Lipids and nanoparticles comprising lipids and uses thereof

Abstract

The present disclosure relates to novel lipids, nanoparticles comprising such lipids and their use for delivering therapeutic agents to a subject or treating and / or preventing a disease in the subject.

Description

Cross-reference

[0001] This application claims the filing date and all rights of U.S. Provisional Patent Application No. 63 / 350,215 filed on June 8, 2022, the entire contents of which are incorporated herein by reference. Background of the Invention 1. Technical Field

[0002] The present disclosure generally relates to the field of drug delivery, and more particularly, to novel nanoparticles formed from novel lipids for delivering therapeutic agents (eg, nucleic acids), and the use of these nanoparticles to treat and / or prevent diseases. 2. Background technology

[0003] Among the various agents used to transfect biologically active agents (e.g., nucleic acids) into cells, the most effective method is generally considered to be lipid nanoparticle (e.g., lipid microparticle)-mediated delivery, because it is the most effective and easiest to use. Lipid nanoparticles are artificial spherical carriers formed by lipid bilayers. In order to deliver molecules to the treatment site, the lipid bilayer will fuse with other bilayers (e.g., cell membranes), thereby delivering the contents of the microparticles into the cell.

[0004] Because of their unique properties, lipid nanoparticles can be used as carriers for drug delivery. Lipid nanoparticles encapsulate an area of ​​aqueous solution in a hydrophobic membrane, and dissolved hydrophilic solutes cannot easily pass through the lipids. Hydrophobic chemicals can be dissolved into the membrane, and in this way, lipid nanoparticles can carry both hydrophobic and hydrophilic molecules. Lipid nanoparticles can be used with other biological agents (e.g., drugs, nucleic acids, etc.), or used to deliver these agents to treat and / or prevent diseases.

[0005] Recently, lipid nanoparticles have been used in COVID-19 mRNA vaccines. mRNA vaccines have been shown to be effective in controlling COVID-19, and many countries are also conducting clinical trial programs to evaluate the effectiveness of mRNA vaccines against other diseases. The advantage of mRNA vaccines is that they can induce sufficient immune responses to protect the host against infectious pathogens, and vaccines can be quickly manufactured to target infectious pathogen variants. However, the poor stability of mRNA vaccines causes difficulties in storage and transportation. For example, the mRNA vaccine manufactured by Moderna and Pfizer-BioNTech to fight COVID-19 can only be stored for 6 months at temperatures between -20°C and -80°C. In addition, the stability of the mRNA vaccine manufactured by Moderna to fight COVID-19 can only be maintained for about 12 hours at room temperature. Similarly, the stability of the mRNA vaccine manufactured by Pfizer-BioNTech to fight COVID-19 is only about 2 hours at room temperature.

[0006] In view of this, the relevant field is in urgent need of a novel lipid molecule that can be used to produce lipid nanoparticles that can deliver drugs (eg, nucleic acids). Summary of the invention

[0007] The present disclosure provides novel cationic lipids that can be used to form novel nanoparticles capable of delivering nucleic acids; and the use of these nanoparticles to treat and / or prevent diseases (e.g., diseases caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)).

[0008] Therefore, the first aspect of the present disclosure is about a lipid having a structure of formula (1), in, R 1 Is the presence or absence of one or more hydroxyl groups, -CH 2 OH, or -N(R) 2 an alkyl or cycloalkyl substituent; m and n are respectively integers between 0 and 12; R 2 and R 3 are hydrogen, alkenyl, R a 、-(C=O)OCH 2 R a 、-O(C=O)R b or -(C=O)OR b ; R a Yes-CR'(COOR') 2 or -CR'(COOR")(COOR'"); and R, R b , R', R", and R'" are each hydrogen or alkyl.

[0009] According to the embodiments of the present disclosure, the lipid having the structure of formula (I) can be any of the following compounds: or

[0010] The second aspect of the present disclosure is to provide a lipid having a structure of formula (II), in, R 1 and R 3are alkyl groups with or without one or more hydroxyl substituents, respectively; R 2 is hydrogen or -O(C=O)R', wherein R' is alkyl; and m and n are integers between 1 and 10 respectively.

[0011] According to a preferred embodiment of the present disclosure, the lipid with the structure of formula (II) can be

[0012] The third aspect of the present disclosure is to provide a lipid having a structure of formula (III), in, m and n are each an integer between 1 and 10; and R 1 , R 2 , R 3 and R 4 are hydrogen or alkyl, respectively.

[0013] According to a preferred embodiment of the present disclosure, the lipid with the structure of formula (III) can be

[0014] Another aspect of the present disclosure is to provide a lipid nanoparticle formed by one or more of the above lipids for delivering a desired active agent (e.g., a nucleic acid of a target protein or a therapeutic agent). The lipid nanoparticle structurally comprises a hydrophilic inner core and a lipid bilayer outer layer formed by one or more lipids of formula (I) to (III).

[0015] In addition, the lipid nanoparticles of the present disclosure further include a therapeutic agent located in the hydrophilic inner core or the lipid bilayer outer layer of the lipid nanoparticle. If the therapeutic agent is hydrophilic, it will be located in the hydrophilic inner core, whereas if it is hydrophobic, it will be located in the lipid outer layer. The therapeutic agent can be a nucleic acid of a target protein.

[0016] Examples of nucleic acids that can be coated in the hydrophilic core of the lipid nanoparticles of the present disclosure include, but are not limited to, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), microRNA (miRNA), transfer RNA (tRNA) or a combination thereof. In certain embodiments, the lipid nanoparticles of the present disclosure further include mRNA of SARS-CoV-2 coated in the hydrophilic core. In other embodiments, the lipid nanoparticles of the present disclosure further include mRNA of dengue virus sheath protein (E protein) coated in the hydrophilic core.

[0017] One or more embodiments of the present disclosure will be described in detail below. After referring to the following detailed description and the scope of claims, the many accompanying features and advantages of the present disclosure will be easily understood.

[0018] It should be understood that the foregoing general description and the following detailed description are merely exemplary and are intended to further illustrate the claimed invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present disclosure and other features, aspects and advantages will become more apparent after referring to the following detailed description of the embodiments, the claims and the accompanying drawings, wherein:

[0020] Figure 1 Expression of Spike protein after administration of SARS-CoV2 spike protein (BA.5) mRNA-LNP complexes in vivo and in vitro (A) ELISA cell data showing successful delivery of mRNA; (B) ELISA assay showing binding activity between immune serum and target; and (C) maximum inhibitory concentration (IC50) from sera from immune animals 50 );

[0021] Figure 2 Expression of Spike protein after administration of SARS-CoV2 Spike protein (WT) mRNA-LNP complex in vivo and in vitro (A) ELISA cell data showing successful delivery of mRNA; (B) ELISA test showing binding activity between immune serum and target; and

[0022] Figure 3 Functional analysis of DENV2 membrane protein (E protein) mRNA-LNP complexes (A) Protein expression, (B) Serum collected 6 weeks after the first vaccination and analyzed by ELSA for specific neutralizing antibodies against dengue virus; and (C) Maximum half inhibitory concentration of serum from immunized animals. DETAILED DESCRIPTION

[0023] In order to make the description of the contents of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention.

[0024] In the present invention, lipid nanoparticles refer to particles consisting of a hydrophilic core coated with a lipid outer layer, which are suitable for treating and / or preventing diseases, wherein if the desired active ingredient (e.g., nucleic acid and / or therapeutic agent) is hydrophilic in nature, it will be located in the hydrophilic core of the particle, otherwise it will be located on the lipid outer layer.

[0025] For the sake of convenience, the terms used in the specification, implementation methods and claims are collectively described here. Unless otherwise specified, the definitions of all technical and scientific terms mentioned in this article are the same as those understood by ordinary skilled persons in this field.

[0026] Unless otherwise specified, nouns in the singular form used herein shall include their plural forms, and nouns in the plural form shall also include their singular forms. In this document and the scope of rights, the singular forms "a" and "an" include their plural forms. In addition, as described, "at least one" and "one or more" mean one, two, three, or more.

[0027] As used herein, "about" generally refers to within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the mean value, as considered by one of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise expressly stated, all ranges, quantities, values ​​and percentages used herein (e.g., to describe the amount of material, the duration of time, the temperature, the operating conditions, the ratio of quantities or the angle of reflection) are understood to be modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying patent claims are approximate values ​​and may be changed as needed. At least these numerical parameters should be understood as the number of significant digits indicated and the values ​​obtained by applying the general rounding method.

[0028] Unless otherwise indicated, any atom, moiety or functional group described herein may be substituted or unsubstituted as long as the valence permits. The term "optionally substituted" refers to substituted or unsubstituted. When the term "substituted" is used to describe a chemical structure or moiety, it means that one or more hydrogen atoms of the chemical structure or moiety are replaced by another atom, moiety or functional group other than hydrogen atoms, such as halogen, -OH, alkyl, aryl, amine, alkylamino and the like. The term "substituted" covers all substituents that are allowed to be substituted in organic compounds, any of which can form stable compounds as described herein. The content of this article covers any and all combinations of such substituents to achieve stable compounds.

[0029] "Alkyl" refers to a straight or branched hydrocarbon having 1 to 21 (e.g., 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) carbon atoms. Examples of alkyl include methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, isobutyl, 2-isopropyl-3-methyl, pentyl, pentane-2-yl, hexyl, isohexyl, heptyl, heptane-2-yl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. Unless otherwise specified, each alkyl group may be optionally substituted, that is, it may be an unsubstituted alkyl group or a substituted alkyl group having one or more substituents. According to certain embodiments, the alkyl group is an unsubstituted C 1-21 In a preferred embodiment, the alkyl group is an octyl group (i.e., -C 8 H 17 In another preferred embodiment, the alkyl group is a decyl group (ie, -C 10 H 21 In other preferred embodiments, the alkyl group is a substituted C 1-12 In a preferred embodiment, the alkyl group is an ethyl group having a hydroxyl substituent (i.e., -C 2 H 5 OH). In another preferred embodiment, the alkyl group is a propyl group having two hydroxyl substituents. In another preferred embodiment, the alkyl group is a propyl group having one amino substituent (i.e., -C 3 H 7 NH 2 In another preferred embodiment, the alkyl group is a propyl group having a dimethylamino substituent (i.e., -C 3 H 6 N(CH 3 ) 2 ).

[0030] "Cycloalkyl" refers to a cycloalkyl group having 3 to 10 ring carbon atoms (i.e., C 3-10 In some embodiments, the cycloalkyl group is a monocyclic, saturated carbocyclic group (i.e., C cycloalkyl) and has no heteroatoms. In some embodiments, the cycloalkyl group is a monocyclic, saturated carbocyclic group (i.e., C cycloalkyl) and has no heteroatoms. In some embodiments, the cycloalkyl group is a monocyclic, saturated carbocyclic group (i.e., C cycloalkyl) having 3 to 10 ring carbon atoms. 3-10 In certain embodiments, the cycloalkyl group is a group having 3 to 8 ring carbon atoms (i.e., C 3-8 In certain embodiments, the cycloalkyl group is a group having 3 to 6 ring carbon atoms (i.e., C 3-6In certain embodiments, the cycloalkyl group is a group having 5 to 6 ring carbon atoms (i.e., C 5-6 In certain embodiments, the cycloalkyl group is a group having 5 to 10 ring carbon atoms (i.e., C 5-10 Cycloalkyl). 5-10 Examples of cycloalkyl groups include cyclopentyl (C 5 ) and cyclohexyl (C 6 ). C 3-6 Examples of the cycloalkyl group include the aforementioned C 5-10 Cycloalkyl and cyclopropyl (C 3 ) and cyclobutyl (C 4 ). C 3-8 Examples of the cycloalkyl group include the aforementioned C 3-6 Cycloalkyl and cycloheptyl (C 7 ) and cyclooctyl (C 8 ). Unless otherwise specified, each cycloalkyl group may be a cycloalkyl group having no substituents (i.e., a cycloalkyl group having no substituents) or a cycloalkyl group having one or more substituents (i.e., a cycloalkyl group having substituents). In certain embodiments, a cycloalkyl group is a C 3-10 Cycloalkyl. The carbocyclic ring itself may be partially saturated. Unless otherwise indicated, each cycloalkyl group may be a cycloalkyl group without a substituent (i.e., a cycloalkyl group without a substituent) or a cycloalkyl group containing one or more substituents (i.e., a cycloalkyl group with a substituent). In certain embodiments, the cycloalkyl group is a C 3-10 In other embodiments, the cycloalkyl group is a substituted C 3-10 Cycloalkyl.

[0031] "Alkenyl" refers to a straight or branched chain hydrocarbon (i.e., C 2-20 In a preferred embodiment, the alkenyl group has 18 carbon atoms (i.e., -C 18 In another preferred embodiment, the alkenyl group is a group having 18 carbon atoms (i.e., -C 18 Unless otherwise specified, each alkenyl group may be an alkenyl group without a substituent (i.e., an unsubstituted alkenyl group) or an alkenyl group containing one or more substituents (i.e., a substituted alkenyl group). In certain embodiments, the alkenyl group is a C 2-20 In other embodiments, the alkenyl group is a substituted C 2-20 Alkenyl.

[0032] It should be noted that compounds with the same chemical formula but different atoms in the order of bonding or in the position of their atoms in space are "isomers" of each other. Isomers whose atoms are arranged in different positions in space are called stereoisomers. Stereoisomers that are not mirror images of each other are called "diastereomers", and those whose mirror images are not superimposable are called "enantiomers". When a compound has an asymmetric center, for example, bonded to four different groups, a pair of mirror images will be produced. The mirror image isomer is characterized by the absolute configuration of its asymmetric center, and is designated as right- or left-racemic (i.e., as a (+) or (-)-isomer) by the R- and S-ordering rules of Cahn and Prelog, or by the polarization of the plane of rotation of the molecule. Chiral compounds can exist alone or as a mixture. When a mixture contains equal proportions of mirror image isomers, it is called a "racemic mixture".

[0033] Unless otherwise specified, the technical or scientific terms used herein have the same meanings as those commonly used or understood by persons skilled in the art in the present invention. In case of any conflict, the definitions in the present invention shall prevail.

[0034] 1. Compounds

[0035] The present disclosure describes a compound having a structure of formula (1),

[0036] In formula (I), R 1 Is the presence or absence of one or more hydroxyl groups, -CH 2 OH, or -N(R) 2 In certain embodiments, R 1 is an ethyl group having a hydroxyl substituent. In other embodiments, R 1 is an ethyl group having two hydroxyl substituents. In a further embodiment, R 1 There is a dimethylamine group (i.e., -N(CH 3 ) 2 In certain embodiments, R 1 is a propyl group having a hydroxyl substituent. In other embodiments, R 1 is a propyl group having two hydroxyl substituents. In a further embodiment, R 1 is a hexyl group having a hydroxyl substituent. In other embodiments, R 1 is a cyclohexyl group having a hydroxyl substituent. In a further embodiment, R 1 There is a -CH 2In a further embodiment, R 1 Yes there is one The substituent is ethyl.

[0037] Alternatively, m and n are each an integer between 0 and 12; and R 2 and R 3 are hydrogen, alkenyl, R a 、-(C=O)OCH 2 R a 、-O(C=O)R b or -(C=O)OR b ; Among them, R a Yes-CR'(COOR') 2 or -CR'(COOR")(COOR'"); and R, R b In certain embodiments, m is 5, n is 7, R 2 and R 3 They are -(C=O)OCH 2 R a , and R a Yes-CR'(COOR') 2 , R' is methyl, and R" is -C 8 H 17 In other embodiments, m and n are 7, R 2 Yes-(C=O)R b , R 3 Yes -(C=O)OCH 2 R a , and R a Yes-CR'(COOR') 2 , R b It is -CH(C 8 H 17 ) 2 , R" is -C 8 H 17 In a further embodiment, m is 10, n is 6, R 2 is hydrogen, R 3 YesR a , and R a Yes-CR'(COOR') 2 , R' is methyl, and R" is -C 8 H 17 In a further embodiment, m is 10, n is 6, R 2 is hydrogen, R 3 YesR a , and R ais -CR'(COOR") (COOR"'), R' is methyl, R" is -C 8 H 17 , and R'" is -C 10 H 21 In other embodiments, m is 0, n is 6, R 2 Yes -CH=CHCH 2 CH=CH(CH 2 ) 4 CH 3 , R 3 YesR a , and R a Yes-CR'(COOR') 2 , R' is methyl, and R" is -C 8 H 17 .

[0038] According to a preferred embodiment of the present disclosure, the lipid having the structure of formula (I) can be any of the following compounds: or

[0039] According to a preferred embodiment of the present disclosure, the lipid having the structure of formula (I) is

[0040] According to a preferred embodiment of the present disclosure, the lipid having the structure of formula (I) is

[0041] The lipid compounds of the present disclosure may also have the structure of formula (II),

[0042] In formula (II), m and n are integers between 1 and 10, R 1 and R 3 In certain embodiments, m and n are 6, R 1 is a propyl group with a hydroxyl substituent, R 3 Then -C 6 H 13 In other embodiments, m is 10, n is 6, R 1 is a propyl group with a hydroxyl substituent, R 3 Then -C 6 H 13.

[0043] Or, R 2 is hydrogen or -O(C=O)R', wherein R' is alkyl. 2 is hydrogen. In other embodiments, R 2 is -O(C=O)R', and R' is -C 6 H 13 .

[0044] According to a preferred embodiment of the present disclosure, the lipid with the structure of formula (II) can be

[0045] The lipid compounds of the present disclosure may also have the structure of formula (III),

[0046] In formula (III), m and n are each an integer between 1 and 10; and R 1 , R 2 , R 3 and R 4 are hydrogen or alkyl, respectively.

[0047] According to a preferred embodiment of the present disclosure, the lipid with the structure of formula (III) can be

[0048] 2. Lipid Nanoparticles

[0049] Another aspect of the present disclosure is to provide a lipid nanoparticle formed by one or more of the above lipids for delivering a desired active agent (e.g., a nucleic acid of a target protein or a therapeutic agent). The lipid nanoparticle structurally comprises a hydrophilic inner core and a lipid bilayer outer layer formed by one or more lipids of formula (I) to (III).

[0050] In addition, the lipid nanoparticles of the present disclosure further include a therapeutic agent located in the hydrophilic inner core or the lipid bilayer outer layer of the lipid nanoparticle. If the therapeutic agent is hydrophilic, it will be located in the hydrophilic inner core, whereas if it is hydrophobic, it will be located in the lipid outer layer. The therapeutic agent can be a nucleic acid of a target protein.

[0051] Examples of nucleic acids that can be coated in the hydrophilic core of the lipid nanoparticles of the present disclosure include, but are not limited to, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), microRNA (miRNA), transfer RNA (tRNA) or a combination thereof. In certain embodiments, the lipid nanoparticles of the present disclosure further include mRNA of SARS-CoV-2 spike protein coated in the hydrophilic core. In other embodiments, the lipid nanoparticles of the present disclosure further include mRNA of dengue virus membrane protein (E protein) coated in the hydrophilic core.

[0052] 3. Application of lipid nanoparticles

[0053] 3.1 Delivery of nucleic acids into cells

[0054] The present disclosure also provides methods for delivering a desired agent into a cell. According to certain embodiments, the lipid nanoparticles of the present disclosure are pre-loaded with a desired nucleic acid (e.g., mRNA of the SARS-CoV-2 spike protein) intended to be delivered to a target cell, and one or more reagents that facilitate contact with the cell or subsequent transfection of the cell. Preferably, the lipid nanoparticles of the present disclosure allow the nucleic acid coated therein to reach the target cell and transfect it. Therefore, after the desired nucleic acid is delivered into the cell, the nucleic acid can encode and produce one or more target proteins in the target cell. The lipid nanoparticles of the present invention can be used to target various types of cells, including, but not limited to, hepatocytes, epidermal cells, hematopoietic cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal stem cells, neural cells, heart cells, adipocytes, smooth muscle cells, skeletal muscle cells, B cells, T cells, white blood cells, granulocytes, fibroblasts, reticulocytes, and the like. According to certain embodiments, the lipid nanoparticles of the present invention pre-loaded with the desired nucleic acid can be successfully taken up by T cells, and transfect T cells to express the desired protein (e.g., SARS-CoV-2 spike protein) encoded by the desired nucleic acid (e.g., mRNA of SARS-CoV-2 spike protein). In certain embodiments, the expression level of the desired protein is higher than that of the control group (i.e., cells not treated with the lipid nanoparticles of the present disclosure).In certain embodiments, the expression level of the desired protein in the target cells (e.g., T cells) is at least 1 to 100,000 times higher than the expression level in the control group cells, for example, about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000 , 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 times; preferably, about 5 to 50,000 times higher than the expression level of the control group, for example, about 5.0 , 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, or 50, More preferably, it is about 10 to 10,000 times higher than the expression level of the control group, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 times higher than the expression level of the control group. In certain embodiments, the expression level of the desired protein in the target cell can be maintained for a period of time, for example, about 1 day, 2 days, 3 days, 4 days, 5 days, 7 days or longer.

[0055] Examples of nucleic acids that can be coated in the hydrophilic inner core of the lipid nanoparticles of the present disclosure include, but are not limited to, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), microRNA (miRNA), transfer RNA (tRNA) or a combination thereof. In certain embodiments, the lipid nanoparticles of the present disclosure further include mRNA of the SARS-CoV-2 spike protein coated in the hydrophilic inner core. In other embodiments, the lipid nanoparticles of the present disclosure further include mRNA of the dengue virus membrane protein (E protein) coated in the hydrophilic inner core.

[0056] 3.2 Treatment of diseases

[0057] The present disclosure also provides a method for treating or preventing a disease in an individual, wherein the method comprises administering an effective amount of the lipid nanoparticles of the present invention to a target tissue of the individual, wherein the lipid nanoparticles have been pre-loaded with a therapeutic agent, so as to treat and / or prevent the disease.

[0058] As used herein, the term "subject" refers to an animal, including but not limited to humans, non-human primates, and rodents, to which the lipid nanoparticles of the present invention pre-loaded with a therapeutic agent can be administered. Generally, the term "subject" herein refers to humans. The therapeutic agent is a nucleic acid analog of a target protein.

[0059] In certain embodiments, the method comprises administering to an individual a lipid nanoparticle of the present invention loaded with a viral nucleic acid, such that the encapsulated viral nucleic acid can be delivered to a target tissue of the individual (e.g., lung, liver, etc.) and expressed in the target tissue as an antigen to induce an immune response in the individual, thereby immunizing the individual and preventing the individual from being subsequently infected by the virus (e.g., SARS) or developing a disease induced by the virus.

[0060] Alternatively, the lipid nanoparticles of the present invention can be formulated into a pharmaceutical formulation together with one or more additional carriers, excipients or stabilizers. The lipid nanoparticles of the present invention can be administered according to existing medical treatment methods and dosages after considering the clinical symptoms of the individual itself, the method and position of administration, the administration period, and the age, sex, weight and other factors related to the clinical condition of the individual. The effective amount of the lipid nanoparticles of the present invention to be applied can be determined based on factors known to those skilled in the art in clinical research, pharmacy, clinical and medical fields. In certain embodiments, the effective amount is an amount that can effectively achieve at least some degree of stabilization, improvement, or removal of disease symptoms, or prevent the disease from continuing to progress. For example, an appropriate dosage refers to an amount that can quickly trigger the expression of the target protein.

[0061] Suitable routes of administration include, for example, oral, rectal, vaginal, mucosal, pulmonary (e.g., intratracheal, inhalation, etc.), or intestinal administration. Parenteral administration routes include, for example, intramuscular, subcutaneous, intramedullary injection, etc., as well as intrathecal, intracerebroventricular, intravenous, intraperitoneal, intranasal or intraocular injection, etc.

[0062] Alternatively, the lipid nanoparticles of the present invention may be administered locally, rather than systemically, for example, by directly injecting the lipid nanoparticles of the present invention into the target tissue. Depending on the desired tissue to be treated and its location, the purpose of local delivery can be achieved in a variety of ways. For example, an aerosol formulation containing the lipid nanoparticles of the present invention may be inhaled into the nasal cavity, trachea or bronchus by inhalation; or the lipid nanoparticles of the present invention may be injected into the symptom or pain site; the lipid nanoparticles of the present invention may also be made into tablets and administered via the mouth, trachea or esophagus; or administered to the stomach or small intestine in the form of a liquid, tablet or capsule; or administered to the eyes in the form of an emulsion, drops or injection.

[0063] In certain embodiments, lipid nanoparticles of the present invention can be formulated into a dosage form that can release nucleic acid in a long-term manner. This type of long-acting release dosage form can be applied to an individual in a longer dosage interval mode. For example, lipid nanoparticles of the present invention can be used once a day, twice a day, or once every two days. In some preferred embodiments, lipid nanoparticles of the present invention are used once a week, twice a week, once every 10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 6 weeks, once every 8 weeks, once every 2 months, once every 3 months, once every 4 months, once every 6 months, once every 8 months, once every 9 months, or once a year. This paper also includes compositions configured to be suitable for storage forms and use (e.g., intramuscular, subcutaneous, etc.) to transmit nucleic acids for a long time.

[0064] Lyophilic compositions comprising one or more lipid nanoparticles disclosed herein are also contemplated. The lyophilic compositions of the present invention may be reconstituted in vitro prior to administration. For example, the lyophilic compositions may be formulated into a suitable dosage form (e.g., a disc-shaped, rod-shaped, or film-shaped subcutaneous dosage form) and rehydrated in vivo by the subject's own body fluids after administration.

[0065] The following will illustrate the content of the present invention through implementation modes (only for the purpose of illustrating the present invention, not for limiting the scope of the present invention). Although these implementation modes are typical implementation modes, those with ordinary skills in the relevant technical field should be able to use other methods and technologies to replace the implementation modes.

[0066] Example

[0067] Materials and Methods

[0068] Evaluation of SARS-CoV2 S protein expression in vitro by ELISA

[0069] The mRNA-LNPs of SARS-CoV2 S protein were infected into 293T cells and incubated in DMEM medium (containing 1% FBS) at 37°C for 24 hours. After transfection, the cells were fixed with 4% triformaldehyde / PB S for 15 minutes, and then permeabilized with 0.1% TX-100 for about 10 minutes. After washing, anti-RBD chimeric antibody (100ng / mL) was added and treated at room temperature for about 1 hour. Then, horseradish peroxidase-conjugated anti-human antibody (1:8000) was added and treated at room temperature for about 1 hour. The cells were washed 3 times with PB S containing 0.1% Tween20 (hereinafter referred to as "PB ST0.1") and then incubated with peroxidase-conjugated goat anti-mouse IgG antibody (1:5000) for 1 hour. After washing three times with PB ST0.1, the cells were developed with 3,3',5,5'-tetramethylbenzidine as a colorimetric reagent. Finally, 3N HCl was added to terminate the reaction, and the absorbance at 450 nm was measured with an ELISA reader.

[0070] Immunization of mice

[0071] At the beginning of the experiment (day 0), 6-8 week old BALB / c mice were immunized with designated mRNA-LNPs (i.e., MC3-LNP, SN 102-LNP, AS-CL05-LNP, AS-CL09-LNP, AS-CL28-LNP, or AS-CL35-LNP, each containing 10 μg of mRNA) by intramuscular injection, followed by booster injections at weeks 2 and 4. At weeks 4, 6, and 8 after the first immunization, sera were collected and tested for binding to FL S-WT or FL S-BA.5 proteins, and their ability to neutralize viruses was confirmed by pseudovirus analysis.

[0072] Pseudovirus neutralization assay

[0073] Serum from experimental animals was collected 6 weeks after the first immunization to determine its ability to neutralize the BA.5 SARS-CoV-2 pseudovirus. SARS-CoV-2 pseudovirus expressing full-length S protein and firefly luminescence was expressed in HEK293T cells overexpressing human ACE2, and pseudovirus neutralization assays were performed. The concentration required to inhibit half of the maximal activity (IC 50 ), IC of each experimental group 50 The values ​​were calculated after three repeated experiments.

[0074] Virus-plaque reduction neutralization (PRNT) assay

[0075] The serum collected from animals injected with DENV 2E mRNA-LNPs was diluted with PBS and incubated with 100 PFU DENV2 at 37°C for 1 hour. The mixture was added to BHK-21 cells and incubated at 37°C for 1 hour. The virus-containing culture medium was removed and incubated for 4 days with DMEM medium containing 2% FBS and 1% methylcellulose. The cells were fixed with 10% formaldehyde and stained with 0.5% crystal violet for about 20 minutes. After washing the culture plate with water, the number of plaques formed at each dilution concentration was calculated. The reduction in plaques was calculated as follows: Percent inhibition = 100x (1-(number of plaques caused by immune serum / number of plaques caused by no added immune serum). The value for a 50% reduction in plaques (PRNT 50 ). DENV2 strain 16881 was used in this experiment.

[0076] Example 1 Synthesis of the compounds of the present invention

[0077] The compounds of the present invention are synthesized according to the steps shown in Scheme 1-5, wherein the amines required in Step 7 of Scheme 1 are listed in Table 1.

[0078] Scheme 1: General procedure for the preparation of disubstituted malonates

[0079] Table 1. Amines required for step 7 of Scheme 1

[0080] Scheme 2: General procedure for the preparation of malonic esters with amino substituents

[0081] Scheme 3: Synthesis of AS-CL-19

[0082] Scheme 4: Preparation of disubstituted malonic esters by asymmetric synthesis

[0083] Scheme 5: Synthesis of AS-CL-30 and AS-CL-31

[0084] Compound 1a: Dioctyl malonate (step 1)

[0085] Malonic acid (2.61 g, 25.08 mmol) and octanol (7.18 g, 55.13 mmol) dissolved in dichloromethane (DCM) were mixed and stirred at 0°C, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (10.58 g, 55.18 mmol) and 4-dimethylaminopyridine (DMAP) (613 mg, 5 mmol) were added to the mixture. The mixture was stirred overnight at room temperature, and the product was washed with 2N HCl and brine, and then dried over magnesium sulfate. After the solvent was completely evaporated, the product was chromatographed on a silica gel column with ethyl acetate / hexane (1 / 20) to obtain compound 1a (7.66 g, 23.32 mmol) as a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.12(t,J=6.6Hz,4H),3.35(s,2H),1.63-1.60(m,4H),1.32-1.26(m,20H),0.86(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ166.7,65.6,41.7,31.7,29.1(x2),28.4,25.8,22.6,14.0.

[0086] Compound 1b: Didecyl malonate

[0087] Compound 1b was synthesized according to the general step 1 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.12(t,J=6.6Hz,4H),3.35(s,2H),1.65-1.60(m,4H),1.33-1.25(m,28H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ166.6,65.6,41.6,31.8,29.4(x2),29.2,29.1,28.4,25.7,22.6,14.0.

[0088] Compound 1c: Diundecyl malonate

[0089] Compound 1c was synthesized according to the general step 1 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.12(t,J=6.6Hz,4H),3.35(s,2H),1.65-1.59(m,4H),1.32-1.25(m,32H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ170.3,65.6,46.3,32.0,29.7,29.6,29.4,29.3,28.6,25.9,22.8,14.2,13.7.

[0090] Compound 2a: Dioctyl 2-methylmalonate (Step 2)

[0091] Tetrahydrofuran (NaH) (430 mg, 10.8 mmol) and dioctyl malonate (4.43 g, 13.5 mmol) dissolved in THF were mixed at 0°C, and then methyl iodide (MeI (0.66 ml, 10.8 mmol) was added. The mixture was stirred overnight at room temperature, and the product was washed with saturated ammonium chloride solution and brine, and then dried over magnesium sulfate. After the solvent was completely evaporated, the product was chromatographed on a silica gel column with ethyl acetate / hexane (1 / 40) to obtain compound 2a (2.45 g, 7.15 mmol) as a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.15-4.08(m,4H),3.42(q,J=7.2Hz,1H),1.65-1.57(m,4H),1.41(d,J=7.2Hz,3H),1.33-1.28(m,20H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ170.2,65.5,46.2,31.8,29.2(x2),28.5,25.8,22.6,14.1,13.6.

[0092] Compound 2b: 2-Methylmalonic acid didecyl ester

[0093] Compound 2b was synthesized according to the general step 2 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.14-4.09(m,4H),3.42(q,J=7.2Hz,1H),1.64-1.60(m,4H),1.41(d,J=7.2Hz,3H),1.32-1.25(m,28H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ170.2,65.5,46.2,31.8,29.5(x2),29.3,29.2,28.5,25.8,22.6,14.1.

[0094] Compound 2c: 2-Methylmalonic acid diundecyl ester

[0095] Compound 2c was synthesized according to the general step 2 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.12-4.07(m,4H),3.40(q,J=7.2Hz,1H),1.61-1.59(m,6H),1.39(d,J=7.2Hz,3H),1.35-1.25(m,30H),0.85(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ170.3,65.6,46.3,32.0,29.7,29.6,29.4,29.3,28.6,25.9,22.8,14.2,13.7.

[0096] Compound 2d: Dioctyl 2-ethylmalonate

[0097] Compound 2d was synthesized according to the general step 2 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.14-4.08(m,4H),3.24(t,J=7.2Hz,1H),1.94-1.89(m,2H),1.64-1.58 (m,4H),1.35-1.26(m,20H),0.95(t,J=7.2Hz,3H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ169.5,65.4,53.6,31.7,29.1,28.5,25.8,22.6,22.2,14.0,11.8.

[0098] Compound 2e: 2-propylmalonate dioctyl ester

[0099] Compound 2e was synthesized according to the general step 2 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.15-4.08(m,4H),3.33(t,J=7.8Hz,1H),1.89-1.85(m,2H),1.64-1.60 (m,4H),1.37-1.27(m,22H),0.93(t,J=7.2Hz,6H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ169.7,65.4,51.9,31.7,30.8,29.1,28.5,25.8,22.6,20.6 14.0,13.7.MS(ESI):m / z[M+Na] + 393.2973for C 22 H 42 O 4 Nah.

[0100] Compound 2f: 2-(6-phenoxy)hexylmalonate dioctyl ester

[0101] Compound 2f was synthesized according to the general step 2 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ7.35-7.26(m,5H),4.49(s,2H),4.15-4.08(m,4H),3.45(t,J=6.6Hz,2H),3.31(t,J=6. 6Hz,1H),1.90-1.86(m,2H),1.64-1.59(m,6H),1.33-1.29(m,26H),0.86(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ169.6,138.5,128.3,127.6,127.5,72.9,70.3,65.4,52.1,31.8,29.6,29.2(x2),29.1,28.7,28.5,27.3,25.9,25.8,22.6,14.1.

[0102] Compound 2g: 2-methylmalonic acid-1-decyl-3-octyl ester

[0103] Compound 2g was synthesized according to the general step 2 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.15-4.08(m,4H),3.42(q,J=7.2Hz,1H),1.65-1.60(m,4H),1.41(d,J=7.2Hz,3H),1.33-1.26(m,24H),0.89-0.87(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ170.2,65.5,64.2,31.9,31.8,29.6,29.3,29.25,29.21,28.5,25.8,22.7,22.6,14.10,14.08,13.6.

[0104] Compound 2h: 2-Methylmalonic acid-1-hexyl-3-octyl ester

[0105] Compound 2h was synthesized according to the general step 2 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.15-4.08(m,4H),3.42(q,J=7.2Hz,1H),1.65-1.60(m,4H),1.41(d,J=7.2Hz,3H),1.30-1.27(m,16H),0.89-0.87(m,6H). 13 C NMR (150MHz, CDCl3) δ170.3,65.5,46.2,31.8,31.4,29.2(x2),28.5,28.4,25.8,25.5,22.6,22.5,14.1,14.0,13.6.

[0106] Compound 2i: 2-Ethylmalonate

[0107] Compound 2i was synthesized according to the general step 2 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.14-4.09(m,4H),3.25(t,J=7.2Hz,1H),1.95-1.90(m,2H),1.64-1.60 (m,4H),1.32-1.25(m,26H),0.96(t,J=7.2Hz,3H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ169.7,65.5,53.7,32.0,29.6(x2),29.4,29.3,28.6,25.9,22.8,22.3,14.2,12.0.

[0108] Compound 3a: 2-(Phenoxy)methyl-2-methylmalonic acid dioctyl ester (step 3)

[0109] NaH (165 mg, 4.13 mmol) and 2-methylmalonic acid octyl ester (1.4 g, 4.08 mmol) dissolved in THF were mixed at 0°C, and then chloromethyl phenyl ether (0.56 ml, 4.08 mmol) was added. The mixture was refluxed overnight at room temperature, and the product was washed with saturated ammonium chloride solution and brine, and then dried over magnesium sulfate. After the solvent was completely evaporated, the product was chromatographed on a silica gel column with ethyl acetate / hexane (1 / 25) to obtain compound 3a (1.4 g, 3.02 mmol, 74%) as a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ7.36-7.27(m,5H),4.53(s,2H),4.10(t,J=6.6Hz,4H),3.81(s,2H),1. 60-1.57(m,4H),1.53(s,3H),1.30-1.25(m,20H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ170.7,138.0,128.3,127.5,127.4,73.4,72.7,65.5,54.9,31.8,29.2(x2),28.5,25.8,22.6,18.5,14.1.

[0110] Compound 3b: 2-(4-phenoxy)butyl-2-methylmalonic acid dioctyl ester

[0111] Compound 3b was synthesized according to the general step 3 disclosed in Scheme 1 to obtain a colorless oil. 1H NMR (600 MHz, CDCl 3 )δ7.35-7.27(m,5H),4.48(s,2H),4.09(t,J=6.6Hz,4H),3.46(t,J=6.6Hz,2H)1.88-1 .86(m,2H),1.65-1.58(m,6H),1.40(s,3H),1.29-1.26(m,22H),0.88(t,J=7.2Hz,6H); 13 C NMR (150 MHz, CDCl 3 )δ172.4,138.5,128.3,127.5,127.4,72.8,69.9,65.3,53.7,35.3,31.7 ,29.9,29.1(x2),28.4,25.8,22.6,21.0,19.8,14.0; MS(ESI):m / z[M+Na] + 527.3703for C 31 H 51 O 5 Nah.

[0112] Compound 3c: 2-(6-phenoxy)hexyl-2-methylmalonate

[0113] Compound 3c was synthesized according to the general step 3 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ7.35-7.27(m,5H),4.49(s,2H),4.09(t,J=6.6Hz,4H),3.45(t,J=6.6Hz,2H),1.85-1 .83(m,2H),1.62-1.57(m,4H),1.39(s,3H),1.38-1.24(m,28H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,138.7,128.3,127.6,127.5,72.9,70.4,65.3,53.8,35.5,31.2,29.7,29.6(x2),29.2,28.5,26.0,25.8,24.3,22.6,19.9,14.1.

[0114] Compound 3d: 2-(8-phenoxy)octyl-2-methylmalonate

[0115] Compound 3d was synthesized according to the general step 3 disclosed in Scheme 1 to obtain a light yellow oil. 1 H NMR (600 MHz, CDCl 3 )δ7.35-7.26(m,5H),4.50(s,2H),4.13-4.06(m,4H),3.45(t,J=6.6Hz,2H),1.85-1. 82(m,2H),1.62-1.55(m,6H),1.39(s,3H),1.33-1.26(m,30H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,138.7,128.3,127.6,127.4,72.8,70.5,65.2,53.8,35.6,31.8,2 9.9,29.7(x2),29.4,29.3,29.2,28.5,26.2,25.9,24.3,22.6,19.9,14.1.

[0116] Compound 3e: 2-(6-phenoxy)hexyl-2-ethylmalonate

[0117] Compound 3e was synthesized according to the general step 3 disclosed in Scheme 1 to obtain a light yellow oil. 1 H NMR (600 MHz, CDCl 3 )δ7.35-7.26(m,5H),4.49(s,2H),4.09(t,J=6.6Hz,4H),3.45(t,J=6.6Hz,2H),1.92(q,J=7.2Hz,2H),1.87-1.8 4(m,2H),1.61-1.58(m,6H),1.38-1.26(m,24H),1.16-1.12(m,2H),0.88(t,J=6.6Hz,6H),0.80(t,J=6.6Hz,3H). 13 C NMR (150 MHz, CDCl 3 )δ171.9,138.5,128.2,127.5,127.3,72.7,70.2,65.0,57.9,31.7,31. 5,29.6,29.5,29.1,29.0,28.4,25.9,25.8,25.1,23.8,22.5,14.0,8.3.

[0118] Compound 3e: 2-(6-phenoxy)hexyl-2-propylmalonate

[0119] Compound 3f was synthesized according to the general step 3 disclosed in Scheme 1 to obtain a light yellow oil. 1 H NMR (600 MHz, CDCl 3 )δ7.35-7.27(m,5H),4.50(s,2H),4.08(t,J=6.6Hz,4H),3.47-3.43(m,2H)1.87-1.82(m,4H),1.61 -1.58(m,6H),1.36-1.26(m,26H),1.17-1.15(m,2H),0.91(t,J=7.2Hz,3H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.0,138.6,128.3,127.5,127.4,72.8,70.3,65.1,57,6,35.5,32.2,3 1.8,29.7,29.6,29.2,29.1,28.5,25.9,25.8,24.0,22.6,17.4,14.4,14.0.

[0120] Compound 3g: 2-(6-phenoxy)hexyl-2-methylmalonic acid-1-decyl-3-octyl ester

[0121] Compound 3g was synthesized according to the general step 3 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ7.34-7.25(m,5H),4.49(s,2H),4.10-4.08(m,4H),3.45(t,J=6.6Hz,2H),1.86-1 .83(m,2H),1.62-1.58(m,6H),1.39(s,3H),1.38-1.19(m,30H),0.89-0.87(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,138.7,128.3,127.6,127.5,72.9,70.4,65.3,53.8,35.6,31.9,31.8,29.8,29.7,229.6,29 .3,29.24,29.22,29.20,28.5,26.0,25.9,24.3,22.7,22.6,19.9,14.11,14.09.MS(ESI):m / z[M+Na]+ 583.4333for C 35 H 60 O 5 Nah.

[0122] Compound 3h: 2-(6-phenoxy)hexyl-2-methylmalonic acid-1-hexyl-3-octyl ester

[0123] Compound 3h was synthesized according to the general step 3 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ7.35-7.27(m,5H),4.49(s,2H),4.09(t,J=6.6Hz,4H),3.45(t,J=6.6Hz,2H)1.85- 1.83(m,2H),1.62-1.59(m,6H),1.38(s,3H),1.32-1.26(m,22H),0.89-0.87(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.5,138.6,128.3,127.6,127.4,72.8,70.3,65.2,53.7,35.5,31.7,31.3,29.7,29.6,29 .1(x2),28.5,28.4,25.9,25.8,25.5,24.2,22.6,22.5,19.8,14.0,13.9.MS(ESI):m / z[M+Na] + 527.3712for C 31 H 52 O 5 Nah.

[0124] Compound 3i: 2-(6-phenoxy)hexyl-2-methylmalonic acid didecyl ester

[0125] Compound 3i was synthesized according to the general step 3 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ7.35-7.27(m,5H),4.48(s,2H),4.09(t,J=6.6Hz,4H),3.46(t,J=6.6Hz,2H),1.95-1.87( m,4H),1.65-1.57(m,6H),1.38-1.26(m,30H),0.88(t,J=6.6Hz,6H),0.80(t,J=6.6Hz,3H). 13 C NMR (150 MHz, CDCl 3 )δ172.0,138.6,128.4,127.6,127.5,73.0,70.0,65.2,58.1,31.9,31.5,30.1 ,29.6(x2),29.4,29.3,28.6,25.9,25.2,22.7,20.7,14.1,8.5.MS(ESI):m / z[M + +H] + 575.4675for C 36 H 63 O 5 .

[0126] Compound 4a: 2-(Hydroxymethyl)-2-methylmalonic acid didecyl ester (step 4)

[0127] A mixture of dioctyl 2-(phenoxy)methyl)-2-methylmalonate (1.4 g, 3.02 mmol) and platinum / carbon (catalyst) dissolved in acetic acid / methanol was stirred overnight at room temperature in the presence of hydrogen. After filtration and evaporation of the solvent, the residue was dissolved in DCM, washed with saturated aqueous sodium bicarbonate solution, and dried over magnesium sulfate. Compound 4a (1.07 g, 2.87 mmol, 95%) was obtained as a colorless oil without further purification. 1 H NMR (600 MHz, CDCl 3 )δ4.13(t,J=6.6Hz,4H),3.83(s,2H),1.64-1.59(m,4H),1.43(s,3H),1.28-1.26(m,20H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ171.7,66.9,65.7,55.9,31.8,29.1(x2),28.4,25.8,22.6,17.6,14.1.

[0128] Compound 4b: 2-(4-Hydroxybutyl)-2-methylmalonic acid dioctyl ester

[0129] Compound 4b was synthesized according to the general step 4 disclosed in Scheme 1 to obtain a colorless oil. 11 H NMR (600 MHz, CDCl 3 )δ4.09(t,J=6.6Hz,4H),3.64(t,J=6.6Hz,2H),1.88-1.85(m,2H),1.62-1.55(m,8H),1.40(s,3H),1.29-1.27(m,20H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.5,65.4,62.4,53.8,35.2,32.8,31.8,29.2(x2),28.5,25.8,22.6,20.6,19.9,14.1.

[0130] Compound 4c: 2-(6-Hydroxyhexyl)-2-methylmalonate

[0131] Compound 4c was synthesized according to the general step 4 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.09(t,J=6.6Hz,4H),3.63(t,J=6.6Hz,2H),1.86-1.83(m,2H),1.62-1.54(m,6H),1.39(s,3H),1.31-1.27(m,26H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ65.3,62.9,53.8,35.5,32.7,31.8,29.7(x2),29.2,28.5,25.6,25.5,24.3,22.6,19.9,14.1.

[0132] Compound 4d: 2-(8-Hydroxyoctyl)-2-methylmalonic acid dioctyl ester

[0133] Compound 4d was synthesized according to the general step 4 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.11-4.07(m,4H),3.63(t,J=6.6Hz,2H),1.85-1.82(m,2H),1.61-1.54(m,6H),1.39(s,3H),1.29-1.27(m,30H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ65.3,63.0,53.8,35.6,32.8,31.8,29.8(x2),29.34,29.3,29.2,28.5,25.9,25.7,24.3,22.6,19.9,14.1.

[0134] Compound 4e: 2-ethyl-2-(6-hydroxyhexyl) dioctyl malonate

[0135] Compound 4e was synthesized according to the general step 4 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.08-4.06(m,4H),3.61-3.59(m,2H),1.89(q,J=7.2Hz,2H),1.85-1.83(m,2H),1.59-1. 52(m,6H),1.32-1.22(m,24H),1.15-1.13(m,2H),0.87-0.85(m,6H),0.79(t,J=7.2Hz,3H). 13 C NMR (150 MHz, CDCl 3 )δ172.1,65.2,63.0,58.1,32.7,31.8,31.7,29.7,29.3,29.2,28.6,25.9,25.7,25.3,24.0,22.7,14.1,8.5.

[0136] Compound 4f: 2-(6-Hydroxyhexyl)-2-propylmalonate

[0137] Compound 4f was synthesized according to the general step 4 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.08(t,J=6.6Hz,4H),3.62(t,J=6.6Hz,2H),1.87-1.82(m,4H),1.64-1.53(m,6H) ,1.36-1.25(m,26H),1.20-1.13(m,2H),0.91(t,J=7.2Hz,3H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.0,65.1,62.9,57.6,34.5,32.6,31.7,29.6,29.2,29.1,28.5,25.8,25.7,25.4,24.0,22.6,17.4,14.4,14.0.

[0138] Compound 4g: 2-(6-hydroxyhexyl) dioctyl malonate

[0139] Compound 4g was synthesized according to the general step 4 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.15-4.08(m,4H),3.63(t,J=6.6Hz,2H),3.31(t,J=7.8Hz,1H),1.91-1 .87(m,2H),1.64-1.53(m,8H),1.35-1.33(m,24H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ169.6,65.4,62.9,52.1,32.6,31.8,29.2(x2),29.0,28.6,28.5,27.3,25.8,25.4,22.6,14.1.

[0140] Compound 4h: 2-(6-Hydroxyhexyl)-2-methylmalonic acid-1-decyl-3-octyl ester

[0141] Compound 4h was synthesized according to the general step 4 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.09-4.07(m,4H),3.61(t,J=6.6Hz,2H),1.85-1.82(m,2H),1.60-1.50(m,6H),1.38(s,3H),1.37-1.21(m,30H),0.88-0.86(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.4,63.0,53.8,35.6,32.7,32.0,31.8,29.7,29.6,29.4,29. 3,29.26,29.24,28.6,25.9,25.6,24.3,22.73,22.70,20.0,14.2,14.1.

[0142] Compound 4i: 2-(6-Hydroxyhexyl)-2-methylmalonic acid-1-hexyl-3-octyl diester

[0143] Compound 4i was synthesized according to the general step 4 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.09(t,J=6.6Hz,4H),3.62(t,J=6.6Hz,2H),1.85-1.83(m,2H),1.62-1.59( m,6H),1.58-1.52(m,2H),1.39(s,3H),1.30-1.26(m,20H),0.89-0.86(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3(x2),62.9,53.8,35.5,32.7,31.8,31.4,29.7,29.2(x2),28.5,28.4,25.9,25.5(x2),24.3,22.6,22.5,19.9,14.1,14.0.

[0144] Compound 4j: 2-ethyl-2-(4-hydroxybutyl)didecylmalonate

[0145] Compound 4j was synthesized according to the general step 4 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.09(t,J=6.6Hz,4H),3.63(t,J=6.6Hz,2H),1.95-1.87(m,4H),1.61-1. 55(m,6H),1.34-1.24(m,30H),0.87(t,J=6.6Hz,6H),0.81(t,J=6.6Hz,3H). 13 C NMR (150 MHz, CDCl 3 )δ171.9,65.2,62.5,58.1,32.8,31.9,31.4,29.6(x2),29.3,29.2,28.5,25.9,25.4,22.7,20.3,14.1,8.5.

[0146] Compound 5a: Dioctyl 2-(((6-bromohexanoyl)oxy)methyl)-2-methylmalonate (Step 5)

[0147] Compound 4a (440 mg, 1.18 mmol) and 6-bromohexanoic acid (203 mg, 1.18 mmol) dissolved in dichloromethane (DCM) were mixed at 0°C, followed by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (275 mg, 1.42 mmol) and 4-dimethylaminopyridine (DMAP) (30 mg, 0.24 mmol). The mixture was stirred overnight at room temperature, and the product was washed with a saturated 2N HCl solution and brine, and then dried over magnesium sulfate. After the solvent was completely evaporated, the product was chromatographed on a silica gel column with ethyl acetate / hexane (1 / 25) to obtain compound 5a (520 mg, 0.95 mmol, 80%) as a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.45(s,2H),4.12(t,J=6.6Hz,4H),3.39(t,J=6.6Hz,2H),2.31(t,J=7.2Hz,2H),1.88-1.83(m, 2H),1.65-1.59(m,6H),1.48(s,3H),1.47-1.44(m,2H),1.43-1.26(m,22H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.8,170.0,66.4,65.9,53.9,33.9,33.4,32.4,31.9,29.3(x2),28.5,27.7,25.9,24.1,22.7,18.4,14.2.

[0148] Compound 5b: 2-(((8-bromooctanoyl)oxy)methyl)-2-methylmalonic acid dioctyl ester

[0149] Compound 5b was synthesized according to the general step 5 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.44(s,2H),4.12(t,J=6.6Hz,4H),3.40(t,J=6.6Hz,2H),2.29(t,J=7.2Hz,2H),1.87-1.82(m, 2H),1.63-1.59(m,6H),1.48(s,3H),1.44-1.40(m,2H),1.33-1.27(m,26H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ173.1,170.0,66.3,65.9,53.9,34.1,33.9,32.8,31.9,29.3(x2),29.0,28.5,28.4,28.0,25.9,24.8,22.7,18.4,14.2.MS(ESI):m / z[M+Na] + 599.2912,[M+Na] 2+ 601.2890 for C 29 H 53 O 6 BrNa.

[0150] Compound 6a: 2-(4-iodobutyl)-2-methylmalonic acid dioctyl ester (step 6)

[0151] Compound 4b (975 mg, 2.202 mmol), triphenylphosphine (PPh 3 ) (635 mg, 2.242 mmol) and imidazole (165 mg, 2.242 mmol) dissolved in dichloromethane (DCM) were added, followed by iodine (670 mg, 2.643 mmol). The reaction mixture was allowed to warm to room temperature and stirred overnight. Saturated sodium thiosulfate (Na 2 S 2 O 3 ) solution and brine, and then dried over magnesium sulfate. After the solvent was completely evaporated, the product was chromatographed on a silica gel column with ethyl acetate / hexane (1 / 25) to obtain compound 6a (1.096 g, 90%) as a colorless oil. 1H NMR (600 MHz, CDCl 3 )δ4.10(t,J=6.6Hz,4H),3.18(t,J=6.6Hz,2H),1.87-1.80(m,4H),1.62-1.58(m,4H),1.41(s,3H),1.34-1.22(m,22H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.3,65.4,53.6,34.4,33.5,31.8,29.2,28.5,25.8,25.3,22.6,19.9,14.1,6.2.

[0152] Compound 6b: 2-(6-iodohexyl)-2-methylmalonic acid dioctyl ester

[0153] Compound 6b was synthesized according to the general step 6 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.10-4.07(m,4H),3.16(t,J=6.6Hz,2H),1.84-1.77(m,4H),1.62-1.57(m,4 H),1.40-1.36(m,2H),1.38(s,3H),1.31-1.20(m,24H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.4,53.8,35.5,33.5,31.8,30.3,29.3,29.2,28.9,28.6,25.9,24.2,22.7,20.0,14.1,6.9.MS(ESI):m / z[M+Na] + 561.2406 for C 25 H 47 O 4 NaI.

[0154] Compound 6c: 2-(8-iodooctyl)-2-methylmalonic acid dioctyl ester

[0155] Compound 6c was synthesized according to the general step 6 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.11-4.04(m,4H),3.16(t,J=7.2Hz,2H),1.83-1.76(m,4H),1.61-1.56(m,4 H),1.37(s,3H),1.37-1.34(m,2H),1.27-1.19(m,28H),0.86(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6 65.3,53.8,35.6,33.5,31.8,30.5,29.8,29.23,29.21(x2),28.55,28.5,25.9,24.3,22.7,19.9,14.1,7.1.MS(ESI):m / z[M+Na] + 603.2876forC 28 H 53 O 4 NaI.

[0156] Compound 6d: 2-ethyl-2-(6-iodohexyl)malonate

[0157] Compound 6d was synthesized according to the general step 6 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.09(t,J=6.6Hz,4H),3.16(t,J=7.2Hz,2H),1.91(q,J=7.2Hz,2H),1.86-1.83(m,2H),1.81-1.78(m,2H),1.62-1 .58(m,4H),1.40-1.37(m,2H),1.33-1.24(m,22H),1.16-1.14(m,2H),0.87(t,J=7.2Hz,6H),0.80(t,J=7.8Hz,3H). 13 C NMR (150 MHz, CDCl 3 )δ172.0,65.2,58.1,33.5,31.9,31.7,30.4,29.3,29.2,28.9,28.6,26.0,25.3,23.9,22.7,14.2,8.5,6.9.

[0158] Compound 6e: 2-(6-iodohexyl)-2-propylmalonate

[0159] Compound 6e was synthesized according to the general step 6 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.09(t,J=6.6Hz,4H),3.16(t,J=6.6Hz,2H),1.88-1.84(m,4H),1.62-1.57(m,6H),1.38-1.26(m,26H),1.18-1.12(m,2H). 13 C NMR (150 MHz, CDCl 3 )δ172.0,65.1,57.6,34.6,33.4,32.2,31.8,30.3,29.2,29.1,28.8,28.5,25.9,23.9,22.6,17.4,14.4,14.1,6.9.

[0160] Compound 6f: 2-(6-iodohexyl) dioctyl malonate

[0161] Compound 6f was synthesized according to the general step 6 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.13-4.06(m,4H),3.30-3.26(m,1H),3.16-3.12(m,2H),1.87-1.85(m,2H) ,1.81-1.76(m,2H),1.61-1.58(m,4H),1.36-1.25(m,26H),0.87-1.83(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ169.6,65.5,52.1,33.4,31.8,30.2,29.2(x2),28.7,28.6,28.2,27.2,25.9,22.7,14.1,6.9.

[0162] Compound 6g: 2-(6-iodohexyl)-2-methylmalonic acid-1-decyl-3-octyl ester

[0163] Compound 6g was synthesized according to the general step 6 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.09-4.03(m,4H),3.13(t,J=6.6Hz,2H),1.82-1.76(m,4H),1.60-1.55(m,4H),1.40-1.32(m,5H),1.31-1.18(m,28H),0.86-0.84(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.5,65.3,53.7,35.5,33.4,31.9,31.8,30.3,29.6,29.3,29.22,29 .21,29.18,28.8,28.5,25.9,24.1,22.7,22.6,19.9,14.11,14.09,6.8.

[0164] Compound 6h: 2-(6-iodohexyl)-2-methylmalonic acid-1-hexyl-3-octyl ester

[0165] Compound 6h was synthesized according to the general step 6 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.11-4.05(m,4H),3.15(t,J=6.6Hz,2H),1.84-1.77(m,4H),1.60-1.56(m,4H),1.40-1.35(m,5H),1.33-1.18(m,20H),0.88-0.85(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.4,53.8,35.5,33.5,31.8,31.4,30.3,29.3,29.2,28.9,28.6,28.5,25.9,25.6,24.2,22.7,22.6,20.0,14.1,14.0,7.0.

[0166] Compound 6i: 2-ethyl-2-(4-iodobutyl) dioctyl malonate

[0167] Compound 6i was synthesized according to the general step 6 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.10(t,J=6.6Hz,4H),3.17(t,J=6.6Hz,2H),1.95-1.91(q,J=6.6Hz,2H),1.87-1.81(m,4 H),1.63-1.58(m,4H),1.32-1.25(m,30H),0.87(t,J=6.6Hz,6H),0.82(t,J=6.6Hz,3H).13C NMR (150MHz, CDCl3) δ171.8,65.4,58.0,33.7,32.0,30.6,29.7(x2),29.4,29.3,28.6,26.0,25.4,25.0,22.8,14.2,8.6,6.3.

[0168] Compound 7a: Dioctyl 2-(((6-((2-hydroxyethyl)amino)hexanoyl)oxy)methyl)-2-methylmalonate (Step 7)

[0169] Compound 5a (490 mg, 0.89 mmol) and KI (150 mg, 0.89 mmol) were mixed in acetonitrile (MeCN) / dichloromethane (DCM) solution at room temperature, and then ethanolamine (1.6 ml, 26.75 mmol) was added. After stirring for 4 hours, the solvent was removed, and the residue was dissolved in DCM. The product was washed with water and brine, and then dried over magnesium sulfate. The product was chromatographed on a silica gel column with methanol (10%) / ammonium hydroxide (1%) dissolved in DCM to obtain compound 7a (325 mg, 0.61 mmol, 69%) as a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.44(s,2H),4.12(t,J=6.6Hz,4H),3.64(t,J=5.4Hz,2H),2.77(t,J=5.4Hz,2H),2.62(t,J=6.6Hz,2H),2.30(t,J=7. 2Hz,2H),2.18(br.s,2H),1.62-1.59(m,6H),1.52-1.49(m,2H),1.48(s,3H),1.36-1.26(m,22H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.9,169.9,66.2,65.8,60.7,53.8,51.2,49.2,33.9,31.7,29.6,29.1(x2),28.4,26.7,25.7,24.6,22.6,18.2,14.0.MS(ESI):m / z[M+H]+ 530.4044for C 29 H 56 NO 7 .

[0170] Compound 7b: 2-(((8-((2-hydroxyethyl)amino)octanoyl)oxy)methyl)-2-methylmalonic acid dioctyl ester

[0171] Compound 7b was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.44(s,2H),4.12(t,J=6.6Hz,4H),3.63(t,J=5.4Hz,2H),2.77(t,J=5.4Hz,2H),2.60(t,J=7.2Hz,2H),2.28 (t,J=7.2Hz,2H),1.63-1.58(m,6H),1.48-1.46(m,2H),1.46(s,3H),1.30-1.26(m,26H),0.88(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ173.2,170.0,66.2,65.9,60.9,53.9,51.0,49.4,34.1,31.8,30.1,29.2(x2),29.1,29.0,28.5,27.1 25.8,24.8,22.7,18.3,14.1.MS(ESI):m / z[M+H] + 558.4363for C 31 H 60 NO 7 .

[0172] Compound 7c: 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester

[0173] 8-Bromooctanoic acid (0.9 g, 4.03 mmol) and heptadecan-9-ol (1.04 g, 4.03 mmol) dissolved in dichloromethane (DCM) were mixed at 0°C, followed by the addition of 1-ethyl-2-(3-dimethylaminopropyl)carbodiimide (EDCI) (0.93 g, 4.84 mmol) and 4-dimethylaminopyridine (DMAP) (0.1 mg, 0.8 mmol). After stirring at room temperature for 4 hours, the product was washed with 2N HCl solution and brine, and dried over magnesium sulfate. After the solvent was completely evaporated, the product was chromatographed on a silica gel column with ethyl acetate / hexane (1 / 20) to obtain compound 7c (1.58 g, 3.42 mmol, 85%) as a colorless oil.

[0174] Compound 7c was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.84-4.79(m,1H),3.60(t,J=5.4Hz,2H),2.71(t,J=5.4Hz,2H),2.56(t,J=7.2Hz,2H),2.22(t ,J=7.2Hz,2H),1.58-1.56(m,2H),1.46-1.45(m,6H),1.27-1.21(m,30H),0.83(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ173.6,74.1,60.6,51.3,49.6,34.6,34.1,31.8,29.9,29.5,29.48,29.2,29.17,29.1,27.1,25.3,25.1,22.6,14.1.MS(ESI):m / z[M+H] + 442.4264for C 27 H 56 NO 3 .

[0175] Compound 7d: 2-(6-((2-hydroxyethyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0176] Compound 7d was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.08(t,J=6.6Hz,4H),3.63(t,J=4.8Hz,2H),2.76(t,J=4.8Hz,2H),2.60(t,J=7.2Hz,2H),1.86-1. 82(m,2H),1.62-1.57(m,4H),1.48-1.44(m,2H),1.38(s,3H),1.31-1.25(m,26H),0.86-0.84(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3,60.8,53.7,51.1,49.5,35.5,31.8,30.0,29.8,29.2(x2),28.5,27.1,25.6,24.3,22.6,19.9,14.1.

[0177] Compound 7e: 2-(8-((2-hydroxyethyl)amino)octyl)-2-methylmalonic acid dioctyl ester

[0178] Compound 7e was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.10-4.04(m,4H),3.64(t,J=4.8Hz,2H),2.76(t,J=4.8Hz,2H),2.61(t,J=7.2Hz,2H),1.83-1.80( m,2H),1.61-1.56(m,4H),1.49-1.45(m,2H),1.37(s,3H),1.34-1.18(m,30H),0.86(t,J=7.2Hz,2H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.2,60.6,53.7,51.1,49.5,35.54,31.8,29.8(x2),29.4,29. 3,29.2(x2),28.5,27.2,25.8,24.3,22.6,19.9,14.0.MS(ESI):m / z[M+H] + 514.4461for C 30 H 60 NO 5 .

[0179] Compound 7f: 2-(4-((3-hydroxypropyl)amino)butyl)-2-methylmalonic acid dioctyl ester

[0180] Compound 7f was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.77(t,J=5.4Hz,2H),2.85(t,J=5.4Hz,2H),2.59(t,J=7.2Hz,2H),1.84-1.81(m,2H), 1.68-1.66(m,2H),1.59-1.56(m,4H),1.48-1.45(m,2H),1.37(s,3H),1.28-1.24(m,22H),0.86(t,J=6.6Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.5,65.4,64.3,53.8,50.0,49.5,35.4,31.8,30.6,30.0,29.2(x2),28.5,25.9,22.7,22.1,19.9,14.1.MS(ESI):m / z[M+H] + 472.3994for C 27 H 54 NO 5 .

[0181] Compound 7g: 2-(6-((3-hydroxypropyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0182] Compound 7g was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.05(t,J=6.6Hz,4H),3.78-3.76(m,2H),2.88-2.86(m,2H),2.61-2.59(m,2H),1.79-1.78(m,2H),1.7 1-1.69(m,2H),1.57-1.54(m,4H),1.48-1.44(m,2H),1.34(s,3H),1.27-1.17(m,26H),0.85-0.83(m,6H). 13 C NMR (150 MHz, CDCl 3)δ172.6,65.3,63.8,53.8,49.6,49.5,35.5,31.8,30.2,29.7,29.3,29.2,29.1,28.5,27.0,25.9,24.3,22.6,19.9,14.1.MS(ESI):m / z[M+H] + 500.4310for C 29 H 58 NO 5 .

[0183] Compound 7h: 2-ethyl-2-(6-((3-hydroxypropyl)amino)hexyl)malonate

[0184] Compound 7h was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.08(t,J=6.6Hz,4H),3.80(t,J=5.4Hz,2H),2.86(t,J=5.4Hz,2H),2.58(t,J=7.2Hz,2H),1.92(q,J=7.2Hz,2H),1.89-1.83(m,2H),1.6 9-1.67(m,2H),1.60-1.58(m,4H),1.48-1.42(m,2H),1.35-1.22(m,24H),1.15-1.12(m,2H),0.87(t,J=6.6Hz,6H),0.80(t,J=7.2Hz,3H). 13 C NMR (150 MHz, CDCl 3 )δ172.1,65.2,64.6,58.1,50.2,49.8,31.9,31.7,30.6,29.9(x2),29. 3(x2),28.6,27.1,26.0,25.3,24.0,22.7,14.2,8.5.MS(ESI):m / z[M+H] + 514.4466for C 30 H 60 NO 5 .

[0185] Compound 7i: 2-(6-((3-hydroxypropyl)amino)hexyl)-2-propylmalonate

[0186] Compound 7i was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1H NMR (600 MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.78(t,J=4.8Hz,2H),2.88(t,J=4.8Hz,2H),2.60(t,J=7.2Hz,2H),1.84-1.81(m,4H),1.72-1.70(m, 2H),1.60-1.56(m,4H),1.50-1.44(m,2H),1.31-1.22(m,24H),1.18-1.10(m,4H),0.9(t,J=7.2Hz,3H),0.86(t,J=6.6Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.1,65.2,64.0,57.7,49.8,49.6,34.6,32.3,31.8,30.4,29.8,29.5,2 9.3,29.2,28.6,27.1,25.9,24.1,22.7,17.5,14.5,14.1.MS(ESI):m / z[M+H] + 528.4623for C 31 H 62 NO 5 .

[0187] Compound 7j: 2-(6-((4-hydroxybutyl)amino)hexyl)-2-propylmalonate

[0188] Compound 7j was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.06(t,J=6.6Hz,4H),3.55(t,J=5.4Hz,2H),2.64(t,J=5.4Hz,2H),2.58(t,J=7.2Hz,2H),1.82-1.7 9(m,2H),1.66-1.56(m,8H),1.50-1.45(m,2H),1.36(s,3H),1.29-1.20(m,26H),0.85(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3)δ172.6,65.4,62.6,53.8,49.6,49.4,35.6,32.6,31.8,29.8,29.5,29.3 ,29.2,28.7,28.6,27.1,25.9,24.3,22.7,19.9,14.1.MS(ESI):m / z[M+H] + 514.4468 for C 30 H 60 NO 5 .

[0189] Compound 7k: 2-(6-((5-hydroxypentyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0190] Compound 7k was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.60(t,J=6.6Hz,2H),2.58(t,J=6.6Hz,2H),2.55(t,J=7.2Hz,2H),1.83-1.80(m,2H),1.60-1.5 4(m,6H),1.51-1.48(m,2H),1.46-1.43(m,2H),1.40-1.37(m,2H),1.36(s,3H),1.28-1.19(m,26H),0.86(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3,62.5,53.8,50.0,49.8,35.6,32.5,31.8,30.0,29.9,29.7,2 9.2(x2),28.5,27.2,25.9,24.3,23.5,22.7,19.9,14.1.MS(ESI):m / z[M+H] + 528.4618forC 31 H 62 NO 5 .

[0191] Compound 71: 2-(6-((6-hydroxyhexyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0192] Compound 71 was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1H NMR (600 MHz, CDCl 3 )δ4.05(t,J=6.6Hz,4H),3.58-3.56(m,2H),2.57-2.53(m,4H),1.81-1.78(m,2 H),1.58-1.42(m,10H),1.35(s,3H),1.34-1.13(m,30H),0.84(t,J=7.2Hz,6H). 13 CNMR (150MHz, CDCl 3 )δ172.6,65.3,62.5,53.8,49.9,49.8,35.5,32.7,31.8,29.8,29.7(x2),29.2(x2),28.5,27.2,27.1,25.9,25.7,24.3,22.6,19.9,14.1.

[0193] Compound 7m: 2-(6-((2,3-dihydroxypropyl)amino)hexyl)-2-methylmalonate

[0194] Compound 7m was synthesized according to the general procedure 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.06(t,J=6.6Hz,4H),3.75-3.72(m,1H),3.66-3.65(m,1H),3.56-3.53(m,1H),2.75-2.73(m,1H),2.65-2.61(m,1H),2.59- 2.52(m,2H),1.82-1.78(m,2H),1.59-1.54(m,4H),1.45-1.42(m,2H),1.34(s,3H),1.28-1.18(m,26H),0.85(t,J=7.2Hz,6H). 13 CNMR (150MHz, CDCl 3 )δ1172.6,69.7,65.8,65.3,53.8,52.4,49.9,35.6,31.8,29.9,29.8,29.2(x2),28.5,27.0,25.9,24.3,22.7,19.9,14.1.MS(ESI):m / z[M+H] + 516.4252forC 29 H 58 NO 6 .

[0195] Compound 7n: 2-(6-(((1s,4s)-4-hydroxycyclohexyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0196] Compound 7n was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.95-3.94(m,1H),2.78-2.71(m,3H),1.82-1.70(m ,8H),1.63-1.52(m,8H),1.36(s,3H),1.32-1.18(m,26H),0.87-0.84(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.4,65.8,65.2,55.4,53.6,46.3,35.4,31.7,31.0,29.6,29.1(x2) ,29.0,28.4,27.0,26.2,25.7,24.2,22.5,19.8,14.0.MS(ESI):m / z[M+H] + 540.4614forC 32 H 62 NO 5 .

[0197] Compound 7o: 2-(6-((3-hydroxypropyl)amino)hexyl)-2-methylmalonic acid-1-decyl-3-octyl ester

[0198] Compound 7o was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.78(t,J=5.4Hz,2H),2.86(t,J=5.4Hz,2H),2.59(t,J=7.2Hz,2H),1.84-1.78(m,2H) ,1.70-1.66(m,2H),1.61-1.55(m,4H),1.46-1.42(m,2H),1.37(s,3H),1.32-1.18(m,30H),0.87-0.85(m,6H). 13 C NMR (150 MHz, CDCl 3)δ172.6,65.3,64.3,53.8,49.9,49.7,35.6,31.9,31.8,30.5,29.8,29.7,29.6,29.3,29.2 6,29.23,29.22,28.5,27.1,25.9,24.3,22.7,22.6,19.9,14.13,14.11.MS(ESI):m / z[M+H] + 528.4619 for C 31 H 62 NO 5 .

[0199] Compound 7p: 2-(6-((3-hydroxypropyl)amino)hexyl)-2-methylmalonic acid-1-hexyl-3-octyl ester

[0200] Compound 7p was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.79(t,J=5.4Hz,2H),2.86(t,J=5.4Hz,2H),2.58(t,J=7.2Hz,2H),1.83-1.80(m,2H) ,1.69-1.66(m,2H),1.60-1.57(m,4H),1.45-1.43(m,2H),1.37(s,3H),1.36-1.19(m,22H),0.87-0.85(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3,64.4,53.8,50.1,49.8,35.6,31.8,31.4,30.5,29.8,29.7,29.24,29.2 2,28.6,28.5,27.1,25.9,25.6,24.3,22.7,22.6,19.9,14.1,14.0.MS(ESI):m / z[M+H] + 472.3987for C 27 H 54 NO 5 .

[0201] Compound 7q: 2-(6-(((1s,4s)-4-hydroxymethyl)cyclohexyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0202] Compound 7q was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.51(d,J=6.6Hz,2H),2.71-2.70(m,1H),2.57(t,J=7.2Hz,2H),1.82-1.8 0(m,2H),1.65-1.54(m,8H),1.51-1.48(m,6H),1.36(s,3H),1.31-1.18(m,26H),0.87-0.84(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.8,65.3,54.4,53.8,46.8,37.8,35.5,31.8,29.8,29.3,29.21,2 9.19,28.5,28.3,27.2,25.9,24.3,24.2,22.7,19.9,14.1.MS(ESI):m / z[M+H] + 554.4787for C 33 H 64 NO 5 .

[0203] Compound 7r: 2-(6-((4-hydroxycyclohexyl)methyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0204] Compound 7r was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.04-4.02(m,4H),3.91(br.s,0.5H),3.49-3.45(m,0.5H),2.55-2.50(m,2H),2.46-2.45(m,1H),2.39-2.38(m,1H),1.93 -1.91(m,1H),1.79-1.75(m,3H),1.67-1.65(m,1H),1.59-1.35(m,12H),1.33(s,3H),1.29-1.15(m,26H),0.83-0.81(m,6H). 13 C NMR (150 MHz, CDCl 3)δ172.5,70.7,66.6,65.2,55.8,55.3,53.7,50.0,49.9,36.8,36.3,35.5,35.2,32.1,31.7,2 9.8,29.7,29.5,29.4,29.2,28.5,27.1,25.8,25.1,24.3,22.6,19.9,14.0.MS(ESI):m / z[M+H] + 554.4775for C 33 H 64 NO 5 .

[0205] Compound 7s: 2-ethyl-2-(4-((3-hydroxypropyl)amino)butyl)malonate

[0206] Compound 7s was synthesized according to the general step 7 disclosed in Scheme 1 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.09-4.05(m,4H),3.78-3.77(m,2H),2.87-2.85(m,2H),2.61(t,J=7.2Hz,2H),1.90(q,J=7.8Hz,2H),1.86-1.83(m,2H ),1.70-1.68(m,2H),1.59-1.55(m,4H),1.50-1.48(m,2H),1.27-1.15(m,30H),0.87-0.84(m,6H),0.79(t,J=7.8Hz,3H). 13 CNMR (150MHz, CDCl 3 )δ171.9,65.3,64.0,58.0,49.8,49.4,31.9,31.6,30.5,29.9,29.6(x2) ,29.4,29.3,28.6.25.9,25.4,22.7,21.8,14.1,8.5.MS(ESI):m / z[M+H] + 542.4770forC 32 H 64 NO 5 .

[0207] Compound AS-CL-01: 2-(11-(2-hydroxyhexyl)-20-methyl-20-(octyloxy)carbonyl)-3,17,21-trioxo-2,18,22-trioxo-11-azatriacontyl)-2-methylmalonic acid dioctyl ester (step 8)

[0208] Compound 6 (86 mg, 0.15 mmol), potassium iodide (26 mg, 0.16 mmol) and potassium carbonate (K 2 CO 3 ) (78 mg, 0.57 mmol) was stirred in MeCN / DCM solution at 30°C overnight and the solvent was removed. The residue was dissolved in DCM, washed with water and brine, and dried over magnesium sulfate. After the solvent was completely evaporated, the product was chromatographed on a silica gel column with 5% methanol / 1% ammonium hydroxide in DCM to obtain compound AS-CL-01 (60 mg, 0.058 mmol, 39%) as a light yellow oil. 1 H NMR (600 MHz, CDCl 3 )δ4.43(s,2H),4.12(t,J=6.6Hz,4H),3.50(t,J=5.4Hz,2H),2.55(t,J=5.4Hz,2H),2.44-2.40(m,4H),2.29- 2.26(m,4H),1.62-1.58(m,12H),1.47(s,6H),1.44-1.38(m,4H),1,29-1.25(m,48H),0.87(t,J=7.2Hz,12H). 13 C NMR (150 MHz, CDCl 3 )δ173.2,173.0,170.0(x2),66.3,65.9(x2),58.4,55.5,53.9(x2),53.8,53.7(x2),34.1,34.0,31.8(x2),29.3,29.2( x4),29.1,28.5(x2),27.3,27.2,27.0,26.9,25.8(x2),24.9,24.8,22.7(x2),18.3(x2),14.1(x2).MS(ESI):m / z[M+H] + 1026.7822 for C 58 H 108 NO 13 .

[0209] Compound AS-CL-02: 2-(((6-((8-heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)hexanoyl)oxy)methyl)-2-methylmalonic acid dioctyl ester

[0210] Compound AS-CL-02 was synthesized according to the general procedure 8 disclosed in Flow 2 to obtain a colorless oil. 1HNMR (600MHz, CDCl 3 )δ 1 H NMR (600 MHz, CDCl 3 )δ4.84-4.81(m,1H),4.42(s,2H),4.10(t,J=6.6Hz,4H),3.48(t,J=5.4Hz,2H),2.53(t,J=5.4Hz,2H),2.40((t,J=7.2Hz,2H) ,2.27-2.23(m,4H),1.61(m,8H),1.48-1.45(m,4H),1.45(s,3H),1.42-1.37(m,6H),1.28-1.22(m,54H),0.86-0.83(m,12H). 13 C NMR (150 MHz, CDCl 3 )δ173.6,173.1,169.9,66.2,65.8(x2),58.3,55.5,53.9,53.8(x2),34.7,34.1,34.0,31.9,31.8,29.52,29.50,29.26,29.22(x2 ),29.17(x2),29.16(x2),29.09,28.4,27.3,27.2,27.1,25.8,25.3,25.1,24.8,22.62,22.61,18.3,14.1(x2).MS(ESI):m / z[M+H] + 938.8015for C 56 H 108 NO 9 .

[0211] Compound AS-CL-03: 2-(8-((8-heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amine)octyl)-2-methylmalonic acid dioctyl ester

[0212] Compound AS-CL-03 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. (600 MHz, CDCl 3 )δ 1 H NMR (600 MHz, CDCl 3)δ4.86-4.82(m,1H),4.10-4.04(m,4H),3.49(t,J=5.4Hz,2H),2.54(t,J=5.4Hz,2H),2.41-2.39(m,4H),2.25(t,J=7.2Hz,2H),1 .82-1.80(m,2H),1.60-1.56(m,6H),1.48-1.47(m,4H),1.43-1.38(m,6H),1.37(s,3H),1.36-1.23(m,58H),0.86-0.84(m,12H). 13 C NMR (150 MHz, CDCl 3 )δ173.6,172.6,74.1,65.3,58.4,55.5,53.9,53.87,53.8,35.6,34.7,34.2,31.9,31.8,29.9,29.6,29.5,29.4,29.3,29.27(x 2),29.24(x2),29.2,28.6,27.5,27.3,27.22,27.2,25.9,25.4,25.2,24.4,22.7,22.67,19.9,14.14,14.11.MS(ESI):m / z[M+H] + 894.8114for C 55 H 108 NO 7 .

[0213] Compound AS-CL-04: 2-(8-((2-hydroxyethyl)(7-methyl-8-octyloxy)-7-((octyloxy)carbonyl)-8-oxooctyl)amine)octyl)-2-methylmalonic acid dioctyl ester

[0214] Compound AS-CL-04 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. (600 MHz, CDCl 3 )δ 1 H NMR (600 MHz, CDCl 3 )δ4.06-4.05(m,4H),3.47(t,J=4.8Hz,2H),2.52(t,J=4.8Hz,2H),2.40-2.37(m,4H),1. 81-1.79(m,4H),1.58-1.54(m,6H),1.37(s,6H),1.35-1.24(m,62H),0.85-0.83(m,12H). 13 C NMR (150 MHz, CDCl 3)δ172.57,172.53,65.26,65.24,58.4,55.5,53.9,53.8,53.77,53.7,35.58,35.55,31.8(x2),29.91,29.89,29.5,29.4, 29.21,29.18,28.5(x2),27.5,27.3,27.2,27.1,25.9(x2),24.4,24.3,22.6(x2),19.9(x2),14.1(x2).MS(ESI):m / z[M+H] + 938.8026for C 56 H 108 NO 9 .

[0215] Compound AS-CL-05: 2-(6-Decyl(2-hydroxyethyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0216] Compound AS-CL-05 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.09-4.07(m,4H),3.50(t,J=5.4Hz,2H),2.55(t,J=5.4Hz,2H),2.42-2.40(m,4H),1.84-1.81( m,2H),1.61-1.57(m,4H),1.43-1.39(m,4H),1.38(s,3H),1.29-1.25(m,40H),0.88-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.4,58.4,55.5,53.9,53.84,53.82,35.6,32.0,31.9,30.0,29.7,29.6,29.4,29.3(x2),29 .2(x2),28.6,27.5,27.3,27.2,27.1,25.9,24.4,22.74,22.7,20.0,14.17,14.14.MS(ESI):m / z[M+H] + 626.5725for C 38 H 76 NO 5 .

[0217] Compound AS-CL-06: 2-(6-((8-heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0218] Compound AS-CL-06 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.85-4.83(m,1H),4.07-4.05(m,4H),3.48(t,J=5.4Hz,2H),2.53(t,J=5.4Hz,2H),2.41-2.38(m,4H),2.25(t,J=7.2Hz,2H),1 .82-1.80(m,2H),1.59-1.55(m,6H),1.48-1.46(m,4H),1.39-1.36(m,4H),1.36(s,3H),1.25-1.23(m,56H),0.86-0.84(m,12H). 13 C NMR (150 MHz, CDCl 3 )δ173.6,172.6,74.1,65.3,58.4,55.5,53.9(x2),53.8,35.6,34.7,34.2,31.9,31.8,30.0,29.6,29.5,29.3,29.27(x2 ),29.24(x2),28.6,27.4,27.3,27.2,27.1,25.9,25.4,25.2,24.4,22.70,22.68,19.9,14.14,14.12.MS(ESI):m / z[M+H] + 866.7800for C 53 H 104 NO 7 .

[0219] Compound AS-CL-07: 2-(((6-(Decyl)(2-hydroxyethyl)amino)hexanoyl)oxy)methyl)-2-methylmalonate

[0220] Compound AS-CL-07 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ 1 H NMR (600 MHz, CDCl 3)δ4.43(s,2H),4.11(t,J=6.6Hz,4H),3.50(t,J=5.4Hz,2H),2.55,(t,J=4.8Hz,2H),2.43-2.40(m,4H),2.28 (t,J=7.2Hz,2H),1.61-1.59(m,6H),1.47(s,3H),1.44-1.40(m,4H),1.27-1.24(m,36H),0.88-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ173.0,170.0,66.3,65.9,58.4,55.5,53.9,53.7,34.0,31.9,31.8,29.7(x2),29.6,29.4(x2), 29.2,28.5,27.5,27.2,27.0,26.9,25.8,24.8,22.7,22.6,18.3,14.15,14.12.MS(ESI):m / z[M+H] + 670.5623for C 39 H 76 NO 7 .

[0221] Compound AS-CL-08: 2-(6-((2-hydroxyethyl)octyl)amino)hexyl)-2-methylmalonate

[0222] Compound AS-CL-08 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.09-4.07(m,4H),3.50(t,J=5.4Hz,2H),2.55(t,J=5.4Hz,2H),2.42-2.40(m,4H),1.84-1.81( m,2H),1.60-1.57(m,4H),1.40-1.37(m,4H),1.37(s,3H),1.29-1.20(m,36H),0.88-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3)δ172.6,65.3,58.4,55.5,53.9,53.84,53.82,35.6,31.9,31.8,29.9,29.6,29.4,29.3,29.2 ,28.6,27.5,27.3,27.2,27.1,25.9,24.4,22.7,22.6,19.9,14.15,14.13.MS(ESI):m / z[M+H] + 598.5402forC 36 H 72 NO 5 .

[0223] Compound AS-CL-09: 2-(6-(Decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate

[0224] Compound AS-CL-09 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.77(t,J=4.8Hz,2H),2.61(t,J=5.4Hz,2H),2.37(t,J=7.2Hz,2H),1.83-1.81(m,2H) ,1.67-1.63(m,2H),1.61-1.56(m,4H),1.46-1.42(m,4H),1.37(s,3H),1.28-1.19(m,40H),0.87-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3,64.9,55.4,54.3,54.2,53.8,35.6,31.9,31.8,30.0,29.7,29.4,29.3(x2),29.2(x 2),28.6,27.8,27.6,27.4,26.9,26.8,25.9,24.4,22.8,22.7,19.9,14.2,14.1.MS(ESI):m / z[M+H] + 640.5868for C 39 H 78 NO 5 .

[0225] Compound AS-CL-10: 2-(6-(Decyl(2-hydroxyethyl)amino)hexyl)-malonic acid dioctyl ester

[0226] Compound AS-CL-10 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.12-4.05(m,4H),3.49(t,J=5.4Hz,2H),3.28(t,J=7.8Hz,1H),2.53(t,J=5.4Hz,2H),2.40(t,J=7.2 Hz,4H),1.87-1.84(m,2H),1.62-1.57(m,4H),1.42-1.36(m,4H),1.29-1.23(m,40H),0.86-0.84(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ169.7,65.5,58.4,55.5,53.9,53.8,52.1,32.0,31.8,29.7,29.6(x2),29.4,29.23(x2),29. 22,28.7,28.6,27.5,27.4,27.2(x2),27.1,25.9,22.7,22.6,14.15,14.12.MS(ESI):m / z[M+H] + 612.5555for C 37 H 74 NO 5 .

[0227] Compound AS-CL-11: 2-(6-(Decyl(3-hydroxypropyl)amino)hexyl)-malonic acid dioctyl ester

[0228] Compound AS-CL-11 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.11-4.05(m,4H),3.74(t,J=5.4Hz,2H),3.27(t,J=7.8Hz,1H),2.59-2.58(m,2H),2.36-2.34(m, 4H),1.86-1.81(m,2H),1.63-1.57(m,6H),1.43-1.39(m,4H),1.31-1.20(m,40H),0.85-0.83(m,9H). 13 C NMR (150 MHz, CDCl 3)δ169.6,65.4,64.8,55.3,54.2,54.1,52.1,31.9,31.8,29.62(x2),29.60(x2),29.3,29.2(x2), 28.7,28.5,27.8,27.5,27.3,27.2,26.8,26.7,25.8,22.7,22.6,14.09,14.07.MS(ESI):m / z[M+H] + 626.5719for C 38 H 76 NO 5 .

[0229] Compound AS-CL-12: 2-(6-((3-dimethylamino)propyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0230] Compound AS-CL-12 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.06(t,J=6.6Hz,4H),2.81(t,J=6.6Hz,2H),2.67(t,J=7.2Hz,2H),2.41(t,J=6.6Hz,2H),2.24(s ,6H),1.82-1.75(m,4H),1.59-1.55(m,6H),1.36(s,3H),1.31-1.24(m,26H),0.85(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.5,65.3,58.8,53.7,49.1(x2),45.5,35.5,31.8,29.6,29.2(x2), 28.7,28.5,26.9,25.9,25.6,24.2,22.7,19.9,14.1.MS(ESI):m / z[M+H] + 527.4779for C 31 H 63 N 2 O 4 .

[0231] Compound AS-CL-13: 2-(6-(Decyl(3-dimethylamino)propyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0232] Compound AS-CL-13 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.06-4.04(m,4H),3.62-3.60(m,2H),3.49-3.45(m,2H),3.34(s,6H),2.72-2.70(m,2H),2.54-2.52(m,2H),1.97-1.92(m,2H) ,1.81-1.77(m,2H),1.72-1.68(m,2H),1.59-1.53(m,4H),1.45-1.42(m,2H),1.35(s,3H),1.35-1.20(m,40H),0.85-0.83(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.5,65.3,64.4,62.8,53.7,51.7,49.9,46.0,35.5,31.8,31.7,29.9,29.8,29.37,29.35,29.2,29 .13(x2),29.12(x2),28.5,27.1,26.2,25.8,24.2,23.5,22.8,22.6,19.8,14.0(x2).MS(ESI):m / z[M+H] + 667.6340for C 41 H 83 N 2 O 4 .

[0233] Compound AS-CL-14: 2-(6-(Decyl(4-hydroxybutyl)amino)hexyl)-2-methylmalonate

[0234] Compound AS-CL-14 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.06(t,J=6.6Hz,4H),3.52-3.51(m,2H),2.42-2.39(m,6H),1.82-1.79(m,2H),1.6 2-1.54(m,8H),1.46-1.42(m,4H),1.35(s,3H),1.26-1.16(m,40H),0.86-0.83(m,9H). 13 C NMR (150 MHz, CDCl 3)δ172.6,65.3,62.6,54.6,53.8,53.7,53.6,35.6,32.7,31.9,31.8,29.9,29.7,29.6,29.5,29.3,29.2 (x2),28.5,27.7,27.5,26.2,25.9,25.8,25.7,24.4,22.7,22.6,19.9,14.13,14.11.MS(ESI):m / z[M+H] + 654.6039forC 40 H 80 NO 5 .

[0235] Compound AS-CL-15: 2-(6-(Decyl(2-hydroxyethyl)amino)hexyl)-2-methylmalonic acid diundecyl ester

[0236] Compound AS-CL-15 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.08(t,J=6.6Hz,4H),3.50(t,J=5.4Hz,2H),2.55(t,J=5.4Hz,2H),2.41(t,J=7.2Hz,4H),1.84-1. 81(m,2H),1.60-1.57(m,4H),1.41-1.38(m,4H),1.37(s,3H),1.29-1.20(m,52H),0.88-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3,58.4,55.5,53.9,53.84,53.80,35.6,32.0,29.9,29.7,29.65,29.60,29 .4,29.3,28.6,27.5,27.3,27.2,27.1,25.9,24.4,22.7,19.9,14.2.MS(ESI):m / z[M+H] + 710.6655for C 44 H 88 NO 5 .

[0237] Compound AS-CL-16: 2-(6-(Decyl(3-hydroxypropyl)amino)hexyl)-2-ethylmalonate

[0238] Compound AS-CL-16 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.08(t,J=6.6Hz,4H),3.77(t,J=4.8Hz,2H),2.62(t,J=4.8Hz,2H),2.39(t,J=7.2Hz,4H),1.90(q,J=7.2Hz,2H),1.85-1.82(m,2H),1 .67-1.65(m,2H),1.61-1.56(m,4H),1.46-1.42(m,4H),1.30-1.24(m,38H),1.15-1.11(m,2H),0.88-0.86(m,9H),0.79(t,J=7.2Hz,3H). 13 C NMR (150 MHz, CDCl 3 )δ172.0,65.2,64.8,58.1,55.3,54.2,54.1,32.0,31.8,31.7,29.9,29.7,29.6,29.4,29.3,29.2(x2), 28.6,27.8,27.6,27.4,26.8,26.7,25.9,25.2,24.0,22.73,22.69,14.2,14.1,8.5.MS(ESI):m / z[M+H] + 654.6036for C 40 H 80 NO 5 .

[0239] Compound AS-CL-17: 2-(6-(decyl(2,3-dihydroxypropyl)amine)hexyl)- Dioctyl 2-methylmalonate

[0240] Compound AS-CL-17 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3)δ4.08(t,J=6.6Hz,4H),3.73-3.68(m,2H),3.48-3.45(m,1H),2.57-2.54(m,1H),2.51-2.46(m,2H),2.41-2.37(m ,3H),1.84-1.81(m,2H),1.61-1.57(m,4H),1.48-1.38(m,4H),1.37(s,3H),1.32-1.18(m,40H),0.88-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,67.3,65.3,64.9,56.8,54.4,54.3,53.8,35.6,31.9,31.8,29.9,29.7,29.6,29.3,29.22(x2 ),29.20,28.5,27.5,27.2,27.1,27.0,25.9,24.3,22.7,22.6,19.9,14.12,12.10.MS(ESI):m / z[M+H] + 656.5822for C 39 H 78 NO 6 .

[0241] Compound AS-CL-18: 2-(6-(Decyl(1s,4s)-4-hydroxycyclohexyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0242] Compound AS-CL-18 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.97(br.s,1H),2.53-2.46(m,5H),1.83-1.80(m,4H),1.68-1.56(m, 8H),1.51-1.41(m,6H),1.37(s,3H),1.31-1.17(m,40H),0.87-0.85(m,9H).MS(ESI):m / z[M+H] + 680.6183for C 42 H 82 NO 5 .

[0243] Compound AS-CL-19: 2-(6-(4-(dimethylamino)butaneamino)hexyl)-2-methylmalonic acid dioctyl ester

[0244] Compound AS-CL-19 was synthesized according to the steps disclosed in Scheme 3. Compound 6b (0.37 g, 0.67 mmol) and potassium phthalimide (0.62 g, 3.35 mmol) were mixed in dimethylformamide (DMF) at room temperature and stirred for about 3 hours. The solvent was removed by filtration and evaporation, and the product was chromatographed on a silica gel column with EA / hexane (1 / 10) to obtain compound 6b-1 (311 mg, 84%) as a light yellow oil. 1 H NMR (600 MHz, CDCl 3 )δ7.84-7.82(m,2H),7.71-7.69(m,2H),4.08(t,4H),3.67(t,2H),1.84-1.81(m,2H) ,1.66-1.64(m,2H),1.61-1.57(m,4H),1.37(s,3H),1.34-1.25(m,26H),0.87(t,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.5,168.4,133.8,132.1,123.1,65.3,53.7,37.9,35.5,31.7,29.5,29.2,28.5,28.4,26.7,26.2,25.8,24.2,22.6,19.8,14.0.

[0245] Compound 6b-1 (311 mg, 0.544 mmol) and hydrazine (50 mg, 1.63 mmol) were mixed in methanol and stirred at reflux for 2 hours. After the solvent evaporated, the residue was dissolved in DCM and filtered. The product was chromatographed on a silica gel column with 10% methanol / DCM / 1% ammonium hydroxide to obtain compound 6b-2 (160 mg, 66%). 1 H NMR (600 MHz, CDCl 3 )δ4.08(t,4H),2.67(t,2H),1.85-1.82(m,2H),1.62-1.57(m,4H),1.43-1.41(m,2H),1.38(s,3H),1.31-1.25(m,26H),0.87(t,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3,53.8,42.1,35.5,33.7,31.9,29.8,29.6,29.5,29.3,29.2,28.5,26.7,25.9,24.3,22.7,19.9,14.1.

[0246] 4-(Dimethylamino)butyric acid (0.071 g, 0.426 mmol), thionyl chloride (SOCl) were stirred at 0°C. 2 ) (0.101 g, 0.852 mmol) and 2 drops of DMF in dichloromethane (DCM) solution overnight. After the solvent evaporated, the residue was reacted with compound 6b-2 in DCM at 0°C, and triethylamine (Et 3 N). The progress of the reaction was monitored by thin layer chromatography (TLC). After the solvent was evaporated, the product was chromatographed on a silica gel column with 10% methanol / DCM / 1% ammonium hydroxide to obtain compound AS-Cl-19 (76 mg, 45%). 1 H NMR (600 MHz, CDCl 3 )δ6.41(br.s,1H),4.08(t,J=6.6Hz,4H),3.20(q,J=6.6Hz,2H),2.31(t,J=7.2Hz,2H),2.24(4,J=7.2Hz,2H),2.21(s,6H),1.84 -1.82(m,2H),1.81-1.76(m,2H),1.61-1.58(m,4H),1.47-1.45(m,2H),1.38(s,3H),1.31-2.20(m,26H),0.87(t,J=7.2Hz,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.7,172.4,65.2,58.7,53.6,45.1,39.3,35.4,34.7,31.7,29.5,29. 1,29.0,28.4,26.6,25.7,24.1,23.1,22.5,19.8,14.0.MS(ESI):m / z[M+H] + 555.4727for C 32 H 63 N 2 O 5 .

[0247] Compound AS-CL-20: 2-(6-(Decyl(5-hydroxypentyl)amino)hexyl)-2-methylmalonate

[0248] Compound AS-CL-20 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3)δ4.07(t,J=6.6Hz,4H),3.63-3.61(m,2H),2.52-2.46(m,6H),1.83-1.80(m,2H ),1.60-1.46(m,12H),1.40-1.34(m,5H),1.31-1.18(m,40H),0.87-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.4,62.6,53.9,53.8,53.7,35.6,32.4,31.9,31.8,29.8,29.7,29.6,29.5,29.4,29.3,29.2 ,28.6,27.6,27.3,26.1,26.0(x2),25.9,24.3,23.7,22.72,22.68,19.9,14.2,14.1.MS(ESI):m / z[M+H] + 668.6194for C 41 H 82 NO 5 .

[0249] Compound AS-CL-21: 2-(6-((6-decanoyloxy)hexyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate

[0250] Compound AS-CL-21 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.08(t,J=6.6Hz,4H),4.05(t,J=6.6Hz,2H),3.78(t,J=5.4Hz,2H),2.62(t,J=5.4Hz,2H),2.41-2.38(m,4H),2.28(t,J= 7.2Hz,2H),1.84-1.81(m,2H),1.67-1.58(m,10H),1.47-1.41(m,4H),1.38(s,3H),1.37-1.20(m,42H),0.88-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3)δ174.1,172.6,65.4,64.8,64.3,55.3,54.2,54.1,53.8,35.6,34.5,31.9,31.8,30.0,29.5,29.3(x2),29.26(x2),29 .24(x2),28.7,28.6,27.8,27.4,27.2,26.8,26.7,26.0,25.9,25.1,24.4,22.72,22.70,19.9,14.1.MS(ESI):m / z[M+H] + 754.6549 for C 45 H 88 NO 7 .

[0251] Compound AS-CL-22: 2-(6-(Decyl(3-hydroxypropyl)amino)hexyl)-2-propylmalonate

[0252] Compound AS-CL-22 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.08(t,J=6.6Hz,4H),3.78(t,J=5.4Hz,2H),2.61(t,J=5.4Hz,2H),2.38(t,J=7.8Hz,4H),1.86-1.82(m,4H),1.68-1.63(m ,2H),1.61-1.57(m,4H),1.47-1.41(m,4H),1.32-1.22(m,38H),1.18-1.11(m,4H),0.91(t,J=7.2Hz,3H),0.88-0.86(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.1,65.2,64.9,57.7,55.4,54.3,54.2,34.6,32.4,32.0,31.9,30.0,29.70,29.69,29.67,29.4,29.3,2 9.2,28.6,27.8,27.6,27.4,26.9,26.8,26.0,24.1,22.8,22.7,17.5,14.5,14.18,14.15.MS(ESI):m / z[M+H] + 668.6180forC 41 H 82 NO 5 .

[0253] Compound AS-CL-23: 2-(6-(dodecyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate

[0254] Compound AS-CL-23 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.08(t,J=6.6Hz,4H),3.78(t,J=5.4Hz,2H),2.61(t,J=5.4Hz,2H),2.39-2.37(m,4H),1.84-1.81(m,2H), 1.67-1.64(m,2H),1.62-1.57(m,4H),1.47-1.42(m,4H),1.38(s,3H),1.32-1.20(m,44H),0.88-0.86(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3,64.9,55.4,54.3,54.2,53.8,35.6,32.0,31.8,29.9,29.7(x2),29.4,29.3(x3),29.2 (x2),28.6,27.8,27.6,27.4,26.9,26.8,25.9,24.4,22.7,22.6,19.9,14.2,14.1.MS(ESI):m / z[M+H] + 668.6181for C 41 H 82 NO 5 .

[0255] Compound 9a: Decyloctylmalonate (Step 9)

[0256] Meldrum's acid (also known as isopropylidene malonate) (2.5 g, 17.35 mmol) and octanol (2.26 g, 17.35 mmol) were refluxed in toluene for 4 hours. After the solvent evaporated, the residue was dissolved in DCM, and 1-decanol (4.12 g, 2.6 mmol), EDCI (5 g, 2.6 mmol) and DMAP (0.42 g, 3.47 mmol) were added. The product was chromatographed on a silica gel column with ethyl acetate / hexane (1 / 50) to obtain compound 9a (5.65 g, 90%). 1 H NMR (600 MHz, CDCl 3)δ4.11(t,J=6.6Hz,4H),3.34(s,2H),1.64-1.59(m,4H),1.32-1.24(m,24H),0.87-0.85(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ166.7,65.7,41.7,31.9,31.8,29.6,29.3,29.25,29.20,28.5,25.8,22.7,22.6,14.11,14.08.MS(ESI):m / z[M+Na] + 379.2823for C 21 H 40 O 4 Nah.

[0257] Compound 9b: Hexyloctylmalonate

[0258] Compound 9b was synthesized according to the general step 9 disclosed in Scheme 4 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.10(t,J=6.6Hz,4H),3.33(s,2H),1.61-1.59(m,4H),1.31-1.26(m,16H),0.85-0.84(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ166.7,65.6,41.7,31.8,31.4,29.2(x2),28.5,28.4,25.8,25.5,22.6,22.5,14.1,14.0.MS(ESI):m / z[M+Na] + 323.2190forC 17 H 32 O 4 Nah.

[0259] Compound AS-CL-24: 2-(6-(Decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonic acid-1-decyl-3-octyl ester

[0260] Compound AS-CL-24 was synthesized according to the general steps disclosed in Scheme 4 and Step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.08(t,J=6.6Hz,4H),3.78(t,J=5.4Hz,2H),2.62(t,J=5.4Hz,2H),2.38(t,J=7.8Hz,4H),1.84-1.81(m,2H) ,1.67-1.63(m,2H),1.62-1.57(m,4H),1.47-1.41(m,4H),1.38(s,3H),1.29-1.18(m,44H),0.88-0.86(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.4,64.9,55.4,54.3,54.2,53.8,35.6,32.0,31.9,30.0,29.70,29.69,29.67,29.63,29.4,29.30, 29.28,29.26,28.6,27.8,27.6,27.4,26.9,26.8,25.9,24.4,22.8,22.7,20.0,14.2,14.1.MS(ESI):m / z[M+H] + 668.6192for C 41 H 82 NO.

[0261] Compound AS-CL-25: 2-(6-(Decyl(6-hydroxyhexyl)amino)hexyl)-2-methylmalonate

[0262] Compound AS-Cl-25 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.61(t,J=6.6Hz,2H),2.43-2.39(m,6H),1.83-1.80(m,2H),1. 59-1.53(m,6H),1.45-1.38(m,6H),1.37(s,3H),1.36-1.19(m,44H),0.87-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3)δ172.6,65.4,62.9,54.12,54.01,53.95,53.82,35.6,32.8,32.0,31.8,29.9,29.7,29.6,29.4,29.3,29.2,28 .6,27.7,27.4,27.3,26.64,26.59,26.55,25.9,25.7,24.4,22.73,22.69,19.9,14.2,14.1.MS(ESI):m / z[M+H] + 682.6350 for C 42 H 84 NO 5 .

[0263] Compound AS-CL-26: 2-(6-(Decyl(3-hydroxypropyl)amino)butyl)-2-methylmalonate

[0264] Compound AS-CL-26 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.08(t,J=6.6Hz,4H),3.76(t,J=5.4Hz,2H),2.60(t,J=5.4Hz,2H),2.39-2.36(m,4H),1.86-1.83(m,2H), 1.65-1.63(m,2H),1.60-1.56(m,4H),1.48-1.41(m,4H),1.37(s,3H),1.31-1.18(m,36H),0.87-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.5,65.4,64.8,55.3,54.4,54.0,53.8,35.6,32.0,31.9,29.70,29.67,29.66,29.4,29.3, 29.2,28.6,27.9,27.6,27.2,26.9,25.9,22.8,22.7,22.4,20.0,14.2,14.1.MS(ESI):m / z[M+H] + 612.5555for C 37 H 74 NO 5 ..

[0265] Compound AS-CL-27: 2-(6-(Decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonic acid-1-hexyl-3-octyl ester

[0266] Compound AS-CL-27 was synthesized according to the general steps disclosed in Scheme 4 and the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.08(t,J=6.6Hz,4H),3.78(t,J=5.4Hz,2H),2.61(t,J=5.4Hz,2H),2.38(t,J=7.2Hz,4H),1.84-1.81(m,2H) ,1.66-1.64(m,2H),1.60-1.58(m,4H),1.45-1.42(m,4H),1.38(s,3H),1.32-1.18(m,36H),0.89-0.86(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.4,64.9,55.4,54.3,54.2,53.8,35.6,32.0,31.9,31.5,30.0,29.69,29.68,29.66,29.4,29.3,29.2,28 .6,28.5,27.8,27.6,27.4,26.9,26.8,25.9,25.6,24.4,22.8,22.7,22.6,19.9,14.2,14.1,14.0.MS(ESI):m / z[M+H] + 612.5565for C 37 H 74 NO 5 ..

[0267] Compound AS-CL-28: 2-(6-(Decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonic acid didecyl ester

[0268] Compound AS-CL-28:2 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3)δ4.07(t,J=6.6Hz,4H),3.77(t,J=5.4Hz,2H),2.61(t,J=5.4Hz,2H),2.37(t,J=7.2Hz,4H),1.83-1.81(m,2H) ,1.67-1.63(m,2H),1.61-1.56(m,4H),1.46-1.41(m,4H),1.37(s,3H),1.32-1.18(m,48H),0.87-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3,64.9,55.4,54.3,54.2,53.8,35.6(x2),32.0,30.0,29.69,29.67,29.65,29.61(x2),29.4 (x2),29.3.28.6,27.8,27.6,27.4,26.9,26.8,25.9,24.4,22.7(x2),19.9,14.2(x2).MS(ESI):m / z[M+H] + 696.6505for C 43 H 86 NO 5 .

[0269] Compound AS-CL-29: 2-(6-((6-((2-hexyldecanoyl)oxy)hexyl)(3-hydroxypropyl)amino)hexyl)-2-methylmalonate

[0270] Compound AS-CL-29 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),4.04(t,J=6.6Hz,2H),3.77(t,J=5.4Hz,2H),2.61(t,J=5.4Hz,2H),2.39-2.36(m,4H),2.31-2.2 7(m,1H),1.83-1.81(m,2H),1.66-1.56(m,10H),1.49-1.39(m,6H),1.37(s,3H),1.35-1.17(m,50H),0.87-0.84(m,12H). 13 CNMR (150MHz, CDCl 3)δ167.8,172.6,65.3,64.9,64.1,55.3,54.22,54.19,53.8,45.9,35. 6,32.6(x2),31.9,31.8,31.7,30.0,29.6,29.5,29.3,29.28,29.26,29 .24,28.8,28.6,27.8,27.5,27.48,27.43,27.2,26.9,26.8,26.0,25. 9,24.4,22.72,22.69,22.65,19.9,14.2,14.1(x2).MS(ESI):m / z[M+H] + 838.7495for C 51 H 100 NO 7 .

[0271] Compound AS-CL-30: Bis-(6-((6-decanoyl)oxy)hexyl)(3-hydroxypropyl)amino)hexyl)-2,2'-dihexylmalonate

[0272] Compound AS-CL-30 was synthesized according to the steps disclosed in Scheme 5.

[0273] Malonic acid (2 g, 19.21 mmol), 6-bromohexane-1-ol (7.65 g, 42.26 mmol), EDCI (8.1 g, 42.26 mmol) and DMAP (0.47 g, 3.84 mmol) were mixed in a DCM solution at room temperature and stirred overnight. After washing with 2N HCl and removing the solvent by evaporation, the product was chromatographed on a silica gel column with ethyl acetate / hexane (1 / 10) to obtain compound 10 (4.18 g, 50%) as a light yellow oil. 1 H NMR (600 MHz, CDCl 3 )δ4.14(t,J=6.6Hz,4H),3.40(t,J=6.6Hz,4H),3.36(s,2H),1.88-1.84(m,4H),1.69-1.64(m,4H),1.50-1.44(m,4H),1.41-1.35(m,4H). 13 C NMR (150 MHz, CDCl 3 )δ166.6,65.3,41.5,33.6,32.5,28.2,27.6,24.9.

[0274] Compound 10 (1.07 g, 2.49 mmol) and 1-iodohexane (2.11 g, 9.95 mmol) were mixed at room temperature and stirred overnight. Then, NaH (0.2 g, 5 mmol) was added and stirred at room temperature for 4 hours. The mixture was washed with saturated ammonium chloride solution. After the solvent evaporated, the product was chromatographed on a silica gel column with ethyl acetate / hexane (1 / 25) to obtain compound 11 (1 g, 67%) as a yellow oil. 1 H NMR (600 MHz, CDCl 3 )δ4.10(t,J=6.6Hz,4H),3.40(t,J=6.6Hz,2H),1.86-1.83(m,8H),1.65-1.60(m,4H),1.48 -1.43(m,4H),1.38-1.33(m,4H),1.33-1.22(m,12H),1.17-1.09(m,4H),0.88-0.86(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ172.0,64.8,57.7,33.5,32.6,32.2,31.5,29.5,28.3,27.7,25.1,23.9,22.5,14.0.MS(ESI):m / z[M+Na] + 619.1967,[M+Na] 2+ 621.1942and[M+Na] 4+ 623.1930 for C 27 H 50 O 4 Br 2 Nah.

[0275] Compound 11 (160 mg, 0.267 mmol), potassium iodide (90 mg, 0.534 mmol) and 3-amino-1-propanol (1 g, 13.37 mmol) were mixed in a MeCN / DCM solution and stirred at room temperature for 4 hours. After the solvent evaporated, the residue was dissolved in DCM and washed with water and brine. After removing the solvent again, the residue was mixed with 6-bromohexyl dodecanoate (212 mg, 0.633 mmol), potassium iodide (KI) (105 mg, 0.633 mmol) and potassium carbonate (KCO) and the mixture was stirred for 4 hours. 2 CO 3 ) (320 mg, 1.15 mmol) was mixed in MeCN / DCM and heated at 50°C overnight. After the solvent evaporated, it was concentrated on a silica gel column with 5% MeOH / DCM / 1% NH 4 OH The product was chromatographed to obtain compound AS-Cl-30 (180 mg, 57%) as a colorless oil. 1H NMR (600 MHz, CDCl 3 )δ4.06(t,J=6.6Hz,4H),4.03(t,J=6.6Hz,4H),3.76(br.s,4H),2.60(t,J=5.4Hz,4H),2.37(t,J=7.8Hz,8H),2.26(t,J=7.8 Hz,4H),1.84-1.81(m,4H),1.65-1.56(m,16H),1.48-1.42(m,8H),1.37-1.24(m,52H),1.14-1.07(m,4H),0.86-0.84(m,6H). 13 C NMR (150 MHz, CDCl 3 )δ174.0,172.1,65.0,64.8,64.3,57.7,55.3,54.2(x2),34.4,32.3,31.9,31.6,29.6,29.5,29.3(x2),29.2 (x2),28.7,28.6,27.9,27.2(x2),26.8,26.0,25.9,25.0,24.0,22.7,22.6,14.14,14.09.MS(ESI):m / z[M+H] + 1095.9487forC 65 H 127 N 2 O 10 .

[0276] Compound AS-CL-31: Bis-(6-(decyl(3-hydroxypropyl)amino)hexyl-2,2'-dihexylmalonate

[0277] AS-CL-31 was synthesized according to the similar steps of AS-CL-30 to obtain a colorless oil. 1 H NMR (600 MHz, CDCl 3 )δ4.09-4.06(m,4H),3.78-3.76(m,4H),2.62-2.38(m,4H),2.38(br.s,8H),1.85-1.83(m,4H),1.66-1.65 (m,4H),1.63-1.58(m,4H),1.49-1.41(m,8H),1.36-1.22(m,48H),1.14-1.11(m,4H),0.86-0.85(m,12H). 13 C NMR (150 MHz, CDCl 3)δ172.2,65.1,64.9,57.8,55.3,54.3,54.2,32.3,32.0,31.6,29.7(x2),29.66,29.6,29.4 ,28.6,27.9,27.6,27.2,26.9,26.8,26.0,24.0,22.8,22.6,14.2,14.1.MS(ESI):m / z[M+H] + 868.0482forC 53 H 107 N 2 O 6 .

[0278] Compound AS-CL-32: 2-(6-(Decyl((1s,4s)-4-(hydroxymethyl)cyclohexyl)amino)hexyl)-2-methylmalonic acid dioctyl ester

[0279] Compound AS-CL-32 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.07(t,J=6.6Hz,4H),3.59(d,J=7.2Hz,2H),2.53-2.45(m,5H),1.83-1.80(m,2H),1.78-1.71( m,2H),1.61-1.55(m,6H),1.45-1.38(m,6H),1.37(s,3H),1.32-1.18(m,42H),0.87-0.85(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.6,65.3,64.4,59.4,53.8,50.6,50.4,36.1,35.6,31.9,31.8,29.9,29.7,29.6,29.4,29. 24,29.22,28.6,27.6,27.4,25.9,24.6,24.4,22.72,22.67,19.9,14.2,14.1.MS(ESI):m / z[M+H] + 694.6339forC 43 H 84 NO 5 .

[0280] Compound AS-CL-33: 2-(6-(Decyl((4-(hydroxycyclohexyl)methyl)amino)hexyl)-2-methylmalonate

[0281] Compound AS-CL-33 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.07-4.01(m,4H),3.88(br.s,0.5H),3.48-3.46(m,0.5H),2.27-2.25(m,4H),2.13-2.12(m,1H),2.06-2 .05(m,1H),1.92-1.90(m,1H),1.80-1.77(m,3H),1.63-1.48(m,7H),1.41-1.14(m,48H),0.84-1.81(m,9H). 13 C NMR (150 MHz, CDCl 3 )δ172.5,71.2,67.3,65.2,61.0,60.2,54.9,54.8,54.7,53.7,35.6,35.5,35.4,34.7,32.2,31.9,31.8,29.9,29.7,29.6,29 .3,29.2,29.1,28.5,27.52,27.49,27.3,27.2,27.1,27.0,25.8,25.6,24.3,22.7,22.6,19.8,14.1,14.0.MS(ESI):m / z[M+H] + 694.6353for C 43 H 84 NO 5 .

[0282] Compound AS-CL-34: 2-ethyl-2(4-((6-((2-hexyldecanoyl)oxy)hexyl)(3-hydroxypropyl)amino)butyl)malonate

[0283] Compound AS-CL-34 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.09-4.02(m,6H),3.76-3.75(m,2H),2.61(br.s,2H),2.39(br.s,4H),2.29-2.27(m,1H),1.90(q,J=7.8H z,2H),1.87-1.84(m,2H),1.65-1.55(m,10H),1.47-1.12(m,60H),0.87-0.85(m,12H),0.79(t,J=7.8Hz,3H).13 C NMR (150 MHz, CDCl 3 )δ176.7,171.9,65.2,64.6,64.0,58.0,55.1,54.3,54.0,45.9,32.6,31.93,31.90,31.7,29.6(x2),29.5,29.4,2 9.3,29.27,28.7,28.6,27.9,27.5,27.4,27.2,26.8,26.0,25.9,25.3,22.7,22.6,22.1,14.1.MS(ESI):m / z[M+H] + 880.7963for C 54 H 106 NO 7 .

[0284] Compound AS-CL-35: 2-(4-(Decyl(3-hydroxypropyl)amino)butyl)-2-ethylmalonate

[0285] Compound AS-CL-35 was synthesized according to the general step 8 disclosed in Scheme 2 to obtain a colorless oil. 1 HNMR (600MHz, CDCl 3 )δ4.09-4.06(m,4H),3.76(t,J=4.8Hz,2H),2.64(br.s,2H),2.43-2.40(m,4H),1.90(q,J=7.8Hz,2H),1.87-1.84(m,2H),1.68-1 .66(m,2H),1.60-1.56(m,4H),1.50-1.45(m,4H),1.28-1.24(m,42H),1.16-1.10(m,2H),0.87-0.85(m,9H),0.79(J=7.8Hz,3H). 1 H NMR (600 MHz, CDCl 3 )δ171.9,65.3,64.4,58.1,55.0,54.2,53.9,32.0,31.7,29.7,29.6(x2),29.4,29.3 ,28.6,27.8,27.5,27.0,26.7,25.9,25.4,22.7,22.1,14.1,8.5.MS(ESI):m / z[M+H] + 682.6341for C 42 H 84 NO 5 .

[0286] Example 2 Preparation of Lipid Nanoparticles Loaded with Nucleic Acids and Analysis of Their Characteristics

[0287] 2.1 Preparation of mRNA-loaded lipid nanoparticles (mRNA-LNP)

[0288] LNPs were prepared using commercial lipids (e.g., MC3-DMA, SM-102) or lipids of the present invention (AS-CL05, AS-CL09, AS-CL28, or AS-CL35) with a total lipid concentration of 50 mM. The lipids were dissolved in ethanol and then mixed with other specified ingredients according to a ratio to prepare LNPs, wherein the molar ratio of lipid (MC3-DMA, SM-102, AS-CL05, AS-CL09, AS-CL28, or AS-CL35) / DSPC / cholesterol / DMG-PEG2000 was 50 / 10 / 38.1 / 1.5.

[0289] Nanoassmblr TM (Precision NanoSystems) microfluidic mixing device assembles LNPs and mRNA together. The LNPs of the present invention are used to encapsulate two different mRNAs: (i) mRNAs from wild-type and Omicron BA.5 SARS-CoV-2 spike proteins; and (ii) mRNAs from DENV2 serotype membrane proteins (E protein) (DENV2 E mRNA). Accordingly, the mRNA was first dissolved in 50 mM sodium acetate buffer (pH 4.5), and then the ratio of lipid:mRNA = 6.5 was used in SparkNanoassmblr TM (Precision NanoSystems) device. 16 μl of the organic phase was mixed with 32 μl of the aqueous phase and injected into 48 μl of PBS (pH 7.4). The LNP was then diluted with 96 μl of DPBS (pH 7.4) and dialyzed with PBS.

[0290] 2.2 Analysis of the physicochemical properties of mRNA-LNPs of Example 2.1

[0291] The mRNA-LNP of Example 2.1 was diluted 100-fold with PBS and then transferred to a 384-well culture plate. The lipid particle size and polydispersity (PDI) were measured by dynamic light scattering (DLS). Each nanoparticle was destroyed with 1% TX-100 to release the mRNA and evaluate the encapsulation efficiency of the nanoparticles.

[0292] Table 2. Physicochemical properties analysis of mRNA-LNP of Example 2.1

[0293] The data in Table 2 confirm that the lipids AS-CL05, AS-CL09, AS-CL28 and AS-CL35 of the present invention can form nanoparticles with other oil components respectively. The physical properties of AS-CL05-LNP, AS-CL09-LNP, AS-CL28-LNP and AS-CL35-LNP, including particle size and polydispersity (PDI), are similar to those of nanoparticles formed by commercial cationic lipids. The average particle size of each mRNA-LNP of the present invention is between 70-90nm, and the PDI is below 0.3, which means that each mRNA-LNP of the present invention can be uniformly dispersed in the solution without particle aggregation.

[0294] 2.3 Protein expression in vivo and in vitro

[0295] To evaluate the extent of mRNA translation and protein expression in vitro, 293T cells were treated with the mRNA-LNP complex of Example 2.1, and then the cells were lysed and the cell lysates were analyzed by flow cytometry. The results showed that each mRNA-LNP complex could induce the transfected cells to express S protein. Among the nanoparticle complexes tested, the LNP formed by the lipid AS-CL09 of the present invention induced the highest protein expression, followed by the nanoparticles formed by MC3, SM102, AS-CL05, AS-CL28 and AS-CL35. The results are shown in Figure 1 A and 2A.

[0296] After confirming that each mRNA-LNP complex can successfully induce protein expression in the cell analysis test, the effect of each mRNA-LNP complex as an immunogen was then explored using BALB / c mice, while the control group of mice received physiological experimental water treatment. The ELISA test was used to evaluate the ability of serum antibodies to bind to wild-type S protein and BA.5 recombinant S protein. The antibodies induced by wild-type mRNA-AS-CL09 LNPs and BA.5mRNA-AS-CL09 LNPs expressed the highest binding affinity. As for the antibodies induced by nanoparticles formed by MC3, SM102, AS-CL05, AS-CL28 and AS-CL35, their binding affinity was slightly inferior to the previous ones. The results are shown in Figure 1 B and 2B.

[0297] 2.4 Ability of serum antibodies to effectively neutralize SARS-CoV-2 variant pseudovirus

[0298] Serum was collected from vaccinated animals and the ability of the serum to neutralize the virus was evaluated using a pseudovirus neutralization test. The results showed that the serum collected from animals immunized with the BA.5mRNA-AS-CL09 LNP complex had the highest neutralizing activity. In addition, in terms of the ability to fight the BA.5 pseudovirus, the neutralizing antibody valence of the serum of animals immunized with the BA.5mRNA-AS-CL09 LNP complex was 9 times higher than that of the neutralizing antibody valence in the serum of animals immunized with MC3 or AS-CL05. In addition, the neutralizing antibodies induced in the bodies of animals immunized with AS-CL09 LNP were also better than those immunized with SM-102LNP ( Figure 1 C and Table 3).

[0299] Table 3. Evaluation of the maximum half inhibitory concentration (IC50) of mRNA-LNP of Example 2.1 using SARS-CV2 pseudovirus 50 )

[0300] 2.5 Comparison of DENV2E mRNA-AS-CL09 LNP complex and DENV2E mRNA-SM-102 LNP complex

[0301] In this embodiment, 293T cells were treated with LNP complexes containing DENV2 E protein mRNA and formed by AS-CL09 or SM-102 lipids, and then the expression of DENV2 E protein in the cells was analyzed by flow cytometry. The results showed that after the cells were transfected with any of the above mRNA-LNPs, DENV2 E protein was successfully expressed (Figure 3A) and the protein expression levels were similar.

[0302] Next, according to the steps described in the "Materials and Methods" section, BALB / c mice were immunized by intramuscular injection of the above mRNA-LNP, and serum was collected at week 6, and the binding capacity of neutralizing antibodies in the serum was analyzed by ELISA test. The results showed that compared with mice vaccinated with DENV2 E mRNA-SM-102LNP complex, the serum of mice inoculated with DENV2 E mRNA-AS-CL09 LNP complex expressed higher binding capacity to DENV2 serotype virus ( Figure 3B). The results of the PRNT test used to analyze the neutralizing activity of DENV antibodies showed that mice injected with the DENV2 E mRNA-AS-CL09 LNP complex had a higher amount of neutralizing antibodies, with a PRNT50 value of approximately 24,725, which was 1.4 times higher than the antibody level in the serum of mice injected with the DENV2 E mRNA-SM-102LNP complex ( Figure 3 C and Table 4).

[0303] Table 4. Evaluation of the maximum half inhibitory concentration (IC50) of DENV2 E mRNA-AS-CL09LNP complexes by PRNT assay 50 )

[0304] Based on the above results, it can be seen that intramuscular injection of mRNA LNP containing SM-02 or mRNA LNP containing AS-CL09 can induce the production of high amounts of neutralizing antibodies in animals.

[0305] In general, the AS-CL05-LNPs, AS-CL09-LNPs, AS-CL28-LNPs, and AS-CL35-LNPs of the present invention can deliver mRNA that encodes the production of SARS-CoV2 S protein or DENV2 E protein into cells or living bodies. In addition, in terms of the efficiency of delivering mRNA, the delivery effects of AS-CL05-LNPs, AS-CL09-LNPs, AS-CL28-LNPs, and AS-CL35-LNPs are similar to the LNP effects formed by other commercial lipids (e.g., MC3, SM-102, or ALC-0315). Therefore, the lipids of the present invention (e.g., AS-CL05, AS-CL09, AS-CL28, and AS-CL35) can be used to manufacture LNPs for delivering genes or proteins.

[0306] Although specific embodiments of the present invention are disclosed in the above implementation modes, they are not intended to limit the present invention. A person having ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on that defined by the accompanying patent application scope.

Claims

1. A lipid having a structure of formula (1), in, R 1 is an alkyl or cycloalkyl group, optionally with or without one or more hydroxyl groups, -CH 2 OH, or -NR 2 ; m and n are respectively integers between 0 and 12; R 2 and R 3 are hydrogen, alkene, R a 、-(C=O)OCH 2 R a , -O(C=O)R b or -(C=O)OR b ; R a Yes-CR'(COOR') 2 or -CR'(COOR")(COOR'"); and R, R b , R', R", and R'" are H or alkyl respectively.

2. The lipid of claim 1, wherein the lipid is selected from the group consisting of the following compounds: and 3. A lipid having a structure of formula (II): in, R 1 and R 3 are alkyl groups optionally with or without one or more hydroxyl substituents; R 2 is hydrogen or -O(C=O)R', wherein R' is alkyl; and m and n are integers between 1 and 10 respectively.

4. The lipid of claim 3, wherein the lipid is any of:

5. A lipid having a structure of formula (III): in, m and n are each an integer between 1 and 10; and R 1 , R 2 , R 3 and R 4 are alkyl groups respectively.

6. The lipid of claim 5, wherein the lipid is 7. A lipid nanoparticle comprising a hydrophilic inner core; and a lipid bilayer outer shell, which is formed by one or more lipids as described in any one of claims 1 to 6.

8. The lipid nanoparticle of claim 7, further comprising a therapeutic agent located in the hydrophilic inner core or on the lipid bilayer outer shell.

9. The lipid nanoparticle of claim 8, wherein the therapeutic agent is a nucleic acid molecule of a viral protein.

10. The lipid nanoparticle of claim 9, wherein the nucleic acid molecule is mRNA of the SARS-CoV-2 spike protein.

11. The lipid nanoparticle of claim 9, wherein the nucleic acid molecule is mRNA of a dengue virus membrane protein.

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