Lipids, nanoparticles containing the same, and uses thereof

Abstract

This specification discloses novel lipids, lipid nanoparticles, and their use for transporting to a subject, or for treating and / or preventing a disease in a subject, a therapeutic agent.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 350,215, filed on June 8, 2022, the entire content of which is incorporated herein by reference.

[0002] 1. Field of the Invention

[0003] The present disclosure generally relates to the field of drug delivery, and more particularly to nanoparticles formed by novel lipids for transporting therapeutic agents such as nucleic acids, and their use in the treatment and / or prevention of diseases.

Background Art

[0004] 2. Description of Related Art

[0005] Among the various reagents used to transfect cells with bioactive agents such as nucleic acids, reagents based on delivery via lipid nanoparticles (e.g., liposomes) are widely recognized as the most effective. This is mainly due to their efficiency and ease of use. Lipid nanoparticles are artificially prepared spherical vesicles consisting of a lipid bilayer. To deliver molecules to the site of action, the lipid bilayer can fuse with other bilayers such as the cell membrane to deliver the contents of the liposome into the cell.

[0006] Lipid nanoparticles are used in drug delivery due to their unique properties. Lipid nanoparticles enclose an aqueous region inside a hydrophobic membrane, and dissolved hydrophilic solutes cannot easily pass through the lipid. Hydrophobic chemicals can dissolve in the membrane, and in this way lipid nanoparticles can carry both hydrophobic and hydrophilic molecules. Lipid nanoparticles can be combined with bioactive agents such as drugs and nucleic acids to deliver these substances for use in the treatment and / or prevention of diseases.

[0007] Recently, lipid nanoparticles have been used in COVID-19 mRNA vaccines. mRNA vaccines have been proven to be effective in controlling COVID-19, and clinical trials are being conducted in many countries to evaluate mRNA vaccines against various other diseases. The advantages of mRNA vaccines are that they can induce a sufficient immune response to protect the host from infectious pathogens, and the vaccines can be manufactured rapidly, enabling targeting of variants of infectious pathogens. However, due to the low thermal stability of mRNA vaccines, there are significant limitations in their storage and distribution. For example, the COVID-19 mRNA vaccines manufactured by Moderna and Pfizer BioNTech can only be stored at -20 °C and -80 °C for 6 months, respectively. Furthermore, Moderna's mRNA COVID-19 vaccine is only stable at room temperature for 12 hours. Similarly, Pfizer BioNTech's mRNA COVID-19 vaccine is only stable at room temperature for 2 hours.

[0008] Therefore, in the related art, there is a need for novel lipid molecules for the manufacture of lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acids).

Summary of the Invention

Problems to be Solved by the Invention

[0009] The present disclosure provides novel cationic lipids for forming nanoparticles for non-viral delivery of nucleic acids, and their use in the treatment and / or prevention of diseases (e.g., infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)).

Means for Solving the Problems

[0010] A first aspect of the present disclosure relates to a lipid of formula (I). Here, JPEG2025523340000002.jpg2050R1 is one or more hydroxyls, -CH2OH, JPEG2025523340000003.jpg728, or an alkyl or cycloalkyl which may be substituted with -NR2 groups, m and n are independently integers from 0 to 12, R2 and R3 are independently H, alkenyl, R a , -(C=O)O(CH2)R a , -O(C=O)R b , or -(C=O)OR b and R a is -CR'(COOR'')2 or -CR'(COOR'')(COOR'''), R, R b , R', R'', and R''' are independently H or alkyl.

[0011] According to an embodiment of the present disclosure, the lipid of formula (I) may be any of the following. JPEG2025523340000004.jpg204138JPEG2025523340000005.jpg179138JPEG2025523340000006.jpg52138

[0012] A second aspect of the present disclosure relates to a lipid of formula (II). JPEG2025523340000007.jpg4065Here, R1 and R3 are independently alkyl which may be substituted with one or more hydroxyl groups, R2 is H or -O(C=O)R', where R' is alkyl, m and n are independently integers from 1 to 10.

[0013] According to an embodiment of the present disclosure, the lipid of formula (II) JPEG2025523340000008.jpg27124may be any of them.

[0014] A third aspect of the present disclosure relates to a lipid of formula (III). JPEG2025523340000009.jpg3880 Here, m and n are independently integers from 1 to 10, R1, R2, R3, and R4 are independently H or alkyl.

[0015] According to an embodiment of the present disclosure, the lipid of formula (III) has the following structure. JPEG2025523340000010.jpg54101

[0016] In a further aspect, there are provided lipid nanoparticles formed by one or more lipids of the present disclosure for delivering a target active ingredient (e.g., a nucleic acid or therapeutic agent of a target protein). The lipid nanoparticles include, in their structure, a hydrophilic core and an outer lipid bilayer shell formed by one or more of the lipids of formulas (I)-(III).

[0017] Furthermore, or optionally, the lipid nanoparticles of the present disclosure further include a therapeutic agent disposed within the hydrophilic core or the outer lipid bilayer shell of the nanoparticles. The therapeutic agent is present within the hydrophilic core in the case of hydrophilicity and within the lipid shell in the case of hydrophobicity. The therapeutic agent may be a nucleic acid of a target protein.

[0018] Examples of nucleic acids that can be encapsulated within the hydrophilic core of the lipid nanoparticles of the present invention 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), and combinations thereof. In some embodiments, the lipid nanoparticles of the present disclosure further include mRNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encapsulated within the hydrophilic core. In other embodiments, the lipid nanoparticles of the present disclosure further include mRNA of the envelope (E) protein of dengue virus encapsulated within the hydrophilic core.

[0019] Details of one or more embodiments of the present disclosure are set forth in the following accompanying description. Other features and advantages of the invention will become apparent from the detailed description and claims.

[0020] It should be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide further explanation of the invention as claimed.

Brief Description of the Drawings

[0021] This specification will be better understood by reading the following detailed description in light of the accompanying drawings.

[0022]

Figure 1

[0023]

Figure 2

[0024]

Figure 3

Modes for Carrying Out the Invention

[0025] The detailed description provided below in connection with the accompanying drawings is intended as a description of the present disclosure and is not intended to represent the only form in which the present disclosure may be constructed or used.

[0026] For the purposes of the present invention, lipid nanoparticles mean particles formed by a hydrophilic core covered with a lipid outer shell, are suitable for use in the treatment and / or prevention of diseases, and the active ingredient of interest (nucleic acid and / or therapeutic agent) is present within the hydrophilic core in the case of hydrophilicity or within the lipid shell in the case of hydrophobicity.

[0027] For convenience, the specific terms used in the specification, examples, and the appended claims are collected herein. Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the technical field to which this disclosure belongs.

[0028] Unless otherwise required by context, singular terms shall be taken to include the plural, and plural terms shall be taken to include the singular. Specifically, as used in this specification and the claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise. Also, as used in this specification and the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more.

[0029] The numerical ranges and parameters setting forth the broad scope of the present invention are approximations, and while the numerical values set forth in the specific examples are reported as precisely as possible, every numerical value inherently contains a certain error resulting from the standard deviation of the measurement in each testing. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean as considered by one of ordinary skill in the art. Unless otherwise indicated in the operating examples or explicitly stated, all numerical ranges, amounts, values, and percentages such as those for amounts of materials, periods of time, temperatures, operating conditions, ratios of amounts, etc. disclosed herein are to be understood as being modified by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and the appended claims are approximations that can vary as required. At the very least, each numerical parameter should be construed in light of the reported significant digits and by applying ordinary rounding techniques.

[0030] The atoms, moieties, or groups described herein may be unsubstituted or substituted, as permitted by their valencies, unless otherwise specified. The term "may be substituted" means either substituted or unsubstituted. Unless otherwise stated, the term "substituted", when used to describe a chemical structure or moiety, refers to a derivative of that structure or moiety in which one or more hydrogen atoms of that structure or moiety are replaced by one or more atoms or groups other than hydrogen, such as halo, hydroxyl, alkyl, aryl, amino, alkylamino, etc. The term "substituted" is intended to include substitution by all acceptable substituents of organic compounds, and any of the substituents described herein form stable compounds. The present invention contemplates any and all such combinations in order to obtain stable compounds.

[0031] The term "alkyl" means a straight-chain or branched hydrocarbon group 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 groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, 2-isopropyl-3-methylbutyl, pentyl, pentan-2-yl, hexyl, isohexyl, heptyl, heptan-2-yl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, and dodecyl. Unless otherwise specified, each example of alkyl may be substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"). In certain embodiments, the alkyl group is unsubstituted C 1-21 alkyl. In one preferred example, the alkyl group is octyl ("-C8H 17 "). In another preferred example, the alkyl group is dodecyl ("-C 10 H 21 "). In other embodiments, the alkyl group is substituted C 1-12 alkyl. In one preferred example, the alkyl group is ethyl substituted with one hydroxy group ("-C2H5OH"). In another preferred example, the alkyl group is propyl substituted with two hydroxy groups. In a further example, the alkyl is propyl substituted with an amino group ("-C3H7NH2"). In a more preferred example, the alkyl group is propyl substituted with dimethylamine ("-C3H6N(CH3)2).

[0032] "Cycloalkyl" refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms ("C 3-10 cycloalkyl") and 0 heteroatoms in a non-aromatic ring system. In certain embodiments, the cycloalkyl group is monocyclic ("monocyclic carbocyclic") or includes a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic alkyl"). In some embodiments, cycloalkyl is a monocyclic saturated carbocyclic group having 3 to 10 ring carbon atoms ("C 3-10is a "cycloalkyl"). In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms ("C 3-8 cycloalkyl"). In some embodiments, the cycloalkyl group has 3 to 6 ring carbon atoms ("C 3-6 cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 6 ring carbon atoms ("C 5-6 cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 10 ring carbon atoms ("C 5-10 cycloalkyl"). C 5-6 Examples of cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C6). C 3-6 Examples of cycloalkyl groups include the aforementioned C 5-6 cycloalkyl group, as well as cyclopropyl (C3) and cyclobutyl (C4). C 3-8 Examples of cycloalkyl groups include the aforementioned C 3-6 cycloalkyl group, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each example of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, the cycloalkyl group is unsubstituted C 3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C 3-10 cycloalkyl. The carbocyclic ring may be partially unsaturated.

[0033] The term "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 20 carbon atoms and one or more carbon-carbon double bonds and no triple bonds ("C 2-20 alkenyl"). In some embodiments, the alkenyl group has 18 carbon atoms ("C 18"(alkenyl") and having one double bond (i.e., oleic acid). In some embodiments, the alkenyl group has 18 carbon atoms and two double bonds (i.e., linoleic acid). Unless otherwise specified, each example of an alkenyl group may be independently substituted, i.e., unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents ("substituted alkenyl"). In certain embodiments, the alkenyl group is unsubstituted C 2-20 alkenyl. In certain embodiments, the alkenyl group is substituted C 2-20 alkenyl.

[0034] It is also understood that compounds having the same molecular formula but different in the nature or the bonding order or arrangement of atoms in space are called "isomers". Isomers that differ in the arrangement of atoms in space are called "stereoisomers". Stereoisomers that are not mirror images of each other are called "diastereomers", and stereoisomers that are mirror images that cannot be superimposed on each other are called "enantiomers". When a compound has an asymmetric center, for example, when bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their asymmetric centers, described by the Cahn and Prelog R- and S-ordering rules, or by the way the molecule rotates plane-polarized light, represented as dextrorotatory or levorotatory ((+) or (-) isomers, respectively). A chiral compound can exist as either individual enantiomers or a mixture thereof. A mixture containing equal amounts of enantiomers is called a "racemic mixture".

[0035] Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, this specification, including definitions, will control.

[0036] 1. Compounds

[0037] The compounds described herein can have the structure of formula (I) below. JPEG2025523340000011.jpg2050

[0038] In formula (I), R1 is alkyl or cycloalkyl which may be substituted with one or more hydroxyls, -CH2OH, JPEG2025523340000012.jpg720, or an -NR2 group. In some embodiments, R1 is ethyl substituted with one hydroxyl group. In other embodiments, R1 is ethyl substituted with two hydroxyl groups. In further embodiments, R1 is ethyl substituted with a dimethylamino group (i.e., -N(CH3)2). In some embodiments, R1 is propyl substituted with one hydroxy group. In further embodiments, R1 is propyl substituted with two hydroxy groups. In some embodiments, R1 is hexyl substituted with one hydroxyl group. In other embodiments, R1 is cyclohexyl substituted with one hydroxyl group. In further embodiments, R1 is cyclohexyl substituted with -CH2OH. In even further embodiments, R1 is JPEG2025523340000013.jpg729 substituted ethyl.

[0039] Furthermore, or alternatively, m and n are independently integers from 0 to 12, and R2 and R3 are independently H, alkenyl, R a 、-(C=O)O(CH2)R a 、-O(C=O)R b 、or -(C=O)OR b wherein R a is -CR'(COOR'')2 or -CR'(COOR'')(COOR'''), and R, R b 、R', R'', and R''' are independently H or alkyl. In some embodiments, m is 5, n is 7, and R2 and R3 are independently -(C=O)O(CH2)R a wherein R a is -CR'(COOR'')2, R' is methyl, and R'' is -C8H 17It is so. In other embodiments, m and n are independently 7, and R2 is -O(C=O)R b and R3 is -(C=O)O(CH2)R a where R a is -CR'(COOR'')2, R' is methyl, and R b is -CH(C8H 17 )2. In a further embodiment, m is 10, n is 6, R2 is H, and R3 is R a (-CR'(COOR')2), R' is methyl, and R'' is -C8H 17 . In yet a further embodiment, m is 10, n is 6, R2 is H, and R3 is R a (-CR'(COOR')2), R' is methyl, and R'' is -C8H 17 . In some embodiments, m is 10, n is 6, R2 is H, and R3 is R a (-CR'(COOR'')(COOR''')), R' is -CH3, R'' is -C8H 17 , and R''' is -C 10 H 21 . In other embodiments, m is 0, n is 6, R2 is -CH=CHCH2CH=CH(CH2)4CH3, and R3 is R a (-CR'(COOR'')2), R' is methyl, and R'' is -C8H 17 .

[0040] According to an embodiment of the present disclosure, the lipid of formula (I) may be any of the following. JPEG2025523340000014.jpg181139JPEG2025523340000015.jpg201139JPEG2025523340000016.jpg52138

[0041] According to a preferred embodiment of the present disclosure, the lipid of formula (I) has the following structure. JPEG2025523340000017.jpg2566

[0042] According to another preferred embodiment of the present disclosure, the lipid of formula (I) has the following structure. JPEG2025523340000018.jpg2476

[0043] The compounds described herein can have the structure of formula (II) below. JPEG2025523340000019.jpg4065

[0044] In formula (II), m and n are independently integers from 1 to 10, and R1 and R3 are independently alkyl optionally substituted with one or more hydroxyl groups. In some embodiments, m and n are independently 6, R1 is propyl substituted with one hydroxy group, and R3 is -C6H 13 is. In other embodiments, m is 10, n is 6, R1 is propyl substituted with one hydroxy group, and R3 is -C6H 13 is.

[0045] Alternatively, or additionally, R2 is H or -O(C=O)R', where R' is alkyl. In some embodiments, R2 is H. In other embodiments, R2 is -O(C=O)R', where R' is -C6H 13 is.

[0046] According to an embodiment of the present disclosure, the lipid of formula (II) may be any of the following. JPEG2025523340000020.jpg27126

[0047] The compounds described herein can have the structure of formula (III) below. JPEG2025523340000021.jpg3880

[0048] In formula (III), m is an integer from 1 to 10. Alternatively, or additionally, X is -(C=O)O-, and R1, R2, R3 and R4 are independently H or alkyl.

[0049] According to an embodiment of the present disclosure, the lipid of formula (III) has the following structure. JPEG2025523340000022.jpg3466

[0050] 2. Lipid nanoparticles

[0051] In a further aspect, lipid nanoparticles formed by one or more of the lipids of the present disclosure for delivering a target active ingredient (e.g., nucleic acid or therapeutic agent of a target protein) are provided. The lipid nanoparticles include, in their structure, a hydrophilic core and an outer lipid bilayer shell formed by one or more of the lipids of formulas (I)-(III).

[0052] Furthermore, or optionally, the lipid nanoparticles can further include a therapeutic agent disposed within the outer lipid bilayer shell or the hydrophilic core. The therapeutic agent is present in the hydrophilic core when hydrophilic and in the lipid shell when hydrophobic. The therapeutic agent may be a nucleic acid of a target protein.

[0053] Examples of nucleic acids that can be encapsulated within the hydrophilic core of the lipid nanoparticles of the present invention 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), and combinations thereof. In some embodiments, the lipid nanoparticles of the present disclosure further include mRNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encapsulated within the hydrophilic core. In further embodiments, the lipid nanoparticles of the present disclosure further include mRNA of the envelope (E) protein of dengue virus encapsulated within the hydrophilic core.

[0054] 3. Use of lipid nanoparticles

[0055] 3.1 Intracellular delivery of nucleic acids

[0056] The present disclosure also provides a method for intracellular delivery of a drug of interest (e.g., a therapeutic agent) to a cell. According to some embodiments of the present disclosure, the lipid nanoparticles of the present invention are pre-loaded with a nucleic acid of interest (e.g., the mRNA of the spike protein of SARS-CoV-2) intended to be delivered to a target cell and one or more agents to facilitate contact with and subsequent transfection of the target cell. Preferably, the lipid nanoparticles of the present invention enable the encapsulated nucleic acid to reach the target cell and then transfect the target cell. Thus, after delivery, the nucleic acid encodes one or more target proteins within the target cell. The lipid nanoparticles and methods of the present invention can be used to target a number of cell types, including but not limited to hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal stem cells, nerve cells, heart cells, adipocytes, vascular smooth muscle cells, skeletal muscle cells, B cells, T cells, white blood cells, granulocytes, fibroblasts, reticulocytes, etc. According to an embodiment of the present disclosure, lipid nanoparticles pre-loaded with a nucleic acid of interest are normally taken up by T cells and then transfected with the T cells to express a protein of interest (e.g., the spike protein of SARS-CoV-2) encoded by the nucleic acid of interest (e.g., the mRNA of the spike protein of SARS-CoV-2) delivered thereto. In certain embodiments, the protein of interest is produced at a higher level than in a control group (i.e., the baseline level of cells not treated with the lipid nanoparticles of the present invention).According to embodiments of the present disclosure, the protein of interest is at least 1 to 100,000 times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 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, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000 times that of the control group, preferably at least 5 to 50,000 times, for example, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 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, 50,000 times that of the control group, more preferably at least 10 to 10,000 times, for example, 10, 20, 30, 40, 50, 55, 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 times that of the control group, and is expressed by target cells (e.g., T cells). In some embodiments, the expression level of the protein of interest remains detectable over a sustained period, such as 1 day, 2 days, 3 days, 4 days, 5 days, 1 week or more.

[0057] Examples of nucleic acids that can be encapsulated within the hydrophilic core of the lipid nanoparticles of the present invention 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), and combinations thereof. In some embodiments, the lipid nanoparticles of the present disclosure further comprise mRNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encapsulated within the hydrophilic core. In further embodiments, the lipid nanoparticles of the present disclosure further comprise mRNA of the envelope (E) protein of dengue virus encapsulated within the hydrophilic core.

[0058] 3.2 Treatment of Diseases

[0059] The present disclosure also provides methods for the treatment and / or prevention of diseases in a subject. The method includes administering to the target tissue of the subject an effective amount of the lipid nanoparticles of the present invention pre-filled with a therapeutic agent to treat and / or prevent the disease.

[0060] As used herein, the term "subject" refers to any animal, including but not limited to, humans, non-human primates, rodents, etc., to which the lipid nanoparticles of the present disclosure pre-filled with a therapeutic agent are administered. Usually, the term "subject" as used herein refers to a human subject. The therapeutic agent may be, for example, a nucleic acid of a target protein.

[0061] In some embodiments, the method includes administering to the subject the lipid nanoparticles of the present disclosure pre-filled with viral nucleic acid, and thus, the encapsulated viral nucleic acid of interest is delivered to the target tissue (e.g., lung, liver, etc.) of the subject, expressed in the target tissue, acts as an antigen, induces a controlled level of immune response in the subject, immunizes the subject, thereby preventing the subject from subsequently being infected with the virus, and / or developing a disease (e.g., severe acute respiratory syndrome, SARS) caused by the viral infection.

[0062] Furthermore, or optionally, the lipid nanoparticles of the present disclosure are formulated in combination with one or more additional carriers, excipients, or stabilizers. The lipid nanoparticles of the present disclosure can be administered according to current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the dosing schedule, the age, sex, weight of the subject, and other factors related to the clinical condition. The effective amount for the purposes of the present disclosure can be determined by relevant factors known to those skilled in the fields of clinical research, pharmacology, clinical, and medical sciences. In some embodiments, the dosage is effective to at least somewhat stabilize, improve, or eliminate the symptoms of the disease or is effective to prevent the progression of the disease. For example, an appropriate amount and dosing regimen is one that causes at least transient protein production.

[0063] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary (e.g., intratracheal, inhalation, etc.), or enteral administration, intramuscular, subcutaneous, intramedullary injection, and parenteral delivery including intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0064] Alternatively, the lipid nanoparticles of the present disclosure can be administered locally rather than systemically, for example, by directly injecting the lipid nanoparticles into the target tissue. Local delivery can be carried out in various ways depending on the target tissue. For example, an aerosol containing the lipid nanoparticles of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery), and the lipid nanoparticles of the present disclosure can be injected into the site where the disease manifests or where pain occurs. Also, the lipid nanoparticles can be provided in lozenges for oral, tracheal, or esophageal administration, or can be provided in the form of a liquid, tablet, or capsule for gastric or intestinal administration, or can also be delivered to the eye by cream, eye drops, or injection.

[0065] In some embodiments, the lipid nanoparticles of the present disclosure are formulated to be suitable for the sustained release of the nucleic acid contained therein. Such sustained-release lipid nanoparticles can be administered to a subject at long dosing intervals. For example, the lipid nanoparticles of the present disclosure can be administered to a subject daily, or twice a day, or every other day. In some preferred embodiments, the lipid nanoparticles are administered to the subject once a week, twice a week, every 10 days, every two weeks, every three weeks, or every four weeks, once a month, every six weeks, every eight weeks, every two months, every three months, every four months, every six months, every eight months, every nine months, or annually. Also contemplated herein are compositions formulated for depot administration (e.g., by intramuscular, subcutaneous, etc.) to deliver or release nucleic acid over a long period of time.

[0066] Also contemplated herein are lyophilized compositions comprising one or more of the lipid nanoparticles disclosed herein. The lyophilized compositions of the present disclosure may be reconstituted prior to administration or may be reconstituted in vivo. For example, the lyophilized composition can be formulated into a suitable dosage form (e.g., an intradermal dosage form such as a disk, rod, or membrane) and administered such that the dosage form is rehydrated in vivo over time by the body fluid of the individual.

[0067] The present invention will be described more specifically with reference to the following examples provided for purposes of illustration and not limitation. These are those that may be commonly used, but other procedures, methodologies, or techniques known to those skilled in the art may be used instead.

[0068] Examples

[0069] Materials and Methods

[0070] Evaluation of in vitro SARS-CoV-2 S protein expression by cell ELISA

[0071] The SARS-CoV-2 S protein mRNA-LNP was individually transfected into 293T cells and cultured in DMEM medium containing 10% FBS at 37 °C for 24 hours. After transfection, the cells were fixed with 4% paraformaldehyde / PBS for 15 minutes and then permeabilized with 0.1% Triton X-100 for 10 minutes. After washing, 100 ng / ml of anti-RBD chimeric antibody was added to the wells at room temperature for 1 hour. Next, horseradish peroxidase-conjugated anti-human antibody (1:8000) was added at room temperature for 1 hour if necessary. The plates were washed three times with PBS containing 0.1% Tween-20 (PBST0.1), and then incubated with peroxidase- AffiniPure goat anti-mouse IgG (H+L) (Jackson ImmunoResearch) (1:5000 dilution) for 1 hour. After washing three times with PBST0.1, a signal was generated using 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution (TMBW-1000-01, SURMODICS). Finally, the reaction was stopped with 3N HCl, and the absorbance at 450 nm was measured with an ELISA reader (Versa Max Tunable Microplate Reader, Molecular Devices).

[0072] Immunization of mice

[0073] Groups of 6- to 8-week-old BALB / c mice were immunized by intramuscular (i.m.) injection of the designated mRNP-LNP (i.e., MC3-LNP, SM102-LNP, AS-CL05-LNP, AS-CL09-LNP, AS-CL28-LNP, or AS-CL35-LNP, each dose containing 10 μg of mRNA) at the start of the experiment (day 0), followed by additional boost injections at 2 and 4 weeks. Serum samples were collected 4, 6, and 8 weeks after the first immunization (day 0) and tested for binding activity to the FLS-WT or FLS-BA.5 protein and neutralization activity by pseudovirus assay.

[0074] Pseudovirus neutralization assay

[0075] Six weeks after the initial boost, blood samples were collected from the mice and sera were used to measure the neutralizing activity against BA.5 SARS-CoV-2 pseudovirus. The pseudovirus neutralization assay was performed using a SARS-CoV-2 pseudotyped lentivirus expressing the full-length S protein and firefly luciferase in HEK293T cells overexpressing human ACE2 (HEK293T / hACE2, purchased from the National RNAi Core Facility, Academia Sinica, Taiwan). The half-maximal inhibitory concentration (IC 50 ) was calculated by non-linear regression using Prism software version 8.1.0 (GraphPad Software Inc.). The mean IC 50 values for each experimental group were determined from three independent experiments.

[0076] Plaque reduction neutralization titer (PRNT) assay

[0077] Serum from animals injected with DENV2 E mRNA-LNP was serially diluted with PBS and pre-incubated with 100 plaque-forming units (PFU) of DENV2 at 37 °C for 1 hour. Next, the mixture was added to pre-seeded BHK-21 cells at 37 °C for 1 hour. The virus-containing medium was removed and replaced with DMEM containing 2% FBS and 1% methylcellulose, and incubated for an additional 4 days. Cells were fixed overnight with 10% formaldehyde and stained with 0.5% crystal violet for 20 minutes. The plates were then washed with tap water and the number of plaques formed at each dilution was counted. Each experiment was repeated three times. Plaque reduction was calculated as follows. Inhibition rate = 100 × [1 - (number of plaques with immune serum / number of plaques without immune serum)]. The 50% plaque reduction (PRNT 50 ) value was calculated using Prism software. DENV2 strain 16881 was used in this study.

[0078] Example 1 Synthesis of the compounds of the present invention

[0079] Generally, the compounds of the present disclosure were synthesized according to the procedures outlined in Schemes 1-5, and the amines required for Step 7 of Scheme 1 are listed in Table 1.

[0080] Scheme 1. General Procedure for Disubstituted Malonic Ester Compounds JPEG2025523340000023.jpg72145

[0081] Table 1. List of Amines JPEG2025523340000024.jpg22155

[0082] Scheme 2. General Alkylation of Amino Malonic Ester Compounds JPEG2025523340000025.jpg25128

[0083] Scheme 3. Synthesis of AS-CL-19 JPEG2025523340000026.jpg39151

[0084] Scheme 4. Asymmetric Synthesis of Disubstituted Malonic Ester Compounds JPEG2025523340000027.jpg23154

[0085] Scheme 5. Synthesis of AS-CL-30 and AS-CL-31 JPEG2025523340000028.jpg64149

[0086] Compound 1a: Dioctyl Malonate (1a) (Step 1) JPEG2025523340000029.jpg1468

[0087] A mixed solution of malonic acid (2.61 g, 25.08 mmol) and octanol (7.18 g, 55.13 mmol) in dichloromethane (DCM) was 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. After stirring overnight at room temperature, the reaction solution was washed with 2N HCl (aqueous solution) and brine, and dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using ethyl acetate / hexane (EA / Hea) (1 / 20) to obtain compound 1a (7.66 g, 23.32 mmol) as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.12 (t, J = 6.6 Hz, 4H), 3.35 (s, 2H), 1.63 - 1.60 (m, 4H), 1.32 - 1.26 (m, 20H), 0.86 (t, J = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 166.7, 65.6, 41.7, 31.7, 29.1 (x2), 28.4, 25.8, 22.6, 14.0.

[0088] Compound 1b: Didecyl malonate JPEG2025523340000030.jpg15128

[0089] Compound 1b was synthesized according to the procedure of general step 1 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.12 (t, J = 6.6 Hz, 4H), 3.35 (s, 2H), 1.65 - 1.60 (m, 4H), 1.33 - 1.25 (m, 28H), 0.87 (t, J = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 166.6, 65.6, 41.6, 31.8, 29.4 (x2), 29.2, 29.1, 28.4, 25.7, 22.6, 14.0.

[0090] Compound 1c: Diundecyl malonate JPEG2025523340000031.jpg14128

[0091] Compound 1c was synthesized according to the procedure of General Step 1 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.12 (t, J = 6.6 Hz, 4H), 3.35 (s, 2H), 1.65 - 1.59 (m, 4H), 1.32 - 1.25 (m, 32H), 0.87 (t, J = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 170.3, 65.6, 46.3, 32.0, 29.7, 29.6, 29.4, 29.3, 28.6, 25.9, 22.8, 14.2, 13.7.

[0092] Compound 2a: Dioctyl 2 - methylmalonate (Step 2) JPEG2025523340000032.jpg1664

[0093] NaH (430 mg, 10.8 mmol) was added to a solution of dioctyl malonate (4.43 g, 13.5 mmol) in THF at 0 °C, and then MeI (0.66 mL, 10.8 mmol) was added. After stirring overnight at room temperature, the reaction solution was washed with saturated NH4Cl (aqueous solution) and brine, and dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using EA / Hea (1 / 40) to obtain compound 2a (2.45 g, 7.15 mmol) as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.15 - 4.08 (m, 4H), 3.42 (q, J = 7.2 Hz, 1H), 1.65 - 1.57 (m, 4H), 1.41 (d, J = 7.2 Hz, 3H), 1.33 - 1.28 (m, 20H), 0.88 (t, J = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 170.2, 65.5, 46.2, 31.8, 29.2 (x2), 28.5, 25.8, 22.6, 14.1, 13.6.

[0094] Compound 2b: Didodecyl 2 - methylmalonate JPEG2025523340000033.jpg1676

[0095] Compound 2b was synthesized according to the procedure of General Step 2 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.14 - 4.09 (m, 4H), 3.42 (q, J = 7.2 Hz, 1H), 1.64 - 1.60 (m, 4H), 1.41 (d, J = 7.2 Hz, 3H), 1.32 - 1.25 (m, 28H), 0.87 (t, J = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 170.2, 65.5, 46.2, 31.8, 29.5 (x2), 29.3, 29.2, 28.5, 25.8, 22.6, 14.1.

[0096] Compound 2c: Diundecyl 2 - methylmalonate JPEG2025523340000034.jpg1785

[0097] Compound 2c was synthesized according to the procedure of General Step 2 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.12 - 4.07 (m, 4H), 3.40 (q, J = 7.2 Hz, 1H), 1.61 - 1.59 (m, 6H), 1.39 (d, J = 7.2 Hz, 3H), 1.35 - 1.25 (m, 30H), 0.85 (t, J = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 170.3, 65.6, 46.3, 32.0, 29.7, 29.6, 29.4, 29.3, 28.6, 25.9, 22.8, 14.2, 13.7.

[0098] Compound 2d: Dioctyl 2 - ethylmalonate JPEG2025523340000035.jpg1765

[0099] Compound 2d was synthesized according to the procedure of General Step 2 in Scheme 1. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.14 - 4.08 (m, 4H), 3.24 (t, J = 7.2 Hz, 1H), 1.94 - 1.89 (m, 2H), 1.64 - 1.58 (m, 4H), 1.35 - 1.26 (m, 20H), 0.95 (t, J = 7.2 Hz, 3H), 0.87 (t, J = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 169.5, 65.4, 53.6, 31.7, 29.1, 28.5, 25.8, 22.6, 22.2, 14.0, 11.8.

[0100] Compound 2e: Dioctyl 2 - propylmalonate JPEG2025523340000036.jpg1972

[0101] Compound 2e was synthesized according to the procedure of General Step 2 in Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.15 - 4.08 (m, 4H), 3.33 (t, J = 7.8 Hz, 1H), 1.89 - 1.85 (m, 2H), 1.64 - 1.60 (m, 4H), 1.37 - 1.27 (m, 22H), 0.93 (t, J = 7.2 Hz, 6H), 0.88 (t, J = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 169.7, 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] + 393.2973 (C 22 H 42 O4Na).

[0102] Compound 2f: Dioctyl 2 - (6 - (benzyloxy)hexyl)malonate JPEG2025523340000037.jpg1869

[0103] Compound 2f was synthesized according to the procedure of General Step 2 in Scheme 1. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 7.35 - 7.26 (m, 5H), 4.49 (s, 2H), 4.15 - 4.08 (m, 4H), 3.45 (t, J = 6.6 Hz, 2H), 3.31 (t, J = 6.6 Hz, 1H), 1.90 - 1.86 (m, 2H), 1.64 - 1.59 (m, 6H), 1.33 - 1.29 (m, 26H), 0.86 (t, J = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 169.6, 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.

[0104] Compound 2g: 1 - Decyl 3 - octyl 2 - methylmalonate JPEG2025523340000038.jpg1947

[0105] Compound 2g was synthesized according to the procedure of General Step 2 in Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.15 - 4.08 (m, 4H), 3.42 (q, J = 7.2 Hz, 1H), 1.65 - 1.60 (m, 4H), 1.41 (d, J = 7.2 Hz, 3H), 1.33 - 1.26 (m, 24H), 0.89 - 0.87 (m, 6H). 13 13C NMR (150 MHz, CDCl3) δ 170.2, 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.

[0106] Compound 2H: 1 - Hexyl 3 - octyl 2 - methylmalonate JPEG2025523340000039.jpg2042

[0107] Compound 2H was synthesized according to the procedure of General Step 2 in Scheme 1. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.15 - 4.08 (m, 4H), 3.42 (q, J = 7.2 Hz, 1H), 1.65 - 1.60 (m, 4H), 1.41 (d, J = 7.2 Hz, 3H), 1.30 - 1.27 (m, 16H), 0.89 - 0.87 (m, 6H). 13 13C NMR (150 MHz, CDCl3) δ 170.3, 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.

[0108] Compound 2i: Didecyl 2 - ethylmalonate JPEG2025523340000040.jpg1878

[0109] Compound 2i was synthesized according to the procedure of General Step 2 of Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.14 - 4.09 (m, 4H), 3.25 (t, J = 7.2 Hz, 1H), 1.95 - 1.90 (m, 2H), 1.64 - 1.60 (m, 4H), 1.32 - 1.25 (m, 26H), 0.96 (t, J = 7.2 Hz, 3H), 0.87 (t, J = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 169.7, 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.

[0110] Compound 3a: Dioctyl 2 - ((benzyloxy)methyl) - 2 - methylmalonate (Step 3) JPEG2025523340000041.jpg2251

[0111] At 0 °C, NaH (165 mg, 4.13 mmol) was added to a solution of dioctyl 2-methylmalonate (1.4 g, 4.08 mmol) in THF, and then benzyl chloromethyl ether (0.56 mL, 4.08 mmol) was added. After stirring overnight under reflux, the reaction solution was washed with saturated NH4Cl (aqueous solution) and brine, and dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using EA / Hex (1 / 25) to obtain compound 3a (1.4 g, 3.02 mmol, 74%) as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 7.36 - 7.27 (m, 5H), 4.53 (s, 2H), 4.10 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 170.7, 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.

[0112] Compound 3b: Dioctyl 2-(4-(benzyloxy)butyl)-2-methylmalonate JPEG2025523340000042.jpg2363

[0113] Compound 3b was synthesized according to the procedure of General Step 3 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 7.35 - 7.27 (m, 5H), 4.48 (s, 2H), 4.09 (t, J = 6.6 Hz, 4H), 3.46 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H); 1313C NMR (150 MHz, CDCl3) δ 172.4, 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] + 527.3703 (C 31 H 51 O5Na).

[0114] Compound 3c: Dioctyl 2-(6-(benzyloxy)hexyl)-2-methylmalonate JPEG2025523340000043.jpg1959

[0115] Compound 3c was synthesized according to the procedure of General Step 3 of Scheme 1. The title compound was obtained as a pale yellow oil. 1 1H NMR (600 MHz, CDCl3) δ 7.35 - 7.27 (m, 5H), 4.49 (s, 2H), 4.09 (t, J = 6.6 Hz, 4H), 3.45 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.6, 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.

[0116] Compound 3d: Dioctyl 2-(8-(benzyloxy)octyl)-2-methylmalonate JPEG2025523340000044.jpg2172

[0117] Compound 3d was synthesized according to the procedure of General Step 3 of Scheme 1. The title compound was obtained as a pale yellow oil. 11H NMR (600 MHz, CDCl3) δ 7.35 - 7.26 (m, 5H), 4.50 (s, 2H), 4.13 - 4.06 (m, 4H), 3.45 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.6, 138.7, 128.3, 127.6, 127.4, 72.8, 70.5, 65.2, 53.8, 35.6, 31.8, 29.9, 29.7 (x2), 29.4, 29.3, 29.2, 28.5, 26.2, 25.9, 24.3, 22.6, 19.9, 14.1.

[0118] Compound 3e: Dioctyl 2-(6-(benzyloxy)hexyl)-2-ethylmalonate JPEG2025523340000045.jpg2163

[0119] Compound 3e was synthesized according to the procedure of General Step 3 in Scheme 1. The title compound was obtained as a pale yellow oil. 1 1H NMR (600 MHz, CDCl3) δ 7.35 - 7.26 (m, 5H), 4.49 (s, 2H), 4.09 (t, J = 6.6 Hz, 4H), 3.45 (t, J = 6.6 Hz, 2H), 1.92 (q, J = 7.2 Hz, 2H), 1.87 - 1.84 (m, 2H), 1.61 - 1.58 (m, 6H), 1.38 - 1.26 (m, 24H), 1.16 - 1.12 (m, 2H), 0.88 (t, J = 6.6 Hz, 6H), 0.80 (t, J = 6.6 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 171.9, 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.

[0120] Compound 3f: Dioctyl 2-(6-(benzyloxy)hexyl)-2-propylmalonate JPEG2025523340000046.jpg2461

[0121] Compound 3f was synthesized according to the procedure of General Step 3 in Scheme 1. The title compound was obtained as a pale yellow oil. 1 H NMR (600 MHz, CDCl3) δ 7.35 - 7.27 (m, 5H), 4.50 (s, 2H), 4.08 (t, J = 6.6 Hz, 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 = 7.2 Hz, 3H), 0.88 (t, J = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 172.0, 138.6, 128.3, 127.5, 127.4, 72.8, 70.3, 65.1, 57,6, 35.5, 32.2, 31.8, 29.7, 29.6, 29.2, 29.1, 28.5, 25.9, 25.8, 24.0, 22.6, 17.4, 14.4, 14.0.

[0122] Compound 3g: 1 - Decyl 3 - octyl 2 - (6 - (benzyloxy)hexyl) - 2 - methylmalonate JPEG2025523340000047.jpg2373

[0123] Compound 3g was synthesized according to the procedure of General Step 3 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 7.34 - 7.25 (m, 5H), 4.49 (s, 2H), 4.10 - 4.08 (m, 4H), 3.45 (t, J = 6.6 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 172.6, 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] + 583.4333 (C 35 H 60 O5Na).

[0124] Compound 3h: 1-Hexyl 3-octyl 2-(6-(benzyloxy)hexyl)-2-methylmalonate JPEG2025523340000048.jpg2062

[0125] Compound 3h was synthesized according to the procedure of General Step 3 in Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 7.35 - 7.27 (m, 5H), 4.49 (s, 2H), 4.09 (t, J = 6.6 Hz, 4H), 3.45 (t, J = 6.6 Hz, 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 13C NMR (150 MHz, CDCl3) δ 172.5, 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] + 527.3712 (C 31 H 52 O5Na).

[0126] Compound 3i: Didodecyl 2-(4-(benzyloxy)butyl)-2-ethylmalonate JPEG2025523340000049.jpg1962

[0127] Compound 3i was synthesized according to the procedure of General Step 3 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 7.35 - 7.27 (m, 5H), 4.48 (s, 2H), 4.09 (t, J = 6.6 Hz, 4H), 3.46 (t, J = 6.6 Hz, 2H), 1.95 - 1.87 (m, 4H), 1.65 - 1.57 (m, 6H), 1.38 - 1.26 (m, 30H), 0.88 (t, J = 6.6 Hz, 6H), 0.80 (t, J = 6.6 Hz, 3H). 13 C NMR (150 MHz, CDCl3) δ 172.0, 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] + 575.4675 (C 36 H 63 O5).

[0128] Compound 4a: Dioctyl 2-(hydroxymethyl)-2-methylmalonate (Step 4) JPEG2025523340000050.jpg2144

[0129] A mixed solution of dioctyl 2-((benzyloxy)methyl)-2-methylmalonate (1.4 g, 3.02 mmol) and Pd / C (catalytic amount) in AcOH / MeOH (4 / 1) was stirred overnight at room temperature under a H2 atmosphere. After filtration and evaporation, the residue was dissolved in DCM, washed with saturated NaHCO3 (aqueous solution), and dried over MgSO4. The desired product 4a (1.07 g, 2.87 mmol, 95%) was obtained as a colorless oil without further purification. 11H NMR (600 MHz, CDCl3) δ 4.13 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 171.7, 66.9, 65.7, 55.9, 31.8, 29.1 (x2), 28.4, 25.8, 22.6, 17.6, 14.1.

[0130] Compound 4b: Dioctyl 2-(4-hydroxybutyl)-2-methylmalonate JPEG2025523340000051.jpg2157

[0131] Compound 4b was synthesized according to the procedure of General Step 4 of Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.09 (t, J = 6.6 Hz, 4H), 3.64 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.5, 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.

[0132] Compound 4c: Dioctyl 2-(6-hydroxyhexyl)-2-methylmalonate JPEG2025523340000052.jpg2268

[0133] Compound 4c was synthesized according to the procedure of General Step 4 of Scheme 1. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.09 (t, J = 6.6 Hz, 4H), 3.63 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 65.3, 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.

[0134] Compound 4d: Dioctyl 2-(8-hydroxyoctyl)-2-methylmalonate JPEG2025523340000053.jpg2379

[0135] Compound 4d was synthesized according to the procedure of General Step 4 of Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.11 - 4.07 (m, 4H), 3.63 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 65.3, 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.

[0136] Compound 4e: Dioctyl 2-ethyl-2-(6-hydroxyhexyl)malonate JPEG2025523340000054.jpg2267

[0137] Compound 4e was synthesized according to the procedure of General Step 4 of Scheme 1. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.08 - 4.06 (m, 4H), 3.61 - 3.59 (m, 2H), 1.89 (q, J = 7.2 Hz, 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 = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 172.1, 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.

[0138] Compound 4f: Dioctyl 2-(6-hydroxyhexyl)-2-propylmalonate JPEG2025523340000055.jpg2463

[0139] Compound 4f was synthesized according to the procedure of General Step 4 of Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.62 (t, J = 6.6 Hz, 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 = 7.2 Hz, 3H), 0.88 (t, J = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.0, 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.

[0140] Compound 4g: Dioctyl 2-(6-hydroxyhexyl)malonate JPEG2025523340000056.jpg2264

[0141] Compound 4g was synthesized according to the procedure of General Step 4 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.15 - 4.08 (m, 4H), 3.63 (t, J = 6.6 Hz, 2H), 3.31 (t, J = 7.8 Hz, 1H), 1.91 - 1.87 (m, 2H), 1.64 - 1.53 (m, 8H), 1.35 - 1.33 (m, 24H), 0.87 (t, J = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 169.6, 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.

[0142] Compound 4h: 1 - Decyl 3 - octyl 2 - (6 - hydroxyhexyl) - 2 - methylmalonate JPEG2025523340000057.jpg2271

[0143] Compound 4h was synthesized according to the procedure of General Step 4 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.09 - 4.07 (m, 4H), 3.61 (t, J = 6.6 Hz, 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, CDCl3) δ 172.6, 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.

[0144] Compound 4i: 1 - Hexyl 3 - octyl 2 - (6 - hydroxyhexyl) - 2 - methylmalonate JPEG2025523340000058.jpg2367

[0145] Compound 4i was synthesized according to the procedure of General Step 4 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.09 (t, J = 6.6 Hz, 4H), 3.62 (t, J = 6.6 Hz, 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, CDCl3) δ 172.6, 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.

[0146] Compound 4j: Didecyl 2 - ethyl - 2 - (4 - hydroxybutyl) malonate JPEG2025523340000059.jpg2162

[0147] Compound 4j was synthesized according to the procedure of General Step 4 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.09 (t, J = 6.6 Hz, 4H), 3.63 (t, J = 6.6 Hz, 2H), 1.95 - 1.87 (m, 4H), 1.61 - 1.55 (m, 6H), 1.34 - 1.24 (m, 30H), 0.87 (t, J = 6.6 Hz, 6H), 0.81 (t, J = 6.6 Hz, 3H). 13 C NMR (150 MHz, CDCl3) δ 171.9, 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.

[0148] Compound 5a: Dioctyl 2 - (((6 - bromohexanoyl)oxy)methyl) - 2 - methylmalonate (Step 5) JPEG2025523340000060.jpg2169

[0149] A mixed solution of compound 4a (440 mg, 1.18 mmol) and 6-bromohexanoic acid (230 mg, 1.18 mmol) in dichloromethane (DCM) was stirred at 0 °C, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (275 mg, 1.42 mmol) and 4-dimethylaminopyridine (DMAP) (30 mg, 0.24 mmol) were added. After stirring overnight at room temperature, the reaction solution was washed with 2N HCl (aqueous solution) and brine, and dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using ethyl acetate / hexane (EA / Hex) (1 / 25) to obtain the desired product 5a (520 mg, 0.95 mmol, 80%) as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.45 (s, 2H), 4.12 (t, J = 6.6 Hz, 4H), 3.39 (t, J = 6.6 Hz, 2H), 2.31 (t, J = 7.2 Hz, 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 = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 172.8, 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.

[0150] Compound 5b: Dioctyl 2-(((8-bromooctanoyl)oxy)methyl)-2-methylmalonate JPEG2025523340000061.jpg2071

[0151] Compound 5b was synthesized according to the procedure of general step 5 in Scheme 1. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.44 (s, 2H), 4.12 (t, J = 6.6 Hz, 4H), 3.40 (t, J = 6.6 Hz, 2H), 2.29 (t, J = 7.2 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 173.1, 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 (C 29 H 53 O6BrNa).

[0152] Compound 6a: Dioctyl 2-(4-iodobutyl)-2-methylmalonate (Step 6) JPEG2025523340000062.jpg2153

[0153] A mixed solution of compound 4b (975 mg, 2.202 mmol), PPh3 (635 mg, 2.242 mmol) and imidazole (165 mg, 2.242 mmol) in DCM was stirred at 0 °C, and then I2 (670 mg, 2.643 mmol) was added. The reaction solution was warmed to room temperature and stirred overnight. After washing with saturated Na2S2O3 (aqueous solution) and brine, the residue was dried over MgSO4 and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography using EA / Hex (1 / 25) to obtain the target product 6a (1.096 g, 90%) as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.10 (t, J = 6.6 Hz, 4H), 3.18 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 1313C NMR (150 MHz, CDCl3) δ 172.3, 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.

[0154] Compound 6b: Dioctyl 2-(6-iodohexyl)-2-methylmalonate JPEG2025523340000063.jpg2160

[0155] Compound 6b was synthesized according to the procedure of General Step 6 of Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.10 - 4.07 (m, 4H), 3.16 (t, J = 6.6 Hz, 2H), 1.84 - 1.77 (m, 4H), 1.62 - 1.57 (m, 4H), 1.40 - 1.36 (m, 2H), 1.38 (s, 3H), 1.31 - 1.20 (m, 24H), 0.87 (t, J = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.6, 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 (C 25 H 47 O4NaI).

[0156] Compound 6c: Dioctyl 2-(8-iodooctyl)-2-methylmalonate JPEG2025523340000064.jpg2065

[0157] Compound 6c was synthesized according to the procedure of General Step 6 of Scheme 1. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.11 - 4.04 (m, 4H), 3.16 (t, J = 7.2 Hz, 2H), 1.83 - 1.76 (m, 4H), 1.61 - 1.56 (m, 4H), 1.37 (s, 3H), 1.37 - 1.34 (m, 2H), 1.27 - 1.19 (m, 28H), 0.86 (t, J = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.6, 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] + 603.2876 (C 28 H 53 O4NaI).

[0158] Compound 6d: Dioctyl 2 - ethyl - 2 - (6 - iodohexyl) malonate JPEG2025523340000065.jpg2162

[0159] Compound 6d was synthesized according to the procedure of General Step 6 in Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.09 (t, J = 6.6 Hz, 4H), 3.16 (t, J = 7.2 Hz, 2H), 1.91 (q, J = 7.2 Hz, 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 = 7.2 Hz, 6H), 0.80 (t, J = 7.8 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 172.0, 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.

[0160] Compound 6e: Dioctyl 2 - (6 - iodohexyl) - 2 - propyl malonate JPEG2025523340000066.jpg2460

[0161] Compound 6e was synthesized according to the procedure of General Step 6 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.09 (t, J = 6.6 Hz, 4H), 3.16 (t, J = 6.6 Hz, 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, CDCl3) δ 172.0, 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.

[0162] Compound 6f: Dioctyl 2-(6-iodohexyl)malonate JPEG2025523340000067.jpg2059

[0163] Compound 6f was synthesized according to the procedure of General Step 6 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.13 - 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, CDCl3) δ 169.6, 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.

[0164] Compound 6g: 1-Decyl 3-octyl 2-(6-iodohexyl)-2-methylmalonate JPEG2025523340000068.jpg2163

[0165] Compound 6g was synthesized according to the procedure of General Step 6 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.09 - 4.03 (m, 4H), 3.13 (t, J = 6.6 Hz, 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, CDCl3) δ 172.5, 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.

[0166] Compound 6h: 1 - hexyl 3 - octyl 2 - (6 - iodohexyl) - 2 - methylmalonate JPEG2025523340000069.jpg2059

[0167] Compound 6h was synthesized according to the procedure of General Step 6 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.11 - 4.05 (m, 4H), 3.15 (t, J = 6.6 Hz, 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, CDCl3) δ 172.6, 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.

[0168] Compound 6i: didecyl 2 - ethyl - 2 - (4 - iodobutyl)malonate JPEG2025523340000070.jpg2163

[0169] Compound 6i was synthesized according to the procedure of General Step 6 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.10 (t, J = 6.6 Hz, 4H), 3.17 (t, J = 6.6 Hz, 2H), 1.95 - 1.91 (q, J = 6.6 Hz, 2H), 1.87 - 1.81 (m, 4H), 1.63 - 1.58 (m, 4H), 1.32 - 1.25 (m, 30H), 0.87 (t, J = 6.6 Hz, 6H), 0.82 (t, J = 6.6 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 171.8, 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.

[0170] Compound 7a: Dioctyl 2 - ((((6 - ((2 - hydroxyethyl)amino)hexanoyl)oxy)methyl) - 2 - methylmalonate (Step 7) JPEG2025523340000071.jpg2075

[0171] A mixed solution of compound 5a (490 mg, 0.89 mmol) and KI (150 mg, 0.89 mmol) in MeCN / DCM was stirred 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 organic solvent was washed with water and brine and dried over MgSO4. The crude product was purified by silica gel column chromatography using 10% MeOH / 1% NH4OH in DCM to give the desired product 7a (325 mg, 0.61 mmol, 69%) as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.44 (s, 2H), 4.12 (t, J = 6.6 Hz, 4H), 3.64 (t, J = 5.4 Hz, 2H), 2.77 (t, J = 5.4 Hz, 2H), 2.62 (t, J = 6.6 Hz, 2H), 2.30 (t, J = 7.2 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.9, 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] + 530.4044 (C 29 H 56 NO7).

[0172] Compound 7b: Dioctyl 2 - ((((8 - ((2 - hydroxyethyl)amino)octanoyl)oxy)methyl)-2 - methylmalonate JPEG2025523340000072.jpg22128

[0173] Compound 7b was synthesized according to the procedure of General Step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.44 (s, 2H), 4.12 (t, J = 6.6 Hz, 4H), 3.63 (t, J = 5.4 Hz, 2H), 2.77 (t, J = 5.4 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 7.2 Hz, 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 = 7.2 Hz, 6H). 1313C NMR (150 MHz, CDCl3) δ 173.2, 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] + 558.4363 (C 31 H 60 NO7).

[0174] Compound 7c: Heptadec-9-yl 8-((2-hydroxyethyl)amino)octanoate JPEG2025523340000073.jpg19128

[0175] A mixed solution of 8-bromooctanoic acid (0.9 g, 4.03 mmol) and heptadec-9-ol (1.04 g, 4.03 mmol) in dichloromethane (DCM) was stirred at 0 °C, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (0.93 g, 4.84 mmol) and 4-dimethylaminopyridine (DMAP) (0.1 mg, 0.8 mmol) were added. After stirring at room temperature for 4 hours, the reaction solution was washed with 2N HCl (aqueous solution) and brine, and dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using ethyl acetate / hexane (EA / Hex) (1 / 20) to obtain the precursor of compound 7c (1.58 g, 3.42 mmol, 85%).

[0176] Compound 7c was synthesized according to the procedure of general step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.84 - 4.79 (m, 1H), 3.60 (t, J = 5.4 Hz, 2H), 2.71 (t, J = 5.4 Hz, 2H), 2.56 (t, J = 7.2 Hz, 2H), 2.22 (t, J = 7.2 Hz, 2H), 1.58 - 1.56 (m, 2H), 1.46 - 1.45 (m, 6H), 1.27 - 1.21 (m, 30H), 0.83 (t, J = 7.2 Hz, 6H). 1313C NMR (150 MHz, CDCl3) δ 173.6, 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] + 442.4264 (C 27 H 56 NO3).

[0177] Compound 7d: Dioctyl 2-(6-((2-hydroxyethyl)amino)hexyl)-2-methylmalonate JPEG2025523340000074.jpg2173

[0178] Compound 7d was synthesized according to the procedure of General Step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.63 (t, J = 4.8 Hz, 2H), 2.76 (t, J = 4.8 Hz, 2H), 2.60 (t, J = 7.2 Hz, 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 13C NMR (150 MHz, CDCl3) δ 172.6, 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.

[0179] Compound 7e: Dioctyl 2-(8-((2-hydroxyethyl)amino)octyl)-2-methylmalonate JPEG2025523340000075.jpg24128

[0180] Compound 7e was synthesized according to the procedure of General Step 7 of Scheme 1. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.10 - 4.04 (m, 4H), 3.64 (t, J = 4.8 Hz, 2H), 2.76 (t, J = 4.8 Hz, 2H), 2.61 (t, J = 7.2 Hz, 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 = 7.2 Hz, 2H). 13 13C NMR (150 MHz, CDCl3) δ 172.6, 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] + 514.4461 (C 30 H 60 NO5).

[0181] Compound 7f: Dioctyl 2-(4-((3-hydroxypropyl)amino)butyl)-2-methylmalonate JPEG2025523340000076.jpg2274

[0182] Compound 7f was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 3.77 (t, J = 5.4 Hz, 2H), 2.85 (t, J = 5.4 Hz, 2H), 2.59 (t, J = 7.2 Hz, 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 = 6.6 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.5, 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] +472.3994(C 27 H 54 NO5).

[0183] Compound 7g: Dioctyl 2-(6-((3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000077.jpg2279

[0184] Compound 7g was synthesized according to the procedure of General Step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.05 (t, J = 6.6 Hz, 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), 1.71 - 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, CDCl3) δ 172.6, 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] + 500.4310(C 29 H 58 NO5).

[0185] Compound 7h: Dioctyl 2-ethyl-2-(6-((3-hydroxypropyl)amino)hexyl)malonate JPEG2025523340000078.jpg2179

[0186] Compound 7h was synthesized according to the procedure of General Step 7 of Scheme 1. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.80 (t, J = 5.4 Hz, 2H), 2.86 (t, J = 5.4 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 1.92 (q, J = 7.2 Hz, 2H), 1.89 - 1.83 (m, 2H), 1.69 - 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 = 6.6 Hz, 6H), 0.80 (t, J = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 172.1, 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] + 514.4466 (C 30 H 60 NO5).

[0187] Compound 7i: Dioctyl 2-(6-((3-hydroxypropyl)amino)hexyl)-2-propylmalonate JPEG2025523340000079.jpg2580

[0188] Compound 7i was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 3.78 (t, J = 4.8 Hz, 2H), 2.88 (t, J = 4.8 Hz, 2H), 2.60 (t, J = 7.2 Hz, 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 = 7.2 Hz, 3H), 0.86 (t, J = 6.6 Hz, 6H). 1313C NMR (150 MHz, CDCl3) δ 172.1, 65.2, 64.0, 57.7, 49.8, 49.6, 34.6, 32.3, 31.8, 30.4, 29.8, 29.5, 29.3, 29.2, 28.6, 27.1, 25.9, 24.1, 22.7, 17.5, 14.5, 14.1. MS (ESI): m / z [M+H] + 528.4623 (C 31 H 62 NO5).

[0189] Compound 7j: Dioctyl 2-(6-((4-hydroxybutyl)amino)hexyl)-2-methylmalonate JPEG2025523340000080.jpg 2284

[0190] Compound 7j was synthesized according to the procedure of General Step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.06 (t, J = 6.6 Hz, 4H), 3.55 (t, J = 5.4 Hz, 2H), 2.64 (t, J = 5.4 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 1.82 - 1.79 (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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.6, 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 (C 30 H 60 NO5).

[0191] Compound 7k: Dioctyl 2-(6-((5-hydroxypentyl)amino)hexyl)-2-methylmalonate JPEG2025523340000081.jpg 24128

[0192] Compound 7k was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 3.60 (t, J = 6.6 Hz, 2H), 2.58 (t, J = 6.6 Hz, 2H), 2.55 (t, J = 7.2 Hz, 2H), 1.83 - 1.80 (m, 2H), 1.60 - 1.54 (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 = 7.2 Hz, 6H). 13 C NMR (150 MHz, CDCl3) δ 172.6, 65.3, 62.5, 53.8, 50.0, 49.8, 35.6, 32.5, 31.8, 30.0, 29.9, 29.7, 29.2 (x2), 28.5, 27.2, 25.9, 24.3, 23.5, 22.7, 19.9, 14.1. MS (ESI): m / z [M + H] + 528.4618 (C 31 H 62 NO5).

[0193] Compound 7l: Dioctyl 2-(6-((6-hydroxyhexyl)amino)hexyl)-2-methylmalonate JPEG2025523340000082.jpg21128

[0194] Compound 7l was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.05 (t, J = 6.6 Hz, 4H), 3.58 - 3.56 (m, 2H), 2.57 - 2.53 (m, 4H), 1.81 - 1.78 (m, 2H), 1.58 - 1.42 (m, 10H), 1.35 (s, 3H), 1.34 - 1.13 (m, 30H), 0.84 (t, J = 7.2 Hz, 6H). 1313C NMR (150 MHz, CDCl3) δ 172.6, 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.

[0195] Compound 7m: Dioctyl 2-(6-((2,3-dihydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000083.jpg2074

[0196] Compound 7m was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.06 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 1172.6, 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] + 516.4252 (C 29 H 58 NO6).

[0197] Compound 7n: Dioctyl 2-(6-(((1s,4s)-4-hydroxycyclohexyl)amino)hexyl)-2-methylmalonate JPEG2025523340000084.jpg2071

[0198] Compound 7n was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 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, CDCl3) δ 172.4, 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] + 540.4614 (C 32 H 62 NO5).

[0199] Compound 7o: 1 - Decyl 3 - octyl 2 - (6 - ((3 - hydroxypropyl)amino)hexyl)-2 - methylmalonate JPEG2025523340000085.jpg2181

[0200] Compound 7o was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 3.78 (t, J = 5.4 Hz, 2H), 2.86 (t, J = 5.4 Hz, 2H), 2.59 (t, J = 7.2 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 172.6, 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, 29.26, 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 (C 31 H 62 NO5).

[0201] Compound 7p: 1-Hexyl 3-octyl 2-(6-((3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000086.jpg2276

[0202] Compound 7p was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 3.79 (t, J = 5.4 Hz, 2H), 2.86 (t, J = 5.4 Hz, 2H), 2.58 (t, J = 7.2 Hz, 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 13C NMR (150 MHz, CDCl3) δ 172.6, 65.3, 64.4, 53.8, 50.1, 49.8, 35.6, 31.8, 31.4, 30.5, 29.8, 29.7, 29.24, 29.22, 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] + 472.3987 (C 27 H 54 NO5).

[0203] Compound 7q: Dioctyl 2-(6-(((1s,4s)-4-(hydroxymethyl)cyclohexyl)amino)hexyl)-2-methylmalonate JPEG2025523340000087.jpg1879

[0204] Compound 7q was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 3.51 (d, J = 6.6 Hz, 2H), 2.71 - 2.70 (m, 1H), 2.57 (t, J = 7.2 Hz, 2H), 1.82 - 1.80 (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, CDCl3) δ 172.6, 65.8, 65.3, 54.4, 53.8, 46.8, 37.8, 35.5, 31.8, 29.8, 29.3, 29.21, 29.19, 28.5, 28.3, 27.2, 25.9, 24.3, 24.2, 22.7, 19.9, 14.1. MS (ESI): m / z [M+H] + 554.4787 (C 33 H 64 NO5).

[0205] Compound 7r: Dioctyl 2-(6-(((4-hydroxycyclohexyl)methyl)amino)hexyl)-2-methylmalonate JPEG2025523340000088.jpg2075

[0206] Compound 7r was synthesized according to the procedure of General Step 7 in Scheme 1. The title compound was obtained as a yellow oil. 11H NMR (600 MHz, CDCl3) δ 4.04 - 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 13C NMR (150 MHz, CDCl3) δ 172.5, 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, 29.8, 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] + 554.4775 (C 33 H 64 NO5).

[0207] Compound 7s: Didecyl 2 - ethyl - 2-(4 - ((3 - hydroxypropyl)amino)butyl)malonate JPEG2025523340000089.jpg21128

[0208] Compound 7s was synthesized according to the procedure of General Step 7 of Scheme 1. The title compound was obtained as a yellow oil. 1 1H NMR (600 MHz, CDCl3) δ 4.09 - 4.05 (m, 4H), 3.78 - 3.77 (m, 2H), 2.87 - 2.85 (m, 2H), 2.61 (t, J = 7.2 Hz, 2H), 1.90 (q, J = 7.8 Hz, 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 = 7.8 Hz, 3H). 1313C NMR (150 MHz, CDCl3) δ 171.9, 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] + 542.4770 (C 32 H 64 NO5).

[0209] Compound AS-CL-01: Dioctyl 2-(11-(2-hydroxyethyl)-20-methyl-20-((octyloxy)carbonyl)-3,17,21-trioxo-2,18,22-trioxa-11-azatriacontyl)-2-methylmalonate (Step 8) JPEG2025523340000090.jpg3371

[0210] A mixed solution of compound 6 (86 mg, 0.15 mmol) and KI (26 mg, 0.16 mmol) in MeCN / DCM was stirred at room temperature, and then compound 7 (75 mg, 0.14 mmol) and K2CO3 (78 mg, 0.57 mmol) were added. After stirring at 30 °C overnight, the solvent was removed, and the residue was dissolved in DCM. The organic solvent was washed with water and brine and dried over MgSO4. The crude product was purified by silica gel column chromatography using 5% MeOH / 1% NH4OH in DCM to give compound AS-CL-01 (60 mg, 0.058 mmol, 39%) as a pale yellow oil. 1 1H NMR (600 MHz, CDCl3) δ 4.43 (s, 2H), 4.12 (t, J = 6.6 Hz, 4H), 3.50 (t, J = 5.4 Hz, 2H), 2.55 (t, J = 5.4 Hz, 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 = 7.2 Hz, 12H). 1313C NMR (150 MHz, CDCl3) δ 173.2, 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 (C 58 H 108 NO 13 )。

[0211] Compound AS-CL-02: Dioctyl 2-(((6-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)hexanoyl)oxy)methyl)-2-methylmalonate JPEG2025523340000091.jpg3172

[0212] Compound AS-CL-02 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.84 - 4.81 (m, 1H), 4.42 (s, 2H), 4.10 (t, J = 6.6 Hz, 4H), 3.48 (t, J = 5.4 Hz, 2H), 2.53 (t, J = 5.4 Hz, 2H), 2.40 ((t, J = 7.2 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 173.6, 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] + 938.8015 (C 56 H 108 NO9).

[0213] Compound AS-CL-03: Dioctyl 2-(8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octyl)-2-methylmalonate JPEG2025523340000092.jpg3275

[0214] Compound AS-CL-03 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.86 - 4.82 (m, 1H), 4.10 - 4.04 (m, 4H), 3.49 (t, J = 5.4 Hz, 2H), 2.54 (t, J = 5.4 Hz, 2H), 2.41 - 2.39 (m, 4H), 2.25 (t, J = 7.2 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 173.6, 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 (x2), 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] + )894.8114 (C 55 H 108 NO7).

[0215] Compound AS-CL-04: Dioctyl 2-(8-((2-hydroxyethyl)(7-methyl-8-(octyloxy)-7-((octyloxy)carbonyl)-8-oxooctyl)amino)octyl)-2-methylmalonate JPEG2025523340000093.jpg3474

[0216] Compound AS-CL-04 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.06 - 4.05 (m, 4H), 3.47 (t, J = 4.8 Hz, 2H), 2.52 (t, J = 4.8 Hz, 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 13C NMR (150 MHz, CDCl3) δ 172.57, 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] +938.8026(C 56 H 108 NO9).

[0217] Compound AS-CL-05: Dioctyl 2-(6-(decyl(2-hydroxyethyl)amino)hexyl)-2-methylmalonate JPEG2025523340000094.jpg2978

[0218] Compound AS-CL-05 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.09 - 4.07 (m, 4H), 3.50 (t, J = 5.4 Hz, 2H), 2.55 (t, J = 5.4 Hz, 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, CDCl3) δ 172.6, 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] + 626.5725(C 38 H 76 NO5).

[0219] Compound AS-CL-06: Dioctyl 2-(6-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)hexyl)-2-methylmalonate JPEG2025523340000095.jpg3575

[0220] Compound AS-CL-06 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil.1 1H NMR (600 MHz, CDCl3) δ 4.85 - 4.83 (m, 1H), 4.07 - 4.05 (m, 4H), 3.48 (t, J = 5.4 Hz, 2H), 2.53 (t, J = 5.4 Hz, 2H), 2.41 - 2.38 (m, 4H), 2.25 (t, J = 7.2 Hz, 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 13C NMR (150 MHz, CDCl3) δ 173.6, 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] + 866.7800 (C 53 H 104 NO7).

[0221] Compound AS-CL-07: Dioctyl 2-(((6-(decyl(2-hydroxyethyl)amino)hexanoyl)oxy)methyl)-2-methylmalonate JPEG2025523340000096.jpg2880

[0222] Compound AS-CL-07 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.43 (s, 2H), 4.11 (t, J = 6.6 Hz, 4H), 3.50 (t, J = 5.4 Hz, 2H), 2.55, (t, J = 4.8 Hz, 2H), 2.43 - 2.40 (m, 4H), 2.28 (t, J = 7.2 Hz, 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 13C NMR (150 MHz, CDCl3) δ 173.0, 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] + 670.5623 (C 39 H 76 NO7).

[0223] Compound AS-CL-08: Dioctyl 2-(6-((2-hydroxyethyl)(octyl)amino)hexyl)-2-methylmalonate JPEG2025523340000097.jpg2875

[0224] Compound AS-CL-08 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.09 - 4.07 (m, 4H), 3.50 (t, J = 5.4 Hz, 2H), 2.55 (t, J = 5.4 Hz, 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 13C NMR (150 MHz, CDCl3) δ 172.6, 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] + 598.5402 (C 36 H 72 NO5).

[0225] Compound AS-CL-09: Dioctyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000098.jpg2775

[0226] Compound AS-CL-09 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)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(150MHz,CDCl3)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(x2), 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] + 640.5868(C 39 H 78 NO5).

[0227] Compound AS-CL-10: Dioctyl 2-(6-(decyl(2-hydroxyethyl)amino)hexyl)malonate JPEG2025523340000099.jpg2977

[0228] Compound AS-CL-10 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.12 - 4.05 (m, 4H), 3.49 (t, J = 5.4 Hz, 2H), 3.28 (t, J = 7.8 Hz, 1H), 2.53 (t, J = 5.4 Hz, 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 13C NMR (150 MHz, CDCl3) δ 169.7, 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] + 612.5555 (C 37 H 74 NO5).

[0229] Compound AS-CL-11: Dioctyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)malonate JPEG2025523340000100.jpg2878

[0230] Compound AS-CL-11 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.11 - 4.05 (m, 4H), 3.74 (t, J = 5.4 Hz, 2H), 3.27 (t, J = 7.8 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 169.6, 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] + 626.5719 (C 38 H 76 NO5).

[0231] Compound AS-CL-12: Dioctyl 2-(6-((3-(dimethylamino)propyl)amino)hexyl)-2-methylmalonate JPEG2025523340000101.jpg2181

[0232] Compound AS-CL-12 was synthesized according to the procedure of General Step 7 of Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.06 (t, J = 6.6 Hz, 4H), 2.81 (t, J = 6.6 Hz, 2H), 2.67 (t, J = 7.2 Hz, 2H), 2.41 (t, J = 6.6 Hz, 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 = 7.2 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.5, 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] + 527.4779 (C 31 H 63 N2O4).

[0233] Compound AS-CL-13: Dioctyl 2-(6-(decyl(3-(dimethylamino)propyl)amino)hexyl)-2-methylmalonate JPEG2025523340000102.jpg2776

[0234] Compound AS-CL-13 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)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(150MHz,CDCl3)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] + 667.6340(C 41 H 83 N2O4).

[0235] Compound AS-CL-14: Dioctyl 2-(6-(decyl(4-hydroxybutyl)amino)hexyl)-2-methylmalonate JPEG2025523340000103.jpg30128

[0236] Compound AS-CL-14 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.06 (t, J = 6.6 Hz, 4H), 3.52 - 3.51 (m, 2H), 2.42 - 2.39 (m, 6H), 1.82 - 1.79 (m, 2H), 1.62 - 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 13C NMR (150 MHz, CDCl3) δ 172.6, 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] + 654.6039 (C 40 H 80 NO5).

[0237] Compound AS-CL-15: Diundecyl 2-(6-(decyl(2-hydroxyethyl)amino)hexyl)-2-methylmalonate JPEG2025523340000104.jpg29128

[0238] Compound AS-CL-15 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.50 (t, J = 5.4 Hz, 2H), 2.55 (t, J = 5.4 Hz, 2H), 2.41 (t, J = 7.2 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 172.6, 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] + 710.6655 (C 44 H 88 NO5).

[0239] Compound AS-CL-16: Dioctyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-ethylmalonate JPEG2025523340000105.jpg 2980

[0240] Compound AS-CL-16 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.77 (t, J = 4.8 Hz, 2H), 2.62 (t, J = 4.8 Hz, 2H), 2.39 (t, J = 7.2 Hz, 4H), 1.90 (q, J = 7.2 Hz, 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 = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 172.0, 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] + 654.6036 (C 40 H 80 NO5).

[0241] Compound AS-CL-17: Dioctyl 2-(6-(decyl(2,3-dihydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000106.jpg31128

[0242] Compound AS-CL-17 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)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(150MHz,CDCl3)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] + 656.5822(C 39 H 78 NO6).

[0243] Compound AS-CL-18: Dioctyl 2-(6-(decyl((1s,4s)-4-hydroxycyclohexyl)amino)hexyl)-2-methylmalonate JPEG2025523340000107.jpg33128

[0244] Compound AS-CL-18 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 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] + 680.6183 (C 42 H 82 NO5).

[0245] Compound AS-CL-19: Dioctyl 2-(6-(4-(dimethylamino)butanamido)hexyl)-2-methylmalonate JPEG2025523340000108.jpg25128

[0246] Compound AS-CL-19 was synthesized according to the procedure of Scheme 3. A mixed solution of compound 6b (0.37 g, 0.67 mmol) and potassium phthalimide (0.62 g, 3.35 mmol) in DMF was stirred at room temperature for 3 h. After filtration and evaporation, the residue was purified by silica gel column chromatography using EA / Hex (1 / 10) to obtain compound 6b-1 (311 mg, 84%). 1 1H NMR (600 MHz, CDCl3) δ 7.84 - 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 13C NMR (150 MHz, CDCl3) δ 172.5, 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.

[0247] A mixed solution of compound 6b-1 (311 mg, 0.544 mmol) and hydrazine (50 mg, 1.63 mmol) in MeOH was stirred under reflux for 2 hours. After evaporation, the residue was dissolved in DCM and filtered. The crude compound was purified by silica gel column chromatography using 10% MeOH / DCM / 1% NH4OH to obtain compound 6b-2 (160 mg, 66%). 1 H NMR (600 MHz, CDCl3) δ 4.08 (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, CDCl3) δ 172.6, 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.

[0248] A mixed solution of 4-(dimethylamino)butanoic acid (0.071 g, 0.426 mmol), SOCl2 (0.101 g, 0.852 mmol), and 2 drops of DMF in DCM was stirred at 0 °C overnight. After evaporation, the residue was reacted with compound 6b-2 in DCM at 0 °C and Et3N was added. The reaction was monitored by TLC. After evaporation, the residue was purified by silica gel column chromatography using 10% MeOH / DCM / 1% NH4OH to obtain compound AS-CL-19 (76 mg, 45%) as a yellow oil. 1 H NMR (600 MHz, CDCl3) δ 6.41 (br.s, 1H), 4.08 (t, J = 6.6 Hz, 4H), 3.20 (q, J = 6.6 Hz, 2H), 2.31 (t, J = 7.2 Hz, 2H), 2.24 (4, J = 7.2 Hz, 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 = 7.2 Hz, 6H). 1313C NMR (150 MHz, CDCl3) δ 172.7, 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] + 555.4727 (C 32 H 63 N2O5).

[0249] Compound AS-CL-20: Dioctyl 2-(6-(decyl(5-hydroxypentyl)amino)hexyl)-2-methylmalonate JPEG2025523340000109.jpg31128

[0250] Compound AS-CL-20 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 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 13C NMR (150 MHz, CDCl3) δ 172.6, 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] + 668.6194 (C 41 H 82 NO5).

[0251] Compound AS-CL-21: Dioctyl 2-(6-((6-(decanoyloxy)hexyl)(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000110.jpg38128

[0252] Compound AS-CL-21 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)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(150MHz,CDCl3)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(C 45 H 88 NO7).

[0253] Compound AS-CL-22: Dioctyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-propylmalonate JPEG2025523340000111.jpg32128

[0254] Compound AS-CL-22 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.78 (t, J = 5.4 Hz, 2H), 2.61 (t, J = 5.4 Hz, 2H), 2.38 (t, J = 7.8 Hz, 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 = 7.2 Hz, 3H), 0.88 - 0.86 (m, 9H). 13 13C NMR (150 MHz, CDCl3) δ 172.1, 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, 29.2, 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] + 668.6180 (C 41 H 82 NO5).

[0255] Compound AS-CL-23: Dioctyl 2-(6-(dodecyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000112.jpg2878

[0256] Compound AS-CL-23 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.78 (t, J = 5.4 Hz, 2H), 2.61 (t, J = 5.4 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 172.6, 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] + 668.6181 (C 41 H 82 NO5).

[0257] Compound 9a: Decyloctyl malonate (Step 9) JPEG2025523340000113.jpg2561

[0258] A solution of malonic acid (2.5 g, 17.35 mmol) and octanol (2.26 g, 17.35 mmol) in toluene was refluxed for 4 h. After evaporation, the residue was dissolved in DCM, and then 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 mixed solution was stirred at room temperature for 4 h. After washing with 2N HCl (aqueous solution) and brine, the crude compound was purified by silica gel column chromatography using EA / Hex (1 / 50) to obtain the target compound 9a (5.65 g, 90%) as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.11 (t, J = 6.6 Hz, 4H), 3.34 (s, 2H), 1.64 - 1.59 (m, 4H), 1.32 - 1.24 (m, 24H), 0.87 - 0.85 (m, 6H). 13 13C NMR (150 MHz, CDCl3) δ 166.7, 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] + 379.2823 (C 21 H 40 O4Na).

[0259] Compound 9b: Hexyloctyl malonate JPEG2025523340000114.jpg2349

[0260] Compound 9b was synthesized according to the procedure of Step 9 in Scheme 4. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.10 (t, J = 6.6 Hz, 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, CDCl3) δ 166.7, 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] + 323.2190 (C 17 H 32 O4Na).

[0261] Compound AS-CL-24: 1-Decyl 3-octyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-methyl malonate JPEG2025523340000115.jpg2882

[0262] Compound AS-CL-24 was synthesized according to the procedure of Scheme 4 and the general procedure of Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.78 (t, J = 5.4 Hz, 2H), 2.62 (t, J = 5.4 Hz, 2H), 2.38 (t, J = 7.8 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 172.6, 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] + 668.6192 (C 41 H 82 NO5).

[0263] Compound AS-CL-25: Dioctyl 2-(6-(decyl(6-hydroxyhexyl)amino)hexyl)-2-methylmalonate JPEG2025523340000116.jpg29128

[0264] Compound AS-CL-25 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 3.61 (t, J = 6.6 Hz, 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 13C NMR (150 MHz, CDCl3) δ 172.6, 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 (C 42 H 84 NO5).

[0265] Compound AS-CL-26: Dioctyl 2-(4-(decyl(3-hydroxypropyl)amino)butyl)-2-methylmalonate JPEG2025523340000117.jpg3282

[0266] Compound AS-CL-26 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)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(150MHz,CDCl3)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] + 612.5555(C 37 H 74 NO5).

[0267] Compound AS-CL-27: 1-Hexyl 3-octyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000118.jpg2980

[0268] Compound AS-CL-27 was synthesized according to the procedure of Scheme 4 and the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.78 (t, J = 5.4 Hz, 2H), 2.61 (t, J = 5.4 Hz, 2H), 2.38 (t, J = 7.2 Hz, 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 13C NMR (150 MHz, CDCl3) δ 172.6, 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] + 612.5565 (C 37 H 74 NO5).

[0269] Compound AS-CL-28: Didecyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000119.jpg29128

[0270] Compound AS-CL-28 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 3.77 (t, J = 5.4 Hz, 2H), 2.61 (t, J = 5.4 Hz, 2H), 2.37 (t, J = 7.2 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 172.6, 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] + 696.6505 (C 43 H 86 NO5).

[0271] Compound AS-CL-29: Dioctyl 2-(6-((6-((2-hexyldecanoyl)oxy)hexyl)(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG2025523340000120.jpg3974

[0272] Compound AS-CL-29 was synthesized according to the procedure of General Step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 4.04 (t, J = 6.6 Hz, 2H), 3.77 (t, J = 5.4 Hz, 2H), 2.61 (t, J = 5.4 Hz, 2H), 2.39 - 2.36 (m, 4H), 2.31 - 2.27 (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). 1313C NMR (150 MHz, CDCl3) δ 167.8, 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] + 838.7495 (C 51 H 100 NO7).

[0273] Compound AS-CL-30: Bis(6-((6-(Decanoyloxy)hexyl)(3-hydroxypropyl)amino)hexyl) 2,2-dihexylmalonate JPEG2025523340000121.jpg4175

[0274] Compound AS-CL-30 was synthesized according to the procedure of Scheme 5.

[0275] A mixed solution of malonic acid (2 g, 19.21 mmol), 6-bromohexan-1-ol (7.65 g, 42.26 mmol), EDCI (8.1 g, 42.26 mmol) and DMAP (0.47 g, 3.84 mmol) in DCM was stirred at room temperature overnight. It was washed with 2N HCl (aqueous solution) and evaporated, and the residue was purified by silica gel column chromatography using EA / Hex (1 / 10) to obtain Compound 10 (4.18 g, 50%) as a pale yellow oil. 1 1H NMR (600 MHz, CDCl3) δ 4.14 (t, J = 6.6 Hz, 4H), 3.40 (t, J = 6.6 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 166.6, 65.3, 41.5, 33.6, 32.5, 28.2, 27.6, 24.9.

[0276] A mixed solution of compound 10 (1.07 g, 2.49 mmol) and 1-iodohexane (2.11 g, 9.95 mmol) was stirred at room temperature, and then NaH (0.2 g, 5 mmol) was added. The reaction solution was stirred at room temperature for 4 hours. It was washed with saturated NH4Cl (aqueous solution) and evaporated, and the residue was purified by silica gel column chromatography using EA / Hex (1 / 25) to obtain compound 11 (1 g, 67%) as a yellow oil. 1 1H NMR (600 MHz, CDCl3) δ 4.10 (t, J = 6.6 Hz, 4H), 3.40 (t, J = 6.6 Hz, 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 13C NMR (150 MHz, CDCl3) δ 172.0, 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+ 621.1942, and [M+Na] 4+ 623.1930 (C 27 H 50 O4Br2Na).

[0277] A mixed solution of compound 11 (160 mg, 0.267 mmol), KI (90 mg, 0.534 mmol), and 3 - amino - 1 - propanol (1 g, 13.37 mmol) in MeCN / DCM was stirred at room temperature for 4 hours. After evaporation, the residue was dissolved in DCM and washed with water and brine. After removing the solvent, the crude product was mixed with 6 - bromohexyldecanoate (212 mg, 0.633 mmol), KI (105 mg, 0.633 mmol), and K2CO3 (320 mg, 1.15 mmol) in MeCN / DCM. The mixed solution was heated at 50 °C overnight. After evaporation, the residue was purified by silica gel column chromatography using 5% MeOH / DCM / 1% NH4OH to obtain the title compound AS - Cl - 30 (180 mg, 57%) as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.06 (t, J = 6.6 Hz, 4H), 4.03 (t, J = 6.6 Hz, 4H), 3.76 (br.s, 4H), 2.60 (t, J = 5.4 Hz, 4H), 2.37 (t, J = 7.8 Hz, 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, CDCl3) δ 174.0, 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] + 1095.9487 (C 65 H 127 N2O 10 )。

[0278] Compound AS - CL - 31: Bis(6 - (decyl(3 - hydroxypropyl)amino)hexyl) 2,2 - dihexylmalonate JPEG2025523340000122.jpg2970

[0279] Compound AS-CL-31 was synthesized according to the procedure of AS-CL-30. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.09 - 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, CDCl3) δ 172.2, 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] + 868.0482 (C 53 H 107 N2O6).

[0280] Compound AS-CL-32: Dioctyl 2-(6-(decyl((1s,4s)-4-(hydroxymethyl)cyclohexyl)amino)hexyl)-2-methylmalonate JPEG2025523340000123.jpg30128

[0281] Compound AS-CL-32 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 4H), 3.59 (d, J = 7.2 Hz, 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). 1313C NMR (150 MHz, CDCl3) δ 172.6, 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] + 694.6339 (C 43 H 84 NO5).

[0282] Compound AS-CL-33: Dioctyl 2-(6-(decyl((4-hydroxycyclohexyl)methyl)amino)hexyl)-2-methylmalonate JPEG2025523340000124.jpg 30128

[0283] Compound AS-CL-33 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1 1H NMR (600 MHz, CDCl3) δ 4.07 - 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 13C NMR (150 MHz, CDCl3) δ 172.5, 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] + 694.6353 (C 43 H 84 NO5).

[0284] Compound AS-CL-34: Didecyl 2-ethyl-2-(4-((6-((2-hexyldecanoyl)oxy)hexyl)(3-hydroxypropyl)amino)butyl)malonate JPEG2025523340000125.jpg3682

[0285] Compound AS-CL-34 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 4.09 - 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 = 7.8 Hz, 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 = 7.8 Hz, 3H). 13 C NMR (150 MHz, CDCl3) δ 176.7, 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, 29.3, 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] + 880.7963 (C 54 H 106 NO7).

[0286] Compound AS-CL-35: Didecyl 2-(4-(decyl(3-hydroxypropyl)amino)butyl)-2-ethylmalonate JPEG2025523340000126.jpg2874

[0287] Compound AS-CL-35 was synthesized according to the procedure of General Step 8 of Scheme 2. The title compound was obtained as a colorless oil. 11H NMR (600 MHz, CDCl3) δ 4.09 - 4.06 (m, 4H), 3.76 (t, J = 4.8 Hz, 2H), 2.64 (br.s, 2H), 2.43 - 2.40 (m, 4H), 1.90 (q, J = 7.8 Hz, 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 = 7.8 Hz, 3H). 13 13C NMR (150 MHz, CDCl3) δ 171.9, 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] + 682.6341 (C 42 H 84 NO5).

[0288] Example 2 Preparation and Characterization of Lipid Nanoparticles Loaded with Nucleic Acids

[0289] 2.1 Preparation of mRNA-Lipid Nanoparticle (mRNA-LNP) Complexes

[0290] LNP composed of commercially available ionizable lipids was formulated at a total lipid concentration of 50 mM with MC3-, SM-102, AS-CL05, AS-CL09, AS-CL28 or AS-CL35. The LNP was formulated with (DLin-MC3-DMA, SM-102, AS-CL05, AS-CL09, AS-CL28 or AS-CL35) / DSP / cholesterol / DMG-PEG2000 in a molar ratio of 50 / 10 / 38.5 / 1.5. Each lipid was dissolved in ethanol and mixed in the organic phase according to a specific molar ratio.

[0291] LNP was prepared using NanoAssmblr TM(Precision NanoSystems) Assembled with the Ignite microfluidic mixing device. Two different mRNA targets, (i) mRNA of SARS-CoV-2 S protein derived from WT and Omicron BA.5, and (ii) mRNA encoding DENV2 serotype envelope (E) protein (DENV2 E mRNA), were encapsulated in LNP. For this purpose, before mixing with Spark NanoAssmblr TM (Precision NanoSystems) Before mixing, dissolve the mRNA in 50 mM sodium acetate buffer (pH 4.5) at a constant NP lipid:mRNA ratio of 6.5. Mix a 16 μL aliquot of the organic phase and a 32 μL aliquot of the aqueous phase and inject into 48 μL of PBS (pH 7.4). Next, dilute the LNP with an additional 96 μL of DPBS (pH 7.4) and dialyze against PBS.

[0292] 2.2 Physicochemical properties of the mRNA-LNP complex

[0293] The mRNA-LNP complex of Example 2.1 was diluted 100-fold with PBS (pH 7.4), transferred to a 384-well microplate, and the size and polydispersity index (PDI) were measured by dynamic light scattering (DLS). The encapsulation efficiency was evaluated by disrupting each complex with 1% Triton X-100 to release the mRNA cargo inside. The results are summarized in Table 2.

[0294] Table 2. Physicochemical properties of the mRNA-LNP complex of Example 2.1 JPEG2025523340000127.jpg65134

[0295] From the data in Table 2, it was confirmed that the cationic lipids of the present invention, AS-CL05, AS-CL09, AS-CL28, and AS-CL35 can each bind to other helper lipids to form nanoparticles. The physical properties such as the size and polydispersity index (PDI) of AS-CL05-LNP, AS-CL09-LNP, AS-CL28-LNP, and AS-CL35-LNP were similar to those of other LNPs composed of commercially available cationic lipids. The average particle size of the mRNA-LNP complex was 70-90 nm, and the PDI value of all complexes was less than 0.3, indicating uniform distribution in the aqueous solution and no aggregation.

[0296] 2.3 Protein Expression In Vitro and In Vivo

[0297] To evaluate mRNA transfection and in vitro protein expression, 293T cells were treated with the mRNA-LNP complexes of Example 2.1, and cell lysates were collected and analyzed by flow cytometry. Each mRNA-LNP complex was found to be able to induce S protein expression in transfected cells. Among the lipids tested, the LNP formed by the cationic lipid AS-CL09 of the present invention showed the highest protein expression level, followed by MC3, SM102, AS-CL05, AS-CL28, and AS-CL35 (Figures 1A and 2A).

[0298] After confirming that each mRNA-LNP complex could normally induce protein expression in a cell-based assay, the immunogenic effect was evaluated in BALB / c mice. Mice administered with physiological saline functioned as a negative control group. An ELISA-based method was performed to evaluate the binding of serum antibodies to the WT recombinant S protein and the BA.5 recombinant S protein. The antibodies induced by WT mRNA-AS-CL09 LNP and BA.5 mRNA-AS-CL09 LNP showed the highest binding efficiency, while the complexes formed by MC3, SM-102, AS-CL05, AS-CL28, and AS-CL35 each induced antibodies with slightly lower binding efficiency (Figures 1B and 2B).

[0299] 2.4 Serum neutralizing activity against SARS-CoV-2 variant pseudoviruses

[0300] Neutralizing activity was evaluated using sera collected from animals after vaccination. In the pseudovirus neutralization assay, sera from mice immunized with the BA.5 mRNA-AS-CL09 LNP complex showed the highest neutralizing ability. Compared with the MC3 and AS-CL05 groups, sera from mice immunized with the BA.5 mRNA-AS-CL09 LNP complex had a 9-fold higher neutralizing antibody titer against the BA.5 pseudovirus. Furthermore, the LNP containing AS-CL09 showed a better neutralizing antibody profile than the SM-102 LNP (Figure 1C and Table 3). Therefore, the type of ionizable lipid in the LNP greatly affects the final result of serum neutralizing activity, and AS-CL09 showed the best performance among the lipids tested.

[0301] Table 3. Evaluation of the half-maximal inhibitory concentration (IC 50 ) of the mRNA-LNP complexes of Example 2.1 against SARS-CoV-2 pseudoviruses JPEG2025523340000128.jpg40156

[0302] 2.5 Comparison of DENV2 E mRNA-AS-CL09 LNP complex and DENV2 E mRNA-SM-102 LNP complex

[0303] In this example, 293T cells were treated with DENV2 E protein mRNA-LNP complexes derived from AS-CL09 or SM-102 lipids, and protein expression was analyzed by flow cytometry using an in-house monoclonal antibody against the DENV2 E protein (DB32-6). As a result, the DENV2 E protein was normally expressed after transfection with either mRNA-LNP complex (Figure 3A), and the protein expression levels were similar for both mRNA-LNP complexes.

[0304] Next, BALB / c mice were immunized with the mRNA-LNP complex by intramuscular injection according to the procedure described in the "Materials and Methods" section, and serum samples were collected at week 6 to evaluate the binding activity of neutralizing antibodies by ELISA. The serum of mice inoculated with DENV2 E mRNA-AS-CL09 LNP was found to have a higher binding activity against DENV2 serotype virus compared to the serum of mice inoculated with DENV2 E mRNA-SM-102LNP (Figure 3B). The plaque reduction neutralization titer (PRNT) assay was used to evaluate the neutralizing activity of DENV antibodies against BHK-21 cells. High levels of neutralizing antibodies were detected in mice injected with DENV2 E mRNA-AS-CL09 LNP. In this sample, the PRNT50 value was approximately 24,725, which was approximately 1.4-fold higher than that of the DENV2 E mRNA-SM-102LNP serum (Figure 3C and Table 4).

[0305] Table 4. Half-maximal inhibitory concentration (IC 50 ) JPEG2025523340000129.jpg41128

[0306] Based on these results, it was concluded that the mRNA-LNP containing the AS-CL09 ionizable lipid showed higher antibody production after intramuscular vaccination compared to the mRNA-LNP complex containing SM-102.

[0307] Collectively, AS-CL05-LNP, AS-CL09-LNP, AS-CL28-LNP, and AS-CL35-LNP can efficiently deliver mRNA encoding the SARS-CoV-2 spike protein or DENV E protein for expression. Furthermore, in terms of mRNA delivery efficiency, AS-CL05-LNP, AS-CL09-LNP, AS-CL28-LNP, and AS-CL35-LNP may be comparable to other LNPs composed of commercially available lipids (i.e., MC3-, SM-102, or ALC-0315). Thus, the cationic lipids of the present invention (e.g., AS-CL05-LNP, AS-CL09-LNP, AS-CL28-LNP, and AS-CL35-LNP) are useful for the manufacture of LNPs for gene and drug delivery.

[0308] The foregoing description of the embodiments is for illustrative purposes only, and it is understood that various changes are possible for those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the present invention. Although the various embodiments of the present invention have been described above with a certain degree of particularity or with reference to one or more individual embodiments, those skilled in the art can make numerous changes to the disclosed embodiments without departing from the spirit or scope of the disclosure.

Claims

**Claim 1** A lipid having the structure of formula (I), wherein R １ is one or more hydroxyls, -CH ２ OH, or -NR ２ alkyl or cycloalkyl which may be substituted with a m and n are independently integers from 0 to 12, R ２ and R ３ are independently H, alkenyl, R ａ , -(C=O)OCH ２ R ａ , -O(C=O)R ｂ , or -(C=O)OR ｂ and R ａ is -CR'(COOR'') ２ or -CR'(COOR'')(COOR'''), and R, R ｂ , R', R'', and R''' are independently H or alkyl, lipid. **Claim 2** The lipid according to claim 1, which is selected from the group consisting of **Claim 3** A lipid having the structure of formula (II), wherein m and n are independently integers from 1 to 10, R １ and R ３ is alkyl which may be independently substituted with one or more hydroxyl groups, R ２ is H or -O(C=O)R', where R' is alkyl, lipid. **Claim 4** The lipid according to claim 3, which is selected from the group consisting of **Claim 5** A lipid having the structure of formula (III), wherein m and n are independently integers from 1 to 10, lipid. **Claim 6** R １ 、 R ２ 、 R ３ and R ４ are independently H or alkyl, The lipid according to claim 5, which is **Claim 7** A lipid nanoparticle having a hydrophilic core and an outer lipid bilayer shell formed by one or more of the lipids according to any one of claims 1 to 6. **Claim 8** The lipid nanoparticle according to claim 7, further comprising a therapeutic agent disposed within the hydrophilic core or the outer lipid bilayer shell of the lipid nanoparticle. **Claim 9** The lipid nanoparticle according to claim 8, wherein the therapeutic agent is a nucleic acid of a viral protein. **Claim 10** The lipid nanoparticle according to claim 9, wherein the nucleic acid is mRNA of the spike protein of SARS-CoV-2. **Claim 11** The lipid nanoparticle according to claim 10, wherein the nucleic acid is mRNA of the envelope protein of dengue virus. ​ ​ ​

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