) in a molar ratio of 49.5:10:39:1.5, dissolving them in anhydrous ethanol, and making up to 237.5 ml. The microfluidic device (MPE-L2) was used to mix and encapsulate in acetic acid-sodium acetate buffer (0.2 mol / L, pH = 5) as the water phase, with a volume ratio of water phase to alcohol phase of 3:1. The encapsulated drug solution was subjected to ultrafiltration replacement under the parameters of TMP of 0.2 bar and liquid flow rate of 300 ml / min, and the dialysis solution used for replacement contained 8 mg / ml of sodium chloride, 0.2 mg / ml of potassium chloride, 0.2 mg / ml of potassium dihydrogen phosphate, 1.15 mg / ml of disodium hydrogen phosphate dihydrate and 80 mg / ml of sucrose. After replacement, sterilization filtration was performed to obtain the I-6-II empty liposome preparation.

[0500] Test method:

[0501] The animal experiment was divided into 5 groups (5 animals / sex / group), and the grouping and dosing were as follows: negative control group (0 dose / animal), empty liposome low-dose group (1 dose / animal) and empty liposome high-dose group (4 doses / animal). The animals in each group were injected intramuscularly once every 2 weeks, for a total of 3 times, i.e., D1 (the first day of administration), D15 (the 15th day after administration) and D29 (the 29th day after administration).

[0502] The animal experiment was divided into 5 groups (5 animals / sex / group), and the grouping and dosing were as follows: negative control group (0 dose / animal), empty liposome low-dose group (1 dose / animal) and empty liposome high-dose group (4 doses / animal). The animals in each group were injected intramuscularly once every 2 weeks, for a total of 3 times, i.e., D1 (the first day of administration), D15 (the 15th day after administration) and D29 (the 29th day after administration).

[0503] The negative control group collected animal blood samples before and 4 hours after the first (D1) and last (D29) administration; the empty liposome group collected animal blood samples at the time points of before administration, 15 min, 1 h, 2 h, 4 h, 8 h, 24 h, 32 h and 48 h after administration of the first (D1) and last (D29) administration, respectively. The content of compound I-6-II in the plasma of cynomolgus monkeys was detected to investigate the exposure of compound I-6-II in cynomolgus monkeys after administration of the animals in each group.

[0504] The main TK parameters of compound I-6-II in the animals in the empty liposome low-dose and high-dose groups after the first administration (D1) and the last administration (D29) are shown in Table 6:

[0505] Table 6 Major TK parameters of compound I-6-II in cynomolgus monkeys in empty liposome low and high dose groups

[0506]

[0507] The test data show that:

[0508] No I-6-II liposome was detected in the plasma samples of the negative control group animals at the first (D1) and last (D29) time points.

[0509] As can be seen from Table 6, compound I-6-II can be rapidly cleared in cynomolgus monkeys, and the plasma drug concentration half-life is 4.68-6.45 hours; compared with the first administration (D1), the Cmax ratio (D29 / D1) of compound I-6-II in the empty liposome group was between 0.58 and 0.84, and the AUC last ratio (D29 / D1) was between 0.68 and 1.02, indicating that after 4 weeks of continuous administration (a total of 3 times), there was no accumulation of compound I-6-II in cynomolgus monkeys.

[0510] In summary, the amino lipid compound provided by the present application has a cycloalkyl structural unit and an additional substituent at the meta position of the amine substituent of the cycloalkyl group, and can be used to deliver bioactive agents into cells, and has one or more of the following advantages over the similar structure amino lipid compounds known in the prior art: good delivery ability, good stability, high safety, can be used to deliver bioactive agents (such as nucleic acids) into cells, improve the protein expression level, more effectively produce an immune response in the body; excellent targeting; can be stored, transported and used at room temperature.

[0511] List of abbreviations

[0512] DIPEA N,N-diisopropylethylamine

[0513] DNA deoxyribonucleic acid

[0514] RNA ribonucleic acid

[0515] DOPE dioleoyl phosphatidyl ethanolamine

[0516] DSPC distearoyl phosphatidyl choline

[0517] PEG2000-DMG 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000

[0518] kD kiloDalton

[0519] PBS phosphate buffered solution

[0520] It will be apparent to those skilled in the art that the present disclosure is not limited to the above-described illustrative embodiments and that it can be embodied in other specific forms without departing from the essential characteristics thereof. It is therefore desired that the embodiments be considered in all respects as illustrative and not restrictive, reference being made to the appended claims as expressing the scope of the present disclosure rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A compound having the structure: ###0001### or a pharmaceutically acceptable salt or stereoisomer thereof.

2. A compound having a structure selected from the group consisting of: ###0002### or a pharmaceutically acceptable salt thereof.

3. A lipid nanoparticle comprising the compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, or the compound of claim 2 or a pharmaceutically acceptable salt thereof.

4. The lipid nanoparticle of claim 3, wherein, The lipid nanoparticle further comprises a biologically active agent.

5. The lipid nanoparticle of claim 4, wherein, The lipid nanoparticle further comprises a helper lipid.

6. The lipid nanoparticle of claim 5, wherein, The lipid nanoparticle further comprises a sterol.

7. The lipid nanoparticle of claim 6, wherein, The lipid nanoparticle further comprises a PEG lipid.

8. Use of the lipid nanoparticle of any one of claims 3 to 7 in the manufacture of a medicament for gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA, wherein the gene therapy is for the treatment of cancer and genetic diseases.

9. The use according to claim 8, wherein the cancer is selected from one or more of lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphatic cancer, blood cancer or prostate cancer; and the genetic disease is selected from one or more of hemophilia, thalassemia, Gaucher's disease.

10. The use according to claim 8, wherein the gene vaccination is for the treatment of cancer, allergy, toxicity and pathogen infection.

11. The use according to claim 10, wherein the pathogen is selected from one or more of a virus, a bacterium or a fungus.

12. Use of the lipid nanoparticle of any one of claims 3 to 7, the compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, or the compound of claim 2 or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the transfer of a nucleic acid, wherein the nucleic acid is RNA, DNA, antisense oligonucleotide.

13. The use according to claim 12, wherein 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).

14. The use according to claim 12, wherein the DNA is a plasmid.

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